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Among visceral metastatic sites, cutaneous melanoma (CM) metastasises initially to the liver in ~14-20% of cases.,Liver metastases in CM patients are associated with both poor prognosis and poor response to immunotherapy.,Histopathological growth patterns (HGPs) of liver metastases of the replacement and desmoplastic type, particularly from colorectal cancer and uveal melanoma (UM), may impart valuable biological and prognostic information.,Here, we have studied HGP in 43 CM liver metastases resected from 42 CM patients along with other prognostic factors from three institutions.,The HGPs (replacement, desmoplastic, pushing) were scored at the metastasis-liver interface with two algorithms: (1) 100% desmoplastic growth pattern (dHGP) and any (≥1%) replacement pattern (any‐rHGP) and (2) >50% dHGP, >50% rHGP or mixed (<50% dHGP and/or rHGP, pushing HGP).,For 1 patient with 2 metastases, an average was taken to obtain 1 final HGP yielding 42 observations from 42 patients. 22 cases (52%) had 100% dHGP whereas 20 (48%) had any replacement.,Cases with rHGP demonstrated vascular co‐option/angiotropism.,With the development of liver metastasis, only rHGP (both algorithms), male gender and positive resection margins predicted diminished overall survival (p = 0.00099 and p = 0.0015; p = 0.034 and p = 0.024 respectively).,On multivariate analysis, only HGP remained significant. 7 of 42 (17%) patients were alive with disease and 21 (50%) died with follow‐up after liver metastases ranging from 1.8 to 42.2 months (mean: 20.4 months, median: 19.0 months). 14 (33%) patients with previously‐treated metastatic disease had no evidence of disease at last follow up.,In conclusion, we report for the first time replacement and desmoplastic HGPs in CM liver metastases and their prognostic value, as in UM and other solid cancers.,Of particular importance, any rHGP significantly predicted diminished overall survival while 100% dHGP correlated with increased survival.,These results contribute to a better understanding of the biology of CM liver metastases and potentially may be utilised in managing patients with these metastases.
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The safety of oral sorafenib up to a maximum protocol-specified dose combined with dacarbazine in patients with metastatic, histologically confirmed melanoma was investigated in a phase I dose-escalation study and the activity of the combination was explored in an open-label phase II study.,In the phase I study, three patients were treated with sorafenib 200 mg twice daily (b.i.d.) plus 1000 mg m−2 dacarbazine on day 1 of a 21-day cycle and 15 patients had the sorafenib dose escalated to 400 mg b.i.d. without reaching the maximum tolerated dose of the combination.,In the phase II study (n=83), the overall response rate was 12% (95% CI: 6, 21): one complete and nine partial, with median response duration of 46.7 weeks.,Stable disease was the best response in 37% median duration was 13.3 weeks.,Median overall survival (OS) was 37.0 weeks (95% CI: 33.9, 46.0).,Oral sorafenib combined with dacarbazine had acceptable toxicity and some antineoplastic activity against metastatic melanoma.
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Mucosal melanoma is an aggressive melanoma with poor prognosis.,We assessed efficacy of pembrolizumab in patients with advanced mucosal melanoma in KEYNOTE-001 (NCT01295827), −002 (NCT01704287), and −006 (NCT01866319).,Patients received pembrolizumab 2 mg/kg every 3 weeks (Q3W) or 10 mg/kg Q2W or Q3W.,Response was assessed by independent central review per RECIST v1.1.,1567 patients were treated and 84 (5%) had mucosal melanoma.,Fifty-one of 84 were ipilimumab-naive.,In patients with mucosal melanoma, the objective response rate (ORR) was 19% (95% CI 11-29%), with median duration of response (DOR) of 27.6 months (range 1.1 + to 27.6).,Median progression-free survival (PFS) was 2.8 months (95% CI 2.7-2.8), with median overall survival (OS) of 11.3 months (7.7-16.6).,ORR was 22% (95% CI 11-35%) and 15% (95% CI 5-32%) in ipilimumab-naive and ipilimumab-treated patients.,Pembrolizumab provides durable antitumour activity in patients with advanced mucosal melanoma regardless of prior ipilimumab.
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Sinonasal mucosal melanoma (SNMM) comprises <1% of all melanomas and lacks well-characterised molecular markers.,Our aim was to determine the frequencies of common mutations and examine their utility as molecular markers in a large series of primary SNMMs.,SNMM patients seen at our institution from August 1991 through July 2016 were identified.,Genomic DNA was extracted from 66 formalin-fixed paraffin-embedded tumours and screened for mutations by direct sequencing.,We investigated the association of mutations with clinicopathological features and survival outcomes.,Overall, 41% (27 out of 66) of the SNMMs harboured mutations.,BRAF and KIT mutations were identified in 8% (five patients) and 5% (three patients) of SNMMs, respectively, whereas NRAS mutations were detected in 30% (20 patients) of SNMMs.,Mutation rates in these oncogenes were similar between SNMMs located in the paranasal sinuses and those in the nasal cavity (30% and 13%, respectively, P=0.09).,In a multivariate analysis, patients with negative margins had significantly better overall survival (hazard ratio 5.43, 95% confidence interval 1.44-21.85, P=0.01) and disease-specific survival (hazard ratio 21.9, 95% confidence interval 3.71-180, P=0.0004).,The mutation status of the tumours showed no association with survival outcomes.,In SNNM, mutation status does not affect survival outcomes, but NRAS mutations are relatively frequent and could be targeted in this disease by MEK inhibitors.
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NRAS mutations are the most common alterations among RAS isoforms in cutaneous melanoma, with patients harboring these aggressive tumors having a poor prognosis and low survival rate.,The main line of treatment for these patients is MAPK pathway‐targeted therapies, such as MEK inhibitors, but, unfortunately, the response to these inhibitors is variable due to tumor resistance.,Identifying genetic modifiers involved in resistance toward MEK‐targeted therapy may assist in the development of new therapeutic strategies, enhancing treatment response and patient survival.,Our whole‐genome CRISPR‐Cas9 knockout screen identified the target Kelch domain‐containing F‐Box protein 42 (FBXO42) as a factor involved in NRAS‐mutant melanoma‐acquired resistance to the MEK1/2 inhibitor trametinib.,We further show that FBXO42, an E3 ubiquitin ligase, is involved in the TAK1 signaling pathway, possibly prompting an increase in active P38.,In addition, we demonstrate that combining trametinib with the TAK1 inhibitor, takinib, is a far more efficient treatment than trametinib alone in NRAS‐mutant melanoma cells.,Our findings thus show a new pathway involved in NRAS‐mutant melanoma resistance and provide new opportunities for novel therapeutic options.
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While response rates to BRAF inhibitiors (BRAFi) are high, disease progression emerges quickly.,One strategy to delay the onset of resistance is to target anti-apoptotic proteins such as BCL-2, known to be associated with a poor prognosis.,We analyzed BCL-2 family member expression levels of 34 samples from 17 patients collected before and 10 to 14 days after treatment initiation with either vemurafenib or dabrafenib/trametinib combination.,The observed changes in mRNA and protein levels with BRAFi treatment led us to hypothesize that combining BRAFi with a BCL-2 inhibitor (the BH3-mimetic navitoclax) would improve outcome.,We tested this hypothesis in cell lines and in mice.,Pretreatment mRNA levels of BCL-2 negatively correlated with maximal tumor regression.,Early increases in mRNA levels were seen in BIM, BCL-XL, BID and BCL2-W, as were decreases in MCL-1 and BCL2A.,No significant changes were observed with BCL-2.,Using reverse phase protein array (RPPA), significant increases in protein levels were found in BIM and BID.,No changes in mRNA or protein correlated with response.,Concurrent BRAF (PLX4720) and BCL2 (navitoclax) inhibition synergistically reduced viability in BRAF mutant cell lines and correlated with down-modulation of MCL-1 and BIM induction after PLX4720 treatment.,In xenograft models, navitoclax enhanced the efficacy of PLX4720.,The combination of a selective BRAF inhibitor with a BH3-mimetic promises to be an important therapeutic strategy capable of enhancing the clinical efficacy of BRAF inhibition in many patients that might otherwise succumb quickly to de novo resistance.,Trial Registrations: ClinicalTrials.gov NCT01006980;,ClinicalTrials.gov NCT01107418;,ClinicalTrials.gov NCT01264380;,ClinicalTrials.gov NCT01248936;,ClinicalTrials.gov NCT00949702;,ClinicalTrials.gov NCT01072175
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In less than 10 years, melanoma treatment has been revolutionized with the approval of tyrosine kinase inhibitors and immune checkpoint inhibitors, which have been shown to have a significant impact on the prognosis of patients with melanoma.,The early steps of this transformation have taken place in research laboratories.,The mitogen-activated protein kinase (MAPK) pathway, phosphoinositol-3-kinase (PI3K) pathway promote the development of melanoma through numerous genomic alterations on different components of these pathways.,Moreover, melanoma cells deeply interact with the tumor microenvironment and the immune system.,This knowledge has led to the identification of novel therapeutic targets and treatment strategies.,In this review, the epidemiological features of cutaneous melanoma along with the biological mechanisms involved in its development and progression are summarized.,The current state-of-the-art of advanced stage melanoma treatment strategies and the currently available evidence of the use of predictive and prognostic biomarkers are also discussed.
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Melanoma is highly resistant to current modalities of therapy, with the extent of pigmentation playing an important role in therapeutic resistance.,Nuclear factor-κB (NF-κB) is constitutively activated in melanoma and can serve as a molecular target for cancer therapy and steroid/secosteroid action.,Cultured melanoma cells were used for mechanistic studies on NF-κB activity, utilising immunofluorescence, western blotting, EMSA, ELISA, gene reporter, and estimated DNA synthesis assays.,Formalin-fixed, paraffin-embedded specimens from melanoma patients were used for immunocytochemical analysis of NF-κB activity in situ.,Novel 20-hydroxyvitamin (20(OH)D3) and classical 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) secosteroids inhibited melanoma cell proliferation.,Active forms of vitamin D were found to inhibit NF-κB activity in nonpigmented cells, while having no effect on pigmented cells.,Treatment of nonpigmented cells with vitamin D3 derivatives inhibited NF-κB DNA binding and NF-κB-dependent reporter assays, as well as inhibited the nuclear translocation of the p65 NF-κB subunit and its accumulation in the cytoplasm.,Moreover, analysis of biopsies of melanoma patients showed that nonpigmented and slightly pigmented melanomas displayed higher nuclear NF-κB p65 expression than highly pigmented melanomas.,Classical 1,25(OH)2D3 and novel 20(OH)D3 hydroxyderivatives of vitamin D3 can target NF-κB and regulate melanoma progression in nonpigmented melanoma cells.,Melanin pigmentation is associated with the resistance of melanomas to 20(OH)D3 and 1,25(OH)2D3 treatment.
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N6-methylation of adenosine (m6A) RNA modification plays important roles in development and tumorigenesis.,The functions and mechanisms of m6A demethylases during cancer immunotherapy is still unclear.,Here we employed melanoma and colon syngeneic mouse models to study the roles of m6A demethylases ALKBH5 and FTO during anti-PD-1 antibody and GVAX vaccination therapy.,We found that ALKBH5 knockout in tumor cells enhances efficacy of immunotherapy and prolonged mouse survival.,ALKBH5 modulates target gene expression and gene splicing, leading to changes of metabolite contents, such as lactate in tumor microenvironment, which regulates suppressive lymphocytes Treg and myeloid-derived suppressor cell accumulations.,Importantly, by using ALKBH5-specific inhibitor, we observed the similar phenotype, indicating future translational application of our findings.,Although immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, many patients do not respond or develop resistance to ICB.,N6-methylation of adenosine (m6A) in RNA regulates many pathophysiological processes.,Here, we show that deletion of the m6A demethylase Alkbh5 sensitized tumors to cancer immunotherapy.,Alkbh5 has effects on m6A density and splicing events in tumors during ICB.,Alkbh5 modulates Mct4/Slc16a3 expression and lactate content of the tumor microenvironment and the composition of tumor-infiltrating Treg and myeloid-derived suppressor cells.,Importantly, a small-molecule Alkbh5 inhibitor enhanced the efficacy of cancer immunotherapy.,Notably, the ALKBH5 gene mutation and expression status of melanoma patients correlate with their response to immunotherapy.,Our results suggest that m6A demethylases in tumor cells contribute to the efficacy of immunotherapy and identify ALKBH5 as a potential therapeutic target to enhance immunotherapy outcome in melanoma, colorectal, and potentially other cancers.
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PD-L1 and PD-L2 are ligands for the PD-1 immune inhibiting checkpoint that can be induced in tumors by interferon exposure, leading to immune evasion.,This process is important for immunotherapy based on PD-1 blockade.,We examined the specific molecules involved in interferon-induced signaling that regulates PD-L1 and PD-L2 expression in melanoma cells.,These studies revealed that the interferon-gamma-JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis primarily regulates PD-L1 expression, with IRF1 binding to its promoter.,PD-L2 responded equally to interferon beta and gamma and is regulated through both IRF1 and STAT3, which bind to the PD-L2 promoter.,Analysis of biopsy specimens from patients with melanoma confirmed interferon signature enrichment and upregulation of gene targets for STAT1/STAT2/STAT3 and IRF1 in anti-PD-1-responding tumors.,Therefore, these studies map the signaling pathway of interferon-gamma-inducible PD-1 ligand expression.,Garcia-Diaz et al. performed a small hairpin RNA screen and genetic and functional studies to map the signaling pathways that result in reactive PD-L1 and PD-L2 on melanoma cells upon interferon gamma exposure.,The authors highlight the importance of the JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis for clinical responses to PD-1 blockade therapy.
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The microphthalmia-associated transcription factor (MITF) is a critical regulator of melanocyte development and differentiation.,It also plays an important role in melanoma where it has been described as a molecular rheostat that, depending on activity levels, allows reversible switching between different cellular states.,Here, we show that MITF directly represses the expression of genes associated with the extracellular matrix (ECM) and focal adhesion pathways in human melanoma cells as well as of regulators of epithelial-to-mesenchymal transition (EMT) such as CDH2, thus affecting cell morphology and cell-matrix interactions.,Importantly, we show that these effects of MITF are reversible, as expected from the rheostat model.,The number of focal adhesion points increased upon MITF knockdown, a feature observed in drug-resistant melanomas.,Cells lacking MITF are similar to the cells of minimal residual disease observed in both human and zebrafish melanomas.,Our results suggest that MITF plays a critical role as a repressor of gene expression and is actively involved in shaping the microenvironment of melanoma cells in a cell-autonomous manner.
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Cell migration underlies metastatic dissemination of cancer cells, and fast “amoeboid” migration in the invasive fronts of tumors is controlled by high levels of actomyosin contractility.,How amoeboid migration is regulated by extracellular signals and sustained over time by transcriptional changes is not fully understood.,Transforming growth factor β (TGF-β) is well known to promote epithelial-to-mesenchymal transition (EMT) and contribute to metastasis, but melanocytes are neural crest derivatives that have undergone EMT during embryonic development.,Surprisingly, we find that in melanoma, TGF-β promotes amoeboid features such as cell rounding, membrane blebbing, high levels of contractility, and increased invasion.,Using genome-wide transcriptomics, we find that amoeboid melanoma cells are enriched in a TGF-β-driven signature.,We observe that downstream of TGF-β, SMAD2 and its adaptor CITED1 control amoeboid behavior by regulating the expression of key genes that activate contractile forces.,Moreover, CITED1 is highly upregulated during melanoma progression, and its high expression is associated with poor prognosis.,CITED1 is coupled to a contractile-rounded, amoeboid phenotype in a panel of 16 melanoma cell lines, in mouse melanoma xenografts, and in 47 human melanoma patients.,Its expression is also enriched in the invasive fronts of lesions.,Functionally, we show how the TGF-β-SMAD2-CITED1 axis promotes different steps associated with progression: melanoma detachment from keratinocytes, 2D and 3D migration, attachment to endothelial cells, and in vivo lung metastatic initial colonization and outgrowth.,We propose a novel mechanism by which TGF-β-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT.,•TGF-β-SMAD promotes amoeboid migration in melanoma•Downstream of TGF-β, the adaptor CITED1 controls actomyosin contractility•Amoeboid features correlate with CITED1 levels in cell lines, xenografts, and patients•TGF-β-SMAD-CITED1 transcriptional network controls melanoma metastatic ability,TGF-β-SMAD promotes amoeboid migration in melanoma,Downstream of TGF-β, the adaptor CITED1 controls actomyosin contractility,Amoeboid features correlate with CITED1 levels in cell lines, xenografts, and patients,TGF-β-SMAD-CITED1 transcriptional network controls melanoma metastatic ability,Cantelli et al. find that, in melanoma, TGF-β-SMAD-CITED1 controls amoeboid behavior through activation of a transcriptional program.,As a result, melanoma cells detach from keratinocytes, increase their invasive potential, and efficiently colonize the lung.,This is a new function of TGF-β, independent of epithelial-to-mesenchymal transition.
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Advanced malignant melanoma represents a public health matter due to its rising incidence and aggressiveness.,Novel therapies such as immunotherapy are showing promising results with improved progression free and overall survival in melanoma patients.,However, novel targeted and immunotherapies could generate atypical patterns of response which are nowadays a big challenge since imaging criteria (ie Recist 1.1) have not been proven to be always reliable to assess response.,Radiomics and in particular texture analysis (TA) represent new quantitative methodologies which could reduce the impact of these limitations providing most robust data in support of clinical decision process.,The aim of this paper was to review the state of the art of radiomics/TA when it is applied to the imaging of metastatic melanoma patients.,Novel cancer therapies are showing promising results with improved progression free and overall survival in patients with advanced melanoma.,Radiomics represents new emerging quantitative methodologies that provide most robust data to support clinical decision in particular when atypical treatment response is present in these patients.,The aim of this paper was to review the state of the art of radiomics when it is applied to the imaging of malignant melanoma patients.
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Metastatic melanoma is one of the most aggressive and drug-resistant cancers with very poor overall survival.,Circulating melanoma cells (CMCs) were first described in 1991.,However, there is no general consensus on the clinical utility of CMC detection, largely due to conflicting results linked to the use of heterogeneous patient populations and different detection methods.,Here, we developed a new EPithelial ImmunoSPOT (EPISPOT) assay to detect viable CMCs based on their secretion of the S100 protein (S100-EPISPOT).,Then, we compared the results obtained with the S100-EPISPOT assay and the CellSearch® CMC kit using blood samples from a homogeneous population of patients with metastatic melanoma.,We found that S100-EPISPOT sensitivity was significantly higher than that of CellSearch®.,Specifically, the percentage of patients with ≥2 CMCs was significantly higher using S100-EPISPOT than CellSearch® (48% and 21%, respectively; p = 0.0114).,Concerning CMC prognostic value, only the CellSearch® results showed a significant association with overall survival (p = 0.006).,However, due to the higher sensitivity of the new S100-EPISPOT assay, it would be interesting to determine whether this functional test could be used in patients with non-metastatic melanoma for the early detection of tumor relapse and for monitoring the treatment response.
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The development of acquired drug resistance hampers the long-term success of B-RAF inhibitor (B-RAFi) therapy for melanoma patients.,Here we show V600EB-RAF copy number gain as a mechanism of acquired B-RAFi resistance in four out of twenty (20%) patients treated with B-RAFi.,In cell lines, V600EB-RAF over-expression and knockdown conferred B-RAFi resistance and sensitivity, respectively.,In V600EB-RAF amplification-driven (vs. mutant N-RAS-driven) B-RAFi resistance, ERK reactivation is saturable, with higher doses of vemurafenib down-regulating pERK and re-sensitizing melanoma cells to B-RAFi.,These two mechanisms of ERK reactivation are sensitive to the MEK1/2 inhibitor AZD6244/selumetinib or its combination with the B-RAFi vemurafenib.,In contrast to mutant N-RAS-mediated V600EB-RAF bypass, which is sensitive to C-RAF knockdown, V600EB-RAF amplification-mediated resistance functions largely independently of C-RAF.,Thus, alternative clinical strategies may potentially overcome distinct modes of ERK reactivation underlying acquired B-RAFi resistance in melanoma.
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Sorafenib monotherapy in patients with metastatic melanoma was explored in this multi-institutional phase II study.,In correlative studies the impact of sorafenib on cyclin D1 and Ki67 was assessed.,Thirty-six patients treatment-naïve advanced melanoma patients received sorafenib 400 mg p.o. twice daily continuously.,Tumor BRAFV600E mutational status was determined by routine DNA sequencing and mutation-specific PCR (MSPCR).,Immunohistochemistry (IHC) staining for cyclin D1 and Ki67 was performed on available pre- and post treatment tumor samples.,The main toxicities included diarrhea, alopecia, rash, mucositis, nausea, hand-foot syndrome, and intestinal perforation.,One patient had a RECIST partial response (PR) lasting 175 days.,Three patients experienced stable disease (SD) with a mean duration of 37 weeks.,Routine BRAFV600E sequencing yielded 27 wild-type (wt) and 6 mutant tumors, whereas MSPCR identified 12 wt and 18 mutant tumors.,No correlation was seen between BRAFV600E mutational status and clinical activity.,No significant changes in expression of cyclin D1 or Ki67 with sorafenib treatment were demonstrable in the 15 patients with pre-and post-treatment tumor samples.,Sorafenib monotherapy has limited activity in advanced melanoma patients.,BRAFV600E mutational status of the tumor was not associated with clinical activity and no significant effect of sorafenib on cyclin D1 or Ki67 was seen, suggesting that sorafenib is not an effective BRAF inhibitor or that additional signaling pathways are equally important in the patients who benefit from sorafenib.,Clinical Trials.gov NCT00119249
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In response to the dynamic intra‐tumor microenvironment, melanoma cells adopt distinct phenotypic states associated with differential expression of the microphthalmia‐associated transcription factor (MITF).,The response to hypoxia is driven by hypoxia‐inducible transcription factors (HIFs) that reprogram metabolism and promote angiogenesis.,HIF1α indirectly represses MITF that can activate HIF1α expression.,Although HIF and MITF share a highly related DNA‐binding specificity, it is unclear whether they co‐regulate subset of target genes.,Moreover, the genomewide impact of hypoxia on melanoma and whether melanoma cell lines representing different phenotypic states exhibit distinct hypoxic responses is unknown.,Here we show that three different melanoma cell lines exhibit widely different hypoxia responses with only a core 23 genes regulated in common after 12 hr in hypoxia.,Surprisingly, under hypoxia MITF is transiently up‐regulated by HIF1α and co‐regulates a subset of HIF targets including VEGFA.,Significantly, we also show that MITF represses itself and also regulates SDHB to control the TCA cycle and suppress pseudo‐hypoxia.,Our results reveal a previously unsuspected role for MITF in metabolism and the network of factors underpinning the hypoxic response in melanoma.
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Intratumoral hypoxia is a well-known feature of solid cancers and constitutes a major contributor to cancer metastasis and poor outcomes including melanoma.,Leucine-rich repeats and Ig-like domains 1 (LRIG1) participate in the aggressive progression of several tumors, where its expression is frequently decreased.,In the present study, hypoxia exposure aggravated melanoma cell invasion, migration, vasculogenic mimicry (VM), and epithelial-mesenchymal transition (EMT).,During this process, LRIG1 expression was also decreased.,Importantly, overexpression of LRIG1 notably counteracted hypoxia-induced invasion, migration, and VM, which was further augmented after LRIG1 inhibition.,Mechanism analysis corroborated that LRIG1 elevation muted hypoxia-induced EMT by suppressing E-cadherin expression and increasing N-cadherin expression.,Conversely, cessation of LRIG1 further potentiated hypoxia-triggered EMT.,Additionally, hypoxia stimulation activated the epidermal growth factor receptor (EGFR)/ERK pathway, which was dampened by LRIG1 up-regulation but further activated by LRIG1 inhibition.,More important, blocking this pathway with its antagonist erlotinib abrogated LRIG1 suppression-induced EMT, and subsequently cell invasion, migration, and VM of melanoma cells under hypoxia.,Together, these findings suggest that LRIG1 overexpression can antagonize hypoxia-evoked aggressive metastatic phenotype by suppressing cell invasion, migration, and VM via regulating EGFR/ERK-mediated EMT process.,Therefore, these findings may provide a promising target for melanoma therapy.
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Tumour spheroid experiments are routinely used to study cancer progression and treatment.,Various and inconsistent experimental designs are used, leading to challenges in interpretation and reproducibility.,Using multiple experimental designs, live-dead cell staining, and real-time cell cycle imaging, we measure necrotic and proliferation-inhibited regions in over 1000 4D tumour spheroids (3D space plus cell cycle status).,By intentionally varying the initial spheroid size and temporal sampling frequencies across multiple cell lines, we collect an abundance of measurements of internal spheroid structure.,These data are difficult to compare and interpret.,However, using an objective mathematical modelling framework and statistical identifiability analysis we quantitatively compare experimental designs and identify design choices that produce reliable biological insight.,Measurements of internal spheroid structure provide the most insight, whereas varying initial spheroid size and temporal measurement frequency is less important.,Our general framework applies to spheroids grown in different conditions and with different cell types.,Tumour spheroid experiments with real-time fluorescent ubiquitination-based cell cycle imaging are interpreted using mathematical modelling and statistical identifiability analysis.,A framework is provided to compare data obtained across different experimental designs and the experimental design choices that provide reliable biological insight are revealed.
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Tumour spheroids are common in vitro experimental models of avascular tumour growth.,Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth.,We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids.,We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region.,Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density.,Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size.,Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.
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To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche.,Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication.,Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma.,Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis.,These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer.,•Profiling of 10 human skin SCCs and matched normals via scRNA-seq, ST, and MIBI•Tumor-specific keratinocytes (TSKs) reside within a fibrovascular niche at leading edges•Distinct ligand-receptor and spatial niche associations for tumor and stromal cells.,•Subpopulation essential tumorigenic gene networks defined by in vivo CRISPR screening,Profiling of 10 human skin SCCs and matched normals via scRNA-seq, ST, and MIBI,Tumor-specific keratinocytes (TSKs) reside within a fibrovascular niche at leading edges,Distinct ligand-receptor and spatial niche associations for tumor and stromal cells.,Subpopulation essential tumorigenic gene networks defined by in vivo CRISPR screening,Integration of high-dimensional multi-omics approaches to characterize human cutaneous squamous cell carcinoma identifies a tumor-specific keratinocyte population as well as the immune infiltrates and heterogeneity at tumor leading edges.
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Cutaneous squamous cell carcinoma (cSCC) is a malignancy of epidermal keratinocytes that is responsible for approximately 20% of skin cancer-related death yearly.,We have previously compared the microRNA (miRNA) expression profile of cSCC to healthy skin and found the dysregulation of miRNAs in human cSCC.,In this study we show that miR-31 is overexpressed in cSCC (n = 68) compared to healthy skin (n = 34) and precancerous skin lesions (actinic keratosis, n = 12).,LNA in situ hybridization revealed that miR-31 was specifically up-regulated in tumor cells.,Mechanistic studies of inhibition of endogenous miR-31 in human metastatic cSCC cells revealed suppressed migration, invasion and colony forming ability, whereas overexpression of miR-31 induced these phenotypes.,These results indicate that miR-31 regulates cancer-associated phenotypes of cSCC and identify miR-31 as a potential target for cSCC treatment.
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CDKN2A is the main high-risk melanoma-susceptibility gene, but it has been poorly assessed in Latin America.,We sought to analyze CDKN2A and MC1R in patients from Latin America with familial and sporadic multiple primary melanoma (SMP) and compare the data with those for patients from Spain to establish bases for melanoma genetic counseling in Latin America.,Genet Med18 7, 727-736.,CDKN2A and MC1R were sequenced in 186 Latin American patients from Argentina, Brazil, Chile, Mexico, and Uruguay, and in 904 Spanish patients.,Clinical and phenotypic data were obtained.,Genet Med18 7, 727-736.,Overall, 24 and 14% of melanoma-prone families in Latin America and Spain, respectively, had mutations in CDKN2A.,Latin American families had CDKN2A mutations more frequently (P = 0.014) than Spanish ones.,Of patients with SMP, 10% of those from Latin America and 8.5% of those from Spain had mutations in CDKN2A (P = 0.623).,The most recurrent CDKN2A mutations were c.-34G>T and p.G101W.,Latin American patients had fairer hair (P = 0.016) and skin (P < 0.001) and a higher prevalence of MC1R variants (P = 0.003) compared with Spanish patients.,Genet Med18 7, 727-736.,The inclusion criteria for genetic counseling of melanoma in Latin America may be the same criteria used in Spain, as suggested in areas with low to medium incidence, SMP with at least two melanomas, or families with at least two cases among first- or second-degree relatives.,Genet Med18 7, 727-736.
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The influence of variants at the 9p21 locus on melanoma risk has been reported through investigation of CDKN2A variants through candidate gene approach as well as by genome wide association studies (GWAS).,In the present study we genotyped, 25 SNPs that tag 273 variants on chromosome 9p21 in 837 melanoma cases and 1154 controls from Spain.,Ten SNPs were selected based on previous associations, reported in GWAS, with either melanocytic nevi or melanoma risk or both.,The other 15 SNPs were selected to fine map the CDKN2A gene region.,All the 10 variants selected from the GWAS showed statistically significant association with melanoma risk.,Statistically significant association with melanoma risk was also observed for the carriers of the variant T-allele of rs3088440 (540 C>T) at the 3’ UTR of CDKN2A gene with an OR 1.52 (95% CI 1.14-2.04).,Interaction analysis between risk associated polymorphisms and previously genotyped MC1R variants, in the present study, did not show any statistically significant association.,Statistical significant association was observed for the interaction between phototypes and the rs10811629 (located in intron 5 of MTAP).,The strongest association was observed between the homozygous carrier of the A-allele and phototype II with an OR of 15.93 (95% CI 5.34-47.54).,Our data confirmed the association of different variants at chromosome 9p21 with melanoma risk and we also found an association of a variant with skin phototypes.
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In recent years, a significant number of studies have investigated the preventive role of vitamin D in a number of different neoplasms.,In this study, we analyze various components of the vitamin D signaling pathways in the human uveal tract and uveal melanoma, including analysis of the expression of vitamin D receptors (VDR), the activating and inactivating hydroxylases, respectively, CYP27B1 and CYP24A1, and the retinoic acid-related orphan receptors (ROR) α (RORα) and γ (RORγ) in these tissues.,We further analyzed the expression of VDR, CYP27B1, CYP24A1, and ROR in relation to melanin levels, clinical stage and prognosis.,Our study indicated that the uveal melanoma melanin level inversely correlated with VDR expression.,We further showed that vitamin D is metabolized in uveal melanoma.,This is significant because until now there has been no paper published, that would describe presence of VDR, hydroxylases CYP27B1 and CYP24A1, and RORα and RORγ in the human uveal tract and uveal melanomas.,The outcomes of our research can contribute to the development of new diagnostic and therapeutic methods in uveal tract disorders, especially in uveal melanoma.,The presented associations between vitamin D signaling elements and uveal melanoma in comparison to uveal tract encourage future clinical research with larger patients’ population.
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Clinical outcome of patients with high-risk melanoma cannot be reliably predicted on the basis of classical histopathological examination.,Our study aimed to determine in melanoma metastases a gene expression profile associated with patient survival, and to identify and validate marker(s) of poor clinical outcome.,Skin and lymph node metastases from melanoma patients (training population) were used to identify candidate prognostic marker(s) based on DNA microarray analysis.,Additional skin metastases (validation population) were used to assess the prognostic value of the first ranked gene by real-time PCR.,We performed microarray analysis in the training population and generated a list of 278 probe sets associated with a shorter survival.,We used the first ranked gene, tyrosinase-related protein 1 (TYRP1), further measured its expression in the validation population by real-time PCR and found it to be significantly correlated with distant metastasis-free survival (DMFS), overall survival (OS) and Breslow thickness.,We also found that it was fairly well conserved in the course of the disease regardless of the delay to metastasis occurrence.,Finally, although Tyrp1 protein (immunohistochemistry (IHC)) was only detected in about half of the samples, we showed that its expression also correlated with Breslow thickness.,Our data indicate that TYRP1 mRNA expression level, at least in skin metastases, is a prognostic marker for melanoma, and is particularly useful when prognostic pathology parameters at the primary lesion are lacking.,Its conserved expression further supports its use as a target for therapy.
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In the search for novel, natural melanogenesis inhibitors, a new sesquiterpene, inularin, was isolated from the flowers of Inula britannica, and the structure was determined using spectroscopic and chemical methods.,The antimelanogenic effects of inularin on B16F10 melanoma cells and zebrafish embryos were evaluated.,Inularin dose-dependently reduced melanocyte-stimulating hormone-induced melanin production and L-DOPA oxidation in B16F10 cells.,Zebrafish embryos were used to confirm the antimelanogenic activity.,Inularin significantly decreased the pigmentation of embryos compared with untreated controls.
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The novel nitrobenzoxadiazole (NBD) derivative MC3181 is endowed with remarkable therapeutic activity in mice bearing both sensitive and vemurafenib-resistant human melanoma xenografts.,Here, we report that subtoxic concentrations of this compound significantly reduced invasiveness of BRAF-V600D mutated WM115 and WM266.4 melanoma cell lines derived from the primary lesion and related skin metastasis of the same patient, respectively.,The strong antimetastatic activity of MC3181 was observed in both 2D monolayer cultures and 3D multicellular tumor spheroids, and confirmed in vivo by the significant decrease in the number of B16-F10 melanoma lung metastases in drug-treated mice.,Our data also show that MC3181 affects the lactate production in the high glycolytic WM266.4 cell line.,To unveil the MC3181 mechanism of action, we analyzed the ability of MC3181 to affect the degree of activation of different MAPK pathways, as well as the expression/activity levels of several proteins involved in angiogenesis, invasion, and survival (i.e.,AP2, MCAM/MUC18, N-cadherin, VEGF and MMP-2).,Our data disclosed both a decrease of the phospho-active form of JNK and an increased expression of the transcription factor AP2, events that occur in the very early phase of drug treatment and may be responsible of the antimetastatic effects of MC3181.
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Ultraviolet radiation (UVR) exposure is a leading cause of skin cancers and an ubiquitous environmental exposure.,However, the molecular mechanisms relating UVR exposure to melanoma is not fully understood.,We aimed to investigate if lifetime UVR exposure could be robustly associated to DNA methylation (DNAm).,We assessed DNAm in whole blood in three data sets (n = 183, 191, and 125) from the Norwegian Woman and Cancer cohort, using Illumina platforms.,We studied genome-wide DNAm, targeted analyses of CpG sites indicated in the literature, global methylation, and accelerated aging.,Lifetime history of UVR exposure (residential ambient UVR, sunburns, sunbathing vacations and indoor tanning) was collected by questionnaires.,We used one data set for discovery and the other two for replication.,One CpG site showed a genome-wide significant association to cumulative UVR exposure (cg01884057) (pnominal = 3.96e-08), but was not replicated in any of the two replication sets (pnominal ≥ 0.42).,Two CpG sites (cg05860019, cg00033666) showed suggestive associations with the other UVR exposures.,We performed extensive analyses of the association between long-term UVR exposure and DNAm.,There was no indication of a robust effect of past UVR exposure on DNAm.
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In Australia, more money is spent on skin cancer than any other malignancy.,Despite this, the mortality rate of melanoma, the deadliest form, has steadily increased over the past 50 years.,Diagnostic imprecision and a lack of complimentary molecular biomarkers are partially responsible for this lack of progress.,Whole-microRNAome profiling was performed on plasma samples from 32 patients with histologically confirmed melanoma and 16 normal controls.,A classification algorithm was trained on these data and independently validated on multiple previously published microRNA data sets, representing (i) melanoma patient- and normal-blood, (ii) melanoma and nevi biopsy tissue, and (iii) cell lines and purified exosomes.,38 circulating microRNAs had biologically and statistically significant differences between melanoma and normal plasma samples (MEL38).,A support vector machine algorithm, trained on these markers, showed strong independent classification accuracy (AUC 0.79-0.94).,A majority of MEL38 genes have been previously associated with melanoma and are known regulators of angiogenesis, metastasis, tumour suppression, and treatment resistance.,MEL38 exhibits disease state specificity and robustness to platform and specimen-type variation.,It has potential to become an objective diagnostic biomarker and improve the precision and accuracy of melanoma detection and monitoring.
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BAP1 has been shown to be a target of both somatic alteration in high-risk ocular melanomas (OM) and germline inactivation in a few individuals from cancer-prone families.,These findings suggest that constitutional BAP1 changes may predispose individuals to metastatic OM and that familial permeation of deleterious alleles could delineate a new cancer syndrome.,To characterize BAP1's contribution to melanoma risk, we sequenced BAP1 in a set of 100 patients with OM, including 50 metastatic OM cases and 50 matched non-metastatic OM controls, and 200 individuals with cutaneous melanoma (CM) including 7 CM patients from CM-OM families and 193 CM patients from CM-non-OM kindreds.,Germline BAP1 mutations were detected in 4/50 patients with metastatic OM and 0/50 cases of non-metastatic OM (8% vs. 0%, p = 0.059).,Since 2/4 of the BAP1 carriers reported a family history of CM, we analyzed 200 additional hereditary CM patients and found mutations in 2/7 CM probands from CM-OM families and 1/193 probands from CM-non-OM kindreds (29% vs.,0.52%, p = .003).,Germline mutations co-segregated with both CM and OM phenotypes and were associated with the presence of unique nevoid melanomas and highly atypical nevoid melanoma-like melanocytic proliferations (NEMMPs).,Interestingly, 7/14 germline variants identified to date reside in C-terminus suggesting that the BRCA1 binding domain is important in cancer predisposition.,Germline BAP1 mutations are associated with a more aggressive OM phenotype and a recurrent phenotypic complex of cutaneous/ocular melanoma, atypical melanocytic proliferations and other internal neoplasms (ie.,COMMON syndrome), which could be a useful clinical marker for constitutive BAP1 inactivation.
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Melanoma cells driven by mutant B-RAF are highly resistant to chemotherapeutic treatments.,Recent Phase 1 results with PLX4032/RG7204/Vemurafenib, which selectively inhibits B-RAF/MEK/ERK1/2 signaling in mutant B-RAF cells, has given encouragement to this struggling field.,Nearly all patients in the phase 1-3 studies saw at least some response and the overall response rates were in between 81 and 48%.,However, despite initial tumor shrinkage, most responders in the trial experienced tumor relapse over time.,These findings indicate that both intrinsic and acquired resistance may affect the clinical efficacy of PLX4032.,It is critical to optimize PLX4032 activity to improve response rates and understand why some patients with the B-RAF mutation do not respond.,We have previously shown that the stemness factor, Forkhead box D3 (FOXD3), is up-regulated following inhibition of B-RAF-MEK signaling in mutant B-RAF melanoma cells.,Here, we show that up-regulation of FOXD3 following treatment with PLX4032 and PLX4720 (the non-clinical tool compound for PLX4032) confers resistance to cell death.,Small interfering RNA (siRNA)-mediated knockdown of FOXD3 significantly enhanced the cell death response after PLX4032/4720 treatment in mutant B-RAF melanoma cell lines.,Additionally, up-regulation of FOXD3 after PLX4720 treatment was attenuated in non-adherent conditions and correlated with enhanced cell death.,Ectopic expression of FOXD3 in non-adherent cells significantly reduced cell death in response to PLX4720 treatment.,Together, these data indicate that up-regulation of FOXD3 is an adaptive response to RAF inhibitors that promotes a state of drug resistance.
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PD-L1 is expressed by a subset of patients with metastatic melanoma (MM) with an unfavorable outcome.,Its expression is increased in cells resistant to BRAF or MEK inhibitors (BRAFi or MEKi).,However, the function and regulation of expression of PD-L1 remain incompletely understood.,After generating BRAFi- and MEKi-resistant cell lines, we observed marked up-regulation of PD-L1 expression.,These cells were characterized by a common gene expression profile with up-regulation of genes involved in cell movement.,Consistently, in vitro they showed significantly increased invasive properties.,This phenotype was controlled in part by PD-L1, as determined after silencing the molecule.,Up-regulation of PD-L1 was due to post-transcriptional events controlled by miR-17-5p, which showed an inverse correlation with PD-L1 mRNA.,Direct binding between miR-17-5p and the 3’-UTR of PD-L1 mRNA was demonstrated using luciferase reporter assays.,In a cohort of 80 BRAF-mutated MM patients treated with BRAFi or MEKi, constitutive expression of PD-L1 in the absence of immune infiltrate, defined the patient subset with the worst prognosis.,Furthermore, PD-L1 expression increased in tissue biopsies after the metastatic lesions became resistant to BRAFi or MEKi.,Lastly, plasmatic miR-17-5p levels were higher in patients with PD-L1+ than PD-L1- lesions.,In conclusion, our findings indicate that PD-L1 expression induces a more aggressive behavior in melanoma cells.,We also show that PD-L1 up-regulation in BRAFi or MEKi-resistant cells is partly due to post-transcriptional mechanisms that involve miR-17-5p, suggesting that miR-17-5p may be used as a marker of PD-L1 expression by metastatic lesions and ultimately a predictor of responses to BRAFi or MEKi.
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MiRNAs are increasingly recognized as biomarkers for the diagnosis of cancers where they are profiled from tumor tissue (intracellular miRNAs) or serum/plasma samples (extracellular miRNAs).,To improve detection of reliable biomarkers from blood samples, we first compiled a healthy reference miRNome and established a well-controlled analysis pipeline allowing for standardized quantification of circulating miRNAs.,Using whole miRNome and custom qPCR arrays, miRNA expression profiles were analyzed in 126 serum, whole blood and tissue samples of healthy volunteers and melanoma patients and in primary melanocyte and keratinocyte cell lines.,We found characteristic signatures with excellent prognostic scores only in late stage but not in early stage melanoma patients.,Upon comparison of melanoma tissue miRNomes with matching serum samples, several miRNAs were identified to be exclusively tissue-derived (miR-30b-5p, miR-374a-5p and others) while others had higher expression levels in serum (miR-3201 and miR-122-5p).,Here we have compiled a healthy and widely applicable miRNome from serum samples and we provide strong evidence that levels of cell-free miRNAs only change significantly at later stages of melanoma progression, which has serious implications for miRNA biomarker studies in cancer.
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Metastatic melanoma is a malignant cancer with generally poor prognosis, with no targeted chemotherapy.,To identify epigenetic changes related to melanoma, we have determined genome-wide methylated CpG island distributions by next-generation sequencing.,Melanoma chromosomes tend to be differentially methylated over short CpG island tracts.,CpG islands in the upstream regulatory regions of many coding and noncoding RNA genes, including, for example, TERC, which encodes the telomerase RNA, exhibit extensive hypermethylation, whereas several repeated elements, such as LINE 2, and several LTR elements, are hypomethylated in advanced stage melanoma cell lines.,By using CpG island demethylation profiles, and by integrating these data with RNA-seq data obtained from melanoma cells, we have identified a co-expression network of differentially methylated genes with significance for cancer related functions.,Focused assays of melanoma patient tissue samples for CpG island methylation near the noncoding RNA gene SNORD-10 demonstrated high specificity.
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The prognosis of cutaneous melanoma (CM) differs for patients with identical clinico-pathological stage, and no molecular markers discriminating the prognosis of stage III individuals have been established.,Genome-wide alterations in DNA methylation are a common event in cancer.,This study aimed to define the prognostic value of genomic DNA methylation levels in stage III CM patients.,Overall level of genomic DNA methylation was measured using bisulfite pyrosequencing at three CpG sites (CpG1, CpG2, CpG3) of the Long Interspersed Nucleotide Element-1 (LINE-1) sequences in short-term CM cultures from 42 stage IIIC patients.,The impact of LINE-1 methylation on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analysis.,Hypomethylation (i.e., methylation below median) at CpG2 and CpG3 sites significantly associated with improved prognosis of CM, CpG3 showing the strongest association.,Patients with hypomethylated CpG3 had increased OS (P = 0.01, log-rank = 6.39) by Kaplan-Meyer analysis.,Median OS of patients with hypomethylated or hypermethylated CpG3 were 31.9 and 11.5 months, respectively.,The 5 year OS for patients with hypomethylated CpG3 was 48% compared to 7% for patients with hypermethylated sequences.,Among the variables examined by Cox regression analysis, LINE-1 methylation at CpG2 and CpG3 was the only predictor of OS (Hazard Ratio = 2.63, for hypermethylated CpG3; 95% Confidence Interval: 1.21-5.69; P = 0.01).,LINE-1 methylation is identified as a molecular marker of prognosis for CM patients in stage IIIC.,Evaluation of LINE-1 promises to represent a key tool for driving the most appropriate clinical management of stage III CM patients.
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Cancer is thought to arise through the accumulation of genomic aberrations evolving under Darwinian selection.,However, it remains unclear when the aberrations associated with metastasis emerge during tumor evolution.,Uveal melanoma (UM) is the most common primary eye cancer and frequently leads to metastatic death, which is strongly linked to BAP1 mutations.,Accordingly, UM is ideally suited for studying the clonal evolution of metastatic competence.,Here we analyze sequencing data from 151 primary UM samples using a customized bioinformatic pipeline, to improve detection of BAP1 mutations and infer the clonal relationships among genomic aberrations.,Strikingly, we find BAP1 mutations and other canonical genomic aberrations usually arise in an early punctuated burst, followed by neutral evolution extending to the time of clinical detection.,This implies that the metastatic proclivity of UM is “set in stone” early in tumor evolution and may explain why advances in primary treatment have not improved survival.,Uveal melanoma (UM), the most common primary eye cancer, is strongly linked to mutations in the tumor suppressor BAP1.,Here, the authors analyze 151 primary UM samples to find that BAP1 and other canonical genomic aberrations arise in an early punctuated burst followed by neutral tumor evolution.
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Since deterioration of the immune apparatus is closely associated with cancer, we examined the effect of aging on the growth and metastasis of intraocular melanomas in mice.,Murine B16LS9 melanoma cells were transplanted into the posterior compartment of the eye (vitreous chamber) and intraocular tumor growth and development of liver metastases were evaluated in young (8-10 weeks of age) and old (>18 months of age) mice.,Liver metastases were also induced by intrasplenic injection of melanoma cells.,Natural killer (NK) cells from the livers of mice harboring liver metastases were evaluated in vitro for their cytolytic activity.,Tumors grew more rapidly in the eyes of young mice than old mice, yet old mice developed significantly more liver metastases.,Increased liver metastasis in old mice was evident even when melanoma cells were injected intrasplenically as a means of bypassing the influence of the ocular immunosuppressive environment.,Increased liver metastases in old mice correlated with reduced cytolytic activity of liver NK cells.,Lethally irradiated young mice reconstituted with bone marrow from old donors developed significantly more liver metastases than young mice reconstituted with bone marrow from young donors, indicating that bone marrow-derived cells were the root cause of the heightened development of metastases in old mice.,Aging affects the growth and metastasis of intraocular melanomas.,Even though intraocular melanomas grow slower in old mice, the development of liver metastases is exacerbated and correlates with a reduction in liver NK cell activity in the old mouse.
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Neoangiogenesis is a recognized hallmark of cancer, granting tumor cells to dispose of metabolic substrates through a newly created vascular supply.,Neoangiogenesis was also confirmed in melanoma, where vascular proliferation is associated with increased aggressiveness and poorer prognosis.,Furthermore, melanoma cells show the so-called vascular mimicry, consisting in the assumption of endothelial-like features inducing the expression of pro-angiogenic receptors and ligands, which take part in the interplay with extracellular matrix (ECM) components and are potentiated by the ECM remodeling and the barrier molecule junction alterations that characterize the metastatic phase.,Although neoangiogenesis was biologically proven and clinically associated with worse outcomes in melanoma patients, in the past anti-angiogenic therapies were employed with poor improvement of the already unsatisfactory results associated with chemotherapic agents.,Among the novel therapies of melanoma, immunotherapy has led to previously unexpected outcomes of treatment, yet there is a still strong need for potentiating the results, possibly by new regimens of combination therapies.,Molecular models in many cancer types showed mutual influences between immune responses and vascular normalization.,Recently, clinical trials are investigating the efficacy of the association between anti-angiogenetic agents and immune-checkpoint inhibitors to treat advanced stage melanoma.,This paper reviews the biological bases of angiogenesis in melanoma and summarizes the currently available clinical data on the use of anti-angiogenetic compounds in melanoma.
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Dysregulation of the microRNA (miRNA) network is a typical feature of many cancers.,However, the key specific miRNAs involved in uveal melanoma carcinogenesis are largely unknown.,RT-qPCR was used to detected miR-652 expression in uveal melanoma tissues and cell lines. miR-652 inhibitor was transfected into uveal melanoma cells to decrease miR-652 expression and determine the biological role of miR-652 by CCK-8 and wound healing assays.,Bioinformatic analysis and dual luciferase reporter assay were used to predict and validate the target gene of miR-652.,HOXA9 siRNA was transfected into cells to confirm that miR-652 relies on regulation of HOXA9 to regulate cell proliferation and migration.,RT-qPCR showed that miR-652 was overexpressed in uveal melanoma cell lines (MUM-2B, MEL270) compared with melanocyte cells (ARPE-19).,Overexpression of miR-652 was also observed in uveal melanoma compared to paired non-tumor tissues.,Downregulation of miR-652 inhibited the cell proliferation ability and migration ability of uveal melanoma cells.,Using bioinformatic analysis, HOXA9 was found to be a potential target gene of miR-652.,The direct regulation of HOXA9 by miR-652 was experimentally validated in uveal melanoma cells by dual luciferase assay and Western blotting.,We also observed that miR-652 promoted HIF-1α signaling via repression of HOXA9 in uveal melanoma cells.,Silencing of HOXA9 attenuated the miR-652 inhibitor decreased cell growth rate and decreased migration ability in uveal melanoma cells.,Our data demonstrate an oncogenic role of miR-652 in uveal melanoma, showing that miR-652 may be a useful biomarker for prediction of prognosis for patients with uveal melanoma.
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Uveal melanoma (UM) is uniformly refractory to all available systemic chemotherapies, thus creating an urgent need for novel therapeutics.,In this study, we investigated the sensitivity of UM cells to ICG-001, a small molecule reported to suppress the Wnt/β-catenin-mediated transcriptional program.,We used a panel of UM cell lines to examine the effects of ICG-001 on cellular proliferation, migration, and gene expression.,In vivo efficacy of ICG-001 was evaluated in a UM xenograft model.,ICG-001 exerted strong antiproliferative activity against UM cells, leading to cell cycle arrest, apoptosis, and inhibition of migration.,Global gene expression profiling revealed strong suppression of genes associated with cell cycle proliferation, DNA replication, and G1/S transition.,Gene set enrichment analysis revealed that ICG-001 suppressed Wnt, mTOR, and MAPK signaling.,Strikingly, ICG-001 suppressed the expression of genes associated with UM aggressiveness, including CDH1, CITED1, EMP1, EMP3, SDCBP, and SPARC.,Notably, the transcriptomic footprint of ICG-001, when applied to a UM patient dataset, was associated with better clinical outcome.,Lastly, ICG-001 exerted anticancer activity against a UM tumor xenograft in mice.,Using in vitro and in vivo experiments, we demonstrate that ICG-001 has strong anticancer activity against UM cells and suppresses transcriptional programs critical for the cancer cell.,Our results suggest that ICG-001 holds promise and should be examined further as a novel therapeutic agent for UM.
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Next generation sequencing of uveal melanoma (UM) samples has identified a number of recurrent oncogenic or loss-of-function mutations in key driver genes including: GNAQ, GNA11, EIF1AX, SF3B1 and BAP1.,To search for additional driver mutations in this tumor type we carried out whole-genome or whole-exome sequencing of 28 tumors or primary cell lines.,These samples have a low mutation burden, with a mean of 10.6 protein changing mutations per sample (range 0 to 53).,As expected for these sun-shielded melanomas the mutation spectrum was not consistent with an ultraviolet radiation signature, instead, a BRCA mutation signature predominated.,In addition to mutations in the known UM driver genes, we found a recurrent mutation in PLCB4 (c.G1888T, p.D630Y, NM_000933), which was validated using Sanger sequencing.,The identical mutation was also found in published UM sequence data (1 of 56 tumors), supporting its role as a novel driver mutation in UM.,PLCB4 p.D630Y mutations are mutually exclusive with mutations in GNA11 and GNAQ, consistent with PLCB4 being the canonical downstream target of the former gene products.,Taken together these data suggest that the PLCB4 hotspot mutation is similarly a gain-of-function mutation leading to activation of the same signaling pathway, promoting UM tumorigenesis.
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Melanoma is notable for its metastatic propensity, lethality in the advanced setting, and association with ultraviolet (UV) exposure early in life1.,To obtain a comprehensive genomic view of melanoma, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA.,A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-UV exposed hairless skin of the extremities (3 and 14 per Mb genome), intermediate in those originating from hair-bearing skin of the trunk (range = 5 to 55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb).,Analysis of whole-genome sequence data identified PREX2 - a PTEN-interacting protein and negative regulator of PTEN in breast cancer2 - as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas.,PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumor formation of immortalized human melanocytes in vivo.,Thus, whole-genome sequencing of human melanoma tumors revealed genomic evidence of UV pathogenesis and discovered a new recurrently mutated gene in melanoma.
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We sequenced 8 melanoma exomes to identify novel somatic mutations in metastatic melanoma.,Focusing on the MAP3K family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9.,Structural modelling predicts that mutations in the kinase domain may affect the activity and regulation of MAP3K5/9 protein kinases.,The position of the mutations and loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely inactivating.,In vitro kinase assay shows reduction in kinase activity in MAP3K5 I780F and MAP3K9 W333X mutants.,Overexpression of MAP3K5 or MAP3K9 mutant in HEK293T cells reduces phosphorylation of downstream MAP kinases.,Attenuation of MAP3K9 function in melanoma cells using siRNA leads to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.
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Actinic keratoses are dysplastic proliferations of keratinocytes with potential for malignant transformation.,Clinically, actinic keratoses present as macules, papules, or hyperkeratotic plaques with an erythematous background that occur on photoexposed areas.,At initial stages, they may be better identified by palpation rather than by visual inspection.,They may also be pigmented and show variable degrees of infiltration; when multiple they then constitute the so-called field cancerization.,Their prevalence ranges from 11% to 60% in Caucasian individuals above 40 years.,Ultraviolet radiation is the main factor involved in pathogenesis, but individual factors also play a role in the predisposing to lesions appearance.,Diagnosis of lesions is based on clinical and dermoscopic examination, but in some situations histopathological analysis may be necessary.,The risk of transformation into squamous cell carcinoma is the major concern regarding actinic keratoses.,Therapeutic modalities for actinic keratoses include topical medications, and ablative and surgical methods; the best treatment option should always be individualized according to the patient.
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Individual skin health attitudes are influenced by various factors, including public education campaigns, mass media, family, and friends.,Evidence-based, educative information materials assist communication and decision-making in doctor-patient interactions.,The present study aims at assessing the prevailing use of skin health information material and sources and their impact on skin health knowledge, motives to tan, and sun protection.,We conducted a questionnaire survey among a representative sample of Austrian residents.,Print media and television were perceived as the two most relevant sources for skin health information, whereas the source physician was ranked third.,Picking the information source physician increased participants’ skin health knowledge (p = 0.025) and sun-protective behavior (p < 0.001).,The study results highlight the demand for targeted health messages to attain lifestyle changes towards photo-protective habits.,Providing resources that encourage pro-active counseling in every-day doctor-patient communication could increase skin health knowledge and sun-protective behavior, and thus, curb the rise in skin cancer incidence rates.
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Melanoma brain metastases (MBMs) are a challenging clinical problem with high morbidity and mortality.,Although first-line dabrafenib-trametinib and ipilimumab-nivolumab have similar intracranial response rates (50%-55%), central nervous system (CNS) resistance to BRAF-MEK inhibitors (BRAF-MEKi) usually occurs around 6 months, and durable responses are only seen with combination immunotherapy.,We sought to investigate the utility of ipilimumab-nivolumab after MBM progression on BRAF-MEKi and identify mechanisms of resistance.,Patients who received first-line ipilimumab-nivolumab for MBMs or second/third line ipilimumab-nivolumab for intracranial metastases with BRAFV600 mutations with prior progression on BRAF-MEKi and MRI brain staging from March 1, 2015 to June 30, 2018 were included.,Modified intracranial RECIST was used to assess response.,Formalin-fixed paraffin-embedded samples of BRAFV600 mutant MBMs that were naïve to systemic treatment (n=18) or excised after progression on BRAF-MEKi (n=14) underwent whole transcriptome sequencing.,Comparative analyses of MBMs naïve to systemic treatment versus BRAF-MEKi progression were performed.,Twenty-five and 30 patients who received first and second/third line ipilimumab-nivolumab, were included respectively.,Median sum of MBM diameters was 13 and 20.5 mm for the first and second/third line ipilimumab-nivolumab groups, respectively.,Intracranial response rate was 75.0% (12/16), and median progression-free survival (PFS) was 41.6 months for first-line ipilimumab-nivolumab.,Efficacy of second/third line ipilimumab-nivolumab after BRAF-MEKi progression was poor with an intracranial response rate of 4.8% (1/21) and median PFS of 1.3 months.,Given the poor activity of ipilimumab-nivolumab after BRAF-MEKi MBM progression, we performed whole transcriptome sequencing to identify mechanisms of drug resistance.,We identified a set of 178 differentially expressed genes (DEGs) between naïve and MBMs with progression on BRAF-MEKi treatment (p value <0.05, false discovery rate (FDR) <0.1).,No distinct pathways were identified from gene set enrichment analyses using Kyoto Encyclopedia of Genes and Genomes, Gene Ontogeny or Hallmark libraries; however, enrichment of DEG from the Innate Anti-PD1 Resistance Signature (IPRES) was identified (p value=0.007, FDR=0.03).,Second-line ipilimumab-nivolumab for MBMs after BRAF-MEKi progression has poor activity.,MBMs that are resistant to BRAF-MEKi that also conferred resistance to second-line ipilimumab-nivolumab showed enrichment of the IPRES gene signature.
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The recent advent of targeted and immune-based therapies has revolutionized the treatment of melanoma and transformed outcomes for patients with metastatic disease.,The majority of patients develop resistance to the current standard-of-care targeted therapy, dual BRAF and MEK inhibition, prompting evaluation of a new combination incorporating a CDK4/6 inhibitor.,Based on promising preclinical data, combined BRAF, MEK and CDK4/6 inhibition has recently entered clinical trials for the treatment of BRAFV600 melanoma.,Interestingly, while BRAF- and MEK-targeted therapy was initially developed on the basis of potent tumor-intrinsic effects, it was later discovered to have significant immune-potentiating activity.,Recent studies have also identified immune-related impacts of CDK4/6 inhibition, though these are less well defined and can be both immune-potentiating and immune-inhibitory.,BRAFV600 melanoma patients are also eligible to receive immunotherapy, specifically checkpoint inhibitors against PD-1 and CTLA-4.,The immunomodulatory activity of BRAF/MEK-targeted therapies has prompted interest in combination therapies incorporating these with immune checkpoint inhibitors, however recent clinical trials investigating this approach have produced variable results.,Here, we summarize the immunomodulatory effects of BRAF, MEK and CDK4/6 inhibitors, shedding light on the prospective utility of this combination alone and in conjunction with immune checkpoint blockade.,Understanding the mechanisms that underpin the clinical efficacy of these available therapies is a critical step forward in optimizing novel combination and scheduling approaches to combat melanoma and improve patient outcomes.
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Acral and mucosal melanomas are extremely rare in Caucasians; however, they are the predominant melanoma subtypes in Asians and other non-Caucasian populations.,Acral and mucosal melanomas share many clinicopathological features, including aggressive phenotypes, similar genetic landscapes, and grim prognoses.,In spite of advances in melanoma management, patients with acral and mucosal melanomas show limited benefit from current therapies.,The rarity of these subtypes of melanoma is a significant factor contributing to the poor understanding of these pathological subtypes and the lack of effective interventions.,Furthermore, the mechanisms contributing to disparities between different types of melanoma remain largely unclear.,Herein, we comprehensively review current knowledge on the clinicopathological characteristics and mutational landscapes of acral and mucosal melanomas, as well as providing an overview of current therapies for patients with these aggressive melanoma subtypes, focusing on available immunotherapeutic interventions.,We also discuss pathological differences between different melanoma subtypes and summarize current knowledge on melanoma disparities between Asians and Caucasians.,Finally, we discuss emerging immunotherapeutic strategies for the treatment of acral and mucosal melanomas, focusing on combination therapies with immune checkpoint inhibitors.,Unraveling the unique features of acral and mucosal melanomas is key for their early diagnosis and for the development of effective therapies.
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Metastasis is the most lethal stage of cancer progression.,The present study aimed to investigate the underlying molecular mechanisms of melanoma metastasis using bioinformatics.,Using the microarray dataset GSE8401 from the Gene Expression Omnibus database, which included 52 biopsy specimens from patients with melanoma metastasis and 31 biopsy specimens from patients with primary melanoma, differentially expressed genes (DEGs) were identified, subsequent to data preprocessing with the affy package, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses.,A protein-protein interaction (PPI) network was constructed.,Mutated genes were analyzed with 80 mutated cases with melanoma from The Cancer Genome Atlas.,The overall survival of key candidate DEGs, which were within a filtering of degree >30 criteria in the PPI network and involved three or more KEGG signaling pathways, and genes with a high mutation frequency were delineated.,The expression analysis of key candidate DEGs, mutant genes and their associated genes were performed on UALCAN.,Of the 1,187 DEGs obtained, 505 were upregulated and 682 were downregulated.,‘Extracellular exosome’ processes, the ‘amoebiasis’ pathway, the ‘ECM-receptor interaction’ pathway and the ‘focal adhesion’ signaling pathway were significantly enriched and identified as important processes or signaling pathways.,The overall survival analysis of phosphoinositide-3-kinase regulator subunit 3 (PIK3R3), centromere protein M (CENPM), aurora kinase A (AURKA), laminin subunit α 1 (LAMA1), proliferating cell nuclear antigen (PCNA), adenylate cyclase 1 (ADCY1), BUB1 mitotic checkpoint serine/threonine kinase (BUB1), NDC80 kinetochore complex component (NDC80) and protein kinase C α (PRKCA) in DEGs was statistically significant.,Mutation gene analysis identified that BRCA1-associated protein 1 (BAP1) had a higher mutation frequency and survival analysis, and its associated genes in the BAP1-associated PPI network, including ASXL transcriptional regulator 1 (ASXL1), proteasome 26S subunit, non-ATPase 3 (PSMD3), proteasome 26S subunit, non ATPase 11 (PSMD11) and ubiquitin C (UBC), were statistically significantly associated with the overall survival of patients with melanoma.,The expression levels of PRKCA, BUB1, BAP1 and ASXL1 were significantly different between primary melanoma and metastatic melanoma.,Based on the present study, ‘extracellular exosome’ processes, ‘amoebiasis’ pathways, ‘ECM-receptor interaction’ pathways and ‘focal adhesion’ signaling pathways may be important in the formation of metastases from melanoma.,The involved genes, including PIK3R3, CENPM, AURKA, LAMA1, PCNA, ADCY1, BUB1, NDC80 and PRKCA, and mutation associated genes, including BAP1, ASXL1, PSMD3, PSMD11 and UBC, may serve important roles in metastases of melanoma.
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We previously identified PRAME as a biomarker for metastatic risk in Class 1 uveal melanomas.,In this study, we sought to define a threshold value for positive PRAME expression (PRAME+) in a large dataset, identify factors associated with PRAME expression, evaluate the prognostic value of PRAME in Class 2 uveal melanomas, and determine whether PRAME expression is associated with aberrant hypomethylation of the PRAME promoter.,Among 678 samples analyzed by qPCR, 498 (73.5%) were PRAME- and 180 (26.5%) were PRAME+.,Class 1 tumors were more likely to be PRAME-, whereas Class 2 tumors were more likely to be PRAME+ (P < 0.0001).,PRAME expression was associated with shorter time to metastasis and melanoma specific mortality in Class 2 tumors (P = 0.01 and P = 0.02, respectively).,In Class 1 tumors, PRAME expression was directly associated with SF3B1 mutations (P < 0.0001) and inversely associated with EIF1AX mutations (P = 0.004).,PRAME expression was strongly associated with hypomethylation at 12 CpG sites near the PRAME promoter.,Analyses included PRAME mRNA expression, Class 1 versus Class 2 status, chromosomal copy number, mutation status of BAP1, EIF1AX, GNA11, GNAQ and SF3B1, and genomic DNA methylation status.,Analyses were performed on 555 de-identified samples from Castle Biosciences, 123 samples from our center, and 80 samples from the TCGA.,PRAME is aberrantly hypomethylated and activated in Class 1 and Class 2 uveal melanomas and is associated with increased metastatic risk in both classes.,Since PRAME has been successfully targeted for immunotherapy, it may prove to be a companion prognostic biomarker.
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Hotspot mutations in the spliceosome gene SF3B1 are reported in ∼20% of uveal melanomas.,SF3B1 is involved in 3′-splice site (3′ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear.,Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1R625/K666 mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3′ss.,Modelling the differential junctions in SF3B1WT and SF3B1R625/K666 cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3’ss-sequence context.,SF3B1WT knockdown or overexpression do not reproduce the SF3B1R625/K666 splice pattern, qualifying SF3B1R625/K666 as change-of-function mutants.,Mutagenesis of predicted branchpoints reveals that the SF3B1R625/K666-promoted splice pattern is a direct result of alternative branchpoint usage.,Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease.,Mutations in the splicing factor SF3B1 are found in uveal melanoma.,Here, Alsafadi et al. use RNA-sequencing data from these cancers and experimental models, and show that mutant SF3B1 promotes alternative branchpoints in a specific gene subset and that the mutant protein gains a new function.
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We describe a new procedure for the parallel mapping of selected metals in histologically characterized tissue samples.,Mapping is achieved via image registration of digital data obtained from two neighbouring cryosections by scanning the first as a histological sample and subjecting the second to laser ablation inductively coupled plasma mass spectrometry.,This computer supported procedure enables determination of the distribution and content of metals of interest directly in the chosen histological zones and represents a substantial improvement over the standard approach, which determines these values in tissue homogenates or whole tissue sections.,The potential of the described procedure was demonstrated in a pilot study that analysed Zn and Cu levels in successive development stages of pig melanoma tissue using MeLiM (Melanoma-bearing-Libechov-Minipig) model.,We anticipate that the procedure could be useful for a complex understanding of the role that the spatial distribution of metals plays within tissues affected by pathological states including cancer.
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Double positive (DP) CD4CD8 Tαβ cells have been reported in normal individuals as well as in different pathological conditions including inflammatory diseases, viral infections and cancer, but their function remains to be elucidated.,We recently reported the increased frequency of DP Tαβ cells in human breast pleural effusions.,This manuscript addresses the question of the existence and above all the role of this non-conventional DP sub-population among tumor associated lymphocytes in melanomas.,We analyzed the intratumoral cell infiltrate in solid metastasis (n = 6) and tumor invaded lymph nodes (n = 26) samples from melanomas patients by multiparametric cytometry.,Here we documented for the first time significant increased frequency of DP T cells in about 60% of melanoma tumors compared to blood samples.,Interestingly, a high proportion of these cells produced TNF-α in response to autologous melanoma cell lines.,Besides, they are characterized by a unique cytokine profile corresponding to higher secretion of IL-13, IL-4 and IL-5 than simple positive T cells.,In deep analysis, we derived a representative tumor-reactive DP T cell clone from a melanoma patient's invaded lymph node.,This clone was restricted by HLA-A*2402 and recognized both autologous and allogeneic tumor cells of various origins as well as normal cells, suggesting that the target antigen was a ubiquitous self antigen.,However, this DP T cell clone failed to kill HLA-A*2402 EBV-transformed B cells, probably due to the constitutive expression of immunoproteasome by these cells.,In conclusion, we can postulate that, according to their broad tumor reactivity and to their original cytokine profile, the tumor associated DP T cells could participate in immune responses to tumors in vivo.,Therefore, the presence of these cells and their role will be crucial to address in cancer patients, especially in the context of immunotherapies.
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The low efficiency of currently-used anti-cancer therapies poses a serious challenge, especially in the case of malignant melanoma, a cancer characterized by elevated invasiveness and relatively high mortality rate.,The role of the tumor microenvironment in the progression of melanoma and its acquisition of resistance to treatment seems to be the main focus of recent studies.,One of the factors that, in normal conditions, aids the organism in its fight against the cancer and, following the malignant transformation, adapts to facilitate the development of the tumor is the immune system.,A variety of cell types, i.e., T and B lymphocytes, macrophages, and dendritic and natural killer cells, as well as neutrophils, support the growth and invasiveness of melanoma cells, utilizing a plethora of mechanisms, including secretion of pro-inflammatory molecules, induction of inhibitory receptors expression, or depletion of essential nutrients.,This review provides a comprehensive summary of the processes regulated by tumor-associated cells that promote the immune escape of melanoma cells.,The described mechanisms offer potential new targets for anti-cancer treatment and should be further studied to improve currently-employed therapies.
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The high mortality rate of melanoma is broadly associated with its metastatic potential.,Tumor cell dissemination is strictly dependent on vascularization; therefore, angiogenesis and lymphangiogenesis play an essential role in metastasis.,Hence, a better understanding of the players of tumor vascularization and establishing them as new molecular biomarkers might help to overcome the poor prognosis of melanoma patients.,Here, we further characterized a linear murine model of melanoma progression and showed that the aggressiveness of melanoma cells is closely associated with high expression of angiogenic factors, such as Vegfc, Angpt2, and Six1, and that blockade of the vascular endothelial growth factor pathway by the inhibitor axitinib abrogates their tumorigenic potential in vitro and in the in vivo chicken chorioallantoic membrane assay.,Furthermore, analysis of The Cancer Genome Atlas data revealed that the expression of the angiogenic factor ANGPT2 (P‐value = 0.044) and the lymphangiogenic receptor VEGFR‐3 (P‐value = 0.002) were independent prognostic factors of overall survival in melanoma patients.,Enhanced reduced representation bisulfite sequencing‐based methylome profiling revealed for the first time a link between abnormal VEGFC, ANGPT2, and SIX1 gene expression and promoter hypomethylation in melanoma cells.,In patients, VEGFC (P‐value = 0.031), ANGPT2 (P‐value < 0.001), and SIX1 (P‐value = 0.009) promoter hypomethylation were independent prognostic factors of shorter overall survival.,Hence, our data suggest that these angio‐ and lymphangiogenesis factors are potential biomarkers of melanoma prognosis.,Moreover, these findings strongly support the applicability of our melanoma progression model to unravel new biomarkers for this aggressive human disease.
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Mitogen activated-protein kinase pathway inhibitors (MAPKis) improve treatment outcome in patients with disseminated BRAFV600 mutant cutaneous malignant melanoma (CMM) but responses are of limited duration due to emerging resistance.,Although extensive research in mechanisms of resistance is being performed, predictive biomarkers for durable responses are still lacking.,We used miRNA qPCR to investigate if different levels of extracellular microvesicle microRNA (EV miRNA) in matched plasma samples collected from patients with metastatic IV BRAFV600 mutated CMM before, during and after therapy with MAPKis could serve as predictive biomarkers.,EV miRNAs were extracted from plasma samples from 28 patients collected before and during therapy, measured by quantitative PCR-array and correlated to therapy outcome.,Increased levels of EV let-7g-5p during treatment compared to before treatment (EV let-7g-5p_delta) were associated with better disease control with MAPKis (odds ratio 8568.4, 95% CI = 4.8-1.5e+07, P = 0.000036).,Elevated levels of EV miR-497-5p during therapy were associated with prolonged progression free survival (PFS) (hazard ratio = 0.27, 95% CI = 0.13-0.52, P <0.000061).,EV miRNAs let-7g-5p and miR-497-5p were identified as putative novel predictive biomarkers of MAPKi treatment benefit in metastatic CMM patients highlighting the potential relevance of assessing EV miRNA during and after treatment to unravel novel mechanisms of resistance.
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The fourth “Melanoma Bridge Meeting” took place in Naples, December 5 to 8th, 2013.,The four topics discussed at this meeting were: Diagnosis and New Procedures, Molecular Advances and Combination Therapies, News in Immunotherapy, and Tumor Microenvironment and Biomarkers.,Until recently systemic therapy for metastatic melanoma patients was ineffective, but recent research in tumor biology and immunology has led to the development of new targeted and immunotherapeutic agents that prolong progression-free survival (PFS) and overall survival (OS).,New therapies, such as mitogen-activated protein kinase (MAPK) pathway inhibitors, like BRAF and MEK inhibitors, as well as other signaling pathways inhibitors, are being tested in metastatic melanoma either as monotherapy or in combination, and have yielded promising results.,Improved survival rates have also been observed with immune therapy for patients with metastatic melanoma.,Immune-modulating antibodies came to the forefront with anti-CTLA-4, programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) pathway blocking antibodies that result in durable responses in a subset of melanoma patients.,Agents targeting other immune inhibitory (e.g., Tim-3) or immune stimulating (e.g., CD137) receptors and other approaches such as adoptive cell transfer demonstrate clinical benefit in melanoma as well.,This meeting’s specific focus was on advances in targeted therapy and immunotherapy.,Both combination targeted therapy approaches and different immunotherapies were discussed.,Similarly to the previous meetings, the importance of biomarkers for clinical application as markers for diagnosis, prognosis and prediction of treatment response was an integral part of the meeting.,Significant consideration was given to issues surrounding the development of novel therapeutic targets as further study of patterns of resistance to both immunologic and targeted drugs are paramount to future drug development to guide existing and future therapies.,The overall emphasis on biomarkers supports novel concepts toward integrating biomarkers into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage.,Translation of the knowledge gained from the biology of tumor microenvironment across different tumors represents a bridge to impact on prognosis and response to therapy in melanoma.
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Cutaneous squamous cell carcinoma (cSCC) has a high tumour mutational burden (50 mutations per megabase DNA pair).,Here, we combine whole-exome analyses from 40 primary cSCC tumours, comprising 20 well-differentiated and 20 moderately/poorly differentiated tumours, with accompanying clinical data from a longitudinal study of immunosuppressed and immunocompetent patients and integrate this analysis with independent gene expression studies.,We identify commonly mutated genes, copy number changes and altered pathways and processes.,Comparisons with tumour differentiation status suggest events which may drive disease progression.,Mutational signature analysis reveals the presence of a novel signature (signature 32), whose incidence correlates with chronic exposure to the immunosuppressive drug azathioprine.,Characterisation of a panel of 15 cSCC tumour-derived cell lines reveals that they accurately reflect the mutational signatures and genomic alterations of primary tumours and provide a valuable resource for the validation of tumour drivers and therapeutic targets.,It is known cutaneous squamous cell carcinoma (cSCC) involves a high tumour mutation burden.,Here the authors identify common cSCC mutated genes, copy number changes, altered pathways and report the presence of a novel mutation signature associated with chronic exposure to the immunosuppressive drug azathioprine.
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Cutaneous squamous cell carcinoma (cSCC) is the second most common skin malignancy and it presents a therapeutic challenge in organ transplant recipient patients.,Despite the need, there are only a few targeted drug treatment options.,Recent studies have revealed a pivotal role played by microRNAs (miRNAs) in multiple cancers, but only a few studies tested their function in cSCC.,Here, we analyzed differential expression of 88 cancer related miRNAs in 43 study participants with cSCC; 32 immunocompetent, 11 OTR patients, and 15 non-lesional skin samples by microarray analysis.,Of the examined miRNAs, miR-135b was the most upregulated (13.3-fold, 21.5-fold; p=0.0001) in both patient groups.,Similarly, the miR-135b expression was also upregulated in three cSCC cell lines when evaluated by quantitative real-time PCR.,In functional studies, inhibition of miR-135b by specific anti-miR oligonucleotides resulted in upregulation of its target gene LZTS1 mRNA and protein levels and led to decreased cell motility and invasion of both primary and metastatic cSCC cell lines.,In contrast, miR-135b overexpression by synthetic miR-135b mimic induced further down-regulation of LZTS1 mRNA in vitro and increased cancer cell motility and invasiveness.,Immunohistochemical evaluation of 67 cSCC tumor tissues demonstrated that miR-135b expression inversely correlated with LZTS1 staining intensity and the tumor grade.,These results indicate that miR-135b functions as an oncogene in cSCC and provide new understanding into its pathological role in cSCC progression and invasiveness.
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National cancer databases document that melanoma is the most aggressive and deadly cutaneous malignancy with worldwide increasing incidence in the Caucasian population.,Around 10% of melanomas occur in families.,Several germline mutations were identified that might help to indicate individuals at risk for preventive interventions and early disease detection.,More than 50% of sporadic melanomas carry mutations in Ras/Raf/mitogen-activated protein kinase (MAPK/MEK) pathway, which may represent aims of novel targeted therapies.,Despite advances in targeted therapies and immunotherapies, the outcomes in metastatic tumor are still unsatisfactory.,Here, we review animal models that help our understanding of melanoma development and treatment, including non-vertebrate, mouse, swine, and other mammal models, with an emphasis on those with spontaneously developing melanoma.,Special attention is paid to the melanoma-bearing Libechov minipig (MeLiM).,This original swine model of hereditary metastatic melanoma enables studying biological processes underlying melanoma progression, as well as spontaneous regression.,Current histological, immunohistochemical, biochemical, genetic, hematological, immunological, and skin microbiome findings in the MeLiM model are summarized, together with development of new therapeutic approaches based on tumor devitalization.,The ongoing study of molecular and immunological base of spontaneous regression in MeLiM model has potential to bring new knowledge of clinical importance.
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We describe a new procedure for the parallel mapping of selected metals in histologically characterized tissue samples.,Mapping is achieved via image registration of digital data obtained from two neighbouring cryosections by scanning the first as a histological sample and subjecting the second to laser ablation inductively coupled plasma mass spectrometry.,This computer supported procedure enables determination of the distribution and content of metals of interest directly in the chosen histological zones and represents a substantial improvement over the standard approach, which determines these values in tissue homogenates or whole tissue sections.,The potential of the described procedure was demonstrated in a pilot study that analysed Zn and Cu levels in successive development stages of pig melanoma tissue using MeLiM (Melanoma-bearing-Libechov-Minipig) model.,We anticipate that the procedure could be useful for a complex understanding of the role that the spatial distribution of metals plays within tissues affected by pathological states including cancer.
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Common acquired melanocytic nevi are benign neoplasms that are composed of small uniform melanocytes and typically present as flat or slightly elevated, pigmented lesions on the skin.,We describe two families with a new autosomal dominant syndrome characterized by multiple skin-colored, elevated melanocytic tumors.,In contrast to common acquired nevi, the melanocytic neoplasms in affected family members ranged histopathologically from epithelioid nevi to atypical melanocytic proliferations that showed overlapping features with melanoma.,Some affected patients developed uveal or cutaneous melanomas.,Segregating with this phenotype, we found inactivating germline mutations of the BAP1 gene.,The majority of melanocytic neoplasms lost the remaining wild-type allele of BAP1 by various somatic alterations.,In addition, we found BAP1 mutations in a subset of sporadic melanocytic neoplasms showing histologic similarities to the familial tumors.,These findings suggest that loss of BAP1 is associated with a clinically and morphologically distinct type of melanocytic neoplasm.
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Because only a small fraction of asbestos-exposed individuals develop malignant mesothelioma1, and because mesothelioma clustering is observed in some families1, we searched for genetic predisposing factors.,We discovered germline mutations in BAP1 (BRCA1-associated protein 1) in two families with a high incidence of mesothelioma.,Somatic alterations affecting BAP1 were observed in familial mesotheliomas, indicating biallelic inactivation.,Besides mesothelioma, some BAP1 mutation carriers developed uveal melanoma.,Germline BAP1 mutations were also found in two of 26 sporadic mesotheliomas: both patients with mutant BAP1 were previously diagnosed with uveal melanoma.,Truncating mutations and aberrant BAP1 expression were common in sporadic mesotheliomas without germline mutations.,These results reveal a BAP1-related cancer syndrome characterized by mesothelioma and uveal melanoma.,We hypothesize that other cancers may also be involved, and that mesothelioma predominates upon asbestos exposure.,These findings will help identify individuals at high risk of mesothelioma who could be targeted for early intervention.
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In this Review, Rambow et al. use melanoma as a model to present a series of theoretical arguments coupled to recent experimental evidence.,The authors discuss key roles for nongenetic state switching at various steps of the evolution of disease progression and therapy resistance.,An incomplete view of the mechanisms that drive metastasis, the primary cause of cancer-related death, has been a major barrier to development of effective therapeutics and prognostic diagnostics.,Increasing evidence indicates that the interplay between microenvironment, genetic lesions, and cellular plasticity drives the metastatic cascade and resistance to therapies.,Here, using melanoma as a model, we outline the diversity and trajectories of cell states during metastatic dissemination and therapy exposure, and highlight how understanding the magnitude and dynamics of nongenetic reprogramming in space and time at single-cell resolution can be exploited to develop therapeutic strategies that capitalize on nongenetic tumor evolution.
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Cell migration underlies metastatic dissemination of cancer cells, and fast “amoeboid” migration in the invasive fronts of tumors is controlled by high levels of actomyosin contractility.,How amoeboid migration is regulated by extracellular signals and sustained over time by transcriptional changes is not fully understood.,Transforming growth factor β (TGF-β) is well known to promote epithelial-to-mesenchymal transition (EMT) and contribute to metastasis, but melanocytes are neural crest derivatives that have undergone EMT during embryonic development.,Surprisingly, we find that in melanoma, TGF-β promotes amoeboid features such as cell rounding, membrane blebbing, high levels of contractility, and increased invasion.,Using genome-wide transcriptomics, we find that amoeboid melanoma cells are enriched in a TGF-β-driven signature.,We observe that downstream of TGF-β, SMAD2 and its adaptor CITED1 control amoeboid behavior by regulating the expression of key genes that activate contractile forces.,Moreover, CITED1 is highly upregulated during melanoma progression, and its high expression is associated with poor prognosis.,CITED1 is coupled to a contractile-rounded, amoeboid phenotype in a panel of 16 melanoma cell lines, in mouse melanoma xenografts, and in 47 human melanoma patients.,Its expression is also enriched in the invasive fronts of lesions.,Functionally, we show how the TGF-β-SMAD2-CITED1 axis promotes different steps associated with progression: melanoma detachment from keratinocytes, 2D and 3D migration, attachment to endothelial cells, and in vivo lung metastatic initial colonization and outgrowth.,We propose a novel mechanism by which TGF-β-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT.,•TGF-β-SMAD promotes amoeboid migration in melanoma•Downstream of TGF-β, the adaptor CITED1 controls actomyosin contractility•Amoeboid features correlate with CITED1 levels in cell lines, xenografts, and patients•TGF-β-SMAD-CITED1 transcriptional network controls melanoma metastatic ability,TGF-β-SMAD promotes amoeboid migration in melanoma,Downstream of TGF-β, the adaptor CITED1 controls actomyosin contractility,Amoeboid features correlate with CITED1 levels in cell lines, xenografts, and patients,TGF-β-SMAD-CITED1 transcriptional network controls melanoma metastatic ability,Cantelli et al. find that, in melanoma, TGF-β-SMAD-CITED1 controls amoeboid behavior through activation of a transcriptional program.,As a result, melanoma cells detach from keratinocytes, increase their invasive potential, and efficiently colonize the lung.,This is a new function of TGF-β, independent of epithelial-to-mesenchymal transition.
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ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination.,Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels.,Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages.,Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression.,Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation.,Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth.,We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.,•Invasive tumor fronts are enriched in rounded-amoeboid cancer cells high in Myosin II•Myosin II activity in these cells regulates an immunomodulatory secretome•The secreted cytokines and chemokines can induce tumor-promoting macrophages•ROCK-Myosin II and IL-1α/NF-κB cross-talk supports this secretory phenotype,Invasive tumor fronts are enriched in rounded-amoeboid cancer cells high in Myosin II,Myosin II activity in these cells regulates an immunomodulatory secretome,The secreted cytokines and chemokines can induce tumor-promoting macrophages,ROCK-Myosin II and IL-1α/NF-κB cross-talk supports this secretory phenotype,Myosin II activation at the tumor edge promotes invasion and also a secretory phenotype that reshapes the local environment and vasculature to support tumor growth.
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Intravital imaging of BRAF-mutant melanoma cells containing an ERK/MAPK biosensor reveals how the tumor microenvironment affects response to BRAF inhibition by PLX4720.,Initially, melanoma cells respond to PLX4720, but rapid reactivation of ERK/MAPK is observed in areas of high stromal density.,This is linked to “paradoxical” activation of melanoma-associated fibroblasts by PLX4720 and the promotion of matrix production and remodeling leading to elevated integrin β1/FAK/Src signaling in melanoma cells.,Fibronectin-rich matrices with 3-12 kPa elastic modulus are sufficient to provide PLX4720 tolerance.,Co-inhibition of BRAF and FAK abolished ERK reactivation and led to more effective control of BRAF-mutant melanoma.,We propose that paradoxically activated MAFs provide a “safe haven” for melanoma cells to tolerate BRAF inhibition.,•BRAF mutant melanoma cells respond to PLX4720 heterogeneously in vivo•BRAF inhibition activates MAFs, leading to FAK-dependent melanoma survival signaling•ECM-derived signals can support residual disease•BRAF and FAK inhibition synergize in pre-clinical models,BRAF mutant melanoma cells respond to PLX4720 heterogeneously in vivo,BRAF inhibition activates MAFs, leading to FAK-dependent melanoma survival signaling,ECM-derived signals can support residual disease,BRAF and FAK inhibition synergize in pre-clinical models,Hirata et al. show that the BRAF inhibitor PLX4720 promotes melanoma-associated fibroblasts in BRAF-mutant melanomas to produce and remodel matrix, leading to integrin β1-FAK-Src signaling and reactivation of ERK and MAPK in melanoma cells.,Co-inhibition of BRAF and FAK blocks ERK reactivation.
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Over two-thirds of melanomas have activating mutations in B-Raf, leading to constitutive activation of the B-Raf/MKK/ERK signaling pathway.,The most prevalent mutation, B-RafV600E, promotes cancer cell behavior through mechanisms that are still incompletely defined.,Here, we used a sensitive microarray profiling platform to compare microRNA (miRNA) expression levels between primary melanocytes and B-RafV600E-positive melanoma cell lines, and between melanoma cells treated in the presence and absence of an MKK1/2 inhibitor.,We identified a network of >20 miRNAs deregulated by B-Raf/MKK/ERK in melanoma cells, the majority of which modulate the expression of key cancer regulatory genes and functions.,Importantly, miRNAs within the network converge on protein regulation and cancer phenotypes, suggesting that these miRNAs might function combinatorially.,We show that miRNAs augment effects on protein repression and cell invasion when co-expressed, and gene-specific latency and interference effects between miRNAs were also observed.,Thus, B-Raf/MKK/ERK controls key aspects of cancer cell behavior and gene expression by modulating a network of miRNAs with cross-regulatory functions.,The findings highlight the potential for complex interactions between coordinately regulated miRNAs within a network.
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A cardinal feature of malignant melanoma is its metastatic propensity.,An incomplete view of the genetic events driving metastatic progression has been a major barrier to rational development of effective therapeutics and prognostic diagnostics for melanoma patients.,In this study, we conducted global genomic characterization of primary and metastatic melanomas to examine the genomic landscape associated with metastatic progression.,In addition to uncovering three genomic subclasses of metastastic melanomas, we delineated 39 focal and recurrent regions of amplification and deletions, many of which encompassed resident genes that have not been implicated in cancer or metastasis.,To identify progression-associated metastasis gene candidates, we applied a statistical approach, Integrative Genome Comparison (IGC), to define 32 genomic regions of interest that were significantly altered in metastatic relative to primary melanomas, encompassing 30 resident genes with statistically significant expression deregulation.,Functional assays on a subset of these candidates, including MET, ASPM, AKAP9, IMP3, PRKCA, RPA3, and SCAP2, validated their pro-invasion activities in human melanoma cells.,Validity of the IGC approach was further reinforced by tissue microarray analysis of Survivin showing significant increased protein expression in thick versus thin primary cutaneous melanomas, and a progression correlation with lymph node metastases.,Together, these functional validation results and correlative analysis of human tissues support the thesis that integrated genomic and pathological analyses of staged melanomas provide a productive entry point for discovery of melanoma metastases genes.
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Adjuvant targeted therapy (TT) improves relapse free survival in patients with resected BRAF mutant stage III melanoma.,The outcomes and optimal management of patients who relapse after adjuvant TT is unknown.,Patients from twenty-one centres with recurrent melanoma after adjuvant TT were included.,Disease characteristics, adjuvant therapy, recurrence, treatment at relapse and outcomes were examined.,Eighty-five patients developed recurrent melanoma; nineteen (22%) during adjuvant TT.,Median time to first recurrence was 18 months and median follow-up from first recurrence was 31 months.,Fifty-eight (68%) patients received immunotherapy (IT) or TT as 1st line systemic therapy at either first or subsequent recurrence and had disease that was assessable for response.,Response to anti-PD-1 (±trial agent), combination ipilimumab-nivolumab, TT rechallenge and ipilimumab monotherapy was 63%, 62% 25% and 10% respectively.,Twenty-eight (33%) patients had died at census, all from melanoma.,Two-year OS was 84% for anti-PD-1 therapy (±trial agent), 92% for combination ipilimumab and nivolumab, 49% for TT and 45% for ipilimumab monotherapy (p = 0.028).,Patients who relapse after adjuvant TT respond well to subsequent anti-PD-1 based therapy and have outcomes similar to those seen when first line anti-PD-1 therapy is used in stage IV melanoma.
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Nivolumab 1 mg/kg plus ipilimumab 3 mg/kg (NIVO1+IPI3) is approved for first-line treatment of patients with advanced melanoma in several countries.,We conducted a phase IIIb/IV study (CheckMate 511) to determine if nivolumab 3 mg/kg plus ipilimumab 1 mg/kg (NIVO3+IPI1) improves the safety profile of the combination.,Patients (N = 360) age 18 years or older with previously untreated, unresectable stage III or IV melanoma were randomly assigned 1:1 to NIVO3+IPI1 or NIVO1+IPI3 once every 3 weeks for four doses.,After 6 weeks, all patients received NIVO 480 mg once every 4 weeks until disease progression or unacceptable toxicity.,The primary end point was a comparison of the incidence of treatment-related grade 3 to 5 adverse events (AEs) between groups.,Secondary end points included descriptive analyses of objective response rate, progression-free survival, and overall survival.,The study was not designed to formally demonstrate noninferiority of NIVO3+IPI1 to NIVO1+IPI3 for efficacy end points.,At a minimum follow-up of 12 months, incidence of treatment-related grade 3 to 5 AEs was 34% with NIVO3+IPI1 versus 48% with NIVO1+IPI3 (P = .006).,In descriptive analyses, objective response rate was 45.6% in the NIVO3+IPI1 group and 50.6% in the NIVO1+IPI3 group, with complete responses in 15.0% and 13.5% of patients, respectively.,Median progression-free survival was 9.9 months in the NIVO3+IPI1 group and 8.9 months in the NIVO1+IPI3 group.,Median overall survival was not reached in either group.,The CheckMate 511 study met its primary end point, demonstrating a significantly lower incidence of treatment-related grade 3-5 AEs with NIVO3+IPI1 versus NIVO1+IPI3.,Descriptive analyses showed that there were no meaningful differences between the groups for any efficacy end point, although longer follow up may help to better characterize efficacy outcomes.
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Cutaneous squamous cell carcinoma (cSCC) is the second most common skin malignancy and it presents a therapeutic challenge in organ transplant recipient patients.,Despite the need, there are only a few targeted drug treatment options.,Recent studies have revealed a pivotal role played by microRNAs (miRNAs) in multiple cancers, but only a few studies tested their function in cSCC.,Here, we analyzed differential expression of 88 cancer related miRNAs in 43 study participants with cSCC; 32 immunocompetent, 11 OTR patients, and 15 non-lesional skin samples by microarray analysis.,Of the examined miRNAs, miR-135b was the most upregulated (13.3-fold, 21.5-fold; p=0.0001) in both patient groups.,Similarly, the miR-135b expression was also upregulated in three cSCC cell lines when evaluated by quantitative real-time PCR.,In functional studies, inhibition of miR-135b by specific anti-miR oligonucleotides resulted in upregulation of its target gene LZTS1 mRNA and protein levels and led to decreased cell motility and invasion of both primary and metastatic cSCC cell lines.,In contrast, miR-135b overexpression by synthetic miR-135b mimic induced further down-regulation of LZTS1 mRNA in vitro and increased cancer cell motility and invasiveness.,Immunohistochemical evaluation of 67 cSCC tumor tissues demonstrated that miR-135b expression inversely correlated with LZTS1 staining intensity and the tumor grade.,These results indicate that miR-135b functions as an oncogene in cSCC and provide new understanding into its pathological role in cSCC progression and invasiveness.
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The aim of this study was to investigate the feasibility of using serum miR-221 as a noninvasive prognostic biomarker for cutaneous malignant melanoma (CMM).,We measured the expression levels of miR-221 in serum samples from 72 CMM patients and 54 healthy controls by real-time quantitative polymerase chain reaction (RT-PCR).,The overall survival (OS) and disease-free survival (DFS) were calculated using the Kaplan-Meier method.,The differences between the survival curves were tested by using the log-rank test.,The COX proportional hazards regression model was used to determine the joint effects of several variables on survival.,The serum miR-221 levels were significantly higher in patients with CMM than in healthy controls (p<0.0001).,Patients with high serum miR-221 levels had a significantly lower 5-year OS rate (22.1% vs.,54.6%; P=0.018) and RFS rate (12.5% vs.,45.2%; P=0.008) than those with low serum miR-221 level.,In a multivariate Cox model, we found that miR-221 expression was an independent predictor of poor 5-year OS (hazards ratio [HR]=3.189, 95% confidence interval [CI]=1.782-6.777, P=0.007) and 5-year DFS (HR=2.119, CI=1.962-8.552, P=0.01) in CMM patients.,Our data indicate that serum miR-221expression level has prognostic value in patients with CMM.
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We previously identified PRAME as a biomarker for metastatic risk in Class 1 uveal melanomas.,In this study, we sought to define a threshold value for positive PRAME expression (PRAME+) in a large dataset, identify factors associated with PRAME expression, evaluate the prognostic value of PRAME in Class 2 uveal melanomas, and determine whether PRAME expression is associated with aberrant hypomethylation of the PRAME promoter.,Among 678 samples analyzed by qPCR, 498 (73.5%) were PRAME- and 180 (26.5%) were PRAME+.,Class 1 tumors were more likely to be PRAME-, whereas Class 2 tumors were more likely to be PRAME+ (P < 0.0001).,PRAME expression was associated with shorter time to metastasis and melanoma specific mortality in Class 2 tumors (P = 0.01 and P = 0.02, respectively).,In Class 1 tumors, PRAME expression was directly associated with SF3B1 mutations (P < 0.0001) and inversely associated with EIF1AX mutations (P = 0.004).,PRAME expression was strongly associated with hypomethylation at 12 CpG sites near the PRAME promoter.,Analyses included PRAME mRNA expression, Class 1 versus Class 2 status, chromosomal copy number, mutation status of BAP1, EIF1AX, GNA11, GNAQ and SF3B1, and genomic DNA methylation status.,Analyses were performed on 555 de-identified samples from Castle Biosciences, 123 samples from our center, and 80 samples from the TCGA.,PRAME is aberrantly hypomethylated and activated in Class 1 and Class 2 uveal melanomas and is associated with increased metastatic risk in both classes.,Since PRAME has been successfully targeted for immunotherapy, it may prove to be a companion prognostic biomarker.
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Elevated levels of cell-free DNA (cfDNA) are frequently observed in tumor patients.,Activating mutations in exon 4 (R183) and exon 5 (Q209) of GNAQ and GNA11 are almost exclusively found in uveal melanoma, thus providing a highly specific marker for the presence of circulating tumor DNA (ctDNA).,To establish a reliable, noninvasive assay that might allow early detection and monitoring of metastatic disease, we determined the proportion of GNAQ or GNA11 mutant reads in cfDNA of uveal melanoma patients by ultradeep sequencing.,Cell-free DNA from 28 uveal melanoma patients with metastases or extraocular growth was isolated and quantified by real-time polymerase chain reaction (PCR) (7-1550 ng DNA/mL plasma).,GNAQ and GNA11 regions of interest were amplified in 22 of 28 patients and ultradeep sequencing of amplicons was performed to detect even low proportions of mutant reads.,We detected Q209 mutations (2-38% mutant reads) in either GNAQ or GNA11 in the plasma of 9 of 22 metastasized patients.,No correlation between the proportion of mutant reads and the concentration of cfDNA could be detected.,Among the nine ctDNA-positive patients, four had metastases in bone, whereas no metastases were detected in the 13 ctDNA-negative patients at this location (P = 0.025).,Furthermore, ctDNA-positive patients tended to be younger at initial diagnosis and show larger metastases.,The results show that ultradeep amplicon sequencing can be used to detect tumor DNA in plasma of metastasized uveal melanoma patients.,It remains to be shown if this approach can be used for early detection of disseminated tumor disease.
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Nivolumab combined with ipilimumab have shown activity in melanoma brain metastasis (MBM).,However, in most of the clinical trials investigating immunotherapy in this subgroup, patients with symptomatic MBM and/or prior local brain radiotherapy were excluded.,We studied the efficacy of nivolumab plus ipilimumab alone or in combination with local therapies regardless of treatment line in patients with asymptomatic and symptomatic MBM.,Patients with MBM treated with nivolumab plus ipilimumab in 23 German Skin Cancer Centers between April 2015 and October 2018 were investigated.,Overall survival (OS) was evaluated by Kaplan-Meier estimator and univariate and multivariate Cox proportional hazard analyses were performed to determine prognostic factors associated with OS.,Three hundred and eighty patients were included in this study and 31% had symptomatic MBM (60/193 with data available) at the time of start nivolumab plus ipilimumab.,The median follow-up was 18 months and the 2 years and 3 years OS rates were 41% and 30%, respectively.,We identified the following independently significant prognostic factors for OS: elevated serum lactate dehydrogenase and protein S100B levels, number of MBM and Eastern Cooperative Oncology Group performance status.,In these patients treated with checkpoint inhibition first-line or later, in the subgroup of patients with BRAFV600-mutated melanoma we found no differences in terms of OS when receiving first-line either BRAF and MEK inhibitors or nivolumab plus ipilimumab (p=0.085).,In BRAF wild-type patients treated with nivolumab plus ipilimumab in first-line or later there was also no difference in OS (p=0.996).,Local therapy with stereotactic radiosurgery or surgery led to an improvement in OS compared with not receiving local therapy (p=0.009), regardless of the timepoint of the local therapy.,Receiving combined immunotherapy for MBM in first-line or at a later time point made no difference in terms of OS in this study population (p=0.119).,Immunotherapy with nivolumab plus ipilimumab, particularly in combination with stereotactic radiosurgery or surgery improves OS in asymptomatic and symptomatic MBM.
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DNA methylation, an epigenetic alteration typically occurring early in cancer development, could aid in the molecular diagnosis of melanoma.,We determined technical feasibility for high-throughput DNA-methylation array-based profiling using formalin-fixed paraffin-embedded tissues for selection of candidate DNA-methylation differences between melanomas and nevi.,Promoter methylation was evaluated in 27 common benign nevi and 22 primary invasive melanomas using a 1505 CpG site microarray.,Unsupervised hierarchical clustering distinguished melanomas from nevi; 26 CpG sites in 22 genes were identified with significantly different methylation levels between melanomas and nevi after adjustment for age, sex, and multiple comparisons and with β-value differences of ≥0.2.,Prediction analysis for microarrays identified 12 CpG loci that were highly predictive of melanoma, with area under the receiver operating characteristic curves of >0.95.,Of our panel of 22 genes, 14 were statistically significant in an independent sample set of 29 nevi (including dysplastic nevi) and 25 primary invasive melanomas after adjustment for age, sex, and multiple comparisons.,This first report of a DNA-methylation signature discriminating melanomas from nevi indicates that DNA methylation appears promising as an additional tool for enhancing melanoma diagnosis.
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Drug resistance remains as one of the major challenges in melanoma therapy.,It is well known that tumour cells undergo phenotypic switching during melanoma progression, increasing melanoma plasticity and resistance to mitogen-activated protein kinase inhibitors (MAPKi).,We investigated the melanoma phenotype switching using a partial reprogramming model to de-differentiate murine melanoma cells and target melanoma therapy adaptation against MAPKi.,Here, we show that partially reprogrammed cells are a less proliferative and more de-differentiated cell population, expressing a gene signature for stemness and suppressing melanocyte-specific markers.,To investigate adaptation to MAPKi, cells were exposed to B-Raf Proto-Oncogene (BRAF) and mitogen-activated protein kinase kinase (MEK) inhibitors.,De-differentiated cells became less sensitive to MAPKi, showed increased cell viability and decreased apoptosis.,Furthermore, T-type calcium channels expression increased in adaptive murine cells and in human adaptive melanoma cells.,Treatment with the calcium channel blocker mibefradil induced cell death, differentiation and susceptibility to MAPKi in vitro and in vivo.,In summary, we show that partial reprogramming of melanoma cells induces de-differentiation and adaptation to MAPKi.,Moreover, we postulated a calcium channel blocker such as mibefradil, as a potential candidate to restore sensitivity to MAPKi in adaptive melanoma cells.
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Allium vegetables such as garlic (Allium sativum L.) are rich in organosulfur compounds that prevent human chronic diseases, including cancer.,Of these, diallyl trisulfide (DATS) exhibits anticancer effects against a variety of tumors, including malignant melanoma.,Although previous studies have shown that DATS increases intracellular calcium (Ca2+) in different cancer cell types, the role of Ca2+ in the anticancer effect is obscure.,In the present study, we investigated the Ca2+ pathways involved in the anti-melanoma effect.,We used melittin, the bee venom that can activate a store-operated Ca2+ entry (SOCE) and apoptosis, as a reference.,DATS increased apoptosis in human melanoma cell lines in a Ca2+-dependent manner.,It also induced mitochondrial Ca2+ (Ca2+mit) overload through intracellular and extracellular Ca2+ fluxes independently of SOCE.,Strikingly, acidification augmented Ca2+mit overload, and Ca2+ channel blockers reduced the effect more significantly under acidic pH conditions.,On the contrary, acidification mitigated SOCE and Ca2+mit overload caused by melittin.,Finally, Ca2+ channel blockers entirely inhibited the anti-melanoma effect of DATS.,Our findings suggest that DATS explicitly evokes Ca2+mit overload via a non-SOCE, thereby displaying the anti-melanoma effect.
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Malignant transformation of melanocytes frequently coincides with an alteration in the expression of cell-cell adhesion molecules (cadherins) and cell-extracellular matrix proteins (integrins).,How these two adhesion systems interplay to impact on cell invasion remains to be described in melanoma.,Cell adhesion networks were localised by immunofluorescence in human primary cutaneous melanoma, metastatic melanoma in the lymph nodes, and melanoma cell lines.,The role of these cell adhesion networks was assessed both in vivo, by analysing their impact on tumour growth in mice, and in vitro, with the use of functional tests including cell aggregation and cell migration.,We found that α2β1 integrin associates with both E-cadherin and N-cadherin to form two adhesive networks, distinguishable by the interaction-or not-of α2β1 integrin with type I collagen.,N-cadherin/α2β1 integrin and E-cadherin/α2β1 integrin networks differently participated towards tumour growth in mice.,The N-cadherin/α2β1 integrin network showed specific involvement in melanoma cell invasion and migration towards type I collagen.,On the other hand, the E-cadherin/α2β1 network regulated cell-cell adhesion.,This suggests that different signalling environments can be generated, depending on the type and/or local concentration of cadherin present in the adhesion complex, which potentially leads to differential cell responses.,Further clarification of how these adhesive networks are regulated is fundamental to understanding important physiological and pathological processes such as morphogenesis, wound healing, tumour invasion and metastasis.
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The RAS-RAF-MEK-ERK pathway is deregulated in over 90% of malignant melanomas and targeting MEK as central kinase of this pathway is currently tested in clinical trials.,However, dose-limiting side effects are observed, and MEK inhibitors that sufficiently reduce ERK activation in patients show a low clinical response.,Apart from dose-limitations, a reason for the low response to MEK targeting drugs is thought to be the up-regulation of counteracting signalling cascades as a direct response to MEK inhibition.,Therefore, understanding the biology of melanoma cells and the effects of MEK inhibition on these cells will help to identify new combinatorial approaches that are more potent and allow for lower concentrations of drug being used.,We have discovered that in melanoma cells MEK inhibition by selumetinib (AZD6244, ARRY-142886) or PD184352 while efficiently suppressing proliferation stimulates increased invasiveness.,Inhibition of MEK suppresses actin-cortex contraction and increases integrin-mediated adhesion.,Most importantly, and surprisingly MEK inhibition results in a significant increase in MMP-2 and MT1-MMP expression.,All together MEK inhibition in melanoma cells induces a ‘mesenchymal’ phenotype that is characterised by protease driven invasion.,This mode of invasion is dependent on integrin-mediated adhesion, and because SRC kinases are main regulators of this process, the SRC kinase inhibitor saracatinib (AZD0530) completely abolished the MEK inhibitor induced invasion.,Moreover, the combination of saracatinib and selumetinib effectively suppressed the growth and invasion of melanoma cells in a 3D environment, suggesting that combined inhibition of MEK and SRC is a promising approach to improve the efficacy of targeting the ERK/MAP kinase pathway in melanoma.
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Although RIPK1 (receptor [TNFRSF]-interacting protein kinase 1) is emerging as a critical determinant of cell fate in response to cellular stress resulting from activation of death receptors and DNA damage, its potential role in cell response to endoplasmic reticulum (ER) stress remains undefined.,Here we report that RIPK1 functions as an important prosurvival mechanism in melanoma cells undergoing pharmacological ER stress induced by tunicamycin (TM) or thapsigargin (TG) through activation of autophagy.,While treatment with TM or TG upregulated RIPK1 and triggered autophagy in melanoma cells, knockdown of RIPK1 inhibited autophagy and rendered the cells sensitive to killing by TM or TG, recapitulating the effect of inhibition of autophagy.,Consistently, overexpression of RIPK1 enhanced induction of autophagy and conferred resistance of melanoma cells to TM- or TG-induced cell death.,Activation of MAPK8/JNK1 or MAPK9/JNK2, which phosphorylated BCL2L11/BIM leading to its dissociation from BECN1/Beclin 1, was involved in TM- or TG-induced, RIPK1-mediated activation of autophagy; whereas, activation of the transcription factor HSF1 (heat shock factor protein 1) downstream of the ERN1/IRE1-XBP1 axis of the unfolded protein response was responsible for the increase in RIPK1 in melanoma cells undergoing pharmacological ER stress.,Collectively, these results identify upregulation of RIPK1 as an important resistance mechanism of melanoma cells to TM- or TG-induced ER stress by protecting against cell death through activation of autophagy, and suggest that targeting the autophagy-activating mechanism of RIPK1 may be a useful strategy to enhance sensitivity of melanoma cells to therapeutic agents that induce ER stress.
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The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAFV600 mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression.,Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors.,However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway.,The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically.,There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS.,In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance.,Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation.,Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs.,This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor.,These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.
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Drug addiction denotes the dependency of tumors on the same therapeutic drugs to which they have acquired resistance.,Observations from cultured cells1-3, animal models4 and patients5-7 raise the possibility that cancer drug addiction can instigate a potential cancer vulnerability, which may be used therapeutically.,However, for this trait to become of clinical interest, it is imperative to first define the underlying mechanism.,Therefore, we performed an unbiased CRISPR-Cas9 knockout screen to functionally mine the genome of melanoma cells that are both resistant and addicted to BRAF inhibition for “addiction genes”.,Here, we describe a signaling pathway comprising ERK2, JUNB and FRA1, disruption of which allows tumor cells to reverse addiction and survive upon treatment discontinuation.,This occurred both in culture and mice, and was irrespective of the acquired drug resistance mechanism.,In melanoma and lung cancer cells, death induced by drug withdrawal was preceded by a specific ERK2-dependent phenotype switch, alongside transcriptional reprogramming reminiscent of EMT.,In melanoma, this caused shutdown of the lineage survival oncoprotein MITF, restoration of which reversed both phenotype switching and drug addiction-associated lethality.,In melanoma patients who had progressed on BRAF inhibition, treatment cessation was followed by increased expression of the phenotype switch-associated receptor tyrosine kinase AXL.,Drug discontinuation synergized with the melanoma chemotherapeutic dacarbazine by further suppressing MITF and its prosurvival target BCL2 while inducing DNA damage.,Our results uncover a pathway driving cancer drug addiction, which may guide alternating therapeutic strategies for enhanced clinical responses of drug-resistant cancers.
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Treatment of BRAF‐mutant melanomas with MAP kinase pathway inhibitors is paradigmatic of the promise of precision cancer therapy but also highlights problems with drug resistance that limit patient benefit.,We use live‐cell imaging, single‐cell analysis, and molecular profiling to show that exposure of tumor cells to RAF/MEK inhibitors elicits a heterogeneous response in which some cells die, some arrest, and the remainder adapt to drug.,Drug‐adapted cells up‐regulate markers of the neural crest (e.g., NGFR), a melanocyte precursor, and grow slowly.,This phenotype is transiently stable, reverting to the drug‐naïve state within 9 days of drug withdrawal.,Transcriptional profiling of cell lines and human tumors implicates a c‐Jun/ECM/FAK/Src cascade in de‐differentiation in about one‐third of cell lines studied; drug‐induced changes in c‐Jun and NGFR levels are also observed in xenograft and human tumors.,Drugs targeting the c‐Jun/ECM/FAK/Src cascade as well as BET bromodomain inhibitors increase the maximum effect (E max) of RAF/MEK kinase inhibitors by promoting cell killing.,Thus, analysis of reversible drug resistance at a single‐cell level identifies signaling pathways and inhibitory drugs missed by assays that focus on cell populations.
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(1) Background: Extracellular nicotinamide phosphoribosyltrasferase (eNAMPT) is released by various cell types with pro-tumoral and pro-inflammatory properties.,In cancer, eNAMPT regulates tumor growth through the activation of intracellular pathways, suggesting that it acts through a putative receptor, although its nature is still elusive.,It has been shown, using surface plasma resonance, that eNAMPT binds to the C-C chemokine receptor type 5 (CCR5), although the physiological meaning of this finding is unknown.,The aim of the present work was to characterize the pharmacodynamics of eNAMPT on CCR5.,(2) Methods: HeLa CCR5-overexpressing stable cell line and B16 melanoma cells were used.,We focused on some phenotypic effects of CCR5 activation, such as calcium release and migration, to evaluate eNAMPT actions on this receptor.,(3) Results: eNAMPT did not induce ERK activation or cytosolic Ca2+-rises alone.,Furthermore, eNAMPT prevents CCR5 internalization mediated by Rantes. eNAMPT pretreatment inhibits CCR5-mediated PKC activation and Rantes-dependent calcium signaling.,The effect of eNAMPT on CCR5 was specific, as the responses to ATP and carbachol were unaffected.,This was strengthened by the observation that eNAMPT inhibited Rantes-induced Ca2+-rises and Rantes-induced migration in a melanoma cell line.,(4) Conclusions: Our work shows that eNAMPT binds to CCR5 and acts as a natural antagonist of this receptor.
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Most melanoma patients with BRAFV600E positive tumors respond well to a combination of BRAF kinase and MEK inhibitors.,However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment.,For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets.,Cellular metabolism is such a promising new target line: mutant BRAFV600E has been shown to affect the metabolism.,Time course experiments and a series of western blots were performed in a panel of BRAFV600E and BRAFWT/NRASmut human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved.,Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested.,We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in BRAFV600E and in BRAFWT/NRASmut harboring cells.,Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation.,BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation.,Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs.,Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells.,In BRAFV600E and BRAFWT/NRASmut melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH.,As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels.,We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells.,Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% NRASmut, 11.9% NF1mut).,The online version of this article (doi:10.1186/s12943-017-0667-y) contains supplementary material, which is available to authorized users.
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To detect and quantify circulating tumour cells (CTCs) in peripheral blood of patients with uveal melanoma primary non-metastatic tumours, and to analyze the possible relationship between CTCs and clinical risk factors.,Prospective study with two clinical groups: 4 patients diagnosed with choroidal nevus and 8 patients with choroidal melanoma prior to treatment.,A single sample of 7.5 mL of peripheral blood was taken and the CTCs were isolated using a CellSearch system that captures positive cells for the CD146 antigen (MUC18).,None of the patients with choroidal nevus showed CTCs in peripheral blood.,More than one CTC/7.5 mL was detected in 50 % of patients with choroidal melanoma prior to treatment.,The higher level of CTC cells in peripheral blood (3/7.5 mL) was detected in the patient with the larger choroidal melanoma which also presented extrascleral extension and epithelioid pathology.,Performing an analysis with the CellSearch system allows to quantify the choroidal melanoma CTCs in peripheral blood.,This finding highlights the potential usefulness of this technique to achieve the correct stratification and monitoring of the treatment.
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Up to 50% of patients with uveal melanoma (UM) develop metastatic disease with limited treatment options.,The immunomodulating agent ipilimumab has shown an overall survival (OS) benefit in patients with cutaneous metastatic melanoma in two phase III trials.,As patients with UM were excluded in these studies, the Dermatologic Cooperative Oncology Group (DeCOG) conducted a phase II to assess the efficacy and safety of ipilimumab in patients with metastatic UM.,We undertook a multicenter phase II study in patients with different subtypes of metastatic melanoma.,Here we present data on patients with metastatic UM (pretreated and treatment-naïve) who received up to four cycles of ipilimumab administered at a dose of 3 mg/kg in 3 week intervals.,Tumor assessments were conducted at baseline, weeks 12, 24, 36 and 48 according to RECIST 1.1 criteria.,Adverse events (AEs), including immune-related AEs were graded according to National Cancer Institute Common Toxicity Criteria (CTC) v.4.0.,Primary endpoint was the OS rate at 12 months.,Forty five pretreated (85%) and eight treatment-naïve (15%) patients received at least one dose of ipilimumab. 1-year and 2-year OS rates were 22% and 7%, respectively.,Median OS was 6.8 months (95% CI 3.7-8.1), median progression-free survival 2.8 months (95% CI 2.5-2.9).,The disease control rate at weeks 12 and 24 was 47% and 21%, respectively.,Sixteen patients had stable disease (47%), none experienced partial or complete response.,Treatment-related AEs were observed in 35 patients (66%), including 19 grade 3-4 events (36%).,One drug-related death due to pancytopenia was observed.,Ipilimumab has very limited clinical activity in patients with metastatic UM.,Toxicity was manageable when treated as per protocol-specific guidelines.,ClinicalTrials.gov NCT01355120
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Melanoma is a life-threatening form of skin cancer with an elevated risk of metastasis and high mortality rates.,The prognosis and clinical outcomes of cancer immunotherapy in melanoma patients are influenced by immune cell infiltration in the tumor microenvironment (TME) and the expression of genetic factors.,Despite reports suggesting that immune-classification may have a better prediction of prognosis compared to the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) TNM-classification, the definition of Immunoscore in melanoma is becoming a difficult challenge.,In this study, we established and verified a 7-gene prognostic signature.,Melanoma patients from the Cancer Genome Atlas (TCGA) were separated into a low-risk group and a high-risk group using the median risk score.,Receiver operating characteristic (ROC) analysis for overall survival (OS) showed that the area under the curve (AUC) was 0.701 for 1 year, 0.726 for 3 years, and 0.745 for 5 years, respectively.,Moreover, a nomogram was constructed as a practical prognostic tool, and the AUC was 0.829 for 3 years, and 0.803 for 5 years, respectively.,Furthermore, we validated the above results in two datasets from the Gene Expression Omnibus (GEO) database and the relationship between 7-gene prognostic signature and immune infiltration estimated.
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The tumor microenvironment (TME) plays a critical role in tumorigenesis, development, and therapeutic efficacy.,Major advances have been achieved in the treatment of various cancers through immunotherapy.,Nevertheless, only a minority of patients have positive responses to immunotherapy, which is partly due to conditions of the immunosuppressive microenvironment.,Therefore, it is essential to identify prognostic biomarkers that reflect heterogeneous landscapes of the TME.,Based upon the ESTIMATE algorithm, we evaluated the infiltrating levels of immune and stromal components derived from patients afflicted by various types of cancer from The Cancer Genome Atlas database (TCGA).,According to respective patient immune and stromal scores, we categorized cases into high‐ and low‐scoring subgroups for each cancer type to explore associations between TME and patient prognosis.,Gene Set Enrichment Analyses (GSEA) were conducted and genes enriched in IFNγ response signaling pathway were selected to facilitate establishment of a risk model for predicting overall survival (OS).,Furthermore, we investigated the associations between the prognostic signature and tumor immune infiltration landscape by using CIBERSORT algorithm and TIMER database.,Among the cancers assessed, the immune scores for skin cutaneous melanoma (SKCM) were the most significantly correlated with patients' survival time (P < .0001).,We identified and validated a five‐IFNγ response‐related gene signature (UBE2L6, PARP14, IFIH1, IRF2, and GBP4), which was closely correlated with the prognosis for SKCM afflicted patients.,Multivariate Cox regression analysis indicated that this risk model was an independent prognostic factor for SKCM.,Tumor‐infiltrating lymphocytes and specific immune checkpoint molecules had notably differential levels of expression in high‐ compared to low‐risk samples.,In this study, we established a novel five‐IFNγ response‐related gene signature that provided a better and increasingly comprehensive understanding of tumor immune landscape, and which demonstrated good performance in predicting outcomes for patients afflicted by SKCM.,We established an effective five‐IFNγ response‐related gene‐based risk score, which has potential to be a novel prognostic signature and may provide insight into tumor immune microenvironment of cutaneous melanoma.
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Therapeutic cancer vaccination is an attractive immune therapy strategy to actively induce T cells that specifically recognize and kill tumour cells in cancer patients.,However, it remains difficult to generate a large number antigen-specific T cells using conventional vaccine carrier systems1,2.,Here we show that α-Al2O3 nanoparticles can act as an antigen carrier to reduce the amount of antigen required by dendritic cells to activate T cells in vitro and in vivo.,We found that α- Al2O3 nanoparticles delivered antigens to autophagosomes in dendritic cells (DCs), which then presented the antigens to T cells through autophagy - the normal degradation process of cell components in cells.,Immunization of mice with α-Al2O3 nanoparticles that are conjugated to either a model tumour antigen or autophagosomes derived from tumour cells resulted in tumour regression.,These results suggest that α-Al2O3 nanoparticles may be a promising adjuvant in the development of therapeutic cancer vaccines.
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The study was performed to investigate the expression of chemokine receptors (CR) on circulating tumor cells (CTC), which may be of importance for organ-specific metastases and cancer treatment since CR are potential drug-targets.,Blood samples from patients with metastatic carcinoma (MC) or melanoma (MM) were enriched for CTC and expression of CR (CXCR4, CCR6, CCR7 and CCR9) was evaluated by flow cytometry.,CTC were detected in 49 of 68 patients (72%) [28 MC; 21 MM] with a median number of 3 CTC (range: 1-94)/10 mL of blood.,CXCR4 was expressed on CTC in 82% (40/49) of patients [median number 1 CTC/10 mL blood; range 1-14] and CCR6 in 29 patients (59%; median 1, range: 1-14).,In MM patients, CCR7 was expressed on CTC in 6 (29%) samples and CCR9 in 12 (57%).,A positive correlation between surface expression of CR and organ-specific metastatic pattern was not observed.,CR were expressed on CTC of patients with solid tumors.,Along with our findings, the observation that CR could be involved in CTC proliferation and migration of tumor cells appoints CTC as potential CR-antagonist therapeutic target.
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Here, we showed that the secretome of senescent melanoma cells drives basal melanoma cells towards a mesenchymal phenotype, with characteristic of stems illustrated by increased level of the prototype genes FN1, SNAIL, OCT4 and NANOG.,This molecular reprogramming leads to an increase in the low-MITF and slow-growing cell population endowed with melanoma-initiating cell features.,The secretome of senescent melanoma cells induces a panel of 52 genes, involved in cell movement and cell/cell interaction, among which AXL and ALDH1A3 have been implicated in melanoma development.,We found that the secretome of senescent melanoma cells activates the STAT3 pathway and STAT3 inhibition prevents secretome effects, including the acquisition of tumorigenic properties.,Collectively, the findings provide insights into how the secretome of melanoma cells entering senescence upon chemotherapy treatments increases the tumorigenicity of naïve melanoma cells by inducing, through STAT3 activation, a melanoma-initiating cell phenotype that could favor chemotherapy resistance and relapse.
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We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas.,Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas.,Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS.,Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas.,This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1P29S) in the highly conserved switch I domain.,Crystal structures, and biochemical and functional studies of RAC1P29S showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration.,These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.
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Sun-exposure is one of the risk factors associated with the development of a cutaneous neoplasm.,In melanoma, the Ras-Raf-MEK-ERK (MAPK) signaling pathway is constitutively activated through multiple mechanisms, including B-RAF mutation.,It has been hypothesized that B-RAF mutations in melanocytic lesions arise from DNA damage induced by ultraviolet (UV) radiation.,However, it is still discussed if B-RAF mutations are associated with melanoma patients exposed to the sun.,Therefore, in the present study, the known B-RAFV600E mutation was analysed in melanoma samples from 30 indoor and 38 outdoor workers.,B-RAFV600E mutation was detected in 52 and 73% of outdoor workers and indoor workers, respectively.,Of note, this mutation was identified in 12 of 14 (85%) melanoma of the trunk diagnosed in indoor workers and in 9 of 19 (47%) samples from outdoor workers (p=0.03).,By analyzing melanomas of other body sites, no statistical difference in the frequency of B-RAFV600E mutation was identified between the groups of workers.,It appears that the mutation detected among indoor workers may be associated with a recreational or intermittent exposure to the sun, as usually the trunk is a sun-protected body site.,Overall, these data indicate that the B-RAFV600E mutation detected in melanoma is not associated with a chronic exposure to the sun.,Mutations detected in other genes may also contribute to melanoma development in the subset of patients exposed to UV radiation.
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We describe a mechanism of tumorigenesis mediated by kinase-dead BRAF in the presence of oncogenic RAS.,We show that drugs that selectively inhibit BRAF drive RAS-dependent BRAF binding to CRAF, CRAF activation, and MEK-ERK signaling.,This does not occur when oncogenic BRAF is inhibited, demonstrating that BRAF inhibition per se does not drive pathway activation; it only occurs when BRAF is inhibited in the presence of oncogenic RAS.,Kinase-dead BRAF mimics the effects of the BRAF-selective drugs and kinase-dead Braf and oncogenic Ras cooperate to induce melanoma in mice.,Our data reveal another paradigm of BRAF-mediated signaling that promotes tumor progression.,They highlight the importance of understanding pathway signaling in clinical practice and of genotyping tumors prior to administering BRAF-selective drugs, to identify patients who are likely to respond and also to identify patients who may experience adverse effects.,► BRAF activates ERK but in some circumstances BRAF inhibitors can induce tumor growth ► BRAF inhibitors drive BRAF-CRAF binding, activating ERK in cells with oncogenic RAS ► Kinase-dead mutants of BRAF have the same effect as BRAF inhibitors ► Oncogenic RAS and kinase-dead BRAF cooperate to induce melanoma in mice
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The expression pattern of Connexins (Cx) 37, 40, 43, 45 and Pannexin 1 (Pnx1) was analyzed immunohistochemically, as well as semi-quantitatively and quantitatively in histological sections of developing 8th- to 12th-week human eyes and postnatal healthy eye, in retinoblastoma and different uveal melanomas.,Expressions of both Cx37 and Cx43 increased during development but diminished in the postnatal period, being higher in the retina than in the choroid.,Cx37 was highly expressed in the choroid of retinoblastoma, and Cx43 in epitheloid melanoma, while they were both increasingly expressed in mixoid melanoma.,In contrast, mild retinal Cx40 expression during development increased to strong in postnatal period, while it was significantly higher in the choroid of mixoid melanoma.,Cx45 showed significantly higher expression in the developing retina compared to other samples, while it became low postnatally and in all types of melanoma.,Pnx1 was increasingly expressed in developing choroid but became lower in the postnatal eye.,It was strongly expressed in epithelial and spindle melanoma, and particularly in retinoblastoma.,Our results indicate importance of Cx37 and Cx40 expression in normal and pathological vascularization, and Cx43 expression in inflammatory response.,Whereas Cx45 is involved in early stages of eye development, Pnx1might influence cell metabolism.,Additionally, Cx43 might be a potential biomarker of tumor prognosis.
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The homeobox containing transcription factor MSX2 is a key regulator of embryonic development and has been implicated to have a role in breast and pancreatic cancer.,Using a selection of two- and three-dimensional in vitro assays and tissue microarrays (TMAs), the clinical and functional relevance of MSX2 in malignant melanoma was explored.,A doxycyline-inducible over-expression system was applied to study the relevance of MSX2 in vitro.,For TMA construction, tumour material from 218 melanoma patients was used.,Ectopic expression of MSX2 resulted in the induction of apoptosis and reduced the invasive capacity of melanoma cells in three-dimensional culture.,MSX2 over-expression was shown to affect several signalling pathways associated with cell invasion and survival.,Downregulation of N-Cadherin, induction of p21 and inhibition of both BCL2 and Survivin were observed.,Cytoplasmic MSX2 expression was found to correlate significantly with increased recurrence-free survival (P=0.008).,Nuclear expression of MSX2 did not result in significant survival correlations, suggesting that the beneficial effect of MSX2 may be independent of its DNA binding activity.,MSX2 may be an important regulator of melanoma cell invasion and survival.,Cytoplasmic expression of the protein was identified as biomarker for good prognosis in malignant melanoma patients.
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In this review, Goding and Arnheiter present the current understanding of MITF's role and regulation in development and disease and highlight key areas where our knowledge of MITF regulation and function is limited.,All transcription factors are equal, but some are more equal than others.,In the 25 yr since the gene encoding the microphthalmia-associated transcription factor (MITF) was first isolated, MITF has emerged as a key coordinator of many aspects of melanocyte and melanoma biology.,Like all transcription factors, MITF binds to specific DNA sequences and up-regulates or down-regulates its target genes.,What marks MITF as being remarkable among its peers is the sheer range of biological processes that it appears to coordinate.,These include cell survival, differentiation, proliferation, invasion, senescence, metabolism, and DNA damage repair.,In this article we present our current understanding of MITF's role and regulation in development and disease, as well as those of the MITF-related factors TFEB and TFE3, and highlight key areas where our knowledge of MITF regulation and function is limited.
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We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas.,Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas.,Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS.,Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas.,This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1P29S) in the highly conserved switch I domain.,Crystal structures, and biochemical and functional studies of RAC1P29S showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration.,These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.
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The understanding of melanoma malignancy mechanisms is essential for patient survival, because melanoma is responsible for ca. 75% of deaths related to skin cancers.,Enhanced formation of invadopodia and extracellular matrix (ECM) degradation are two important drivers of cell invasion, and actin dynamics facilitate protrusive activity by providing a driving force to push through the ECM.,We focused on the influence of epidermal growth factor (EGF), hepatocyte growth factor (HGF) and transforming growth factor β (TGFβ) on melanoma cell invasiveness, since they are observed in the melanoma microenvironment.,All three factors stimulated invasion of A375 and WM1341D cells derived from primary tumor sites.,In contrast, only EGF and HGF stimulated invasion of WM9 and Hs294T cells isolated from lymph node metastasis.,Enhanced formation of invadopodia and ECM degradation underlie the increased amount of invasive cells after stimulation with the tested agents.,Generally, a rise in invasive potential was accompanied by a decrease in actin polymerization state (F:G ratio).,The F:G ratio remained unchanged or was even increased in cell lines from a metastasis treated with TGFβ.,Our findings indicate that the effects of stimulation with EGF, HGF and TGFβ on melanoma cell invasiveness could depend on melanoma cell progression stage.
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We report the association of an inherited variant located upstream of the poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) gene (rs2249844), with survival in 11 BioGenoMEL melanoma cohorts.,The gene encodes a protein involved in a number of cellular processes including single-strand DNA repair.,Survival analysis was conducted for each cohort using proportional hazards regression adjusting for factors known to be associated with survival.,Survival was measured as overall survival (OS) and, where available, melanoma-specific survival (MSS).,Results were combined using random effects meta-analysis.,Evidence for a role of the PARP1 protein in melanoma ulceration and survival was investigated by testing gene expression levels taken from formalin-fixed paraffin-embedded tumors.,A significant association was seen for inheritance of the rarer variant of PARP1, rs2249844 with OS (hazard ratio (HR) = 1.16 per allele, 95% confidence interval (CI) 1.04-1.28, p = 0.005, eleven cohorts) and MSS (HR = 1.20 per allele, 95% CI 1.01-1.39, p = 0.03, eight cohorts).,We report bioinformatic data supportive of a functional effect for rs2249844.,Higher levels of PARP1 gene expression in tumors were shown to be associated with tumor ulceration and poorer OS.,Although staging systems predict outcome fairly well for melanoma, survival still varies among individual patients.,In this meta-analysis, the authors found that inheritance of a rare genetic variant of PARP1 was associated with improved survival of melanoma patients.,Increased expression of PARP1 has been associated with poorer outcome, and depletion of PARP1 may reduce both melanoma growth and angiogenesis.,The identification of this and other germline variants that affect survival may help to identify key biological pathways active in host/tumor interactions, which may lead to the discovery of new therapeutic targets for treating advanced melanoma.
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Uveal melanoma (UM) is highly refractory to treatment with dismal prognosis in advanced stages.,The value of the combined checkpoint blockade with CTLA-4 and PD-1 inhibition in metastatic UM is currently unclear.,Patients with metastatic or unresectable UM treated with ipilimumab in combination with a PD-1 inhibitor were collected from 16 German skin cancer centers.,Patient records of 64 cases were analyzed for response, progression-free survival (PFS), overall survival (OS), and safety.,Clinical parameters and serum biomarkers associated with OS and treatment response were determined with Cox regression modelling and logistic regression.,The best overall response rate to combined checkpoint blockade was 15.6% with 3.1 and 12.5% complete and partial response, respectively.,The median duration of response was 25.5 months (range 9.0-65.0).,Stable disease was achieved in 21.9%, resulting in a disease control rate of 37.5% with a median duration of the clinical benefit of 28.0 months (range 7.0-65.0).,The median PFS was 3.0 months (95% CI 2.4-3.6).,The median OS was estimated to 16.1 months (95% CI 12.9-19.3).,Regarding safety, 39.1% of treated patients experienced a severe, treatment-related adverse event according to the CTCAE criteria (grade 3: 37.5%; grade 4: 1.6%).,The most common toxicities were colitis (20.3%), hepatitis (20.3%), thyreoiditis (15.6%), and hypophysitis (7.8%).,A poor ECOG performance status was an independent risk factor for decreased OS (p = 0.007).,The tolerability of the combined checkpoint blockade in UM may possibly be better than in trials on cutaneous melanoma.,This study implies that combined checkpoint blockade represents the hitherto most effective treatment option available for metastatic UM available outside of clinical trials.
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Next generation sequencing of uveal melanoma (UM) samples has identified a number of recurrent oncogenic or loss-of-function mutations in key driver genes including: GNAQ, GNA11, EIF1AX, SF3B1 and BAP1.,To search for additional driver mutations in this tumor type we carried out whole-genome or whole-exome sequencing of 28 tumors or primary cell lines.,These samples have a low mutation burden, with a mean of 10.6 protein changing mutations per sample (range 0 to 53).,As expected for these sun-shielded melanomas the mutation spectrum was not consistent with an ultraviolet radiation signature, instead, a BRCA mutation signature predominated.,In addition to mutations in the known UM driver genes, we found a recurrent mutation in PLCB4 (c.G1888T, p.D630Y, NM_000933), which was validated using Sanger sequencing.,The identical mutation was also found in published UM sequence data (1 of 56 tumors), supporting its role as a novel driver mutation in UM.,PLCB4 p.D630Y mutations are mutually exclusive with mutations in GNA11 and GNAQ, consistent with PLCB4 being the canonical downstream target of the former gene products.,Taken together these data suggest that the PLCB4 hotspot mutation is similarly a gain-of-function mutation leading to activation of the same signaling pathway, promoting UM tumorigenesis.
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Adoptive T-cell therapy relying on conventional T cells transduced with T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has caused substantial tumor regression in several clinical trials.,However, genetically engineered T cells have been associated with serious side-effects due to off-target toxicities and massive cytokine release.,To obviate these concerns, we established a protocol adaptable to GMP to expand and transiently transfect γ/δ T cells with mRNA.,PBMC from healthy donors were stimulated using zoledronic-acid or OKT3 to expand γ/δ T cells and bulk T cells, respectively.,Additionally, CD8+ T cells and γ/δ T cells were MACS-isolated from PBMC and expanded with OKT3.,Next, these four populations were electroporated with RNA encoding a gp100/HLA-A2-specific TCR or a CAR specific for MCSP.,Thereafter, receptor expression, antigen-specific cytokine secretion, specific cytotoxicity, and killing of the endogenous γ/δ T cell-target Daudi were analyzed.,Using zoledronic-acid in average 6 million of γ/δ T cells with a purity of 85% were generated from one million PBMC.,MACS-isolation and OKT3-mediated expansion of γ/δ T cells yielded approximately ten times less cells.,OKT3-expanded and CD8+ MACS-isolated conventional T cells behaved correspondingly similar.,All employed T cells were efficiently transfected with the TCR or the CAR.,Upon respective stimulation, γ/δ T cells produced IFNγ and TNF, but little IL-2 and the zoledronic-acid expanded T cells exceeded MACS-γ/δ T cells in antigen-specific cytokine secretion.,While the cytokine production of γ/δ T cells was in general lower than that of conventional T cells, specific cytotoxicity against melanoma cell lines was similar.,In contrast to OKT3-expanded and MACS-CD8+ T cells, mock-electroporated γ/δ T cells also lysed tumor cells reflecting the γ/δ T cell-intrinsic anti-tumor activity.,After transfection, γ/δ T cells were still able to kill MHC-deficient Daudi cells.,We present a protocol adaptable to GMP for the expansion of γ/δ T cells and their subsequent RNA-transfection with tumor-specific TCRs or CARs.,Given the transient receptor expression, the reduced cytokine release, and the equivalent cytotoxicity, these γ/δ T cells may represent a safer complementation to genetically engineered conventional T cells in the immunotherapy of melanoma (Exper Dermatol 26: 157, 2017, J Investig Dermatol 136: A173, 2016).,The online version of this article (doi:10.1186/s12885-017-3539-3) contains supplementary material, which is available to authorized users.
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While the morbidity and mortality from cancer are largely attributable to its metastatic dissemination, the integral features of the cascade are not well understood.,The widely accepted hypothesis is that the primary tumor microenvironment induces the epithelial-to-mesenchymal transition in cancer cells, facilitating their escape into the bloodstream, possibly accompanied by cancer stem cells.,An alternative theory for metastasis involves fusion of macrophages with tumor cells (MTFs).,Here we culture and characterize apparent MTFs from blood of melanoma patients.,We isolated enriched CTC populations from peripheral blood samples from melanoma patients, and cultured them.,We interrogated these cultured cells for characteristic BRAF mutations, and used confocal microscopy for immunophenotyping, motility, DNA content and chromatin texture analyses, and then conducted xenograft studies using nude mice.,Morphologically, the cultured MTFs were generally large with many pseudopod extensions and lamellipodia.,Ultrastructurally, the cultured MTFs appeared to be macrophages.,They were rich in mitochondria and lysosomes, as well as apparent melanosomes.,The cultured MTF populations were all heterogeneous with regard to DNA content, containing aneuploid and/or high-ploidy cells, and they typically showed large sheets (and/or clumps) of cytoplasmic chromatin.,This cytoplasmic DNA was found within heterogeneously-sized autophagic vacuoles, which prominently contained chromatin and micronuclei.,Cultured MTFs uniformly expressed pan-macrophage markers (CD14, CD68) and macrophage markers indicative of M2 polarization (CD163, CD204, CD206).,They also expressed melanocyte-specific markers (ALCAM, MLANA), epithelial biomarkers (KRT, EpCAM), as well as the pro-carcinogenic cytokine MIF along with functionally related stem cell markers (CXCR4, CD44).,MTF cultures from individual patients (5 of 8) contained melanoma-specific BRAF activating mutations.,Chromatin texture analysis of deconvoluted images showed condensed DNA (DAPI-intense) regions similar to focal regions described in stem cell fusions.,MTFs were readily apparent in vivo in all human melanomas examined, often exhibiting even higher DNA content than the cultured MTFs.,When cultured MTFs were transplanted subcutaneously in nude mice, they disseminated and produced metastatic lesions at distant sites.,Apparent MTFs are present in peripheral blood of patients with cutaneous melanomas, and they possess the ability to form metastatic lesions when transplanted into mice.,We hypothesize that these MTFs arise at the periphery of primary tumors in vivo, that they readily enter the bloodstream and invade distant tissues, secreting cytokines (such as MIF) to prepare “niches” for colonization by metastasis initiating cells.
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Oncogene-driven metabolic rewiring is an adaptation to low nutrient and oxygen conditions in the tumor microenvironment that enables cancer cells of diverse origin to hyperproliferate.,Aerobic glycolysis and enhanced reliance on glutamine utilization are prime examples of such rewiring.,However, tissue of origin as well as specific genetic and epigenetic changes determines gene expression profiles underlying these metabolic alterations in specific cancers.,In melanoma, activation of the MAPK pathway driven by mutant BRAF or NRAS is a primary cause of malignant transformation.,Activity of the MAPK pathway, as well as other factors, such as HIF1α, Myc and MITF, are among those that control the balance between non-oxidative and oxidative branches of central carbon metabolism.,Here, we discuss the nature of metabolic alterations that underlie melanoma development and affect its response to therapy.
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Drugs that inhibit RAF/MEK signaling, such as vemurafenib, elicit profound but often temporary anti-tumor responses in patients with BRAFV600E melanoma.,Adaptive responses to RAF/MEK inhibition occur on a timescale of hours to days, involve homeostatic responses that reactivate MAP kinase signaling and compensatory mitogenic pathways, and attenuate the anti-tumor effects of RAF/MEK inhibitors.,We profile adaptive responses across a panel of melanoma cell lines using multiplex biochemical measurement, single-cell assays, and statistical modeling and show that adaptation involves at least six signaling cascades that act to reduce drug potency (IC50) and maximal effect (i.e., Emax ≪ 1).,Among these cascades, we identify a role for JNK/c-Jun signaling in vemurafenib adaptation and show that RAF and JNK inhibitors synergize in cell killing.,This arises because JNK inhibition prevents a subset of cells in a cycling population from becoming quiescent upon vemurafenib treatment, thereby reducing drug Emax.,Our findings demonstrate the breadth and diversity of adaptive responses to RAF/MEK inhibition and a means to identify which steps in a signaling cascade are most predictive of phenotypic response.
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BRAF inhibitor (BRAF-I) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months.,Understanding the molecular mechanisms behind BRAF-I responsiveness and acquired resistance is therefore an important issue.,Here we assessed the role of urokinase type plasminogen activator receptor (uPAR) as a potentially valuable biomarker in the acquisition of BRAF-I resistance in V600E mutant melanoma cells.,We examined uPAR and EGFR levels by real time PCR and western blot analysis. uPAR loss of function was realized by knocking down uPAR by RNAi or using M25, a peptide that uncouples uPAR-integrin interaction.,We investigated uPAR-β1integrin-EGFR association by co-immunoprecipitation and confocal immuno-fluorescence analysis.,Acquired resistance to BRAF-I was generated by chronic exposure of cells to vemurafenib.,We proved that uPAR knockdown in combination with vemurafenib inhibits melanoma cell proliferation to greater extent than either treatment alone causing a decrease in AKT and ERK1/2 phosphorylation.,Conversely, we demonstrated that uPAR enforced over-expression results in reduced sensitivity to BRAF inhibition.,Moreover, by targeting uPAR and EGFR interaction with an integrin antagonist peptide we restored vemurafenib responsiveness in melanoma resistant cells.,Furthermore, we found significant detectable uPAR and EGFR levels in tumor biopsies of 4 relapsed patients.,We disclosed an unpredicted mechanism of reduced sensitiveness to BRAF inhibition, driven by elevated levels of uPAR and identified a potential therapeutic strategy to overcome acquired resistance.,Associazione Italiana Ricerca sul Cancro (AIRC); Ente Cassa di Risparmio di Firenze.
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A point mutation in the BRAF gene, leading to a constitutively active form of the protein, is present in 45%-60% of patients and acts as a key driver in melanoma.,Shortly after therapy induction, resistance to MAPK pathway-specific inhibitors develops, indicating that pathway inhibition is circumvented by epigenetic mechanisms.,Here, we mimicked epigenetic modifications in melanoma cells by reprogramming them into metastable induced pluripotent cancer cells (iPCCs) with the ability to terminally differentiate into non-tumorigenic lineages. iPCCs and their differentiated progeny were characterized by an increased resistance against targeted therapies, although the cells harbor the same oncogenic mutations and signaling activity as the parental melanoma cells.,Furthermore, induction of a pluripotent state allowed the melanoma-derived cells to acquire a non-tumorigenic cell fate, further suggesting that tumorigenicity is influenced by the cell state.,•Human melanoma cells reprogrammed toward an iPSC-like state (iPCCs)•iPCCs differentiated into neurons and fibroblasts•iPCC-derived fibroblasts show no tumorigenic potential•iPCCs and iPCC-derived fibroblasts lose oncogene addiction,Human melanoma cells reprogrammed toward an iPSC-like state (iPCCs),iPCCs differentiated into neurons and fibroblasts,iPCC-derived fibroblasts show no tumorigenic potential,iPCCs and iPCC-derived fibroblasts lose oncogene addiction,Aberrant activation of the MAPK pathway is a major cause of melanoma.,Resistance to MAPK pathway-specific inhibitors hampers successful treatment of melanoma.,By reprogramming human melanoma cells toward pluripotency and subsequent differentiation, Utikal and colleagues demonstrate that the tumorigenic phenotype and oncogene addiction are linked to a certain differentiation lineage.
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Melanoma is an aggressive malignancy of melanocytes and most commonly arises in the skin.,In 2002, BRAF gene mutations were identified in melanoma, and this finding resulted in the development of several small-molecule molecular inhibitors that specifically targeted the BRAF V600E mutation.,The development of targeted therapies for advanced-stage melanoma, including tyrosine kinase inhibitors (TKIs) of the BRAF (V600E) kinase, vemurafenib and dabrafenib, have been approved for the treatment of advanced melanoma leading to improved clinical outcomes.,However, the development of BRAF inhibitor (BRAFi) resistance has significantly reduced the therapeutic efficacy after prolonged treatment.,Recent studies have identified the molecular mechanisms for BRAFi resistance.,This review aims to describe the impact of BRAFi resistance on the pathogenesis of melanoma, the current status of molecular pathways involved in BRAFi resistance, including intrinsic resistance, adaptive resistance, and acquired resistance.,This review will discuss how an understanding of the mechanisms associated with BRAFi resistance may aid the identification of useful strategies for overcoming the resistance to BRAF-targeted therapy in patients with advanced-stage melanoma.
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Durable remissions are observed in a fraction of metastatic melanoma patients treated with high-dose interleukin-2 (HD IL-2).,Early studies reported overall (OR) and complete response (CR) rates of 16% and 8% respectively.,Toxicity limited use to specialized centers with standardized protocols.,We report on 243 patients treated at the University of Pittsburgh in a non-intensive care unit (ICU) oncology specialty setting.,Clinical and radiological data were collected on 243 patients treated between 1992 and 2015.,Each HD IL-2 cycle was given over 5 days, cycles were repeated after 9 days and courses (2 cycles) were repeated every 6-9 weeks in patients with stable or responding disease, for up to 3 courses total.,Influence of baseline characteristics on outcomes was assessed using Kaplan-Meier estimates and Cox proportional hazards analysis.,Two hundred forty-three patients received 692 cycles (5270 doses) between 1992 and 2015.,Two hundred thirty-seven patients were evaluable for response: OR rate 18.1% with CR rate 8.0%.,Median overall survival (OS) 9.6 months in the entire cohort but 64.9 months in responders.,Median number of cycles delivered was 2,and median number of doses per cycle was 8.,Toxicity was consistent with prior reports.,HD IL-2 required ICU transfers in 11 patients and 1 death was attributed to HD IL-2.,Pre-treatment lactate dehydrogenase (LDH) levels correlated significantly with progression-free survival [1-2× upper limit normal (ULN) HR 1.95; >2× ULN HR 2.32] and overall survival (1-2× ULN HR 1.67; >2× ULN 2.49).,Response to HD IL-2 and site of metastatic disease also correlated significantly with progression-free and overall survival.,In this large series of patients spanning more than two decades, OR/CR rates with HD IL-2 were 18.1%/8.0% respectively.,Toxicity data was consistent with prior reports.,Pre-treatment LDH values and site(s) of metastatic disease may be useful markers to select patients at greater likelihood of benefit to HD IL-2 therapy.,The online version of this article (10.1186/s40425-017-0279-5) contains supplementary material, which is available to authorized users.
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The discovery of the BRAFV600E mutation led to the development of vemurafenib (PLX4032), a selective BRAF inhibitor specific to the kinase, for the treatment of metastatic melanomas.,However, initial success of the drug was dampened by the development of acquired resistance.,Melanoma was shown to relapse in patients following treatment with vemurafenib which eventually led to patients’ deaths.,It has been proposed that mechanisms of resistance can be due to (1) reactivation of the mitogen-activated protein kinase (MAPK) signalling pathway via secondary mutations, amplification or activation of target kinase(s), (2) the bypass of oncogenic pathway via activation of alternative signalling pathways, (3) other uncharacterized mechanisms.,Studies showed that receptor tyrosine kinases (RTK) such as PDGFRβ, IGF1R, EGFR and c-Met were overexpressed in melanoma cells.,Along with increased secretion of growth factors such as HGF and TGF-α, this will trigger intracellular signalling cascades.,This review discusses the role MAPK and Phosphatidylinositol-3-kinase-protein kinase B-mammalian target of rapamycin (PI3K-AKT-mTOR) pathways play in the mechanism of resistance of melanomas.
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BRAFV600E-mutant malignant melanomas depend on RAF/MEK/ERK (MAPK) signaling for tumor cell growth1.,RAF and MEK inhibitors show remarkable clinical efficacy in BRAFV600E melanoma2, 3; however, resistance to these agents remains a formidable challenge2, 4.,Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations.,Here, we performed systematic gain-of-function resistance studies by expressing >15,500 genes individually in a BRAFV600E melanoma cell line treated with RAF, MEK, ERK, or combined RAF/MEK inhibitors.,These studies revealed a cyclic AMP-dependent melanocytic signaling network not previously associated with drug resistance, including G-protein coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB).,Preliminary analysis of biopsies from BRAFV600E melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF/MEK-inhibition but restored in relapsing tumors.,Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF.,Combined treatment with MAP kinase pathway and histone deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance.,Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF/MEK/ERK inhibition, which may be overcome by combining signaling- and chromatin-directed therapeutics.
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Melanoma is one of the most aggressive skin cancers.,The 5-year survival rate of stage III melanoma patients ranges from 93% (IIIA) to 32% (IIID) with a high risk of recurrence after complete surgery.,The introduction of target and immune therapies has dramatically improved the overall survival, but the identification of patients with a high risk of relapse who will benefit from adjuvant therapy and the determination of the best treatment choice remain crucial.,Currently, patient prognosis is based on clinico-pathological features, highlighting the urgent need of predictive and prognostic markers to improve patient management.,In recent years, many groups have focused their attention on identifying molecular biomarkers with prognostic and predictive potential.,In this review, we examined the main candidate biomarkers reported in the literature.
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Originally described as interpatient variability, tumour heterogeneity has now been demonstrated to occur intrapatiently, within the same lesion, or in different lesions of the same patient.,Tumour heterogeneity involves both genetic and epigenetic changes.,Intrapatient heterogeneity is responsible for generating subpopulations of cancer cells which undergo clonal evolution with time.,Tumour heterogeneity develops also as a consequence of the selective pressure imposed by the immune system.,It has been demonstrated that tumour heterogeneity and different spatiotemporal interactions between all the cellular compontents within the tumour microenvironment lead to cancer adaptation and to therapeutic pressure.,In this context, the recent advent of single cell analysis approaches which are able to better study tumour heterogeneity from the genomic, transcriptomic and proteomic standpoint represent a major technological breakthrough.,In this review, using metastatic melanoma as a prototypical example, we will focus on applying single cell analyses to the study of clonal trajectories which guide the evolution of drug resistance to targeted therapy.
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Desmoplastic melanoma (DM) is a rare subtype of melanoma characterized by dense fibrous stroma, resistance to chemotherapy and a lack of actionable driver mutations, but is highly associated with ultraviolet light DNA damage.1 We analysed 60 patients with advanced DM treated with programmed cell death 1 (PD-1) or PD-1 ligand (PD-L1) blocking antibody therapy.,Objective tumour responses were observed in 42 of the 60 patients (70%, 95% confidence interval 57-81%), including 19 patients (32% overall) with a complete response.,Whole-exome sequencing revealed a high mutational load and frequent NF-1 mutations (14 out of 17 cases).,Immunohistochemistry (IHC) analysis from 19 DM and 13 non-DM revealed a higher percentage of PD-L1 positive cells in the tumour parenchyma in DM (p = 0.04), highly associated with increased CD8 density and PD-L1 expression in the tumour invasive margin.,Therefore, patients with advanced DM derive significant clinical benefit from PD-1/PD-L1 immune checkpoint blockade therapy despite being a cancer defined by its dense desmoplastic fibrous stroma.,The benefit is likely derived from the high mutational burden and a frequent pre-existing adaptive immune response limited by PD-L1 expression.
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Congenital melanocytic naevi (CMN) are a known risk factor for melanoma, with the greatest risk currently thought to be in childhood.,There has been controversy over the years about the incidence of melanoma, and therefore over the clinical management of CMN, due partly to the difficulties of histological diagnosis and partly to publishing bias towards cases of malignancy.,Large cohort studies have demonstrated that melanoma risk in childhood is related to the severity of the congenital phenotype.,New understanding of the genetics of CMN offers the possibility of improvement in diagnosis of melanoma, identification of those at highest risk, and new treatment options.,We review the world literature and our centre's experience over the last 25 years, including the molecular characteristics of melanoma in these patients and new melanoma incidence and outcome data from our prospective cohort.,Management strategies are proposed for presentation of suspected melanoma of the skin and the central nervous system in patients with CMN, including use of oral mitogen‐activated protein kinase kinase inhibitors in NRAS‐mutated tumours.,What's already known about this topic?,Multiple congenital melanocytic naevi (CMN) are the greatest risk factor for paediatric melanoma.Different clinical phenotypes have different risks of malignancy; however, the overall absolute risk for all types of CMN taken together is low.CMN can develop proliferative nodules that can cause diagnostic uncertainty and lead to repeated resections.Histology in patients with CMN is difficult and often requires specialist review.Melanoma in CMN is highly aggressive.,Multiple congenital melanocytic naevi (CMN) are the greatest risk factor for paediatric melanoma.,Different clinical phenotypes have different risks of malignancy; however, the overall absolute risk for all types of CMN taken together is low.,CMN can develop proliferative nodules that can cause diagnostic uncertainty and lead to repeated resections.,Histology in patients with CMN is difficult and often requires specialist review.,Melanoma in CMN is highly aggressive.,What does this study add?,In our prospective cohort, the strongest statistical risk factor for all‐site melanoma in childhood is an abnormal screening MRI of the central nervous system (CNS) in the first months of life, and in this group the incidence is 12%.,Where melanoma does arise in children with multiple CMN, a primary in the CNS is at least as common as in the skin.CNS melanoma currently has 100% mortality, but oral mitogen‐activated protein kinase kinase inhibition in NRAS‐mutation mosaic patients may improve symptom control.Management strategies are proposed for the presentation of a possible malignancy in the skin or CNS.,In our prospective cohort, the strongest statistical risk factor for all‐site melanoma in childhood is an abnormal screening MRI of the central nervous system (CNS) in the first months of life, and in this group the incidence is 12%.,Where melanoma does arise in children with multiple CMN, a primary in the CNS is at least as common as in the skin.,CNS melanoma currently has 100% mortality, but oral mitogen‐activated protein kinase kinase inhibition in NRAS‐mutation mosaic patients may improve symptom control.,Management strategies are proposed for the presentation of a possible malignancy in the skin or CNS.
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Metastatic melanoma is the most aggressive and difficult to treat type of skin cancer, with a survival rate of less than 10%.,Metastatic melanoma has conventionally been considered very difficult to treat; however, recent progress in understanding the cellular and molecular mechanisms involved in the tumorigenesis, metastasis and immune escape have led to the introduction of new therapies.,These include targeted molecular therapy and novel immune-based approaches such as immune checkpoint blockade (ICB), tumor-infiltrating lymphocytes (TILs), and genetically engineered T-lymphocytes such as chimeric antigen receptor (CAR) T cells.,Among these, CAR T cell therapy has recently made promising strides towards the treatment of advanced hematological and solid cancers.,Although CAR T cell therapy might offer new hope for melanoma patients, it is not without its shortcomings, which include off-target toxicity, and the emergence of resistance to therapy (e.g., due to antigen loss), leading to eventual relapse.,The present review will not only describe the basic steps of melanoma metastasis, but also discuss how CAR T cells could treat metastatic melanoma.,We will outline specific strategies including combination approaches that could be used to overcome some limitations of CAR T cell therapy for metastatic melanoma.
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The effect of apatinib on the formation of vasculogenic mimicry (VM) was studied in a malignant melanoma cell line.,MUM-2B cells cultured in three-dimensional Matrigel were treated with varying concentrations (0, 0.01, 0.05, 0.1, 0.5 μmol/L) of apatinib to test its effect on VM in vitro, followed by MTT proliferation and transwell invasion assays to determine the effect of apatinib on cell proliferation and invasion of MUM-2B cells.,In vivo, we used a melanoma cancer model to test the effect of short-term apatinib (100, 200, 300 mg/kg) treatment on VM.,Western blotting, immunohistochemistry staining, and CD31-PAS dual staining were performed to assess the expression of VEGFR-2, ERK-1/2, PI3K, and MMP-2, and formation of VM.,The results showed apatinib-treated groups formed a lesser number of VM in 3D matrigel, while the cell viability in MTT proliferation assay and the number of migration cells in transwell invasion assay were significantly lower in apatinib-treated groups.,In addition, short-term apatinib treatment inhibited angiogenesis, VM formation, and tumor growth in models of melanoma cancer.,Mice in apatinib-treated groups showed a markedly reduced expression of VEGFR-2, ERK-1/2, PI3K, and MMP-2.,In summary, apatinib could inhibit the expression of VEGFR-2, and downregulate the ERK1/2/PI3K/MMP-2 signaling cascade, which may be one of the underlying mechanisms by which apatinib inhibits angiogenesis and the development of VM in models of melanoma cancer, and restrains the formation of VM by MUM-2B cells.,Apatinib shows inhibitory effects on cell proliferation and invasion of MUM-2B cells, which is a close relationship with the VM.
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DNA methylation at CpG dinucleotides is modified in tumorigenesis with potential impact on transcriptional activity.,We used the Illumina 450 K platform to evaluate DNA methylation patterns of 50 metastatic melanoma tumors, with matched gene expression data.,We identified three different methylation groups and validated the groups in independent data from The Cancer Genome Atlas.,One group displayed hypermethylation of a developmental promoter set, genome-wide demethylation, increased proliferation and activity of the SWI/SNF complex.,A second group had a methylation pattern resembling stromal and leukocyte cells, over-expressed an immune signature and had improved survival rates in metastatic tumors (p < 0.05).,A third group had intermediate methylation levels and expressed both proliferative and immune signatures.,The methylation groups corresponded to some degree with previously identified gene expression phenotypes.,Melanoma consists of divergent methylation groups that are distinguished by promoter methylation, proliferation and content of immunological cells.,The online version of this article (doi:10.1186/s12920-015-0147-4) contains supplementary material, which is available to authorized users.
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MicroRNAs (miRNAs) are endogenously expressed, small noncoding RNAs that inhibit gene expression by binding to target mRNAs.,Recent studies have revealed that miRNAs function as tumor suppressors or oncogenes.,In the present study, we investigated the role of miRNA-34b/c in uveal melanoma.,Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect the expression level of miR-34b/c in uveal melanoma cells and primary samples.,Subsequently, uveal melanoma cell proliferation was examined by the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl] -2H-tetrazolium, inner salt) assay, clone formation assay, and flow cytometry.,Cell apoptosis was measured by caspase3/7 assay.,Cell migration was evaluated by transwell migration assay.,The target of miR-34b/c was predicted by bioinformatics and validated by luciferase assay.,In addition, the effect of miR-34b/c on c-Met, cell cycle-related proteins, v-akt murine thymoma viral oncogene homolog (Akt) and extracellular signal-regulated kinase (ERK) pathway was determined by western blotting.,miR-34b/c expression, which was dramatically decreased in uveal melanoma cells and clinical samples, can be upregulated by doxorubicin and epigenetic drugs.,The transfection of miR-34b/c into uveal melanoma cells leads to a significant reduction in cell growth and migration. miR-34b/c caused cell cycle G1 arrest rather than the induction of apoptosis.,Met proto-oncogene (c-Met) was identified as a target of miR-34b/c in uveal melanoma cells.,Furthermore, miR-34b/c was confirmed to downregulate the expression of c-Met, p-Akt, and cell cycle-related proteins by western blotting.,Our results demonstrate that both miR-34b and miR-34c act as tumor suppressors in uveal melanoma cell proliferation and migration through the downregulation of multiple targets.
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Cancer immunotherapy restores and/or enhances effector function of CD8+ T cells in the tumor microenvironment1,2.,CD8+ T cells activated by cancer immunotherapy execute tumor clearance mainly by inducing cell death through perforin-granzyme- and Fas/Fas ligand-pathways3,4.,Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent lipid peroxide accumulation5,6.,Although it was mechanistically illuminated in vitro7,8, emerging evidence has shown that ferroptosis may be implicated in a variety of pathological scenarios9,10.,However, the involvement of ferroptosis in T cell immunity and cancer immunotherapy is unknown.,Here, we find that immunotherapy-activated CD8+ T cells enhance ferroptosis-specific lipid peroxidation in tumor cells, and in turn, increased ferroptosis contributes to the anti-tumor efficacy of immunotherapy.,Mechanistically, interferon gamma (IFNγ) released from CD8+ T cells downregulates expression of SLC3A2 and SLC7A11, two subunits of glutamate-cystine antiporter system xc-, restrains tumor cell cystine uptake, and as a consequence, promotes tumor cell lipid peroxidation and ferroptosis.,In preclinical models, depletion of cyst(e)ine by cyst(e)inase in combination with checkpoint blockade synergistically enhances T cell-mediated anti-tumor immunity and induces tumor cell ferroptosis.,Expression of system xc- is negatively associated with CD8+ T cell signature, IFNγ expression, and cancer patient outcome.,Transcriptome analyses before and during nivolumab therapy reveal that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8.,Thus, T cell-promoted tumor ferroptosis is a novel anti-tumor mechanism.,Targeting tumor ferroptosis pathway constitutes a therapeutic approach in combination with checkpoint blockade.
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Melanoma aggressiveness determines its growth and metastatic potential.,This study aimed at identifying new molecular pathways controlling melanoma cell malignancy.,Ten metastatic melanoma cell lines were characterized by their proliferation, migration and invasion capabilities.,The most representative cells were also characterized by spheroid formation assay, gene- and protein- expression profiling as well as cytokines secretion and the most relevant pathways identified through bioinformatic analysis were tested by in silico transcriptomic validation on datasets generated from biopsies specimens of melanoma patients.,Further, matrix metalloproteases (MMPs) activity was tested by zymography assays and TNF-alpha role was validated by anti-TNF cell-treatment.,An aggressiveness score (here named Melanoma AGgressiveness Score: MAGS) was calculated by measuring proliferation, migration, invasion and cell-doubling time in10human melanoma cell lines which were clustered in two distinct groups, according to the corresponding MAGS.,SK-MEL-28 and A375 cell lines were selected as representative models for the less and the most aggressive phenotype, respectively.,Gene-expression and protein expression data were collected for SK-MEL-28 and A375 cells by Illumina-, multiplex x-MAP-and mass-spectrometry technology.,The collected data were subjected to an integrated Ingenuity Pathway Analysis, which highlighted that cytokine/chemokine secretion, as well as Cell-To-Cell Signaling and Interaction functions as well as matrix metalloproteases activity were significantly different in these two cell types.,The key role of these pathways was then confirmed by functional validation.,TNF role was confirmed by exposing cells to the anti-TNF Infliximab antibody.,Upon such treatment melanoma cells aggressiveness was strongly reduced.,Metalloproteases activity was assayed, and their role was confirmed by comparing transcriptomic data from cutaneous melanoma patients (n = 45) and benign nevi (n = 18).,Inflammatory signals such as TNF and MMP-2 activity are key intrinsic players to determine melanoma cells aggressiveness suggesting new venue sin the identification of novel molecular targets with potential therapeutic relevance.,The online version of this article (10.1186/s13046-018-0982-1) contains supplementary material, which is available to authorized users.
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Uveal melanoma (UM) is the most common intraocular malignancy in adults.,Despite improvements in surgical, radiation and chemotherapy treatments, the overall survival of UM and prognosis remain poor.,In the present study, we hypothesized that Sirtuin 1 and 2 (SIRT1/2), class III histone deacetylases (HDACs), were critical in controlling the destiny of bulk tumor cells and cancer stem cells (CSCs) of UM.,We testified this hypothesis in four lines of UM cells (92.1, Mel 270, Omm 1 and Omm 2.3).,Our results showed that inhibition of SIRT1/2 by Tenovin-6 induced apoptosis in UM cells by activating the expression of tumor suppressor genes such as p53 and elevating reactive oxygen species (ROS).,Tenovin-6 inhibited the growth of UM cells.,Tenovin-6 and vinblastine was synergistic in inducing apoptosis of UM cell line 92.1 and Mel 270.,Furthermore, Tenovin-6 eliminated cancer stem cells in 92.1 and Mel 270 cells.,In conclusion, our findings suggest that Tenovin-6 may be a promising agent to kill UM bulk tumor cells and CSCs.
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Up to 50% of patients with uveal melanoma develop metastatic disease with poor prognosis.,Regional, mainly liver-directed, therapies may induce limited tumor responses but do not improve overall survival.,Response rates of metastatic uveal melanoma (MUM) to systemic chemotherapy are poor.,Insights into the molecular biology of MUM recently led to investigation of new drugs.,In this study, to compare response rates of systemic treatment for MUM we searched Pubmed/Web of Knowledge databases and ASCO website (1980-2013) for “metastatic/uveal/melanoma” and “melanoma/eye.”,Forty studies (one case series, three phase I, five pilot, 22 nonrandomized, and two randomized phase II, one randomized phase III study, data of three expanded access programs, three retrospective studies) with 841 evaluable patients were included in the numeric outcome analysis.,Complete or partial remissions were observed in 39/841 patients (overall response rate [ORR] 4.6%; 95% confidence intervals [CI] 3.3-6.3%), no responses were observed in 22/40 studies.,Progression-free survival ranged from 1.8 to 7.2, median overall survival from 5.2 to 19.0 months as reported in 21/40 and 26/40 studies, respectively.,Best responses were seen for chemoimmunotherapy (ORR 10.3%; 95% CI 4.8-18.7%) though mainly in first-line patients.,Immunotherapy with ipilimumab, antiangiogenetic approaches, and kinase inhibitors have not yet proven to be superior to chemotherapy.,MEK inhibitors are currently investigated in a phase II trial with promising preliminary data.,Despite new insights into genetic and molecular background of MUM, satisfying systemic treatment approaches are currently lacking.,Study results of innovative treatment strategies are urgently awaited.,Forty clinical studies on metastatic uveal melanoma were reviewed regarding responses to systemic treatments.,New insights into genetic and molecular background led to investigation of new substances but promising in vitro data have not yet been translated into satisfying treatment responses; however, preliminary results of ongoing studies are highly encouraging.
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Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer associated with poor survival outcomes in patients with distant metastatic disease (mMCC).,In an initial analysis from JAVELIN Merkel 200, a phase 2, prospective, open-label, single-arm trial in mMCC, avelumab-a human anti-programmed death-ligand 1 (PD-L1) monoclonal antibody-showed promising efficacy and a safety profile that was generally manageable and tolerable.,Here, we report the efficacy of avelumab after ≥1 year of follow-up in patients with distant mMCC that had progressed following prior chemotherapy for metastatic disease.,Patients received avelumab 10 mg/kg by 1-h intravenous infusion every 2 weeks until confirmed disease progression, unacceptable toxicity, or withdrawal.,The primary endpoint was best overall response.,Secondary endpoints included duration of response (DOR), progression-free survival (PFS), and overall survival (OS).,Patients (N = 88) were followed for a minimum of 12 months.,The confirmed objective response rate was 33.0% (95% CI, 23.3%-43.8%; complete response: 11.4%).,An estimated 74% of responses lasted ≥1 year, and 72.4% of responses were ongoing at data cutoff.,Responses were durable, with the median DOR not yet reached (95% CI, 18.0 months-not estimable), and PFS was prolonged; 1-year PFS and OS rates were 30% (95% CI, 21%-41%) and 52% (95% CI, 41%-62%), respectively.,Median OS was 12.9 months (95% CI, 7.5-not estimable).,Subgroup analyses suggested a higher probability of response in patients receiving fewer prior lines of systemic therapy, with a lower baseline disease burden, and with PD-L1-positive tumors; however, durable responses occurred irrespective of baseline factors, including tumor Merkel cell polyomavirus status.,With longer follow-up, avelumab continues to show durable responses and promising survival outcomes in patients with distant mMCC whose disease had progressed after chemotherapy.,Clinicaltrials.gov identifier: NCT02155647.
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Merkel cell carcinoma (MCC) is a rare but highly aggressive cutaneous neuroendocrine carcinoma, associated with the Merkel cell polyomavirus (MCPyV) in 80% of cases.,To define the genetic basis of MCCs, we performed exome sequencing of 49 MCCs.,We show that MCPyV-negative MCCs have a high mutation burden (median of 1121 somatic single nucleotide variants (SSNVs) per-exome with frequent mutations in RB1 and TP53 and additional damaging mutations in genes in the chromatin modification (ASXL1, MLL2, and MLL3), JNK (MAP3K1 and TRAF7), and DNA-damage pathways (ATM, MSH2, and BRCA1).,In contrast, MCPyV-positive MCCs harbor few SSNVs (median of 12.5 SSNVs/tumor) with none in the genes listed above.,In both subgroups, there are rare cancer-promoting mutations predicted to activate the PI3K pathway (HRAS, KRAS, PIK3CA, PTEN, and TSC1) and to inactivate the Notch pathway (Notch1 and Notch2).,TP53 mutations appear to be clinically relevant in virus-negative MCCs as 37% of these tumors harbor potentially targetable gain-of-function mutations in TP53 at p.R248 and p.P278.,Moreover, TP53 mutational status predicts death in early stage MCC (5-year survival in TP53 mutant vs wild-type stage I and II MCCs is 20% vs. 92%, respectively; P = 0.0036).,Lastly, we identified the tumor neoantigens in MCPyV-negative and MCPyV-positive MCCs.,We found that virus-negative MCCs harbor more tumor neoantigens than melanomas or non-small cell lung cancers (median of 173, 65, and 111 neoantigens/sample, respectively), two cancers for which immune checkpoint blockade can produce durable clinical responses.,Collectively, these data support the use of immunotherapies for virus-negative MCCs.
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Although immune checkpoint inhibitors (ICIs) have achieved unprecedented results in melanoma, the biological features of the durable responses initiated by these drugs remain unknown.,Here we show the genetic and phenotypic changes induced by treatment with programmed cell death-1 (PD-1) blockade in a genetically engineered mouse model of melanoma driven by oncogenic BRAF.,In this controlled system anti-PD-1 treatment yields responses in ~35% of the tumors, and prolongs survival in ~27% of the animals.,We identify increased stroma remodeling and reduced expression of proliferation markers as features associated with prolonged response.,These traits are corroborated in two independent early on-treatment anti-PD-1 melanoma patient cohorts.,These insights into the biological responses of tumors to ICI provide a strategy for identification of durable response early during the course of treatment and could improve patient stratification for checkpoint inhibitory drugs.,Identification of clinical relevant biomarkers to predict response to immune checkpoint blockade (ICB) in melanoma remains challenging.,Here, the authors show that stroma remodelling and reduced cell division are associated with durable response to anti-PD1 in a mouse model of melanoma and in ICB-treated patients.
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People with pale skin, red hair, freckles, and an inability to tan-the “redhair/fairskin” phenotype- are at highest risk of developing melanoma, compared to all other pigmentation types1.,Genetically, this phenotype is frequently the product of inactivating polymorphisms in the Melanocortin 1 receptor (MC1R) gene.,MC1R encodes a cAMP stimulating G-protein coupled receptor that controls pigment production.,Minimal receptor activity, as in redhair/fairskin polymorphisms, produces red/yellow pheomelanin pigment, while increasing MC1R activity stimulates production of black/brown eumelanin2.,Pheomelanin has weak UV shielding capacity relative to eumelanin and has been shown to amplify UVA-induced reactive oxygen species (ROS) 3-5.,Several observations, however, complicate the assumption that melanoma risk is completely UV dependent.,For example, unlike non-melanoma skin cancers, melanoma is not restricted to sun-exposed skin and UV signature mutations are infrequently oncogenic drivers6.,While linkage of melanoma risk to UV exposure is beyond doubt, UV-independent events are also likely to play a significant role1,7.,Here, we introduced into mice carrying an inactivating mutation in the Mc1r gene (who exhibit a phenotype analogous to redhair/fairskin humans), a conditional, melanocyte-targeted allele of the most commonly mutated melanoma oncogene, BRafV600E.,We observed a high incidence of invasive melanomas without providing additional gene aberrations or UV exposure.,To investigate the mechanism of UV-independent carcinogenesis, we introduced an albino allele, which ablates all pigment production on the Mc1r e/e background.,Selective absence of pheomelanin synthesis was protective against melanoma development.,In addition, normal Mc1re/e mouse skin was found to have significantly greater oxidative DNA and lipid damage than albino-Mc1re/e mouse skin.,These data suggest that the pheomelanin pigment pathway produces UV-independent carcinogenic contributions to melanomagenesis by a mechanism of oxidative damage.,While UV protection remains important, additional strategies may be required for optimal melanoma prevention.
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The conserved serine/threonine kinase mTOR (the mammalian target of rapamycin), a downstream effector of the PI3K/AKT pathway, forms two distinct multiprotein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to rapamycin, activates S6K1 and 4EBP1, which are involved in mRNA translation.,It is activated by diverse stimuli, such as growth factors, nutrients, energy and stress signals, and essential signalling pathways, such as PI3K, MAPK and AMPK, in order to control cell growth, proliferation and survival. mTORC2 is considered resistant to rapamycin and is generally insensitive to nutrients and energy signals.,It activates PKC-α and AKT and regulates the actin cytoskeleton.,Deregulation of multiple elements of the mTOR pathway (PI3K amplification/mutation, PTEN loss of function, AKT overexpression, and S6K1, 4EBP1 and eIF4E overexpression) has been reported in many types of cancers, particularly in melanoma, where alterations in major components of the mTOR pathway were reported to have significant effects on tumour progression.,Therefore, mTOR is an appealing therapeutic target and mTOR inhibitors, including the rapamycin analogues deforolimus, everolimus and temsirolimus, are submitted to clinical trials for treating multiple cancers, alone or in combination with inhibitors of other pathways.,Importantly, temsirolimus and everolimus were recently approved by the FDA for the treatment of renal cell carcinoma, PNET and giant cell astrocytoma.,Small molecules that inhibit mTOR kinase activity and dual PI3K-mTOR inhibitors are also being developed.,In this review, we aim to survey relevant research, the molecular mechanisms of signalling, including upstream activation and downstream effectors, and the role of mTOR in cancer, mainly in melanoma.
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Cell-type specific signalling determines cell fate under physiological conditions, but it is increasingly apparent that also in cancer development the impact of any given oncogenic pathway on the individual cancer pathology is dependent on cell-lineage specific molecular traits.,For instance in colon and liver cancer canonical Wnt signalling produces increased cytoplasmic and nuclear localised beta-catenin, which correlates with invasion and poor prognosis.,In contrast, in melanoma increased cytoplasmic and nuclear beta-catenin is currently emerging as a marker for good prognosis and thus appears to have a different function compared to other cancer types; however this function is unknown.,We discovered that in contrast to its function in other cancers, in melanoma, beta-catenin blocks invasion.,We demonstrate that this opposing role of nuclear beta-catenin in melanoma is mediated through MITF, a melanoma-specific protein that defines the lineage background of this cancer type.,Downstream of beta-catenin MITF not only suppresses the Rho-GTPase regulated cell-morphology of invading melanoma cells, but also interferes with beta-catenin induced expression of the essential collagenase MT1-MMP, thus affecting all aspects of an invasive phenotype.,Importantly, overexpression of MITF in invasive colon cancer cells modifies beta-catenin directed signalling and induces a ‘melanoma-phenotype’.,In summary, the cell type specific presence of MITF in melanoma affects beta-catenin’s pro-invasive properties otherwise active in colon or liver cancer.,Thus our study reveals the general importance of considering cell-type specific signalling for the accurate interpretation of tumour markers and ultimately for the design of rational therapies.
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Melanoma is the deadliest cutaneous neoplasm.,To prevent metastasis, early diagnosis and surgical treatment is vital.,Long non-coding RNAs (lncRNAs) may serve as biomarkers and therapeutic targets in tumors.,We investigated the molecular mechanisms of lncRNA KCNQ1OT1 in melanoma.,Real time PCR demonstrated that KCNQ1OT1 expression is up-regulated in melanoma tissues and cells.,KCNQ1OT1 promoted cell proliferation and metastasis in melanoma.,By directly bindin to miR-153, KCNQ1OT1 acted as a competing endogenous RNA (ceRNA) to de-repress MET expression.,Our results may provide the basis for a novel strategy for early detection and/or treatment of melanoma.
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Melanoma is notorious for its propensity to metastasize, which makes treatment extremely difficult.,Receptor tyrosine kinase c-Met is activated in human melanoma and is involved in melanoma progression and metastasis.,Hepatocyte growth factor (HGF)-mediated activation of c-Met signaling has been suggested as a therapeutic target for melanoma metastasis.,Quercetin is a dietary flavonoid that exerts anti-metastatic effect in various types of cancer including melanoma.,In a previous report, we demonstrated that quercetin inhibited melanoma cell migration and invasion in vitro, and prevented melanoma cell lung metastasis in vivo.,In this study, we sought to determine the involvement of HGF/c-Met signaling in the anti-metastatic action of quercetin in melanoma.,Transwell chamber assay was conducted to determine the cell migratory and invasive abilities.,Western blotting was performed to determine the expression levels and activities of c-Met and its downstream molecules.,And immunoblotting was performed in BS3 cross-linked cells to examine the homo-dimerization of c-Met.,Quantitative real-time PCR analysis was carried out to evaluate the mRNA expression level of HGF.,Transient transfection was used to overexpress PAK or FAK in cell models.,Student’s t-test was used in analyzing differences between two groups.,Quercetin dose-dependently suppressed HGF-stimulated melanoma cell migration and invasion.,Further study indicated that quercetin inhibited c-Met phosphorylation, reduced c-Met homo-dimerization and decreased c-Met protein expression.,The effect of quercetin on c-Met expression was associated with a reduced expression of fatty acid synthase.,In addition, quercetin suppressed the phosphorylation of c-Met downstream molecules including Gab1 (GRB2-associated-binding protein 1), FAK (Focal Adhesion Kinase) and PAK (p21-activated kinases).,More importantly, overexpression of FAK or PAK significantly reduced the inhibitory effect of quercetin on the migration of the melanoma cells.,Our findings suggest that suppression of the HGF/c-Met signaling pathway contributes to the anti-metastatic action of quercetin in melanoma.,The online version of this article (doi:10.1186/s12943-015-0367-4) contains supplementary material, which is available to authorized users.
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Cutaneous melanoma is a highly aggressive cancer with a propensity for distant metastasis to various organs.,In contrast, melanoma arising in pigmented uveal layers of the eye metastasizes mostly in the liver.,The mechanisms of these metastases, which are ultimately resistant to therapy, are still unclear.,Metastasis via intravascular dissemination of tumour cells is widely accepted as a central paradigm.,However, we have previously described an alternative mode of tumour dissemination, extravascular migratory metastasis, based on clinical and experimental data.,This mechanism is characterised by the interaction of cancer cells with the abluminal vascular surface, which defines angiotropism.,Here, we employed our 3D co-culture approach to monitor cutaneous and uveal human melanoma cells dynamics in presence of vascular tubules.,Using time-lapse microscopy, we evaluated angiotropism, the migration of tumour cells along vascular tubules and the morphological changes occurring during these processes.,Cutaneous and uveal melanoma cells were injected in zebrafish embryos in order to develop xenografts.,Employing in vivo imaging coupled with 3D reconstruction, we monitored the interactions between cancer cells and the external surface of zebrafish vessels.,Overall, our results indicate that cutaneous and uveal melanoma cells spread similarly along the abluminal vascular surfaces, in vitro and in vivo.
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Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge.,Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task.,Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action.,We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system.,FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention.,Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.,Pertussis toxin is used extensively for perturbing Gαi/o pathways in the study of physiology and disease, but an equivalent inhibitor of Gαq signalling is not currently available to the research community.,Here the authors characterize FR900359 as a specific Gq inhibitor and demonstrate its utility to dissect GPCR signalling and its potential to inhibit melanoma cells.
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Nivolumab, a monoclonal antibody of immune checkpoint programmed death 1 on T cells (PD-1), combined with ipilimumab, an immune checkpoint cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, as combination therapy on the one hand and nivolumab as monotherapy on the other, have both demonstrated improved efficacy compared with ipilimumab alone in the CheckMate 067 study.,However, the combination resulted in a higher frequency of grade 3/4 adverse events (AEs), which could result in diminished health-related quality of life (HRQoL).,Here we report analyses of HRQoL for patients with advanced melanoma in clinical trial CheckMate 067.,HRQoL was assessed at weeks 1 and 5 per 6-week cycle for the first 6 months, once every 6 weeks thereafter, and at two follow-up visits using the European Organization for Research and Treatment of Care Core Quality of Life Questionnaire and the EuroQoL Five Dimensions Questionnaire.,In addition to the randomised population, patient subgroups, including BRAF mutation status, partial or complete response, treatment-related AEs of grade 3/4, and those who discontinued due to any reason and due to an AE, were investigated.,Nivolumab and ipilimumab combination and nivolumab alone both maintained HRQoL, and no clinically meaningful deterioration was observed over time compared with ipilimumab.,In addition, similar results were observed across patient subgroups, and no clinically meaningful changes in HRQoL were observed during follow-up visits for patients who discontinued due to any cause.,These results further support the clinical benefit of nivolumab monotherapy and nivolumab and ipilimumab combination therapy in patients with advanced melanoma.,The finding that the difference in grade 3/4 AEs between the arms did not translate into clinically meaningful differences in the reported HRQoL may be relevant in the clinical setting.,NCT01844505.
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In an international, randomized Phase III trial ipilimumab demonstrated a significant overall survival benefit in previously treated advanced melanoma patients.,This report summarizes health-related quality of life (HRQL) outcomes for ipilimumab with/without gp100 vaccine compared to gp100 alone during the clinical trial’s 12 week treatment induction period.,The Phase III clinical trial (MDX010-20) was a double-blind, fixed dose study in 676 previously treated advanced unresectable stage III or IV melanoma patients.,Patients were randomized 3:1:1 to receive either ipilimumab (3 mg/kg q3w x 4 doses) + gp100 (peptide vaccine; 1 mg q3w x 4 doses; ipilimumab plus gp100, n = 403); gp100 vaccine + placebo (gp100 alone, n = 136); or ipilimumab + placebo (ipilimumab alone, n = 137).,The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) assessed HRQL.,Baseline to Week 12 changes in EORTC QLQ-C30 function, global health status, and symptom scores were analyzed for ipilimumab with/without gp100 vaccine compared to gp100 alone.,Mean change in scores were categorized “no change” (0-5), “a little” (5-10 points), “moderate” (10-20 points), and “very much” (>20).,In the ipilimumab plus gp100 and ipilimumab alone groups, mean changes from baseline to Week 12 generally indicated “no change” or “a little” impairment across EORTC QLQ-C30 global health status, function, and symptom subscales.,Significant differences in constipation, favoring ipilimumab, were observed (p < 0.05).,For ipilimumab alone arm, subscales with no or a little impairment were physical, emotional, cognitive, social function, global health, nausea, pain, dyspnea, constipation, and diarrhea subscales.,For the gp100 alone group, the observed changes were moderate to large for global health, role function, fatigue, and for pain.,Ipilimumab with/without gp100 vaccine does not have a significant negative HRQL impact during the treatment induction phase relative to gp100 alone in stage III or IV melanoma patients.,Clinicaltrials.gov identification number NCT00094653
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The optimal treatment sequence for patients with advanced BRAF V600 mutant melanoma is unknown.,BRAF/MEK inhibition (BRAF/MEKi), single agent anti‐PD‐1 (aPD‐1) antibodies and combination immune checkpoint inhibition with nivolumab and ipilimumab (niv/ipi) are all approved; however, they have not been prospectively compared.,Therefore, we sought to compare overall survival of patients with advanced BRAF mutant melanoma treated with either front‐line BRAF/MEKi, aPD‐1, or niv/ipi.,Patients with advanced BRAF mutant melanoma who had received BRAF/MEKi, niv/ipi, or aPD‐1 in the front‐line setting were identified from a nationwide database comprising de‐identified patient‐level structured and unstructured data derived from electronic health records.,Survival was compared using Kaplan‐Meier curves and log‐rank analysis.,Univariate and multivariate Cox regression models were used to measure the effect of front‐line treatment, age (>64 or not), LDH (elevated or not), and Eastern Cooperative Oncology Group (ECOG) performance status (>1 or not) on survival.,Five hundred and sixty seven patients with advanced disease and treated with front‐line aPD‐1 (n = 162), BRAF/MEKi (n = 297) or niv/ipi (n = 108) were identified.,With a median follow‐up of 22.4 months, median overall survival (OS) for patients treated with front‐line niv/ipi was not reached (NR) while median OS for patients treated with aPD‐1 or BRAF/MEKi was 39.5 months and 13.2 months, respectively.,Front‐line treatment with PD‐1 and niv/ipi were associated with statistically longer survival than BRAF/MEKi in multivariate analyses.,In our real‐world retrospective analysis, patients with advanced BRAF mutant melanoma treated with front‐line niv/ipi or aPD‐1 had longer survival compared to those treated with front‐line BRAF/MEKi.,Real‐world overall survival of patients with advanced BRAF mutant melanoma treated with front‐line BRAF/MEK inhibitors, anti‐PD‐1 antibodies, or nivolumab/ipilimumab.
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Activated RAS promotes dimerization of members of the RAF kinase family1-3.,ATP-competitive RAF inhibitors activate ERK signaling4-7 by transactivating RAF dimers4.,In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity.,This tumor-specific inhibition of ERK signaling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbor mutant BRAF(V600E)8.,However, resistance invariably develops.,Here, we identify a novel resistance mechanism.,We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61kd variant form of BRAF(V600E) that lacks exons 4-8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) exhibits enhanced dimerization in cells with low levels of RAS activation, as compared to full length BRAF(V600E).,In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signaling is resistant to the RAF inhibitor.,Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib.,Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumors of six of 19 patients with acquired resistance to vemurafenib.,These data support the model that inhibition of ERK signaling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.
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Basal cell carcinoma (BCC) is the most common human cancer and represents a growing public health care problem.,Several tumor suppressor genes and proto-oncogenes have been implicated in BCC pathogenesis, including the key components of the Hedgehog pathway, PTCH1 and SMO, the TP53 tumor suppressor, and members of the RAS proto-oncogene family.,Aberrant activation of the Hedgehog pathway represents the molecular driver in basal cell carcinoma pathogenesis, with the majority of BCCs carrying somatic point mutations, mainly ultraviolet (UV)-induced, and/or copy-loss of heterozygosis in the PTCH1 gene.,Recent advances in sequencing technology allowed genome-scale approaches to mutation discovery, identifying new genes and pathways potentially involved in BCC carcinogenesis.,Mutational and functional analysis suggested PTPN14 and LATS1, both effectors of the Hippo-YAP pathway, and MYCN as new BCC-associated genes.,In addition, emerging reports identified frequent non-coding mutations within the regulatory promoter sequences of the TERT and DPH3-OXNAD1 genes.,Thus, it is clear that a more complex genetic network of cancer-associated genes than previously hypothesized is involved in BCC carcinogenesis, with a potential impact on the development of new molecular targeted therapies.,This article reviews established knowledge and new hypotheses regarding the molecular genetics of BCC pathogenesis.
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Photodynamic therapy (PDT) is a widely used technique for epithelial skin cancer treatment. 5-aminolevulinic acid (5-ALA) is a drug currently used for PDT and is a hydrophilic molecule at its physiological pH, and this limits its capacity to cross the stratum corneum of skin.,Since skin penetration is a key factor in the efficacy of topical 5-ALA-mediated PDT, numerous strategies have been proposed to improve skin penetration.,Yet this problem is still ongoing.,The results of a previous study showed a low rate of 5-ALA encapsulated in liposomes (5.7%) that were 400 nm in size.,In the present study, we used 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes as vehicles and tested their delivery efficacy of 5-ALA-medicated PDT both in vitro and in vivo.,Our data shows that 5-ALA encapsulated in 0.1 or 0.5% DPPC liposomes (5-ALA/DPPC) had a better encapsulated rate (15~16%) and were smaller in size (84~89 nm).,We found the 5-ALA/DPPC formulation reduced cell viability, mitochondria membrane potential, and enhanced intracellular ROS accumulation as compared to 5-ALA alone in melanoma cells.,Furthermore, the 5-ALA/DPPC formulation also had better skin penetration ability as compared to the 5-ALA in our ex vivo data by assaying 5-ALA converted into protoporphyrin IX (PpIX) in the skin of the mice that were experimented on.,In melanoma xenograft models, 5-ALA/DPPC enhanced PpIX accumulation only in tumor tissue but not normal skin.,In conclusion, we found DPPC liposomes to be good carriers for 5-ALA delivery and believe that they may prove useful in 5-ALA-mediated PDT in the future.
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With a constantly increasing incidence, cutaneous melanoma has raised the need for a better understanding of its complex microenvironment that may further guide therapeutic options.,Melanoma is a model tumor in immuno-oncology.,Inflammation represents an important hallmark of cancer capable of inducing and sustaining tumor development.,The inflammatory process also orchestrates the adaptative immunosuppression of tumor cells that helps them to evade immune destruction.,Besides its role in proliferation, angiogenesis, and apoptosis, cyclooxygenase-2 (COX-2) is a well-known promoter of immune suppression in melanoma.,COX-2 inhibitors are closely involved in this condition.,This review attempts to answer two controversial questions: is COX-2 a valuable prognostic factor?,Among all COX-2 inhibitors, is celecoxib a suitable adjuvant in melanoma therapy?
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Stress has an emerging role in cancer and targeting stress-related β-adrenergic receptors (AR) has been proposed as a potential therapeutic approach in melanoma.,Here we report that β3-AR expression correlates with melanoma aggressiveness.,In addition, we highlight that β3-AR expression is not only restricted to cancer cells, but it is also expressed in vivo in stromal, inflammatory and vascular cells of the melanoma microenvironment.,Particularly, we demonstrated that β3-AR can (i) instruct melanoma cells to respond to environmental stimuli, (ii) enhance melanoma cells response to stromal fibroblasts and macrophages, (iii) increase melanoma cell motility and (iv) induce stem-like traits.,Noteworthy, β3-AR activation in melanoma accessory cells drives stromal reactivity by inducing pro-inflammatory cytokines secretion and de novo angiogenesis, sustaining tumor growth and melanoma aggressiveness. β3-ARs also play a mandatory role in the recruitment to tumor sites of circulating stromal cells precursors, in the differentiation of these cells towards different lineages, further favoring tumor inflammation, angiogenesis and ultimately melanoma malignancy.,Our findings validate selective β3-AR antagonists as potential promising anti-metastatic agents.,These could be used to complement current therapeutic approaches for melanoma patients (e.g. propranolol) by targeting non-neoplastic stromal cells, hence reducing therapy resistance of melanoma.
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Tumor associated inflammation predicts response to immune checkpoint blockade in human melanoma.,Current theories on regulation of inflammation center on anti-tumor T cell responses.,Here we show that tumor associated B cells are vital to melanoma associated inflammation.,Human B cells express pro- and anti-inflammatory factors and differentiate into plasmablast-like cells when exposed to autologous melanoma secretomes in vitro.,This plasmablast-like phenotype can be reconciled in human melanomas where plasmablast-like cells also express T cell-recruiting chemokines CCL3, CCL4, CCL5.,Depletion of B cells in melanoma patients by anti-CD20 immunotherapy decreases tumor associated inflammation and CD8+ T cell numbers.,Plasmablast-like cells also increase PD-1+ T cell activation through anti-PD-1 blockade in vitro and their frequency in pretherapy melanomas predicts response and survival to immune checkpoint blockade.,Tumor associated B cells therefore orchestrate and sustain melanoma inflammation and may represent a predictor for survival and response to immune checkpoint blockade therapy.,The regulation of tumor inflammation is incompletely understood and the role of B cells is unclear.,Here, the authors show that a specific subtype of B cells is induced in melanoma and required to recruit T lymphocytes and elicit inflammation.
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In melanoma, therapies with inhibitors to oncogenic BRAFV600E are highly effective but responses are often short-lived due to the emergence of drug-resistant tumor subpopulations.,We describe here a mechanism of acquired drug resistance through the tumor microenvironment, which is mediated by human tumor-associated B cells.,Human melanoma cells constitutively produce the growth factor FGF-2, which activates tumor-infiltrating B cells to produce the growth factor IGF-1.,B-cell-derived IGF-1 is critical for resistance of melanomas to BRAF and MEK inhibitors due to emergence of heterogeneous subpopulations and activation of FGFR-3.,Consistently, resistance of melanomas to BRAF and/or MEK inhibitors is associated with increased CD20 and IGF-1 transcript levels in tumors and IGF-1 expression in tumor-associated B cells.,Furthermore, first clinical data from a pilot trial in therapy-resistant metastatic melanoma patients show anti-tumor activity through B-cell depletion by anti-CD20 antibody.,Our findings establish a mechanism of acquired therapy resistance through tumor-associated B cells with important clinical implications.,Resistance to BRAFV600E inhibitors often occurs in melanoma patients.,Here, the authors describe a potential mechanism of acquired drug resistance mediated by tumor-associated B cells-derived IGF-1.
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Reflectance confocal microscopy (RCM) imaging can be used to diagnose and subtype basal cell carcinoma (BCC) but relies on individual morphologic pattern recognition that might vary among users.,We assessed the inter‐rater and intrarater agreement of RCM in correctly diagnosing and subtyping BCC.,In this prospective study, we evaluated the inter‐rater and intrarater agreement of RCM on BCC presence and subtype among three raters with varying experience who independently assessed static images of 48 RCM cases twice with four‐week interval (T1 and T2).,Histopathologic confirmation of presence and subtype of BCC from surgical excision specimen was defined as the reference standard.,The inter‐rater agreement of RCM for BCC presence showed an agreement of 82% at T1 and 84% at T2.,The agreements for subtyping BCC were lower (52% for T1 and 47% for T2).,The intrarater agreement of RCM for BCC presence showed an observed agreement that varied from 79% to 92%.,The observed agreements for subtyping varied from 56% to 71%.,In conclusion, our results show that RCM is reliable in correctly diagnosing BCC based on the assessment of static RCM images.,RCM could potentially play an important role in BCC management if accurate subtyping will be achieved.,Therefore, future clinical studies on reliability and specific RCM features for BCC subtypes are required.
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Even though progress has been made, the detection of melanoma still poses a challenge.,In light of this situation, the Nevisense electrical impedance spectroscopy (EIS) system (SciBase AB, Stockholm, Sweden) was designed and shown to have the potential to be used as an adjunct diagnostic tool for melanoma detection.,To assess the effectiveness and safety of the Nevisense system in the distinction of benign lesions of the skin from melanoma with electrical impedance spectroscopy.,This multicentre, prospective, and blinded clinical study was conducted at five American and 17 European investigational sites.,All eligible skin lesions in the study were examined with the EIS-based Nevisense system, photographed, removed by excisional biopsy and subjected to histopathological evaluation.,A postprocedure clinical follow-up was conducted at 7 ± 3 days from the initial measurement.,A total of 1951 patients with 2416 lesions were enrolled into the study; 1943 lesions were eligible and evaluable for the primary efficacy end point, including 265 melanomas - 112 in situ and 153 invasive melanomas with a median Breslow thickness of 0·57 mm [48 basal cell carcinomas (BCCs) and seven squamous cell carcinomas (SCCs)].,The observed sensitivity of Nevisense was 96·6% (256 of 265 melanomas) with an exact one-sided 95% lower confidence bound estimated at 94·2% and an observed specificity of 34·4%, and an exact two-sided 95% confidence bound estimated at 32·0-36·9%.,The positive and negative predictive values of Nevisense were 21·1% and 98·2%, respectively.,The observed sensitivity for nonmelanoma skin cancer was 100% (55 of 48 BCCs and seven SCCs) with an exact two-sided 95% confidence bound estimated at 93·5-100·0%.,Nevisense is an accurate and safe device to support clinicians in the detection of cutaneous melanoma.,Although progress has been made in the detection of melanoma it still poses a challenge.,Electrical impedance spectroscopy (EIS) may potentially be used as a diagnostic aid for the detection of melanoma.,In the largest international prospective study of its kind in melanoma detection, the EIS system Nevisense was shown to be both accurate and safe in the lesion cohort studied.,In the absence of a perfect gold standard, the accuracy of a device should be compared with the consensus diagnosis from multiple experts.
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Cancer persister cells tolerate anticancer drugs and serve as the founders of acquired resistance and cancer relapse.,Here we show that a subpopulation of BRAFV600 mutant melanoma cells that tolerates exposure to BRAF and MEK inhibitors undergoes a reversible remodelling of mRNA translation that evolves in parallel with drug sensitivity.,Although this process is associated with a global reduction in protein synthesis, a subset of mRNAs undergoes an increased efficiency in translation.,Inhibiting the eIF4A RNA helicase, a component of the eIF4F translation initiation complex, abrogates this selectively increased translation and is lethal to persister cells.,Translation remodelling in persister cells coincides with an increased N6-methyladenosine modification in the 5′-untranslated region of some highly translated mRNAs.,Combination of eIF4A inhibitor with BRAF and MEK inhibitors effectively inhibits the emergence of persister cells and may represent a new therapeutic strategy to prevent acquired drug resistance.,Melanoma persister cells are tolerant to anti-BRAF and anti-MEK inhibition and can trigger cancer relapse.,Here the authors show that a subset of N6-methyladenosine modified mRNAs is translationally activated in persister cells.,This preferential translation can be abrogated via eIF4A inhibition.
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Melanoma patients carry a high risk of developing brain metastases, and improvements in survival are still measured in weeks or months.,Durable disease control within the brain is impeded by poor drug penetration across the blood-brain barrier, as well as intrinsic and acquired drug resistance.,Augmented mitochondrial respiration is a key resistance mechanism in BRAF-mutant melanomas but, as we show in this study, this dependence on mitochondrial respiration may also be exploited therapeutically.,We first used high-throughput pharmacogenomic profiling to identify potentially repurposable compounds against BRAF-mutant melanoma brain metastases.,One of the compounds identified was β-sitosterol, a well-tolerated and brain-penetrable phytosterol.,Here we show that β-sitosterol attenuates melanoma cell growth in vitro and also inhibits brain metastasis formation in vivo.,Functional analyses indicated that the therapeutic potential of β-sitosterol was linked to mitochondrial interference.,Mechanistically, β-sitosterol effectively reduced mitochondrial respiratory capacity, mediated by an inhibition of mitochondrial complex I.,The net result of this action was increased oxidative stress that led to apoptosis.,This effect was only seen in tumor cells, and not in normal cells.,Large-scale analyses of human melanoma brain metastases indicated a significant role of mitochondrial complex I compared to brain metastases from other cancers.,Finally, we observed completely abrogated BRAF inhibitor resistance when vemurafenib was combined with either β-sitosterol or a functional knockdown of mitochondrial complex I.,In conclusion, based on its favorable tolerability, excellent brain bioavailability, and capacity to inhibit mitochondrial respiration, β-sitosterol represents a promising adjuvant to BRAF inhibitor therapy in patients with, or at risk for, melanoma brain metastases.,The online version of this article (10.1186/s40478-019-0712-8) contains supplementary material, which is available to authorized users.
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Unlike nuclear nucleolin, surface-expressed and cytoplasmic nucleolin exhibit Tn antigen.,Here, we show localization-dependent differences in the glycosylation and proteolysis patterns of nucleolin.,Our results provide evidence for different paths of nucleolin proteolysis in the nucleus, in the cytoplasm, and on the cell surface.,We found that full-length nucleolin and some proteolytic fragments coexist within live cells and are not solely the result of the preparation procedure.,Extranuclear nucleolin undergoes N- and O-glycosylation, and unlike cytoplasmic nucleolin, membrane-associated nucleolin is not fucosylated.,Here, we show for the first time that nucleolin and endogenous galectin-3 exist in the same complexes in the nucleolus, the cytoplasm, and on the cell surface of melanoma cells.,Assessments of the interaction of nucleolin with galectin-3 revealed nucleolar co-localization in interphase, suggesting that galectin-3 may be involved in DNA organization and ribosome biogenesis.,Supplementary material is available for this article at 10.2478/s11658-014-0206-4 and is accessible for authorized users.
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Advances in melanoma treatment through targeted inhibition of oncogenic BRAF are limited owing to the development of acquired resistance.,The involvement of BRAFV600E in metabolic reprogramming of melanoma cells provides a rationale for co-targeting metabolism as a therapeutic approach.,We examined the effects of dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, on the growth and metabolic activity of human melanoma cell lines.,The combined effect of DCA and the BRAF inhibitor vemurafenib was investigated in BRAFV600E -mutated melanoma cell lines.,Vemurafenib-resistant cell lines were established in vitro and their sensitivity to DCA was tested.,DCA induced a reduction in glycolytic activity and intracellular ATP levels, and inhibited cellular growth.,Co-treatment of BRAFV600E-mutant melanoma cells with DCA and vemurafenib induced a greater reduction in intracellular ATP levels and cellular growth than either compound alone.,In addition, melanoma cells with in vitro acquired resistance to vemurafenib retained their sensitivity to DCA.,These results suggest that DCA potentiates the effect of vemurafenib through a cooperative attenuation of energy production.,Furthermore, the demonstration of retained sensitivity to DCA in melanoma cells with acquired resistance to vemurafenib could have implications for melanoma treatment.,The online version of this article (doi:10.1186/s12967-014-0247-5) contains supplementary material, which is available to authorized users.
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Erasing histone H3 trimethylation marks suppresses the metastasis of human melanomas.,Metabolic reprogramming is a major factor in transformation, and particular metabolic phenotypes correlate with oncogenotype, tumor progression, and metastasis.,By profiling metabolites in 17 patient-derived xenograft melanoma models, we identified durable metabolomic signatures that correlate with biological features of the tumors.,BRAF mutant tumors had metabolomic and metabolic flux features of enhanced glycolysis compared to BRAF wild-type tumors.,Tumors that metastasized efficiently from their primary sites had elevated levels of metabolites related to protein methylation, including trimethyllysine (TML).,TML levels correlated with histone H3 trimethylation at Lys9 and Lys27, and methylation at these sites was also enhanced in efficiently metastasizing tumors.,Erasing either of these marks by genetically or pharmacologically silencing the histone methyltransferase SETDB1 or EZH2 had no effect on primary tumor growth but reduced cellular invasiveness and metastatic spread.,Thus, metabolite profiling can uncover targetable epigenetic requirements for the metastasis of human melanoma cells.
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Statins induces cell cycle arrest, apoptosis, reduction of angiogenic factors, inhibition of the endothelial growth factor, impairing tissue adhesion and attenuation of the resistance mechanisms.,The aim of this study was evaluate the anti-tumoral activity of simvastatin in a B16F10 melanoma-mouse model.,Melanoma cells were treated with different concentrations of simvastatin and assessed by viability methods.,Melanoma cells (5 × 104) were implanted in two month old C57Bl6/J mice.,Around 7 days after cells injection, the oral treatments were started with simvastatin (5 mg/kg/day, p.o.).,Tumor size, hematological and biochemical analyses were evaluated.,Simvastatin at a concentration of 0.8 μM, 1.2 μM and 1.6 μM had toxic effect.,Concentration of 1.6 μM induced a massive death in the first 24 h of incubation.,Simvastatin at 0.8 μM induces early cell cycle arrest in G0/G1, followed by increase of hypodiploidy.,Tumor size were evaluated and the difference of treated group and control, after ten days, demonstrates that simvastatin inhibited the tumor expansion in 68%.,Simvastatin at 1.6 μM, presented cytototoxicity after 72 h of treatment, with an intense death.,In vivo, simvastatin being potentially useful as an antiproliferative drug, with an impairment of growth after ten days.
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The CheckMate 066 trial investigated nivolumab monotherapy as first-line treatment for patients with previously untreated BRAF wild-type advanced melanoma.,Five-year results are presented herein.,In this multicenter, double-blind, phase III study, 418 patients with previously untreated, unresectable, stage III/IV, wild-type BRAF melanoma were randomly assigned 1:1 to receive nivolumab 3 mg/kg every 2 weeks or dacarbazine 1,000 mg/m2 every 3 weeks.,The primary end point was overall survival (OS), and secondary end points included progression-free survival (PFS), objective response rate (ORR), and safety.,Patients were followed for a minimum of 60 months from the last patient randomly assigned (median follow-up, 32.0 months for nivolumab and 10.9 months for dacarbazine).,Five-year OS rates were 39% with nivolumab and 17% with dacarbazine; PFS rates were 28% and 3%, respectively.,Five-year OS was 38% in patients randomly assigned to dacarbazine who had subsequent therapy, including nivolumab (n = 37).,ORR was 42% with nivolumab and 14% with dacarbazine; among patients alive at 5 years, ORR was 81% and 39%, respectively.,Of 42 patients treated with nivolumab who had a complete response (20%), 88% (37 of 42) were alive as of the 5-year analysis.,Among 75 nivolumab-treated patients alive and evaluable at the 5-year analysis, 83% had not received subsequent therapy; 23% were still on study treatment, and 60% were treatment free.,Safety analyses were similar to the 3-year report.,Results from this 5-year analysis confirm the significant benefit of nivolumab over dacarbazine for all end points and add to the growing body of evidence supporting long-term survival with nivolumab mono-therapy.,Survival is strongly associated with achieving a durable response, which can be maintained after treatment discontinuation, even without subsequent systemic therapies.
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Knowledge of key drivers and therapeutic targets in mucosal melanoma is limited due to the paucity of comprehensive mutation data on this rare tumor type.,To better understand the genomic landscape of mucosal melanoma, here we describe whole genome sequencing analysis of 67 tumors and validation of driver gene mutations by exome sequencing of 45 tumors.,Tumors have a low point mutation burden and high numbers of structural variants, including recurrent structural rearrangements targeting TERT, CDK4 and MDM2.,Significantly mutated genes are NRAS, BRAF, NF1, KIT, SF3B1, TP53, SPRED1, ATRX, HLA-A and CHD8.,SF3B1 mutations occur more commonly in female genital and anorectal melanomas and CTNNB1 mutations implicate a role for WNT signaling defects in the genesis of some mucosal melanomas.,TERT aberrations and ATRX mutations are associated with alterations in telomere length.,Mutation profiles of the majority of mucosal melanomas suggest potential susceptibility to CDK4/6 and/or MEK inhibitors.,Mucosal melanomas are challenging to treat partly because so little is known about the genetic drivers underpinning them.,Here, the authors perform a genomic landscape analysis of samples collected from three continents, revealing a potential role for CDK4/6 or MEK inhibition in the treatment of the disease.
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Phosphoinositide-dependent kinase-1 (PDK-1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and PKC, and is implicated in important cellular processes including survival, metabolism and tumorigenesis.,In large cohorts of nevi and melanoma samples, PDK1 expression was significantly higher in primary melanoma, compared with nevi, and was further increased in metastatic melanoma.,PDK1 expression suffices for its activity, due to auto-activation, or elevated phosphorylation by phosphoinositide 3'-OH-kinase (PI 3-K).,Selective inactivation of Pdk1 in the melanocytes of BrafV600E::Pten−/− or BrafV600E::Cdkn2a−/−::Pten−/− mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-HT.,Melanoma invasion and metastasis were significantly reduced or completely prevented by Pdk1 deletion.,Administration of the PDK1 inhibitor GSK2334470 (PDKi) effectively delayed melanomagenesis and metastasis in BrafV600E::Pten−/− mice.,Pdk1−/− melanomas exhibit a marked decrease in the activity of AKT, P70S6K and PKC.,Notably, PDKi was as effective in inhibiting AGC kinases and colony forming efficiency of melanoma with Pten WT genotypes.,Gene expression analyses identified Pdk1-dependent changes in FOXO3a-regulated genes and inhibition of FOXO3a restored proliferation and colony formation of Pdk1−/− melanoma cells.,Our studies provide direct genetic evidence for the importance of PDK1, in part through FOXO3a-dependent pathway, in melanoma development and progression.
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GNAQ mutations at codon 209 have been recently identified in approximately 50% of uveal melanomas (UM) and are reported to be oncogenic through activating the MAPK/Erk1/2 pathway.,Protein kinase C (PKC) is a component of signaling from GNAQ to Erk1/2.,Inhibition of PKC might regulate GNAQ mutation-induced Erk1/2 activation, resulting in growth inhibition of UM cells carrying GNAQ mutations.,UM cells carrying wild type or mutant GNAQ were treated with the PKC inhibitor enzastaurin.,Effects on proliferation, apoptosis, and signaling events were evaluated.,Enzastaurin downregulated the expression of several PKC isoforms including PKCβII PKCθ, PKCε and/or their phosphorylation in GNAQ mutated cells.,Downregulation of these PKC isoforms in GNAQ mutated cells by shRNA resulted in reduced viability.,Enzastaurin exhibited greater antiproliferative effect on GNAQ mutant cells than wild type cells through induction of G1 arrest and apoptosis.,Enzastaurin-induced G1 arrest was associated with inhibition of Erk1/2 phosphorylation, downregulation of cyclin D1, and accumulation of cyclin dependent kinase inhibitor p27Kip1.,Furthermore, enzastaurin reduced the expression of antiapoptotic Bcl-2 and survivin in GNAQ mutant cells.,Inhibition of Erk1/2 phosphorylation with a MEK specific inhibitor enhanced the sensitivity of GNAQ wild type cells to enzastaurin, accompanied by p27Kip1 accumulation and/or inhibition of enzastaurin-induced survivin and Bcl-2 upregulation.,PKC inhibitors such as enzastaurin have activity against UM cells carrying GNAQ mutations through inhibition of the PKC/Erk1/2 pathway and induction of G1 arrest and apoptosis.,Inhibition of the PKC pathway provides a basis for clinical investigation in patients with UM.
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The updated American Joint Committee on Cancer (AJCC) staging criteria for melanoma remain unable to identify high‐risk stage I tumour subsets.,To determine the utility of epidermal autophagy and beclin 1 regulator 1 (AMBRA1)/loricrin (AMLo) expression as a prognostic biomarker for AJCC stage I cutaneous melanoma.,Peritumoral AMBRA1 expression was evaluated in a retrospective discovery cohort of 76 AJCC stage I melanomas.,AMLo expression was correlated with clinical outcomes up to 12 years in two independent powered, retrospective validation and qualification cohorts comprising 379 AJCC stage I melanomas.,Decreased AMBRA1 expression in the epidermis overlying primary melanomas in a discovery cohort of 76 AJCC stage I tumours was associated with a 7‐year disease‐free survival (DFS) rate of 81·5% vs. 100% survival with maintained AMBRA1 (P < 0·081).,Following an immunohistochemistry protocol for semi‐quantitative analysis of AMLo, analysis was undertaken in validation (n = 218) and qualification cohorts (n = 161) of AJCC stage I melanomas.,Combined cohort analysis revealed a DFS rate of 98·3% in the AMLo low‐risk group (n = 239) vs. 85·4% in the AMLo high‐risk cohort (n = 140; P < 0·001).,Subcohort multivariate analysis revealed that an AMLo hazard ratio (HR) of 4·04 [95% confidence interval (CI) 1·69-9·66; P = 0·002] is a stronger predictor of DFS than Breslow depth (HR 2·97, 95% CI 0·93-9·56; P = 0·068) in stage IB patients.,Loss of AMLo expression in the epidermis overlying primary AJCC stage I melanomas identifies high‐risk tumour subsets independently of Breslow depth.,What's already known about this topic?,There is an unmet clinical need for biomarkers of early‐stage melanoma.Autophagy and beclin 1 regulator 1 (AMBRA1) is a proautophagy regulatory protein with known roles in cell proliferation and differentiation, and is a known tumour suppressor.Loricrin is a marker of epidermal terminal differentiation.,There is an unmet clinical need for biomarkers of early‐stage melanoma.,Autophagy and beclin 1 regulator 1 (AMBRA1) is a proautophagy regulatory protein with known roles in cell proliferation and differentiation, and is a known tumour suppressor.,Loricrin is a marker of epidermal terminal differentiation.,What does this study add?,AMBRA1 has a functional role in keratinocyte/epidermal proliferation and differentiation.The combined decrease/loss of peritumoral AMBRA1 and loricrin is associated with a significantly increased risk of metastatic spread in American Joint Committee on Cancer (AJCC) stage I tumours vs. melanomas, in which peritumoral AMBRA1 and loricrin are maintained, independently of Breslow depth.,AMBRA1 has a functional role in keratinocyte/epidermal proliferation and differentiation.,The combined decrease/loss of peritumoral AMBRA1 and loricrin is associated with a significantly increased risk of metastatic spread in American Joint Committee on Cancer (AJCC) stage I tumours vs. melanomas, in which peritumoral AMBRA1 and loricrin are maintained, independently of Breslow depth.,What is the translational message?,The integration of peritumoral epidermal AMBRA1/loricrin biomarker expression into melanoma care guidelines will facilitate more accurate, personalized risk stratification for patients with AJCC stage I melanomas, thereby facilitating stratification for appropriate follow‐up and informing postdiagnostic investigations, including sentinel lymph node biopsy, ultimately resulting in improved disease outcomes and rationalization of healthcare costs.,The integration of peritumoral epidermal AMBRA1/loricrin biomarker expression into melanoma care guidelines will facilitate more accurate, personalized risk stratification for patients with AJCC stage I melanomas, thereby facilitating stratification for appropriate follow‐up and informing postdiagnostic investigations, including sentinel lymph node biopsy, ultimately resulting in improved disease outcomes and rationalization of healthcare costs.,https://doi.org/10.1111/bjd.18658 available online,Linked Editorial:https://doi.org/10.1111/bjd.18555
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Nodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome.,Similarly to other tumors, permissive microenvironment is essential for melanoma progression.,Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue.,Here, we study the effect of melanoma cells on human primary keratinocytes (HPK).,Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC).,Comparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed.,Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively.,Differentially expressed candidate genes were verified by RT-qPCR.,Biological activity of candidate proteins was assessed on cultured HPK.,Epidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases.,This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10.,We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK.,This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies.,We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes.,We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro.,This interaction further highlights the role of intercellular interactions in melanoma.,The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.,The online version of this article (doi:10.1186/1476-4598-14-1) contains supplementary material, which is available to authorized users.
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Melanoma is a heterogeneous tumor with different subpopulations showing different proliferation rates.,Slow-cycling cells were previously identified in melanoma, but not fully biologically characterized.,Using the label-retention method, we identified a subpopulation of slow-cycling cells, defined as label-retaining cells (LRC), with strong invasive properties.,We demonstrate through live imaging that LRC are leaving the primary tumor mass at a very early stage and disseminate to peripheral organs.,Through global proteome analyses, we identified the secreted protein SerpinE2/protease nexin-1 as causative for the highly invasive potential of LRC in melanomas.
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This study investigated the role of cancer/testis antigen DDX53 in regulating cancer stem cell-like properties.,DDX53 shows co-expression with CD133, a marker for cancer stem cells.,DDX53 directly regulates the SOX-2 expression in anticancer drug-resistant Malme3MR cells.,DDX53 and miR-200b were found to be involved in the regulation of tumor spheroid forming potential of Malme3M and Malme3MR cells.,Furthermore, the self-renewal activity and the tumorigenic potential of Malme3MR-CD133 (+) cells were also regulated by DDX53.,A miR-200b inhibitor induced the direct regulation of SOX-2 by DDX53 We therefore, conclude that DDX53 may serve as an immunotherapeutic target for regulating cancer stem-like properties of melanomas.
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Sphere formation in conditioned serum‐free culture medium supplemented with epidermal growth factor and basic fibroblast growth factor (tumorospheres) is considered useful for the enrichment of cancer stem‐like cells, also known as tumor‐initiating cells.,We used a gene expression microarray to investigate the gene expression profile of melanoma cancer stem‐like cells (MCSLCs).,The results showed that MCSLCs highly expressed the following Notch signaling pathway molecules: Notch3 (NM_008716), Notch4 (NM_010929), Dtx4 (NM_172442), and JAG2 (NM_010588).,Immunofluorescence staining showed tumorosphere cells highly expressed Notch4.,Notch4high B16F10 cells were isolated by FACS, and Western blotting showed that high Notch4 expression is related to the expression of epithelial-mesenchymal transition (EMT)‐associated proteins.,Reduced invasive and migratory properties concomitant with the downregulation of the EMT markers Twist1, vimentin, and VE‐cadherin and the overexpression of E‐cadherin was observed in human melanoma A375 and MUM‐2B cells.,In these cells, Notch4 was also downregulated, both by Notch4 gene knockdown and by application of the γ‐secretase inhibitor, DAPT.,Mechanistically, the re‐overexpression of Twist1 by the transfection of cells with a Twist1 expression plasmid led to an increase in VE‐cadherin expression and a decrease in E‐cadherin expression.,Immunohistochemical analysis of 120 human melanoma tissues revealed a significant correlation between the high expression of Notch4 and the metastasis of melanoma.,Taken together, our findings indicate that Notch4+ MCSLCs trigger EMT and promote the metastasis of melanoma cells.
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It is well established that inflammation promotes cancer, including melanoma, although the exact mechanisms involved are less known.,In this study, we tested the hypothesis that inflammatory factors affect the cancer stem cell (CSC) compartment responsible for tumor development and relapse.,Using an inducible histone 2B-GFP fusion protein as a tracer of cell divisional history, we determined that tumor necrosis factor (TNF), which is a classical pro-inflammatory cytokine, enlarged the CSC pool of GFP-positive label-retaining cells (LRCs) in tumor-like melanospheres.,Although these cells acquired melanoma stem cell markers, including ABCB5 and CD271, and self-renewal ability, they lost their capacity to differentiate, as evidenced by the diminished MelanA expression in melanosphere cells and the loss of pigmentation in a skin equivalent model of human melanoma.,The undifferentiated cell phenotype could be reversed by LY294002, which is an inhibitor of the PI3K/AKT signaling pathway, and this reversal was accompanied by a significant reduction in CSC phenotypic markers and functional properties.,Importantly, the changes induced by a transient exposure to TNF were long-lasting and observed for many generations after TNF withdrawal.,We conclude that pro-inflammatory TNF targets the quiescent/slow-cycling melanoma SC compartment and promotes PI3K/AKT-driven expansion of melanoma SCs most likely by preventing their asymmetrical self-renewal.,This TNF effect is maintained and transferred to descendants of LRC CSCs and is manifested in the absence of TNF, suggesting that a transient exposure to inflammatory factors imprints long-lasting molecular and/or cellular changes with functional consequences long after inflammatory signal suppression.,Clinically, these results may translate into an inflammation-triggered accumulation of quiescent/slow-cycling CSCs and a post-inflammatory onset of an aggressive tumor.
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Dynamic cellular heterogeneity underlies melanoma progression and therapy resistance.,Advances in single-cell technologies have revealed an increasing number of tumor and microenvironment cell states in melanoma, but little is understood about their function in vivo.,Zebrafish models are a powerful system for discovery, live imaging, and functional investigation of cell states throughout melanoma progression and treatment.,By capturing dynamic melanoma states in living animals, zebrafish have the potential to resolve the complexity of melanoma heterogeneity from a single cell through disease processes within the context of the whole body, revealing novel cancer biology and therapeutic targets.
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Current treatment modalities for disseminated cutaneous malignant melanoma (CMM) improve survival; however, relapses are common.,A number of receptor tyrosine kinases (RTKs) including EGFR and MET have been reported to be involved in CMM metastasis and in the development of resistance to therapy, targeting the mitogen-activated protein kinase (MAPK pathway).,IHC analysis showed that patients with higher MET protein expression had a significantly shorter overall survival.,In addition, silencing of MET caused an upregulation of EGFR and p-AKT, which was abrogated by concomitant silencing of MET and EGFR in CMM cells resistant to MAPK-targeting drugs.,We therefore explored novel treatment strategies using clinically approved drugs afatinib (ERBB family inhibitor) and crizotinib (MET inhibitor), to simultaneously block MET and ERBB family RTKs.,The effects of the combination were assessed in cell culture and spheroid models using established CMM and patient-derived short-term cell lines, and an in vivo xenograft mouse model.,The combination had a synergistic effect, promoting cell death, concomitant with a potent downregulation of migratory and invasive capacity independent of their BRAF/NRAS mutational status.,Furthermore, the combination attenuated tumor growth rate, as ascertained by the significant reduction of Ki67 expression and induced DNA damage in vivo.,Importantly, this combination therapy had minimal therapy-related toxicity in mice.,Lastly, the cell cycle G2 checkpoint kinase WEE1 and the RTK IGF1R, non-canonical targets, were altered upon exposure to the combination.,Knockdown of WEE1 abrogated the combination-mediated effects on cell migration and proliferation in BRAF mutant BRAF inhibitor-sensitive cells, whereas WEE1 silencing alone inhibited cell migration in NRAS mutant cells.,In summary, our results show that afatinib and crizotinib in combination is a promising alternative targeted therapy option for CMM patients, irrespective of BRAF/NRAS mutational status, as well as for cases where resistance has developed towards BRAF inhibitors.
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Costimulation of T cell responses with monoclonal antibody agonists (mAb-AG) targeting 4-1BB showed robust anti-tumor activity in preclinical models, but their clinical development was hampered by low efficacy (Utomilumab) or severe liver toxicity (Urelumab).,Here we show that isotype and intrinsic agonistic strength co-determine the efficacy and toxicity of anti-4-1BB mAb-AG.,While intrinsically strong agonistic anti-4-1BB can activate 4-1BB in the absence of FcγRs, weak agonistic antibodies rely on FcγRs to activate 4-1BB.,All FcγRs can crosslink anti-41BB antibodies to strengthen co-stimulation, but activating FcγR-induced antibody-dependent cell-mediated cytotoxicity compromises anti-tumor immunity by deleting 4-1BB+ cells.,This suggests balancing agonistic activity with the strength of FcγR interaction as a strategy to engineer 4-1BB mAb-AG with optimal therapeutic performance.,As a proof of this concept, we have developed LVGN6051, a humanized 4-1BB mAb-AG that shows high anti-tumor efficacy in the absence of liver toxicity in a mouse model of cancer immunotherapy.,Agonistic 4-1BB antibodies developed for cancer immunotherapy have suffered from either hepatotoxicity or insufficient anti-cancer activity.,Here the authors determine the contribution of FcγR binding and agonistic strength to these outcomes, and engineer a 4-1BB antibody with potent anti-tumor effect and no liver toxicity in mice.
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There is growing evidence that tripartite motif-containing protein 44 (TRIM44) plays crucial role in tumor development.,However, the underlying mechanism of this deubiquitinating enzyme remains unclear.,Large clinical samples were used to detect TRIM44 expression and its associations with clinicopathological features and prognosis.,Gain- and loss-of-function experiments in cell lines and mouse xenograft models were performed to elucidate the function and underlying mechanisms of TRIM44 induced tumor progression.,Co-immunoprecipitation (Co-IP) assays and mass spectrometric analyses were applied to verify the interacting proteins of TRIM44.,We found that TRIM44 was commonly amplified in melanoma tissues compared with paratumoral tissues.,TRIM44 expression also positively correlated with more aggressive clinicopathological features, such as Breslow depth (p = 0.025), distant metastasis (p = 0.012), and TNM stage (p = 0.002).,Importantly, we found that TRIM44 was an independent indicator of prognosis for melanoma patients.,Functionally, overexpression of TRIM44 facilitated cell invasion, migration, apoptosis resistance and proliferation in vitro, and promoted lung metastasis and tumorigenic ability in vivo.,Importantly, high level of TRIM44 induced melanoma cell epithelial-mesenchymal transition (EMT), which is one of the most important mechanisms for the promotion of tumor metastasis.,Mechanistically, high levels of TRIM44 increased the levels of p-AKT (T308) and p-mTOR (S2448), and a specific AKT inhibitor inhibited TRIM44-induced tumor progression.,Co-IP assays and mass spectrometric analyses indicated that TRIM44 overexpression induces cell EMT through activating AKT/mTOR pathway via directly binding and stabilizing TOLL-like receptor 4 (TLR4), and TLR4 interference impeded TRIM44 induced tumor progression.,Moreover, we demonstrated that TRIM44 is the target of miR-26b-5p, which is significantly downregulated in melanoma tissues and may be responsible for the overexpression of TRIM44.,TRIM44, regulated by miR-26b-5p, promotes melanoma progression by stabilizing TLR4, which then activates the AKT/mTOR pathway.,TRIM44 shows promise as a prognostic predictor and a therapeutic target for melanoma patients.,The online version of this article (10.1186/s13046-019-1138-7) contains supplementary material, which is available to authorized users.
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Photodynamic therapy (PDT) is an established treatment option for low-risk basal cell carcinoma (BCC).,BCC is the most common human cancer and also a convenient cancer in which to study PDT treatment.,This review clarifies challenges to researchers evident from the clinical use of PDT in BCC treatment.,It outlines the context of PDT and how PDT treatments for BCC have been developed hitherto.,The sections examine the development of systemic and subsequently topical photosensitizers, light delivery regimens, and the use of PDT in different patient populations and subtypes of BCC.,The outcomes of topical PDT are discussed in comparison with alternative treatments, and topical PDT applications in combination and adjuvant therapy are considered.,The intention is to summarize the clinical relevance and expose areas of research need in the BCC context, ultimately to facilitate improvements in PDT treatment.
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Actinic keratoses are dysplastic proliferations of keratinocytes with potential for malignant transformation.,Clinically, actinic keratoses present as macules, papules, or hyperkeratotic plaques with an erythematous background that occur on photoexposed areas.,At initial stages, they may be better identified by palpation rather than by visual inspection.,They may also be pigmented and show variable degrees of infiltration; when multiple they then constitute the so-called field cancerization.,Their prevalence ranges from 11% to 60% in Caucasian individuals above 40 years.,Ultraviolet radiation is the main factor involved in pathogenesis, but individual factors also play a role in the predisposing to lesions appearance.,Diagnosis of lesions is based on clinical and dermoscopic examination, but in some situations histopathological analysis may be necessary.,The risk of transformation into squamous cell carcinoma is the major concern regarding actinic keratoses.,Therapeutic modalities for actinic keratoses include topical medications, and ablative and surgical methods; the best treatment option should always be individualized according to the patient.
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T cell senescence and exhaustion are major barriers to successful cancer immunotherapy.,Here we show that miR-155 increases CD8+ T cell antitumor function by restraining T cell senescence and functional exhaustion through epigenetic silencing of drivers of terminal differentiation. miR-155 enhances Polycomb repressor complex 2 (PRC2) activity indirectly by promoting the expression of the PRC2-associated factor Phf19 through downregulation of the Akt inhibitor, Ship1.,Phf19 orchestrates a transcriptional program extensively shared with miR-155 to restrain T cell senescence and sustain CD8+ T cell antitumor responses.,These effects rely on Phf19 histone-binding capacity, which is critical for the recruitment of PRC2 to the target chromatin.,These findings establish the miR-155-Phf19-PRC2 as a pivotal axis regulating CD8+ T cell differentiation, thereby paving new ways for potentiating cancer immunotherapy through epigenetic reprogramming of CD8+ T cell fate.,The inability of T cells to properly mount anti-tumour immunity underlies failed cancer immune surveillance or therapy.,Here the authors show that a microRNA, miR-155, suppresses Ship1 phosphatase expression to modulate epigenetic reprogramming of CD8 T cell differentiation via the Phf19/PRC2 axis, thereby implicating a novel aspect of cancer immunity regulation.
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While recent years have seen a revolution in the treatment of metastatic cutaneous melanoma, no treatment has yet been able to demonstrate any prolonged survival in metastatic uveal melanoma.,Thus, metastatic uveal melanoma remains a disease with an urgent unmet medical need.,Reports of treatment with immune checkpoint inhibitors have thus far been disappointing.,Based on animal experiments, it is reasonable to hypothesize that the effect of immunotherapy may be augmented by epigenetic therapy.,Proposed mechanisms include enhanced expression of HLA class I and cancer antigens on cancer cells, as well as suppression of myeloid suppressor cells.,The PEMDAC study is a multicenter, open label phase II study assessing the efficacy of concomitant use of the PD1 inhibitor pembrolizumab and the class I HDAC inhibitor entinostat in adult patients with metastatic uveal melanoma.,Primary endpoint is objective response rate.,Eligible patients have histologically confirmed metastatic uveal melanoma, ECOG performance status 0-1, measurable disease as per RECIST 1.1 and may have received any number of prior therapies, with the exception of anticancer immunotherapy.,Twenty nine patients will be enrolled.,Patients receive pembrolizumab 200 mg intravenously every third week in combination with entinostat 5 mg orally once weekly.,Treatment will continue until progression of disease or intolerable toxicity or for a maximum of 24 months.,The PEMDAC study is the first trial to assess whether the addition of an HDAC inhibitor to anti-PD1 therapy can yield objective anti-tumoral responses in metastatic UM.,ClinicalTrials.gov registration number: NCT02697630.,(Registered 3 March 2016).,EudraCT registration number: 2016-002114-50.
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The tumor immune contexture plays a major role for the clinical outcome of patients.,High densities of CD45RO+ T helper 1 cells and CD8+ T cells are associated with improved survival of patients with various cancer entities.,In contrast, a higher frequency of tumor-infiltrating M2 macrophages is correlated with poor prognosis.,Recent studies provide evidence that the tumor immune architecture also essentially contributes to the clinical efficacy of immune checkpoint inhibitor (CPI) therapy in patients.,Pretreatment melanoma samples from patients who experienced a clinical response to anti-programmed cell death protein 1 (PD-1) treatment show higher densities of infiltrating CD8+ T cells compared to samples from patients that progressed during therapy.,Anti-PD-1 therapy results in an increased density of tumor-infiltrating T lymphocytes in treatment responders.,In addition, elevated frequencies of melanoma-infiltrating TCF7+CD8+ T cells are correlated with beneficial clinical outcome of anti-PD-1-treated patients.,In contrast, a high density of tumor-infiltrating, dysfunctional PD-1+CD38hi CD8+ cells in melanoma patients is associated with anti-PD-1 resistance.,Such findings indicate that comprehensive tumor immune contexture profiling prior to and during CPI therapy may lead to the identification of underlying mechanisms for treatment response or resistance, and the design of improved immunotherapeutic strategies.,Here, we focus on studies exploring the impact of intratumoral T and B cells at baseline on the clinical outcome of CPI-treated cancer patients.,In addition, recent findings demonstrating the influence of CPIs on tumor-infiltrating lymphocytes are summarized.
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Quantitative assessment of key proteins that control the tumor-immune interface is one of the most formidable analytical challenges in immunotherapeutics.,We developed a targeted MS platform to quantify programmed cell death-1 (PD-1), programmed cell death 1 ligand 1 (PD-L1), and programmed cell death 1 ligand 2 (PD-L2) at fmol/microgram protein levels in formalin fixed, paraffin-embedded sections from 22 human melanomas.,PD-L1 abundance ranged 50-fold, from ∼0.03 to 1.5 fmol/microgram protein and the parallel reaction monitoring (PRM) data were largely concordant with total PD-L1-positive cell content, as analyzed by immunohistochemistry (IHC) with the E1L3N antibody.,PD-1 was measured at levels up to 20-fold lower than PD-L1, but the abundances were not significantly correlated (r2 = 0.062, p = 0.264).,PD-1 abundance was weakly correlated (r2 = 0.3057, p = 0.009) with the fraction of lymphocytes and histiocytes in sections.,PD-L2 was measured from 0.03 to 1.90 fmol/microgram protein and the ratio of PD-L2 to PD-L1 abundance ranged from 0.03 to 2.58.,In 10 samples, PD-L2 was present at more than half the level of PD-L1, which suggests that PD-L2, a higher affinity PD-1 ligand, is sufficiently abundant to contribute to T-cell downregulation.,We also identified five branched mannose and N-acetylglucosamine glycans at PD-L1 position N192 in all 22 samples.,Extent of PD-L1 glycan modification varied by ∼10-fold and the melanoma with the highest PD-L1 protein abundance and most abundant glycan modification yielded a very low PD-L1 IHC estimate, thus suggesting that N-glycosylation may affect IHC measurement and PD-L1 function.,Additional PRM analyses quantified immune checkpoint/co-regulator proteins LAG3, IDO1, TIM-3, VISTA, and CD40, which all displayed distinct expression independent of PD-1, PD-L1, and PD-L2.,Targeted MS can provide a next-generation analysis platform to advance cancer immuno-therapeutic research and diagnostics.
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The transcription factor NRF2 is the major mediator of oxidative stress responses and is closely connected to therapy resistance in tumors harboring activating mutations in the NRF2 pathway.,In melanoma, such mutations are rare, and it is unclear to what extent melanomas rely on NRF2.,Here we show that NRF2 suppresses the activity of the melanocyte lineage marker MITF in melanoma, thereby reducing the expression of pigmentation markers.,Intriguingly, we furthermore identified NRF2 as key regulator of immune-modulating genes, linking oxidative stress with the induction of cyclooxygenase 2 (COX2) in an ATF4-dependent manner.,COX2 is critical for the secretion of prostaglandin E2 and was strongly induced by H2O2 or TNFα only in presence of NRF2.,Induction of MITF and depletion of COX2 and PGE2 were also observed in NRF2-deleted melanoma cells in vivo.,Furthermore, genes corresponding to the innate immune response such as RSAD2 and IFIH1 were strongly elevated in absence of NRF2 and coincided with immune evasion parameters in human melanoma datasets.,Even in vitro, NRF2 activation or prostaglandin E2 supplementation blunted the induction of the innate immune response in melanoma cells.,Transcriptome analyses from lung adenocarcinomas indicate that the observed link between NRF2 and the innate immune response is not restricted to melanoma.
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Targeted therapies with MAPK inhibitors (MAPKi) are faced with severe problems of resistance in BRAF‐mutant melanoma.,In parallel to the acquisition of genetic mutations, melanoma cells may also adapt to the drugs through phenotype switching.,The ZEB1 transcription factor, a known inducer of EMT and invasiveness, is now considered as a genuine oncogenic factor required for tumor initiation, cancer cell plasticity, and drug resistance in carcinomas.,Here, we show that high levels of ZEB1 expression are associated with inherent resistance to MAPKi in BRAFV 600‐mutated cell lines and tumors.,ZEB1 levels are also elevated in melanoma cells with acquired resistance and in biopsies from patients relapsing while under treatment.,ZEB1 overexpression is sufficient to drive the emergence of resistance to MAPKi by promoting a reversible transition toward a MITF low/p75high stem‐like and tumorigenic phenotype.,ZEB1 inhibition promotes cell differentiation, prevents tumorigenic growth in vivo, sensitizes naive melanoma cells to MAPKi, and induces cell death in resistant cells.,Overall, our results demonstrate that ZEB1 is a major driver of melanoma cell plasticity, driving drug adaptation and phenotypic resistance to MAPKi.
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The tumor immune contexture plays a major role for the clinical outcome of patients.,High densities of CD45RO+ T helper 1 cells and CD8+ T cells are associated with improved survival of patients with various cancer entities.,In contrast, a higher frequency of tumor-infiltrating M2 macrophages is correlated with poor prognosis.,Recent studies provide evidence that the tumor immune architecture also essentially contributes to the clinical efficacy of immune checkpoint inhibitor (CPI) therapy in patients.,Pretreatment melanoma samples from patients who experienced a clinical response to anti-programmed cell death protein 1 (PD-1) treatment show higher densities of infiltrating CD8+ T cells compared to samples from patients that progressed during therapy.,Anti-PD-1 therapy results in an increased density of tumor-infiltrating T lymphocytes in treatment responders.,In addition, elevated frequencies of melanoma-infiltrating TCF7+CD8+ T cells are correlated with beneficial clinical outcome of anti-PD-1-treated patients.,In contrast, a high density of tumor-infiltrating, dysfunctional PD-1+CD38hi CD8+ cells in melanoma patients is associated with anti-PD-1 resistance.,Such findings indicate that comprehensive tumor immune contexture profiling prior to and during CPI therapy may lead to the identification of underlying mechanisms for treatment response or resistance, and the design of improved immunotherapeutic strategies.,Here, we focus on studies exploring the impact of intratumoral T and B cells at baseline on the clinical outcome of CPI-treated cancer patients.,In addition, recent findings demonstrating the influence of CPIs on tumor-infiltrating lymphocytes are summarized.
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High dose interleukin-2 (IL-2) is active against metastatic melanoma and renal cell carcinoma, but treatment-associated toxicity and expansion of suppressive regulatory T cells (Tregs) limit its use in patients with cancer.,Bempegaldesleukin (NKTR-214) is an engineered IL-2 cytokine prodrug that provides sustained activation of the IL-2 pathway with a bias to the IL-2 receptor CD122 (IL-2Rβ).,Here we assess the therapeutic impact and mechanism of action of NKTR-214 in combination with anti-PD-1 and anti-CTLA-4 checkpoint blockade therapy or peptide-based vaccination in mice.,NKTR-214 shows superior anti-tumor activity over native IL-2 and systemically expands anti-tumor CD8+ T cells while inducing Treg depletion in tumor tissue but not in the periphery.,Similar trends of intratumoral Treg dynamics are observed in a small cohort of patients treated with NKTR-214.,Mechanistically, intratumoral Treg depletion is mediated by CD8+ Teff-associated cytokines IFN-γ and TNF-α.,These findings demonstrate that NKTR-214 synergizes with T cell-mediated anti-cancer therapies.,Interleukin-2 can induce an anti-tumour response, but is associated with toxicity.,Here, the authors demonstrate that an engineered interleukin-2 promotes intratumoral T regulatory cell depletion while enhancing effective anti-tumour CD8+ T cell responses that result in potent tumor suppression.
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The indoleamine 2,3-dioxygenase (IDO) pathway is a key counter-regulatory mechanism that, in cancer, is exploited by tumors to evade antitumor immunity.,Indoximod is a small-molecule IDO pathway inhibitor that reverses the immunosuppressive effects of low tryptophan (Trp) and high kynurenine (Kyn) that result from IDO activity.,In this study, indoximod was used in combination with a checkpoint inhibitor (CPI) pembrolizumab for the treatment for advanced melanoma.,Patients with advanced melanoma were enrolled in a single-arm phase II clinical trial evaluating the addition of indoximod to standard of care CPI approved for melanoma.,Investigators administered their choice of CPI including pembrolizumab (P), nivolumab (N), or ipilimumab (I).,Indoximod was administered continuously (1200 mg orally two times per day), with concurrent CPI dosed per US Food and Drug Administration (FDA)-approved label.,Between July 2014 and July 2017, 131 patients were enrolled.,(P) was used more frequently (n=114, 87%) per investigator’s choice.,The efficacy evaluable population consisted of 89 patients from the phase II cohort with non-ocular melanoma who received indoximod combined with (P).,The objective response rate (ORR) for the evaluable population was 51% with confirmed complete response of 20% and disease control rate of 70%.,Median progression-free survival was 12.4 months (95% CI 6.4 to 24.9).,The ORR for Programmed Death-Ligand 1 (PD-L1)-positive patients was 70% compared with 46% for PD-L1-negative patients.,The combination was well tolerated, and side effects were similar to what was expected from single agent (P).,In this study, the combination of indoximod and (P) was well tolerated and showed antitumor efficacy that is worth further evaluation in selected patients with advanced melanoma.
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Metastatic mucosal melanoma responds poorly to anti-programmed cell death-1 (PD-1) monotherapy.,Vascular endothelial growth factor (VEGF) has been shown to play an important immunosuppressive role in the tumor microenvironment.,The combination of VEGF inhibition and PD-1 blockade provides therapeutic opportunities for patients refractory to either therapy alone.,We conducted a single-center, phase IB trial evaluating the safety and preliminary efficacy of toripalimab, a humanized immunoglobulin G4 monoclonal antibody against PD-1 in combination with the VEGF receptor inhibitor axitinib in patients with advanced melanoma, including patients with chemotherapy-naïve mucosal melanomas (88%).,Patients received toripalimab at 1 or 3 mg/kg via intravenous infusion every 2 weeks, in combination with axitinib 5 mg orally twice a day, in a dose-escalation and cohort-expansion study until confirmed disease progression, unacceptable toxicity, or voluntary withdrawal.,The primary objective was safety.,Secondary objectives included efficacy, pharmacokinetics, pharmacodynamics, immunogenicity, and tumor tissue biomarkers.,Thirty-three patients were enrolled.,No dose-limiting toxicities were observed.,Ninety-seven percent of patients experienced treatment-related adverse events (TRAEs).,The most common TRAEs were mild (grade 1 or 2) and included diarrhea, proteinuria, hand and foot syndrome, fatigue, AST or ALT elevation, hypertension, hypo- or hyperthyroidism, and rash.,Grade 3 or greater TRAEs occurred in 39.4% of patients.,By the cutoff date, among 29 patients with chemotherapy-naïve mucosal melanoma, 14 patients (48.3%; 95% CI, 29.4% to 67.5%) achieved objective response, and the median progression-free survival time was 7.5 months (95% CI, 3.7 months to not reached) per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1.,The combination of toripalimab plus axitinib was tolerable and showed promising antitumor activity in patients with treatment-naïve metastatic mucosal melanoma.,Patients enrolled in this study were all Asian, and this combination therapy must be validated in a randomized phase III trial that includes a non-Asian population before it can become a standard of care.
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Skin cancer has always been and remains the leader among all tumors in terms of occurrence.,One of the main factors responsible for skin cancer, natural and artificial UV radiation, causes the mutations that transform healthy cells into cancer cells.,These mutations inactivate apoptosis, an event required to avoid the malignant transformation of healthy cells.,Among these deadliest of cancers, melanoma and its ‘younger sister’, Merkel cell carcinoma, are the most lethal.,The heavy toll of skin cancers stems from their rapid progression and the fact that they metastasize easily.,Added to this is the difficulty in determining reliable margins when excising tumors and the lack of effective chemotherapy.,Possibly the biggest problem posed by skin cancer is reliably detecting the extent to which cancer cells have spread throughout the body.,The initial tumor is visible and can be removed, whereas metastases are invisible to the naked eye and much harder to eliminate.,In our opinion, antisense oligonucleotides, which can be used in the form of targeted ointments, provide real hope as a treatment that will eliminate cancer cells near the tumor focus both before and after surgery.
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The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway promotes melanoma tumor growth and survival while suppressing autophagy, a catabolic process through which cells collect and recycle cellular components to sustain energy homeostasis in starvation.,Conversely, inhibitors of the PI3K/AKT/mTOR pathway, in particular the mTOR inhibitor temsirolimus (CCI-779), induce autophagy, which can promote tumor survival and thus, these agents potentially limit their own efficacy.,We hypothesized that inhibition of autophagy in combination with mTOR inhibition would block this tumor survival mechanism and hence improve the cytotoxicity of mTOR inhibitors in melanoma.,Here we found that melanoma cell lines of multiple genotypes exhibit high basal levels of autophagy.,Knockdown of expression of the essential autophagy gene product ATG7 resulted in cell death, indicating that survival of melanoma cells is autophagy-dependent.,We also found that the lysosomotropic agent and autophagy inhibitor hydroxychloroquine (HCQ) synergizes with CCI-779 and led to melanoma cell death via apoptosis.,Combination treatment with CCI-779 and HCQ suppressed melanoma growth and induced cell death both in 3-dimensional (3D) spheroid cultures and in tumor xenografts.,These data suggest that coordinate inhibition of the mTOR and autophagy pathways promotes apoptosis and could be a new therapeutic paradigm for the treatment of melanoma.
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BRAF-mutant melanoma patients respond to BRAF inhibitors and MEK inhibitors (BRAFi/MEKi), but drug-tolerant cells persist, which may seed disease progression.,Adaptive activation of receptor tyrosine kinases (RTKs) has been associated with melanoma cell drug tolerance following targeted therapy.,While co-targeting individual RTKs can enhance the efficacy of BRAFi/MEKi effects, it remains unclear how to broadly target multiple RTKs to achieve more durable tumour growth inhibition.,The blockage of adaptive RTK responses by the new BET inhibitor (BETi), PLX51107, was measured by RPPA and Western blot.,Melanoma growth was evaluated in vitro by colony assay and EdU staining, as well as in skin reconstructs, xenografts and PDX models following BRAFi, MEKi and/or PLX51107 treatment.,Treatment with PLX51107 limited BRAFi/MEKi upregulation of ErbB3 and PDGFR-β expression levels.,Similar effects were observed following BRD2/4 depletion.,In stage III melanoma patients, expression of BRD2/4 was strongly correlated with ErbB3.,PLX51107 enhanced the effects of BRAFi/MEKi on inhibiting melanoma growth in vitro, in human skin reconstructs and in xenografts in vivo.,Continuous triple drug combination treatment resulted in significant weight loss in mice, but intermittent BETi combined with continuous BRAFi/MEKi treatment was tolerable and improved durable tumour inhibition outcomes.,Together, our data suggest that intermittent inhibition of BET proteins may improve the duration of responses following BRAFi/MEKi treatment in BRAF-mutant melanoma.
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Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5).,Mouse models have been broadly utilized to study tissue morphogenesis in vivo.,However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans.,Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis.,The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types.,The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis.,Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction.,The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin.,As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes.,Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo.,Recently, dermal stem cells have been identified in the human dermis (6).,These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.
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Adoptive T cell transfer (ACT) with ex vivo-expanded tumor-reactive T cells proved to be successful for the treatment of metastatic melanoma patients.,Mixed lymphocyte tumor cell cultures (MLTC) can be used to generate tumor-specific T cells for ACT; however, in a number of cases tumor-reactive T cell, expansion is far from optimal.,We hypothesized that this is due to tumor intrinsic and extrinsic factors and aimed to identify and manipulate these factors so to optimize our clinical, GMP-compliant MLTC protocol.,We found that the tumor cell produced IDO and/or galectin-3, and the accumulation of CD4+CD25hiFoxP3+ T cells suppressed the expansion of tumor-specific T cells in the MLTC.,Strategies to eliminate CD4+CD25hiFoxP3+ T cells during culture required the depletion of the whole CD4+ T cell population and were found to be undesirable.,Blocking of IDO and galectin-3 was feasible and resulted in improved efficiency of the MLTC.,Implementation of these findings in clinical protocols for ex vivo expansion of tumor-reactive T cells holds promise for an increased therapeutic potential of adoptive cell transfer treatments with tumor-specific T cells.,The online version of this article (doi:10.1007/s00262-017-1995-x) contains supplementary material, which is available to authorized users.
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Anti-CTLA-4 antibody induces selective depletion of T reg cells within tumor lesions in a manner that is dependent on the presence of Fc gamma receptor-expressing macrophages within the tumor microenvironment.,Treatment with monoclonal antibody specific for cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an inhibitory receptor expressed by T lymphocytes, has emerged as an effective therapy for the treatment of metastatic melanoma.,Although subject to debate, current models favor a mechanism of activity involving blockade of the inhibitory activity of CTLA-4 on both effector (T eff) and regulatory (T reg) T cells, resulting in enhanced antitumor effector T cell activity capable of inducing tumor regression.,We demonstrate, however, that the activity of anti-CTLA-4 antibody on the T reg cell compartment is mediated via selective depletion of T reg cells within tumor lesions.,Importantly, T reg cell depletion is dependent on the presence of Fcγ receptor-expressing macrophages within the tumor microenvironment, indicating that T reg cells are depleted in trans in a context-dependent manner.,Our results reveal further mechanistic insight into the activity of anti-CTLA-4-based cancer immunotherapy, and illustrate the importance of specific features of the local tumor environment on the final outcome of antibody-based immunomodulatory therapies.
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Analysis of melanomas for actionable mutations has become the standard of care.,Recently, a classification scheme has been proposed that categorizes BRAF mutations based on their mechanisms for activation of the MAPK pathway.,In this analysis BRAF, KIT, NRAS, and PIK3CA mutations were examined by next generation sequencing (NGS) in 446 melanomas in a clinical diagnostic setting.,KRAS and HRAS were also analyzed to elucidate coexisting BRAF and RAS mutations.,BRAF mutations were categorized into class-1 (kinase-activated, codon 600), class-2 (kinase-activated, non-codon 600) and class-3 (kinase-impaired), based on the newly proposed classification scheme.,NGS demonstrated high analytic sensitivity.,Among 355 mutations detected, variant allele frequencies were 2-5% in 21 (5.9%) mutations and 2-10% in 47 (13%) mutations.,Mutations were detected in BRAF (42%), NRAS (25%), KIT (4.9%) and PIK3CA (2.7%).,The incidence of class-1, class-2 and class-3 mutations were 33% (26% p.V600E and 6.1% p.V600K), 3.1 and 4.9% respectively.,With a broader reportable range of NGS, class-1, class-2 and class-3 mutations accounted for 77, 7.4 and 12% of all BRAF mutations.,Class-3 mutations, commonly affecting codons 594, 466 and 467, showed a higher incidence of coexisting RAS mutations, consistent with their RAS-dependent signaling.,Significant association with old age and primary tumors of head/neck/upper back suggest chronic solar damage as a contributing factor for melanomas harboring BRAF p.V600K or class-3 mutations.,This study categorizes the range, frequency, coexisting driver mutations and clinical characteristics of the three classes of BRAF mutations in a large cohort of melanomas in a clinical diagnostic setting.,Further prospective studies are warranted to elucidate the clinical outcomes and benefits of newly developed targeted therapy in melanoma patients carrying each class of BRAF mutation.,The online version of this article (10.1186/s12885-019-5864-1) contains supplementary material, which is available to authorized users.
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Metastatic melanoma is the most aggressive and obstinate skin cancer with poor prognosis.,Variant novel applicable regimens have emerged during the past decades intensively, while the most profound approaches are oncogene-targeted therapy and T-lymphocyte mediated immunotherapy.,Although targeted therapies generated remarkable and rapid clinical responses in the majority of patients, acquired resistance was developed promptly within months leading to tumor relapse.,By contrast, immunotherapies elicited long-term tumor regression.,However, the overall response rate was limited.,In view of the above, either targeted therapy or immunotherapy cannot elicit durable clinical responses in large range of patients.,Interestingly, the advantages and limitations of these regimens happened to be complementary.,An increasing number of preclinical studies and clinical trials proved a synergistic antitumor effect with the combination of targeted therapy and immunotherapy, implying a promising prospect for the treatment of metastatic melanoma.,In order to achieve a better therapeutic effectiveness and reduce toxicity in patients, great efforts need to be made to illuminate multifaceted interplay between targeted therapy and immunotherapy.
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Metastatic uveal melanoma is less well understood than its primary counterpart, has a distinct biology compared to skin melanoma, and lacks effective treatments.,Here we genomically profile metastatic tumors and infiltrating lymphocytes.,BAP1 alterations are overrepresented and found in 29/32 of cases.,Reintroducing a functional BAP1 allele into a deficient patient-derived cell line, reveals a broad shift towards a transcriptomic subtype previously associated with better prognosis of the primary disease.,One outlier tumor has a high mutational burden associated with UV-damage.,CDKN2A deletions also occur, which are rarely present in primaries.,A focused knockdown screen is used to investigate overexpressed genes associated withcopy number gains.,Tumor-infiltrating lymphocytes are in several cases found tumor-reactive, but expression of the immune checkpoint receptors TIM-3, TIGIT and LAG3 is also abundant.,This study represents the largest whole-genome analysis of uveal melanoma to date, and presents an updated view of the metastatic disease.,The genetics of uveal melanoma has mainly been studied in primary tumours.,In this study, the authors perform whole genome sequencing as well as immune cell profiling of biopsy samples obtained from metastatic uveal melanoma patients, providing an updated genomic landscape of these advanced lesions.
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Recent experimental studies have demonstrated an essential role for the Hippo-Yes-associated protein (YAP) pathway in GNAQ/GNA11-induced tumorigenesis in uveal melanoma (UM).,However, the association between YAP activity and clinical outcomes remains elusive.,We investigated possible associations between YAP activity and clinicopathological features including survival outcomes in patients with UM using The Cancer Genome Atlas (TCGA) cohort and our local cohort.,We estimated YAP activity by mRNA expression levels, Gene Set Variation Analysis (GSVA) for the TCGA cohort, and immunohistochemical YAP staining for the local cohort.,In the TCGA cohort, most clinicopathological features including tumor stage, mitotic counts, mutation of genes, and tumor sizes did not significantly differ between low and high YAP activity groups.,In the local cohort, YAP nuclear-positive staining was observed in 30 (42%) of 72 patients with primary UM.,UM-specific survival was not significantly different between tumors with low and high YAP activities.,Unlike mesothelioma cells harboring a mutation of negative regulators of YAP, the survival of multiple UM cell lines was not significantly reduced by YAP/TAZ depletion.,Our results suggest that the effect of YAP on development, growth, and invasion of UM in actual patients is less than previously demonstrated in experimental studies.
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Cutaneous malignant melanoma is an aggressive cancer of melanocytes with a strong propensity to metastasize.,We posit that melanoma cells acquire metastatic capability by adopting an embryonic-like phenotype, and that a lineage approach would uncover metastatic melanoma biology.,Using a genetically engineered mouse model to generate a rich melanoblast transcriptome dataset, we identify melanoblast-specific genes whose expression contribute to metastatic competence and derive a 43-gene signature that predicts patient survival.,We identify a melanoblast gene, KDELR3, whose loss impairs experimental metastasis.,In contrast, KDELR1 deficiency enhances metastasis, providing the first example of different disease etiologies within the KDELR-family of retrograde transporters.,We show that KDELR3 regulates the metastasis suppressor, KAI1, and report an interaction with the E3 ubiquitin-protein ligase gp78, a regulator of KAI1 degradation.,Our work demonstrates that the melanoblast transcriptome can be mined to uncover targetable pathways for melanoma therapy.,Metastatic cells can mimic many of the phenotypic behaviors of embryonic cells.,Here, the authors generate a melanoblast-specific transcriptome using a genetically engineered mouse model and identify KDELR3 as a pro-metastasis gene in melanoma.
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The overall 5-year survival for melanoma is 91%.,However, if distant metastasis occurs (stage IV), cure rates are < 15%.,Hence, melanoma detection in earlier stages (stages I-III) maximises the chances of patient survival.,We measured the expression of a panel of 17 microRNAs (miRNAs) (MELmiR-17) in melanoma tissues (stage III; n = 76 and IV; n = 10) and serum samples (collected from controls with no melanoma, n = 130; and patients with melanoma (stages I/II, n = 86; III, n = 50; and IV, n = 119)) obtained from biobanks in Australia and Germany.,In melanoma tissues, members of the ‘MELmiR-17’ panel were found to be predictors of stage, recurrence, and survival.,Additionally, in a minimally-invasive blood test, a seven-miRNA panel (MELmiR-7) detected the presence of melanoma (relative to controls) with high sensitivity (93%) and specificity (≥ 82%) when ≥ 4 miRNAs were expressed.,Moreover, the ‘MELmiR-7’ panel characterised overall survival of melanoma patients better than both serum LDH and S100B (delta log likelihood = 11, p < 0.001).,This panel was found to be superior to currently used serological markers for melanoma progression, recurrence, and survival; and would be ideally suited to monitor tumour progression in patients diagnosed with early metastatic disease (stages IIIa-c/IV M1a-b) to detect relapse following surgical or adjuvant treatment.,•A seven-miRNA panel (MELmiR-7) detected the presence of melanoma with high sensitivity (93%) and specificity (≥ 82%).,•In serially collected stage IV specimens, members of the ‘MELmiR-7’ panel confirmed tumour progression in 100% of cases.,•The ‘MELmiR-7’ panel is superior to currently used serological markers for melanoma progression, recurrence, and survival.,A seven-miRNA panel (MELmiR-7) detected the presence of melanoma with high sensitivity (93%) and specificity (≥ 82%).,In serially collected stage IV specimens, members of the ‘MELmiR-7’ panel confirmed tumour progression in 100% of cases.,The ‘MELmiR-7’ panel is superior to currently used serological markers for melanoma progression, recurrence, and survival.
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Targeted therapies with MAPK inhibitors (MAPKi) are faced with severe problems of resistance in BRAF‐mutant melanoma.,In parallel to the acquisition of genetic mutations, melanoma cells may also adapt to the drugs through phenotype switching.,The ZEB1 transcription factor, a known inducer of EMT and invasiveness, is now considered as a genuine oncogenic factor required for tumor initiation, cancer cell plasticity, and drug resistance in carcinomas.,Here, we show that high levels of ZEB1 expression are associated with inherent resistance to MAPKi in BRAFV 600‐mutated cell lines and tumors.,ZEB1 levels are also elevated in melanoma cells with acquired resistance and in biopsies from patients relapsing while under treatment.,ZEB1 overexpression is sufficient to drive the emergence of resistance to MAPKi by promoting a reversible transition toward a MITF low/p75high stem‐like and tumorigenic phenotype.,ZEB1 inhibition promotes cell differentiation, prevents tumorigenic growth in vivo, sensitizes naive melanoma cells to MAPKi, and induces cell death in resistant cells.,Overall, our results demonstrate that ZEB1 is a major driver of melanoma cell plasticity, driving drug adaptation and phenotypic resistance to MAPKi.
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Genomic locus at chromosome 9p21 that contains the CDKN2A and CDKN2B tumor suppressor genes is inactivated through mutations, deletions and promoter methylation in multiple human cancers.,Additionally, the locus encodes an anti-sense RNA (ANRIL).,Both hemizygous and homozygous deletions at the locus targeting multiple genes are fairly common in different cancers.,We in this study investigated breakpoints in five melanoma cell lines, derived from metastasized tumors, with previously identified homozygous deletions using array comparative genomic hybridization (aCGH).,For breakpoint mapping, we used primer approximation multiplex PCR (PAMP) and inverse PCR techniques.,Our results showed that three cell lines carried complex rearrangements.,In two other cell lines, with focal deletions of 141 kb and 181 kb, we identified fusion gene products, involving MTAP and ANRIL.,We also confirmed the complex rearrangements and focal deletions in DNA from tumor tissues corresponding to three cell lines.,The rapid amplification of 3′cDNA ends (3′RACE) carried out on transcripts resulted in identification of three isoforms of MTAP-ANRIL fusion gene.,Screening of cDNA from 64 melanoma cell lines resulted in detection of fusion transcripts in 13 (20%) cell lines that involved exons 4-7 of the MTAP and exon 2 or 5 of the ANRIL genes.,We also detected fusion transcripts involving MTAP and ANRIL in two of the seven primary melanoma tumors with focal deletion at the locus.,The results from the study, besides identifying complex rearrangements involving CDKN2A locus, show frequent occurrence of fusion transcripts involving MTAP and ANRIL genes.
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A vast array of tumor-derived genetic, proteomic and cellular components are constantly released into the circulation of cancer patients.,These molecules including circulating tumor DNA and RNA, proteins, tumor and immune cells are emerging as convenient and accurate liquid biomarkers of cancer.,Circulating cancer biomarkers provide invaluable information on cancer detection and diagnosis, prognosticate patient outcomes, and predict treatment response.,In this era of effective molecular targeted treatments and immunotherapies, there is now an urgent need to implement use of these circulating biomarkers in the clinic to facilitate personalized therapy.,In this review, we present recent findings in circulating melanoma biomarkers, examine the challenges and promise of evolving technologies used for liquid biomarker discovery, and discuss future directions and perspectives in melanoma biomarker research.
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Objective To estimate the burden of melanoma resulting from sunbed use in western Europe.,Design Systematic review and meta-analysis.,Data sources PubMed, ISI Web of Science (Science Citation Index Expanded), Embase, Pascal, Cochrane Library, LILACS, and MedCarib, along with published surveys reporting prevalence of sunbed use at national level in Europe.,Study selection Observational studies reporting a measure of risk for skin cancer (cutaneous melanoma, squamous cell carcinoma, basal cell carcinoma) associated with ever use of sunbeds.,Results Based on 27 studies ever use of sunbeds was associated with a summary relative risk of 1.20 (95% confidence interval 1.08 to 1.34).,Publication bias was not evident.,Restricting the analysis to cohorts and population based studies, the summary relative risk was 1.25 (1.09 to 1.43).,Calculations for dose-response showed a 1.8% (95% confidence interval 0% to 3.8%) increase in risk of melanoma for each additional session of sunbed use per year.,Based on 13 informative studies, first use of sunbeds before age 35 years was associated with a summary relative risk of 1.87 (1.41 to 2.48), with no indication of heterogeneity between studies.,By using prevalence data from surveys and data from GLOBOCAN 2008, in 2008 in the 15 original member countries of the European Community plus three countries that were members of the European Free Trade Association, an estimated 3438 cases of melanoma could be attributable to sunbed use, most (n=2341) occurring among women.,Conclusions Sunbed use is associated with a significant increase in risk of melanoma.,This risk increases with number of sunbed sessions and with initial usage at a young age (<35 years).,The cancerous damage associated with sunbed use is substantial and could be avoided by strict regulations.
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Melanoma is the deadliest skin cancer.,Despite improvements in the understanding of the molecular mechanisms underlying melanoma biology and in defining new curative strategies, the therapeutic needs for this disease have not yet been fulfilled.,Herein, we provide evidence that the Activating Molecule in Beclin-1-Regulated Autophagy (Ambra1) contributes to melanoma development.,Indeed, we show that Ambra1 deficiency confers accelerated tumor growth and decreased overall survival in Braf/Pten-mutated mouse models of melanoma.,Also, we demonstrate that Ambra1 deletion promotes melanoma aggressiveness and metastasis by increasing cell motility/invasion and activating an EMT-like process.,Moreover, we show that Ambra1 deficiency in melanoma impacts extracellular matrix remodeling and induces hyperactivation of the focal adhesion kinase 1 (FAK1) signaling, whose inhibition is able to reduce cell invasion and melanoma growth.,Overall, our findings identify a function for AMBRA1 as tumor suppressor in melanoma, proposing FAK1 inhibition as a therapeutic strategy for AMBRA1 low-expressing melanoma.,The absence of scaffold protein Ambra1 leads to hyperproliferation and growth in mouse models.,Here the authors show that Ambra1 deficiency accelerates melanoma growth and increases metastasis in mouse models of melanoma through FAK1 hyperactivation.
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Different 3D-cell culture approaches with varying degrees of complexity have been developed to serve as melanoma models for drug testing or mechanistic studies.,While these 3D-culture initiatives are already often superior to classical 2D approaches, they are either composed of only melanoma cells or they are so complex that the behavior of individual cell types is hard to understand, and often they are difficult to establish and expensive.,This study used low-attachment based generation of spheroids composed of up to three cell types.,Characterization of cells and spheroids involved cryosectioning, immunofluorescence, FACS, and quantitative analyses.,Statistical evaluation used one-way ANOVA with post-hoc Tukey test or Student’s t-test.,The tri-culture model allowed to track cellular behavior in a cell-type specific manner and recapitulated different characteristics of early melanoma stages.,Cells arranged into a collagen-IV rich fibroblast core, a ring of keratinocytes, and groups of highly proliferating melanoma cells on the outside.,Regularly, some melanoma cells were also found to invade the fibroblast core.,In the absence of melanoma cells, the keratinocyte ring stratified into central basal-like and peripheral, more differentiated cells.,Conversely, keratinocyte differentiation was clearly reduced upon addition of melanoma cells.,Treatment with the cytostatic drug, docetaxel, restored keratinocyte differentiation and induced apoptosis of external melanoma cells.,Remaining intact external melanoma cells showed a significantly increased amount of ABCB5-immunoreactivity.,In the present work, a novel, simple spheroid-based melanoma tri-culture model composed of fibroblasts, keratinocytes, and melanoma cells was described.,This model mimicked features observed in early melanoma stages, including loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced increase of ABCB5 expression in external melanoma cells.,The online version of this article (10.1186/s12885-019-5606-4) contains supplementary material, which is available to authorized users.
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