Vertical Growth Phase Melanomas Research Articles

Intracellular mechanics and TBX3 expression jointly dictate the spreading mode of melanoma cells in 3D environments

Cell stiffness and T-box transcription factor 3 (TBX3) expression have been identified as biomarkers of melanoma metastasis in 2D environments. This study aimed to determine how mechanical and biochemical properties of melanoma cells change during cluster formation in 3D environments. Vertical growth phase (VGP) and metastatic (MET) melanoma cells were embedded in 3D collagen matrices of 2 and 4 mg/ml collagen concentrations, representing low and high matrix stiffness. Mitochondrial fluctuation, intracellular stiffness, and TBX3 expression were quantified before and during cluster formation. In isolated cells, mitochondrial fluctuation decreased and intracellular stiffness increased with increase in disease stage from VGP to MET and increased matrix stiffness. TBX3 was highly expressed in soft matrices but diminished in stiff matrices for VGP and MET cells. Cluster formation of VGP cells was excessive in soft matrices but limited in stiff matrices, whereas for MET cells it was limited in soft and stiff matrices. In soft matrices, VGP cells did not change the intracellular properties, whereas MET cells exhibited increased mitochondrial fluctuation and decreased TBX3 expression. In stiff matrices, mitochondrial fluctuation and TBX3 expression increased in VGP and MET, and intracellular stiffness increased in VGP but decreased in MET cells. The findings suggest that soft extracellular environments are more favourable for tumour growth, and high TBX3 levels mediate collective cell migration and tumour growth in the earlier VGP disease stage but play a lesser role in the later metastatic stage of melanoma.

TERT Promoter Mutational Analysis as an Ancillary Diagnostic Tool for Diagnostically Challenging Melanocytic Neoplasms.

Telomerase reverse transcriptase promoter mutations (TPMs) have been shown to be common in melanoma and uncommon in benign nevi. To assess the use of TPMs as an ancillary diagnostic tool, we report the concordance of the TPM status with the final diagnosis in clinical cases with distinct differential diagnostic scenarios: dysplastic nevus versus melanoma, atypical Spitz nevus versus melanoma, atypical deep penetrating nevus (DPN) versus melanoma, and atypical blue nevus versus malignant blue nevus. In a control cohort, we found a positive TPM in 51/70 (73%) of the total melanomas with the highest frequency in vertical growth phase melanoma cases. Conversely, only 2/35 (6%) dysplastic nevi in our control cases were TPM-positive and b were severely atypical dysplastic nevi. Our clinical cohort of 257 cases had a positive TPM in 24% of cases diagnosed as melanoma and in 1% of cases with a benign diagnosis. The overall concordance of the TPM status with the final diagnosis was 86%. The TPM status had the greatest concordance (95%) with the final diagnosis in the atypical DPN versus melanoma group, with the rest of the groups ranging between 50% and 88%. Overall, our results suggest that TPMs are most useful in the differential diagnosis of atypical DPN versus melanoma. It also has some value in the differential diagnosis of atypical Spitz tumor versus melanoma and dysplastic nevus versus melanoma, whereas in our cohort, it did not contribute meaningfully to differentiating malignant blue nevus and atypical blue nevus.

Abstract A001: Shared gene expression and immune pathway changes associated with progression from nevi to melanoma

Abstract There is a need to identify biomarkers of melanoma progression to assist the development of chemoprevention strategies to lower melanoma incidence. In this study, we assessed the feasibility of creating a molecular signature for melanomagenesis using three publicly available RNA sequencing and microarray expression datasets. We performed differential expression and regularized regression analyses across nevi and melanoma samples to identify consistent genes associated with melanomagenesis. The regularized regression models demonstrated that a small number of genes could successfully distinguish between nevi and melanoma, providing evidence for the feasibility of creating a molecular signature. Differential expression analysis identified consistent upregulation of C1QB, CXCL9, CXCL10, DFNA5 (GSDME), FCGR1B, and PRAME in melanoma and consistent downregulation of SCGB1D2 in melanoma compared to nevi. Additionally, each of these genes demonstrated a linear association with the progression from benign nevi to dysplastic nevi, to radial growth phase melanoma to vertical growth phase melanoma, providing additional evidence for their role in melanomagenesis. Subsequent pathway analysis demonstrated significant enrichment of immune-related pathways among the differentially expressed genes. Overall, this study 1) demonstrates the feasibility of creating a gene signature for melanomagenesis and 2) highlights genes and pathways of interest for melanoma progression. We are in the process of generating a new dataset with benign nevi, dysplastic nevi, and melanoma with which to build and validate a molecular signature of melanoma. Citation Format: Elizabeth S. Borden, Anngela C. Adams, Kenneth H. Buetow, Melissa A. Wilson, Julie E. Bauman, Clara Curiel-Lewandrowski, H.-H. Sherry Chow, Bonnie J. LaFleur, Karen Taraszka Hastings. Shared gene expression and immune pathway changes associated with progression from nevi to melanoma [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A001.

Abstract A006: Shared gene expression and immune pathway changes associated with progression from nevi to melanoma

Abstract There is a need to identify biomarkers of melanoma progression to assist the development of chemoprevention strategies to lower melanoma incidence. In this study, we assessed the feasibility of creating a molecular signature for melanomagenesis using three publicly available RNA sequencing and microarray expression datasets. We performed differential expression and regularized regression analyses across nevi and melanoma samples to identify consistent genes associated with melanomagenesis. The regularized regression models demonstrated that a small number of genes could successfully distinguish between nevi and melanoma, providing evidence for the feasibility of creating a molecular signature. Differential expression analysis identified consistent upregulation of C1QB, CXCL9, CXCL10, DFNA5 (GSDME), FCGR1B, and PRAME in melanoma and consistent downregulation of SCGB1D2 in melanoma compared to nevi. Additionally, each of these genes demonstrated a linear association with the progression from benign nevi to dysplastic nevi, to radial growth phase melanoma to vertical growth phase melanoma, providing additional evidence for their role in melanomagenesis. Subsequent pathway analysis demonstrated significant enrichment of immune-related pathways among the differentially expressed genes. Overall, this study 1) demonstrates the feasibility of creating a gene signature for melanomagenesis and 2) highlights genes and pathways of interest for melanoma progression. We are in the process of generating a new dataset with benign nevi, dysplastic nevi, and melanoma with which to build and validate a molecular signature of melanoma. Citation Format: Elizabeth S. Borden, Anngela C. Adams, Kenneth H. Buetow, Melissa A. Wilson, Julie E. Bauman, Clara Curiel-Lewandrowski, H.-H. Sherry Chow, Bonnie J. LaFleur, Karen Taraszka Hastings. Shared gene expression and immune pathway changes associated with progression from nevi to melanoma [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A006.

Shared Gene Expression and Immune Pathway Changes Associated with Progression from Nevi to Melanoma.

Simple SummaryMelanoma is a deadly skin cancer, and the incidence of melanoma is rising. Chemoprevention, using small molecule drugs to prevent the development of cancer, is a key strategy that could reduce the burden of melanoma on society. The long-term goal of our study is to develop a gene signature biomarker of progression from nevi to melanoma. We found that a small number of genes can distinguish nevi from melanoma and identified shared genes and immune-related pathways that are associated with progression from nevi to melanoma across independent datasets. This study demonstrates (1) a novel approach to aid melanoma chemoprevention trials by using a gene signature as a surrogate endpoint and (2) the feasibility of determining a gene signature biomarker of melanoma progression.There is a need to identify molecular biomarkers of melanoma progression to assist the development of chemoprevention strategies to lower melanoma incidence. Using datasets containing gene expression for dysplastic nevi and melanoma or melanoma arising in a nevus, we performed differential gene expression analysis and regularized regression models to identify genes and pathways that were associated with progression from nevi to melanoma. A small number of genes distinguished nevi from melanoma. Differential expression of seven genes was identified between nevi and melanoma in three independent datasets. C1QB, CXCL9, CXCL10, DFNA5 (GSDME), FCGR1B, and PRAME were increased in melanoma, and SCGB1D2 was decreased in melanoma, compared to dysplastic nevi or nevi that progressed to melanoma. Further supporting an association with melanomagenesis, these genes demonstrated a linear change in expression from benign nevi to dysplastic nevi to radial growth phase melanoma to vertical growth phase melanoma. The genes associated with melanoma progression showed significant enrichment of multiple pathways related to the immune system. This study demonstrates (1) a novel application of bioinformatic approaches to aid clinical trials of melanoma chemoprevention and (2) the feasibility of determining a gene signature biomarker of melanomagenesis.

Assessing the Prognostic Significance of Tumor-Infiltrating Lymphocytes in Patients With Melanoma Using Pathologic Features Identified by Natural Language Processing

Although tumor-infiltrating lymphocytes (TILs) are an important histopathologic characteristic reflecting host immune response in patients with melanoma, their prognostic value remains controversial. Because manual review of medical records is labor intensive, a survival analysis using a large patient cohort with comprehensive clinical and histopathologic characteristics is lacking. To assess the prognostic significance of TILs among patients with cutaneous melanoma using a large cohort established through natural language processing (NLP) algorithms. This retrospective cohort study analyzed the medical records of 14 436 patients with cutaneous melanoma at Brigham and Women's Hospital between June 1, 2004, and December 31, 2019. Patients were followed up to death or censored at their last clinical visit. The primary outcome was overall survival (OS). Survival analysis was conducted using Kaplan-Meier curves, the log-rank test, and Cox proportional hazards regression analysis. A total of 14 436 patients with cutaneous melanoma were identified in the institution's pathology information system. Using NLP, we established a study cohort of 2624 patients (1462 men [55.7%]; median age, 61 years [interquartile range, 50-72 years]) who had vertical growth phase melanoma with TIL status scored. Absent TILs were identified in 434 patients (16.5%), nonbrisk TILs in 1916 patients (73.0%), and brisk TILs in 274 patients (10.4%). The 5-year survival rate was 71.0% (95% CI, 65.5%-76.9%) among patients with an absence of TILs, 73.8% (95% CI, 71.1%-76.5%) among patients with nonbrisk TILs, and 85.2% (95% CI, 80.0%-90.7%) among patients with brisk TILs. Brisk TILs were significantly associated with improved OS (adjusted hazard ratio, 0.63; 95% CI, 0.42-0.95; P = .03; 14.2% OS advantage at 5 years), and nonbrisk TILs were not associated with improved OS (adjusted hazard ratio, 0.87; 95% CI, 0.68-1.11; P = .25), compared with the absence of TILs. This study provides evidence based on a large patient cohort from a single institution that suggests that brisk TILs represent an independent prognostic factor for OS among patients with primary cutaneous melanoma. The study also suggests that NLP is a highly efficient tool to facilitate large-scale analyses that involve free-text clinical data.

527 Expression of OPN3 correlating to tumor initiation and development in cutaneous melanoma

The function of OPN3 in melanoma remains largely unknown. To assess the biological role of OPN3 in the initiation and development of cutaneous melanoma, we first systematically investigated the expression patterns of OPN3 gene from human melanoma based on the available three independent gene expression datasets (GEO GSE3189, GSE8401 and GSE4587). We found that the OPN3 mRNA was upregulated in human melanoma when compared to normal skin and nevus tissue samples, especially in vertical growth phase melanoma. Additionally, no statistically significant difference was evident between the primary melanoma and metastasis of melanoma tissues. By the weighted correlation network and protein-protein association networks analysis, it suggested that OPN3 might play a role in the initiation and development of cutaneous melanoma. Next, to further illustrate the possible mechanisms by which OPN3 mediates melanoma cell tumorigenicity, RNA-seq analysis was performed on low expression of OPN3 and untreated M14 human skin melanoma cells. In this present study, our heatmap revealed that 5029 differentially expressed genes (DEGs) were upregulated, while 4495 DEGs were downregulated. Notably, in GO (Gene Ontology) term enrichment analysis our results indicated that the DEGs were evidently enriched in post-transcriptional gene regulation and cell cycle regulation, suggesting that OPN3 was mainly involved in proliferation and cell cycle regulation. The KEGG pathway analysis, OPN3 was dramatically enriched in cell cycle regulation process, neurotrophin signaling pathway and mRNA surveillance pathway. Taken together, we here showed that high expression of OPN3 can promote melanoma initiation and development by regulating cell cycle progression and tumor-associated signaling pathway.

TBX3 Promotes Melanoma Migration by Transcriptional Activation of ID1, which Prevents Activation of E-Cadherin by MITF

In melanoma, a phenotype switch from proliferation to invasion underpins metastasis, the major cause of melanoma-associated death. The transition from radial to vertical growth phase (invasive) melanoma is characterized by downregulation of both E-cadherin (CDH1) and MITF and upregulation of the key cancer-associated gene TBX3 and the phosphatidylinositol 3 kinase signaling pathway. Yet, whether and how these diverse events are linked remains poorly understood. Here, we show that TBX3 directly promotes expression of ID1, a dominant-negative regulator of basic helix-loop-helix transcription factors, and that ID1 decreases MITF binding and upregulation of CDH1. Significantly, we show that TBX3 activation of ID1 is necessary for TBX3 to enhance melanoma cell migration, and the mechanistic links between TBX3, ID1, MITF, and invasion revealed here are reflected in their expression in human melanomas. Our results reveal that melanoma migration is promoted through a TBX3-ID1-MITF-E-cadherin axis and that ID1-mediated repression of MITF activity may reinforce maintenance of an MITFLow phenotype associated with disease progression and therapy resistance.

Role of TGF‑β in the motility of ShcD‑overexpressing 293 cells.

The newly identified Src homology and collagen (Shc) family member ShcD was observed to be upregulated in 50% of vertical growth phase and metastatic melanomas. The aim of the present study was to investigate the mechanism by which ShcD mediates cell motility. 293 cell lines were altered to stably express GFP (GF) or GFP-ShcD (G5). Treatment of the cells with transforming growth factor (TGF)β2 promoted extracellular signal-regulated kinase (ERK) phosphorylation and, to a lesser extent, Smad2 phosphorylation in GFP-ShcD-expressing cells but not in GFP-overexpressing cells. GFP-ShcD-expressing cells exhibited upregulated expression of certain epithelial-mesenchymal transition-related genes, such as snail family transcriptional repressor 1 and SLUG, than GFP-expressing cells. Higher levels of ERK were found in the nuclear fraction of GFP-ShcD-expressing cells than that of GFP-expressing cells. Overall, GFP-ShcD-expressing cells demonstrated enhanced migration compared with GFP-expressing cells. A slight increase in cell migration was observed in both cell lines (GF and G5) when the cells were allowed to migrate towards conditioned medium derived from TGFβ2-treated GFP-ShcD expressing cells. Collectively, ShcD upregulation was proposed to induce cell migration by affecting the expression of certain epithelial-mesenchymal transition-related genes. Thus, our findings may improve understanding of the role of ShcD in cell migration.

Biophysical and Biochemical Characteristics as Complementary Indicators of Melanoma Progression

The multistep character of cancer progression makes it difficult to define a unique biomarker of the disease. Interdisciplinary approaches, combining various complementary techniques, especially those operating at a nanoscale level, potentially accelerate characterization of cancer cells or tissue properties. Here, we study a relation between the surface and biomechanical properties of melanoma cells, measured by mass spectrometry (ToF-SIMS) and atomic force microscopy (AFM). In total, seven cell lines have been studied. Six of them were melanoma cells derived from various stages of tumor progression: (1) WM115 cells derived from a 55 year old female skin melanoma at a vertical growth phase (VGP) in the primary melanoma site, (2) WM793 cells established from the vertical growth phase (VGP) of a primary skin melanoma lesion, (3) WM266-4 cells established from a cutaneous skin metastasis detected in the same patient as WM115 cells, (4) WM239 cells derived from a cutaneous skin metastasis, (5) 1205Lu cells originated from a lung metastasis diagnosed in the same patient as WM793 cells, and (6) A375P-cells were derived from a solid malignant tumor located in the lung. As a reference cell line, human epidermal melanocytes from adult skin (primary cell line HEMa-LP) were used. Results reveal low, medium, and large deformability of melanoma cells originating from vertical growth phase (VGP), and skin and lung metastasis, respectively. These changes were accompanied by distinct outcome from principal component analysis (PCA). In relation to VGP melanoma cells, cells from skin and lung metastasis reveal similar or significantly different surface properties. The largest deformability difference observed for cells from VGP and lung metastasis was accompanied by the largest separation of unspecific changes in their surface properties. In this way, we show the evidence that biomechanical and surface biochemical properties of cells change in parallel, indicating a potential of being used as nanobiophysical fingerprints of melanoma progression.

846 Resistance to apoptosis and PD-L1 induction in melanoma is associated with suppressed aryl hydrocarbon receptor signaling

Metastatic melanoma is an aggressive form of skin cancer with a 5-year survival rate of 15% to 20%. Unlike primary melanoma, metastatic disease is difficult to treat due to acquisition of chemoresistance and immune evasion through PD-L1 expression. Highlighting the intrinsic differences in signaling networks that are overactive or suppressed between primary and metastatic melanoma will provide essential cues to understanding mechanisms of therapeutic resistance and immune evasion. We detected reduced cytochrome p450 enzymes, Cyp1a1 and Cyp1b1, in vertical growth phase melanoma cell line, WM793, and its metastatic variant, 1205Lu, compared to primary melanoma cells WM1552c. Since their expression is directly controlled by aryl hydrocarbon receptor (AhR), whose expression was also decreased in 1205Lu and WM793 cells, we tested whether reduced AhR signaling in melanoma cells confers chemoresistance and immune evasion properties. We used paclitaxel (ptx)-mediated apoptosis and interferon gamma (IFNg)-mediated PD-L1 induction as end points to evaluate chemotherapeutic resistance and immune evasion, respectively. 1205Lu and WM793 cells were resistant to ptx-mediated apoptosis (<10%) and PD-L1 expression increased by 3 to 6-fold following IFNg treatment. WM1552c cells, however, were sensitive to ptx-mediated apoptosis (>25%) with minimal induction of PD-L1 (1.5-fold) following IFNg treatment. Treatment with AhR antagonist, CH223191, prior to ptx decreased the percentage of apoptotic cells to <10% in WM1552 cells. IFNg-induced PD-L1 expression also increased by 3-folds in WM1552c cells following AhR inhibition although the mean fluorescence intensity was still lower than 1205Lu cells. Our data indicates that a suppressed AhR signaling in vertical phase and metastatic melanoma cells may have been evolved to resist apoptosis to chemotherapy and promote immune evasion via enhanced PD-L1 induction. Elucidating components of AhR pathway that are affected can be of high therapeutic value for metastatic melanoma.

Abstract 1072: Potential biomarkers correlated with poor survival rate of melanoma patients

Abstract Malignant melanoma is an extremely aggressive tumor, responsible for 90% of skin cancer mortality. Widespread metastasis is the main cause of death in melanoma patients. Therefore, the identification of molecules related to the degree of melanoma malignancy could be used as prognostic biomarkers. In the present study, using a murine model of melanoma progression, different genes, such as Snai1, Chd1, Gata4, Id4, Cbs, Vav1, and Itga4, were first identified as presenting aberrant DNA methylation in their promoters along melanoma progression by Methylight analysis. The gene expression pattern and epigenetic regulation were validated in these murine cell lines by RT-qPCR and 5AzaCdR treatment, respectively. The genes Angpt2 and Fgf1 were also identified as differentially expressed along tumor progression in this murine model. The expression profile of these genes was also evaluated in primary human melanocytes and RGP (radial growth phase), VGP (vertical growth phase) and metastatic melanoma cell lines. Clinical and gene expression data from 460 patients were obtained from TCGA (The Cancer Genome Atlas). Using an univariate test, patients presenting tumors with low levels of Chd1, Vav1, Id4 and Fgf1, and high levels of Snai1 and Angpt2 were shown to exhibit poor overall survival rate. Indeed, the multivariate analysis using Cox regression model identified high Snai1 (HR=1.217; 95% CI 1.075-1.377; p=0.002) and Angpt2 (HR=1.186; 95% CI 1.059-1.329; p=0.003) expression and low Chd1 (HR=0.757; 95% CI 0.575-0.996; p=0.047), Fgf1 (HR=0.858; 95% CI 0.780-0.944; p=0.002) and Vav1 (HR=0.859; 95% CI 0.764-0.967; p=0.011) expression as independent prognostic factors for the overall survival rate of patients with melanoma. Also using a multivariate analysis, the low levels of Id4 promoter methylation (HR=0.689; 95% CI 0.531-0.891; p=0.045) was shown to be an independent predictive factor for poor survival rate, although no correlation was found between Id4 expression and survival rate. In conclusion, the upregulation of Snai1 and Angpt2, and the downregulation of Chd1, Vav1 and Fgf1 are associated with unfavorable survival outcome, indicating they might be valuable prognostic biomarkers for melanoma. Supported by CNPq and FAPESP. Citation Format: Alice S. Morais, Ana C. Monteiro, Fernando Andrade, Lucas Goedert, Enilza Espreáfico, Regine Schneider-Stock, André Fujiota, Miriam G. Jasiulionis. Potential biomarkers correlated with poor survival rate of melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1072.

ETS1, nucleolar and non-nucleolar TERT expression in nevus to melanoma progression.

TERT (telomerase reverse transcriptase) is the catalytic component of telomerase. TERT shows little expression in normal somatic cells but is commonly re-expressed in cancers, facilitating immortalization. Recently-discovered TERT promoter mutations create binding sites for ETS-family transcription factors to upregulate TERT. ETS1 is reported to be important for TERT upregulation in melanoma. However it is unclear when in melanoma progression TERT and ETS1 proteins are expressed. To elucidate this question, ETS1 and TERT immunohistochemistry were performed on a panel of benign (n=27) and dysplastic nevi (n=34), radial growth phase (n=29), vertical growth phase (n=25) and metastatic melanomas (n=27). Lesions were scored by percentage of positive cells. ETS1 was readily detectable in all lesions, but not in normal melanocytes. TERT was located in either the nucleolus, the nucleoplasm (non-nucleolar) or both. Non-nucleolar TERT increased in prevalence with progression, from 19% of benign nevi to 78% of metastases. It did not however correlate with cell proliferation (Ki-67 immunostaining), nor differ significantly in prevalence between primary melanomas with or without a TERT promoter mutation. These results demonstrate that ETS1 is expressed very early in melanoma progression, and interestingly only non-nucleolar TERT correlates clearly in prevalence with melanoma progression. It can be acquired at various stages and by mechanisms other than promoter mutations.

Abstract 1037: Progression from melanocytic nevi to melanoma is associated with increased genomic mutations in a UV-induced mouse model of human melanoma

Abstract Melanoma is the deadliest form of skin cancer with approximately 132,000 cases worldwide each year. Benign melanocytic nevi are nearly universal, and although progression of nevi to melanoma is very rare, 20-50% of melanoma appear to arise from a pre-existing nevus. UV exposure, particularly childhood sunburn, is believed to play an important role in the development of melanocytic nevi and melanoma, but the exact mechanism is unknown. Alterations in MAPK pathway genes, especially NRAS and BRAF, are common in both benign nevi and melanoma, but approximately 1/3 of melanomas do not have an identified driver mutation. Studying nevus initiation and progression prospectively in the human population is impractical due to the long latency to progression and repeated UV exposures Our laboratory has developed a hepatocyte growth factor (HGF) genetically engineered mouse model with “humanized” junctional distribution of melanocytes on an iDCT-GFP background with melanocyte-specific GFP expression, allowing melanocytic lesions to be tracked through percutaneous GFP imaging. Following a single relevant dose of UV modeling childhood sunburn, HGF iDCT-GFP develop discrete, small melanocytic lesions consistent with nevi. Most nevi remain stable over the lifetime of the mouse, but around 1 in 30 progress to melanoma usually starting at 6-12 months. The melanocytic lesions are histologically similar to human nevi and melanoma, label with melanocyte markers and tumors are transplantable into syngeneic mice. Melanomas that arise in the model are heterogeneous and include radial growth phase and vertical growth phase tumors and sometimes metastasize to liver and lung. Exome sequencing of 28 nevi and melanomas show that vertical growth phase melanomas have approximately 3x more mutations than radial growth phase melanomas or nevi. The increased number of mutations in vertical growth phase tumors is due to an increase in C&amp;gt;T transitions despite the lack of additional UV exposure. Interestingly, melanocytic nevi and melanomas with DNA repair pathway mutations average 3x more mutations than lesions without mutations in these pathways. Melanomas sometime contain mutations in hotspot locations from human melanomas, including GNAQ, but most do not have a previously identified dominant driver. Genes potentially involved in the initiation of melanocytic lesions or progression to aggressive melanomas and relevant to human melanoma have been identified and are being functionally tested using CRISPR to introduce point mutations or knock out genes and in vitro skin reconstitution assays. Identification of novel drivers and pathways involved in non-BRAF, non-NRAS melanoma has the potential to uncover biomarkers and new therapeutic targets to improve clinical outcomes for melanoma patients. Citation Format: Helen Michael, Chi-Ping Day, Howard Yang, Aleksandra Michalowski, Maxwell Lee, Glenn Merlino. Progression from melanocytic nevi to melanoma is associated with increased genomic mutations in a UV-induced mouse model of human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1037. doi:10.1158/1538-7445.AM2017-1037

Functional expression of olfactory receptors in human primary melanoma and melanoma metastasis.

We identified the olfactory receptor 51E2 in human melanoma and have measured both OR51E2 mRNA and protein expression in melanoma tissue sections. qPCR analysis revealed that the receptor is upregulated in melanoma cells compared to normal melanocytes, indicating that OR51E2 may play a role in early melanoma development and progression. Activation of endogenous OR51E2 in cultured cells derived from metastatic and vertical-growth phase (VGP) by its ligand β-ionone results in an increase in the intracellular Ca2+ concentration. RNAi experiments showed that the β-ionone-induced Ca2+ signal depends on the activation of OR51E2. Furthermore, OR51E2 activation inhibits the growth of VGP melanoma cells via apoptotic processes. Cell motility assays revealed that treatment with β-ionone decreases the migration of VGP melanoma cells. Overall, our data demonstrates that OR51E2 is involved in the regulation of cell proliferation and migration, suggesting that it may serve as a novel target for melanoma therapy.

Cross-talk between Dopachrome Tautomerase and Caveolin-1 Is Melanoma Cell Phenotype-specific and Potentially Involved in Tumor Progression

l-Dopachrome tautomerase (l-DCT), also called tyrosinase-related protein-2 (TRP-2), is a melanoma antigen overexpressed in most chemo-/radiotherapeutic stress-resistant tumor clones, and caveolin-1 (CAV1) is a main regulator of numerous signaling processes. A structural and functional relationship between DCT and CAV1 is first presented here in two human amelanotic melanoma cell lines, derived from vertical growth phase (MelJuSo) and metastatic (SKMel28) melanomas. DCT co-localizes at the plasma membrane with CAV1 and Cavin-1, another molecular marker for caveolae in both cell phenotypes. Our novel structural model proposed for the DCT-CAV1 complex, in addition to co-immunoprecipitation and mass spectrometry data, indicates a possible direct interaction between DCT and CAV1. The CAV1 control on DCT gene expression, DCT post-translational processing, and subcellular distribution is cell phenotype-dependent. DCT is a modulator of CAV1 stability and supramolecular assembly in both cell phenotypes. During autocrine stimulation, the expressions of DCT and CAV1 are oppositely regulated; DCT increases while CAV1 decreases. Sub-confluent MelJuSo clones DCT(high)/CAV1(low) are proliferating and acquire fibroblast-like morphology, forming massive, confluent clusters as demonstrated by immunofluorescent staining and TissueFAXS quantitative image cytometry analysis. CAV1 down-regulation directly contributes to the expansion of MelJuSo DCT(high) subtype. CAV1 involved in the perpetuation of cell phenotype-overexpressing anti-stress DCT molecule supports the concept that CAV1 functions as a tumor suppressor in early stages of melanoma. DCT is a regulator of the CAV1-associated structures and is possibly a new molecular player in CAV1-mediated processes in melanoma.

Solamargine triggers cellular necrosis selectively in different types of human melanoma cancer cells through extrinsic lysosomal mitochondrial death pathway.

BackgroundPrevious reports showed that the Steroidal Glycoalkaloid Solamargine inhibited proliferation of non-melanoma skin cancer cells. However, Solamargine was not tested systematically on different types of melanoma cells and was not simultaneously tested on normal cells either. In this study we aimed to investigate the effect of Solamargine and the mechanism involved in inhibiting the growth of different types of melanoma cells.MethodsSolamargine effect was tested on normal cells and on another three melanoma cell lines. Vertical growth phase metastatic and primary melanoma cell lines WM239 and WM115, respectively and the radial growth phase benign melanoma cells WM35 were used. The half inhibitory concentration IC50 of Solamargine was determined using Alamarblue assay. The cellular and subcellular changes were assessed using light and Transmission Electron Microscope, respectively. The percentage of cells undergoing apoptosis and necrosis were measured using Flow cytometry. The different protein expression was detected and measured using western blotting. The efficacy of Solamargine was determined by performing the clonogenic assay. The data collected was analyzed statistically on the means of the triplicate of at least three independent repeated experiments using one-way ANOVA test for parametric data and Kruskal–Wallis for non-parametric data. Differences were considered significant when the P values were less than 0.05.ResultsHereby, we demonstrate that Solamargine rapidly, selectively and effectively inhibited the growth of metastatic and primary melanoma cells WM239 and WM115 respectively, with minimum effect on normal and benign WM35 cells. Solamargine caused cellular necrosis to the two malignant melanoma cell lines (WM115, WM239), by rapid induction of lysosomal membrane permeabilization as confirmed by cathepsin B upregulation which triggered the extrinsic mitochondrial death pathway represented by the release of cytochrome c and upregulation of TNFR1. Solamargine disrupted the intrinsic apoptosis pathway as revealed by the down regulation of hILP/XIAP, resulting in caspase-3 cleavage, upregulation of Bcl-xL, and Bcl2, and down regulation of Apaf-1 and Bax in WM115 and WM239 cells only. Solamargine showed high efficacy in vitro particularly against the vertical growth phase melanoma cells.ConclusionOur findings suggest that Solamargine is a promising anti-malignant melanoma drug which warrants further attention.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0287-4) contains supplementary material, which is available to authorized users.

Elastic staining does not assist detection of venous invasion in cutaneous melanoma

The aim of the present study was to determine the benefit of orcein elastic staining of primary cutaneous melanoma specimens in detecting venous invasion. Primary cutaneous melanomas in vertical growth phase were assessed for vascular invasion. All tumour blocks were stained with haematoxylin and eosin (H&E) and orcein. The cases were reviewed by two pathologists. Vascular invasion was not identified more frequently on orcein stained slides than on H&E stained ones. Elastosis and periappendiceal elastic fibres interfered with vascular invasion detection with elastic staining. Based on our study, we conclude that elastic stains such as orcein do not improve the detection rate of venous invasion in primary cutaneous melanomas.

A Novel Fully Humanized 3D Skin Equivalent to Model Early Melanoma Invasion.

Metastatic melanoma remains incurable, emphasizing the acute need for improved research models to investigate the underlying biologic mechanisms mediating tumor invasion and metastasis, and to develop more effective targeted therapies to improve clinical outcome. Available animal models of melanoma do not accurately reflect human disease and current in vitro human skin equivalent models incorporating melanoma cells are not fully representative of the human skin microenvironment. We have developed a robust and reproducible, fully humanized three-dimensional (3D) skin equivalent comprising a stratified, terminally differentiated epidermis and a dermal compartment consisting of fibroblast-generated extracellular matrix. Melanoma cells incorporated into the epidermis were able to invade through the basement membrane and into the dermis, mirroring early tumor invasion in vivo. Comparison of our novel 3D melanoma skin equivalent with melanoma in situ and metastatic melanoma indicates that this model accurately recreates features of disease pathology, making it a physiologically representative model of early radial and vertical growth-phase melanoma invasion.

The BRAFV600E inhibitor, PLX4032, increases type I collagen synthesis in melanoma cells

Vertical growth phase (VGP) melanoma is frequently metastatic, a process mediated by changes in gene expression, which are directed by signal transduction pathways in the tumor cells. A prominent signaling pathway is the Ras-Raf-Mek-Erk MAPK pathway, which increases expression of genes that promote melanoma progression. Many melanomas harbor a mutation in this pathway, BRAFV600E, which constitutively activates MAPK signaling and expression of downstream target genes that facilitate tumor progression. In BRAFV600E melanoma, the small molecule inhibitor, vemurafenib (PLX4032), has revolutionized therapy for melanoma by inducing rapid tumor regression. This compound down-regulates the expression of many genes. However, in this study, we document that blocking the Ras-Raf-Mek-Erk MAPK pathway, either with an ERK (PLX4032) or a MEK (U1026) signaling inhibitor, in BRAFV600E human and murine melanoma cell lines increases collagen synthesis in vitro and collagen deposition in vivo. Since TGFß signaling is a major mediator of collagen synthesis, we examined whether blocking TGFß signaling with a small molecule inhibitor would block this increase in collagen. However, there was minimal reduction in collagen synthesis in response to blocking TGFß signaling, suggesting additional mechanism(s), which may include activation of the p38 MAPK pathway. Presently, it is unclear whether this increased collagen synthesis and deposition in melanomas represent a therapeutic benefit or an unwanted “off target” effect of inhibiting the Ras-Raf-Erk-Mek pathway.