Phenotype Of Melanoma Cells Research Articles

SOCS1 favors the epithelial-mesenchymal transition in melanoma, promotes tumor progression and prevents antitumor immunity by PD-L1 expression

Silencing of SOCS1 protein with shRNAi lentivirus (shR-SOCS1) led to partial reversion of the tumorigenic phenotype of B16F10-Nex2 melanoma cells. SOCS1 silencing inhibited cell migration and invasion as well as in vitro growth by cell cycle arrest at S phase with increased cell size and nuclei. Down-regulation of SOCS1 decreased the expression of epidermal growth factor receptor, Ins-Rα, and fibroblast growth factor receptors. The present work aimed at analyzing the SOCS1 cell signaling and expression of proteins relevant to tumor development. An RNA microarray analysis of B16F10-Nex2 melanoma cells with SOCS1 silenced by shRNAi-SOCS1 was undertaken in comparison with cells transduced with the empty vector. Among 609 differentially expressed genes, c-Kit, Met and EphA3 cytokine/tyrosine-kinase (TK) receptors were down regulated. A significant decrease in the expression of TK receptors, the phosphorylation of mediators of ERK1/2 and p38 pathways and STAT3 (S727) were observed. Subcutaneous immunization with shR-SOCS1-transduced viable tumor cells rendered protection against melanoma in a syngeneic model, with decreased expression of PD-L1 and of matrix metallo-proteinases (MMPs) and CD-10 in those cells. The present work shows the role of SOCS1 in murine melanoma development and the potential of SOCS1-silenced tumor cells in raising an effective anti-melanoma immune response.

Reprogramming Factors Remodel Melanoma Cell Phenotype by Changing Stat3 Expression.

The limited availability of melanoma stem cells is a major challenge for therapeutic reagent screening and study of molecular mechanisms. It has been shown that induced expression of four stem cell factors (Oct4, Sox2, Klf4, and c-Myc) changes the phenotype of osteosarcoma and breast cancer cells to osteosarcoma stem cells and breast cancer stem cells, respectively. The present study aimed to explore whether these four factors might change the phenotype of melanoma cells to melanoma stem cells and, if so, to examine the possible molecular signal involved. Melanoma B16-F10 cells were transfected with the plasmid TetO-FUW-OSKM which contains cDNA expressing four factors, driven by the Tet-On element. We found that expression of the four transcription factors was highly induced by DOX in the stable melanoma cell clones. Further studies confirmed that induced expression of these factors remodeled the phenotype of the melanoma cells to melanoma stem cells (MSCs). This conclusion was supported by the evidence that induced expression of these factors increased the numbers of tumor-initiating cells, (namely MSCs), both in an in vitro cell culture system and in a mouse in vivo model. The conclusion was further supported by the observation that the induction of these factors exclusively increased the mRNA of signal transducer and activator of transcription 3 which has been reported to play a crucial role in stem cell maintenance. Thus, phenotypic remodeling of melanoma cells following the induction of these four factors provided a simple and optimal means to constantly obtain MSCs for screening new therapeutic reagents. The result also reveals that Stat3 may be a crucial link between the induction of the four factors and the cell remodeling, suggesting its potential role as a target to fight melanoma.

Hmgb1 inhibits Klotho expression and malignant phenotype in melanoma cells by activating NF-κB.

The molecular and cellular mechanisms behind the involvement of inflammation in melanoma have not been fully elucidated. In this study, knockdown of Hmgb1 expression increased apoptosis, reduced invasion and p-NF-κB expression, but increased Klotho protein level in melanoma tumor cells. The effect of Hmgb1 knockdown was overcome by LPS. Introduction of exogenous Hmgb1 significantly decreased apoptosis, increased invasion, elevated p-NF-κB, but lowered Klotho protein level in melanoma cells. The effect of exogenous Hmgb1 was agonized by NF-κB inhibitor CAPE. Hmgb1 knockdown activated, but exogenous Hmgb1 inactivated, p-IGF1R/p-PI3K p-85/p-Akt/p-mTOR signaling. Knockdown of Klotho gene expression significantly decreased apoptosis, increased invasion in melanoma cells, and inhibited xenograft A375 tumor growth. A significantly high percentage of cells stained positive for p-NF-κB, but negative for Klotho, in melanoma tissues compared to normal and benign skin tissues. The positive p-NF-κB and negative Klotho protein expression correlated with poor prognosis in melanoma patients. Multivariate analysis revealed an independent association between p-NF-κB / Klotho protein level and overall survival. In conclusion, Hmgb1 can inhibit Klotho gene expression and malignant phenotype in melanoma cells through activation of NF-κB signaling.

Abstract 14: A switch in metabolic phenotype in vemurafenib-resistant melanoma cells to increased amino acid dependence serves as an alternate mechanism for survival

Abstract Despite considerable progress in the understanding of melanoma, and an increase in overall survival, resistance ultimately develops in most patients treated with BRAF inhibitors. To study the molecular mechanism(s) involved in acquired resistance, we generated a Vemurafenib-resistant A2-1b cell line in the BRAFV600E mutated SK-Mel28 melanoma cell background. Viability assays established IC50 of parental SK-Mel28 cells as 0.2 μM, while IC50>12 μM was noted for Vemurafenib-resistant A2-1b cells. Utilizing a quantitative proteomic strategy, profiling of A2-1b versus SK-Mel28 cells resulted in positive identification of 1720 proteins (≤1% FDR). Analysis of this data set identified a subset of 13 proteins, including Enolase 2, Triose-phosphate isomerase and Glyceraldehyde-3-phosphate dehydrogenase with a role in glycolysis, gluconeogenesis and NADH repair, to be differentially regulated in resistant cells. Additional studies validated significantly decreased Enolase 1 and 2 protein expression, suppressed glucose uptake and reduced lactic acid levels in resistant A2-1b cells. The pentose phosphate pathway, in contrast to citric acid cycle intermediates, was unaffected while the latter was significantly suppressed in resistant cells. The energy status of cells was similar as evidenced by equivalent total adenylate, guanylate, NADH, NADP+ and NADPH levels. Flux experiments measuring extracelluar acidification and oxygen consumption rates demonstrated that resistant A2-1b cells are unable to efficiently utilize intrinsic or exogenous fatty acids to meet energy demands. However, A2-1b cells maintained in media deprived of arginine and lysine exhibited increased basal and maximal respiration indicative of an energy reserve that relies on amino acid metabolism. Finally, metabolomics studies showing an overall increase in the absolute concentrations of gluconeogenic and ketogenic amino acids along with significantly elevated S6Kinase expression signify a shift from glucose to amino acids as the major energy source, in resistant A2-1b cells. Our data are consistent with a model of prolonged treatment response in which de-repression of ERK-MAPK pathway activity upon emergence of resistance to Vemurafenib is associated with adaptive changes in cellular metabolism. Sensitivity to BRAF inhibitors in the context of melanoma may not be defined by a reliance on glycolysis for survival, and that switch in metabolic phenotype can serve as an alternate mechanism for cell survival. Citation Format: Deeba N. Syed, Rahul K. Lall, Yong Wei Soon, Ahmed Aljohani, Changan Guo, Feng Liu, Frank L. Meyskens, James M. Ntambi, Hasan Mukhtar. A switch in metabolic phenotype in vemurafenib-resistant melanoma cells to increased amino acid dependence serves as an alternate mechanism for survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 14.

Abstract 927: Fibroblast-induced switching to the invasive phenotype and PI3K-mTOR signaling protects melanoma cells from BRAF inhibitors

Abstract Phenotypic heterogeneity of cancer cells can reason diversity in therapy responses within the same tumor, which might influence the overall efficacy of the treatment. Tumor stroma is an important contributor to intratumoral heterogeneity and can play a significant modulatory role in therapy response/resistance. Through studies on biological mechanisms of stroma-promoted resistance, novel targets could be identified for combination therapies aimed to eradicate both stroma-dependent and independent counterparts of the tumor. In this study we explore how the efficacy of the BRAF inhibitor (BRAFi) vemurafenib, a targeted agent commonly used against BRAF-mutant malignant melanoma, is modulated by stromal cells. By using multiple co-culture systems and experimental metastasis models, we showed that in the presence of lung fibroblasts, adjacent melanoma cells respond poorly to BRAFi. The protective influence of stroma was associated with stroma-induced changes in the melanoma cell phenotype, which was mapped by global gene expression and proteome analysis. We revealed that under the influence of fibroblasts, melanoma cells underwent a phenotype transition to the invasive, mesenchymal-like state characterized by down-regulation of melanocytic markers (MITF and its targets), up-regulation of receptor tyrosine kinases (RTKs)/RTK-linked signaling (like AXL or PDGFR activating down-stream PI3K) and elevation of extracellular-matrix fibronectin. We propose that these alterations allow melanoma cells to utilize alternative signaling pathways, like RTK-PI3K-mTOR instead of BRAF-driven MAPK, which reduces sensitivity to BRAFi. This scenario is further supported by the observations that: i) upon BRAFi treatment, stroma-protected melanoma maintained high levels of phospho-ribosomal protein S6 (pS6), a mTOR effector protein; ii) inhibition of mTOR or the upstream pathway PI3K together with BRAF eradicated pS6high subpopulations and enhanced the anti-proliferative effect in stroma-interacting melanoma; iii) the benefit of mTOR and BRAF co-inhibition was also seen in early-stage lung metastases in vivo. In conclusion, our findings signify the importance of stromal cells, specifically lung fibroblasts, in regulating melanoma cell phenotype and signaling, which impairs response to BRAFi. The stroma-induced invasive phenotype determinants that facilitate RTK-PI3K-mTOR signaling (e.g. AXL, PDGFR or fibronectin-binding integrins) could represent potential targets for overcoming stroma-mediated resistance to BRAFi. Currently, we are testing BRAFi in combination with several novel inhibitors of the identified RTKs and performing a cancer drug sensitivity screen to reveal the most effective drug combinations against stroma-interacting melanoma cells. Citation Format: Kotryna Seip, Karianne Giller Fleten, Anna Barkovskaya, Vigdis Nygaard, Mads Haugen Haugen, Birgit Øvstebø Engesæter, Gunhild Mari Mælandsmo, Lina Prasmickaite. Fibroblast-induced switching to the invasive phenotype and PI3K-mTOR signaling protects melanoma cells from BRAF inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 927.

LncRNA GAS5 is a critical regulator of metastasis phenotype of melanoma cells and inhibits tumor growth in vivo.

The present study intended to demonstrate the effects of long noncoding RNA growth arrest-specific transcript 5 (GAS5) on the migration and invasion of melanoma cells. We first detected the expression of GAS5 among four kinds of melanoma cell lines, followed by constructing GAS5-knocked down and overexpressed stable cells. Next, we evaluated the effects of GAS5 on cell migration and invasion using wound healing and gelatin zymography assays. Finally, melanoma cells with different GAS5 expression were injected into nude mice, and the tumor volumes were recorded and tumor tissues were analyzed after sacrificing the mice. This study systematically examined the function of GAS5 in mediating melanoma metastasis and revealed that GAS5 plays an anticancer role in melanoma via regulating gelatinase A and B, both in vitro and in vivo.

Phenotypic tumour cell plasticity as a resistance mechanism and therapeutic target in melanoma

Despite the recent success of MAPK and immune checkpoint inhibitors in advanced melanoma, intrinsic and acquired resistance mechanisms determine the efficacy of these therapeutic approaches. Therapy resistance in melanoma is not solely driven by genetic evolution, but also by epigenetically driven adaptive plasticity. Melanoma cells are shifting between different transcriptional programs, cell cycle states and differentiation phenotypes reflecting a highly dynamic potential to adapt to various exogenous stressors including immune attack or cancer therapies. This review will focus on the dynamic interconversion and overlap between different melanoma cell phenotypes in the context of therapy resistance and a dynamically changing multicellular microenvironment.

Enhanced OXPHOS, glutaminolysis and β-oxidation constitute the metastatic phenotype of melanoma cells.

Tumours display different cell populations with distinct metabolic phenotypes. Thus, subpopulations can adjust to different environments, particularly with regard to oxygen and nutrient availability. Our results indicate that progression to metastasis requires mitochondrial function. Our research, centered on cell lines that display increasing degrees of malignancy, focused on metabolic events, especially those involving mitochondria, which could reveal which stages are mechanistically associated with metastasis. Melanocytes were subjected to several cycles of adhesion impairment, producing stable cell lines exhibiting phenotypes representing a progression from non-tumorigenic to metastatic cells. Metastatic cells (4C11+) released the highest amounts of lactate, part of which was derived from glutamine catabolism. The 4C11+ cells also displayed an increased oxidative metabolism, accompanied by enhanced rates of oxygen consumption coupled to ATP synthesis. Enhanced mitochondrial function could not be explained by an increase in mitochondrial content or mitochondrial biogenesis. Furthermore, 4C11+ cells had a higher ATP content, and increased succinate oxidation (complex II activity) and fatty acid oxidation. In addition, 4C11+ cells exhibited a 2-fold increase in mitochondrial membrane potential (ΔΨmit). Consistently, functional assays showed that the migration of cells depended on glutaminase activity. Metabolomic analysis revealed that 4C11+ cells could be grouped as a subpopulation with a profile that was quite distinct from the other cells investigated in the present study. The results presented here have centred on how the multiple metabolic inputs of tumour cells may converge to compose the so-called metastatic phenotype.

Directed Dedifferentiation Using Partial Reprogramming Induces Invasive Phenotype in Melanoma Cells.

The combination of cancer-focused studies and research related to nuclear reprogramming has gained increasing importance since both processes-reprogramming towards pluripotency and malignant transformation-share essential features. Studies have revealed that incomplete reprogramming of somatic cells leads to malignant transformation indicating that epigenetic regulation associated with iPSC generation can drive cancer development [J Mol Cell Biol 2011;341-350; Cell 2012;151:1617-1632; Cell 2014;156:663-677]. However, so far it is unclear whether incomplete reprogramming also affects cancer cells and their function. In the context of melanoma, dedifferentiation correlates to therapy resistance in mouse studies and has been documented in melanoma patients [Nature 2012;490:412-416; Clin Cancer Res 2014;20:2498-2499]. Therefore, we sought to investigate directed dedifferentiation using incomplete reprogramming of melanoma cells. Using a murine model we investigated the effects of partial reprogramming on the cellular plasticity of melanoma cells. We demonstrate for the first time that induced partial reprogramming results in a reversible phenotype switch in melanoma cells. Partially reprogrammed cells at day 12 after transgene induction display elevated invasive potential in vitro and increased lung colonization in vivo. Additionally, using global gene expression analysis of partially reprogrammed cells, we identified SNAI3 as a novel invasion-related marker in human melanoma. SNAI3 expression correlates with tumor thickness in primary melanomas and thus, may be of prognostic value. In summary, we show that investigating intermediate states during the process of reprogramming melanoma cells can reveal novel insights into the pathogenesis of melanoma progression. We propose that deeper analysis of partially reprogrammed melanoma cells may contribute to identification of yet unknown signaling pathways that can drive melanoma progression.

Genomic profiling of invasive melanoma cell lines by array comparative genomic hybridization.

Malignant melanoma is one of the most aggressive human cancers. Invasion of cells is the first step in metastasis, resulting in cell migration through tissue compartments. We aimed to evaluate genomic alterations specifically associated with the invasive characteristics of melanoma cells. Matrigel invasion assays were used to determine the invasive properties of cell lines that originated from primary melanomas. Array comparative genomic hybridization analyses were carried out to define the chromosome copy number alterations (CNAs). Several recurrent CNAs were identified by array comparative genomic hybridization that affected melanoma-related genes. Invasive primary cell lines showed high frequencies of CNAs, including the loss of 7q and gain of 12q chromosomal regions targeting PTPN12, ADAM22, FZD1, TFPI2, GNG11, COL1A2, SMURF1, VGF, RELN and GLIPR1 genes. Gain of the GDNF (5p13.1), GPAA1, PLEC and SHARPIN (8q24.3) genes was significantly more frequent in invasive cell lines compared with the noninvasive ones. Importantly, copy number gains of these genes were also found in cell lines that originated from metastases, suggesting their role in melanoma metastasis formation. The present study describes genomic differences between invasive and noninvasive melanoma cell lines that may contribute toward the aggressive phenotype of human melanoma cells.

Abstract A146: The role of SOCS-1 in stimulating melanoma development through tyrosine-kinase receptors and MAPK pathways and prevents antitumour immunity by PD-L1 expression

Abstract Knowledge on the regulatory pathways involved in melanoma development and progression has advanced significantly in recent years. It is now recognized that multiple signaling pathways that affect motility, growth control, invasion, metabolism, cytokine release and the ability to escape the immune response, regulate melanoma development. Silencing of SOCS-1 protein, a negative regulator of Jak/Stat pathway, leads to partial reversion of the tumorigenic phenotype of B16F10-Nex2 melanoma cells. SOCS-1 silencing with shRNAi lentivirus (shR-SOCS-1) inhibits in vitro growth by cell cycle regulation with S phase arrest and increased size of cells and nuclei. It also inhibited tumor cell migration and invasion. Down-regulation of SOCS-1 decreased the expression of epidermal growth factor receptor (mainly the phosphorylated-R), Ins-Rα, and fibroblast growth factor receptors. Based on the results of SOCS-1 silencing, the present work aims at analyzing the expression of proteins relevant to tumor development, searching for signaling pathways related with SOCS-1. A protein microarray analysis of murine melanoma cells silenced for SOCS-1 by shRNAi-SOCS-1 lentivirus transduction was undertaken using as controls, cells transduced with the empty vector. Among 614 differentially expressed genes, c-kit, met and EphA3 cytokine/tyrosine-kinase (TK) receptors were downregulated. A significant decrease in the expression of TK receptors as observed by Western blotting of B16 shR-SOCS-1 extracts showed that SOCS-1 positively regulates the MAPK signaling pathways in these cells. A decrease in the phosphorylation of mediators of ERK1/2 and p38 pathways and of STAT3 (S727) was observed. These results demonstrate that SOCS-1 plays an important role in tumor development and progression up-regulating a number of growth factor receptors in murine melanoma cells. Interestingly, p-CREB (S133) and total AP-2 alpha were up-regulated in the SOCS-1-deficient B16F10-Nex2 cells. In vivo experiments using a syngeneic model and a prophylactic protocol showed that the subcutaneous immunization with shR-SOCS-1-transduced viable tumor cells, rendered protection against either subcutaneously grafted or metastatic murine melanoma cells, with significant reduction in tumor growth and lung metastatic nodules. Such immunization increased the expression of CD11c, CD80, CD86 and MHC-II in splenic dendritic cells, as compared to DCs from a group that received only B16F10-Nex2 cells. Of notice was the decreased expression of PD-L1 in SOCS-1-silenced B16F10-Nex2 cells (B16shR-SOCS-1). Such molecular phenotype may have a role in the immune response and protective effect of subcutaneous immunization with these cells, rather than with wild-type B16F10-Nex2 cells. By overexpressing PD-L1, WT-melanoma cells negatively regulates T-cell proliferation in the tumor environment. Down regulation of PD-L1 is compatible with inactive ERK. The present results show the important role of SOCS-1 in murine melanoma development and the potential of SOCS-1-silenced tumor cells in raising an effective anti-tumor immune response, inducing protection even against a metastatic melanoma challenge. Funded by FAPESP 2010/51423-0 and 2012/17473-6. Citation Format: Rodrigo Berzaghi, Esq., Vera SC Maia, II, Felipe V. Pereira, III, Filipe Manegatti De Melo, Luiz R. Travassos, Luiz R. Travassos. The role of SOCS-1 in stimulating melanoma development through tyrosine-kinase receptors and MAPK pathways and prevents antitumour immunity by PD-L1 expression. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A146.

Tribbles breaking bad: TRIB2 suppresses FOXO and acts as an oncogenic protein in melanoma.

TRIB2 (tribbles homolog 2) encodes one of three members of the tribbles family in mammals. These members share a Trb (tribbles) domain, which is homologous to protein serine-threonine kinases, but lack the active site lysine. The tribbles proteins interact and modulate the activity of signal transduction pathways in a number of physiological and pathological processes. TRIB2 has been identified as an oncogene that inactivates the transcription factor CCAAT/enhancer-binding protein α (C/EBPα) and causes acute myelogenous leukaemia (AML). Recent research provided compelling evidence that TRIB2 can also act as oncogenic driver in solid tumours, such as lung and liver cancer. In particular, our recent work demonstrated that TRIB2 is dramatically overexpressed in malignant melanomas compared with normal skin and promotes the malignant phenotype of melanoma cells via the down-regulation of FOXO (forkhead box protein O) tumour suppressor activity invitro and invivo. TRIB2 was found to be expressed in normal skin, but its expression consistently increased in benign nevi, melanoma and was highest in samples from patients with malignant melanoma. The observation that TRIB2 strongly correlates with the progression of melanocyte-derived malignancies suggests TRIB2 as a meaningful biomarker to both diagnose and stage melanoma. In addition, interfering with TRIB2 activity might be a therapeutic strategy for the treatment of several different tumour types.

The CASC15 Long Intergenic Noncoding RNA Locus Is Involved in Melanoma Progression and Phenotype Switching

In recent years, considerable advances have been made in the characterization of protein-coding alterations involved in the pathogenesis of melanoma. However, despite their growing implication in cancer, little is known about the role of long non-coding RNAs in melanoma progression. We hypothesized that copy number alterations of intergenic non-protein coding domains could help identify long intergenic non-coding RNAs (lincRNAs) associated with metastatic cutaneous melanoma. Among several candidates, our approach uncovered the chromosome 6p22.3 CASC15 lincRNA locus as a frequently gained genomic segment in metastatic melanoma tumors and cell lines. The locus was actively transcribed in metastatic melanoma cells, and up-regulation of CASC15 expression was associated with metastatic progression to brain metastasis in a mouse xenograft model. In clinical specimens, CASC15 levels increased during melanoma progression and were independent predictors of disease recurrence in a cohort of 141 patients with AJCC stage III lymph node metastasis. Moreover, siRNA knockdown experiments revealed that CASC15 regulates melanoma cell phenotype switching between proliferative and invasive states. Accordingly, CASC15 levels correlated with known gene signatures corresponding to melanoma proliferative and invasive phenotypes. These findings support a key role for CASC15 in metastatic melanoma.

Abstract 3129: miR-340 is a modulator of oncogenic signaling in melanoma

Abstract Melanoma is responsible for only 4% of skin cancer diagnoses; nonetheless 80% of skin cancer deaths are attributed to this aggressive skin cancer each year. Growing evidence points to an important role miRNAs may play in the aberrant cellular signaling of cancers, including melanoma. microRNAs (miRNAs) are highly conserved 22-25nt non-coding RNAs involved in regulating RNA stability and translation and are predicted to regulate ∼60% of the human transcriptome. We are investigating the tumor suppressor role miR-340 plays in the development and progression of melanoma. A limited number of miR-340 targets were previously characterized, however, it is unlikely the effects of miR-340 on the melanoma phenotype are the result of directly targeting a few mRNAs, as miRNAs are likely to target upwards of 100 mRNAs each. Using an in silico target identification and pathway mapping system, we identified a multitude of potential miR-340 mRNA targets that encode proteins distributed amongst a variety of cellular signaling pathways. In silico analysis revealed a high proportion of miR-340 predicted mRNA targets that encode for members of the MAPK signaling pathway. Our results show that miR-340 modulated most of the assayed mRNAs that encode MAPK components. We also demonstrate that miR-340 inhibits the tumorigenic phenotype of melanoma cells. We recently developed a novel method to identify direct targets of miRNAs. Using this method, we demonstrate miR-340 directly targets mRNAs that encode protein kinases of the MAPK pathway. Further direct target identification is crucial toward understanding the role miR-340-directed regulation plays in triggering pleiotropic effects during melanoma development and progression. Elucidating the roles aberrant gene regulatory networks play in melanoma is critical for understanding the disease and developing treatment options to improve the melanoma prognosis. Supported by NIH grants ES007015 (to A.M.P.S.), AR063361 (to V.S.S.) and CA191550 (to V.S.S.). Citation Format: Ashley M. Poenitzsch Strong, Emily G. Adochio, Vijayasaradhi Setaluri, Vladimir S. Spiegelman. miR-340 is a modulator of oncogenic signaling in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3129. doi:10.1158/1538-7445.AM2015-3129

Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion

Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells.

Cyclin-dependent kinase 2 (CDK2) is a key mediator for EGF-induced cell transformation mediated through the ELK4/c-Fos signaling pathway.

Cyclin dependent kinase 2 (CDK2) is a known regulator in the cell cycle control of the G1/S and S/G2 transitions. However, the role of CDK2 in tumorigenesis is controversial. Evidence from knockout mice as well as colon cancer cell lines indicated that CDK2 is dispensable for cell proliferation. In this study, we found that ectopic CDK2 enhances Ras (G12V)-induced foci formation and knocking down CDK2 expression dramatically decreases EGF-induced cell transformation mediated through the down-regulation of c-fos expression. Interestingly, CDK2 directly phosphorylates ELK4 at Thr194 and Ser387 and regulates ELK4 transcriptional activity, which serves as a mechanism to regulate c-fos expression. In addition, ELK4 is over-expressed in melanoma and knocking down ELK4 or CDK2 expression significantly attenuated the malignant phenotype of melanoma cells. Taken together, our study reveals a novel function of CDK2 in EGF-induced cell transformation and the associated signal transduction pathways. This indicates that CDK2 is a useful molecular target for chemoprevention and therapy against skin cancer.

Interleukin-8 is a key mediator of FKBP51-induced melanoma growth, angiogenesis and metastasis.

Background:FKBP51 is overexpressed in melanoma and impacts tumour cell properties. However, its comprehensive role in melanoma pathogenesis and underlying mechanism(s) remain elusive.Methods:FKBP51 was stably silenced in aggressive melanoma cell lines and its effect examined in vitro and in mouse model. Histological/immunohistochemical analyses were performed to confirm metastasis, angiogenesis and neutrophil infiltration. Gene expression was analyzed by qRT–PCR, immunoblot and/or ELISA. NF-κB transcriptional activity and promoter binding were monitored by luciferase-based promoter-reporter and ChIP assays, respectively. Interleukin (IL)-8 inhibition was achieved by gene silencing or neutralising-antibody treatment.Results:FKBP51 silencing reduced melanoma growth, metastasis, angiogenesis and neutrophil infiltration and led to IL-8 downregulation through NF-κB suppression in cell lines and tumour xenografts. IL-8 inhibition drastically decreased growth, migration and invasiveness of FKPB51-overexpressing cells; whereas its treatment partially restored the suppressed phenotypes of FKBP51-silenced melanoma cells. Interleukin-8 depletion in conditioned medium (CM) of FKBP51-overexpressing melanoma cells inhibited endothelial cell proliferation and capillary-like structure formation, whereas its treatment promoted these effects in endothelial cells cultured in CM of FKBP51-silenced melanoma cells.Conclusions:FKBP51 promotes melanoma growth, metastasis and angiogenesis, and IL-8 plays a key role in these processes. Thus, targeting of FKBP51 or its upstream or downstream regulatory pathways could lead to effective therapeutic strategies against melanoma.

MicroRNA dysregulation in uveal melanoma: a new player enters the game.

Uveal melanoma is the second most common form of melanoma and a predominant intraocular malignant tumor in adults. The development of uveal melanoma is a multistep process involving genetic and epigenetic alteration of proto-oncogenes and tumor-suppressor genes. Recent discoveries have shed a new light on the involvement of a class of noncoding RNA known as microRNAs (miRNAs) in uveal melanoma. A lot of miRNAs show differential expressions in uveal melanoma tissues and cell lines. Genes coding for these miRNAs have been characterized as novel oncogene and tumor-suppressor genes based on findings that these miRNAs control malignant phenotypes of uveal melanoma cells. Several studies have confirmed that dysregulation of miRNAs promotes cell-cycle progression, confers resistance to apoptosis, and enhances invasiveness and metastasis. Moreover, several miRNAs have also been shown to correlate with uveal melanoma initiation and progression, and thus may be used as biomarkers for early diagnosis and prognosis. Elucidating the biological aspects of miRNA dysregulation may help us better understand the pathogenesis of uveal melanoma and promote the development of miRNA directed-therapeutics against this disease.

Anti-tumor properties of cis-resveratrol methylated analogs in metastatic mouse melanoma cells.

Resveratrol (E-3,5,4'-trihydroxystilbene) is a polyphenol found in red wine that has been shown to have multiple anti-cancer properties. Although cis-(Z)- and trans-(E)-isomers of resveratrol occur in nature, the cis form is not biologically active. However, methylation at key positions of the cis form results in more potent anti-cancer properties. This study determined that synthetic cis-polymethoxystilbenes (methylated analogs of cis-resveratrol) inhibited cancer-related phenotypes of metastatic B16 F10 and non-metastatic B16 F1 mouse melanoma cells. In contrast with cis- or trans-resveratrol and trans-polymethoxystilbene which were ineffective at 10μM, cis-polymethoxystilbenes inhibited motility and proliferation of melanoma cells with low micromolar specificity (IC50<10μM). Inhibitory effects by cis-polymethoxystilbenes were significantly stronger with B16 F10 cells and were accompanied by decreased expression of β-tubulin and pleckstrin homology domain-interacting protein, a marker of metastatic B16 cells. Thus, cis-polymethoxystilbenes have potential as chemotherapeutic agents for metastatic melanoma.

Cilengitide downmodulates invasiveness and vasculogenic mimicry of neuropilin 1 expressing melanoma cells through the inhibition of αvβ5 integrin.

During melanoma progression, tumour cells show increased adhesiveness to the vascular wall, invade the extracellular matrix (ECM) and frequently form functional channels similar to vascular vessels (vasculogenic mimicry). These properties are mainly mediated by the interaction of integrins with ECM components. Since we had previously identified neuropilin 1 (NRP-1), a coreceptor of vascular endothelial growth factor A (VEGF-A), as an important determinant of melanoma aggressiveness, aims of this study were to identify the specific integrins involved in the highly invasive phenotype of NRP-1 expressing cells and to investigate their role as targets to counteract melanoma progression. Melanoma aggressiveness was evaluated in vitro as cell ability to migrate through an ECM layer and to form tubule-like structures using transfected cells. Integrins relevant to these processes were identified using specific blocking antibodies. The αvβ5 integrin was found to be responsible for about 80% of the capability of NRP-1 expressing cells to adhere on vitronectin. In these cells αvβ5 expression level was twice higher than in low-invasive control cells and contributed to the ability of melanoma cells to form tubule-like structures on matrigel. Cilengitide, a potent inhibitor of αν integrins activation, reduced ECM invasion, vasculogenic mimicry and secretion of VEGF-A and metalloproteinase 9 by melanoma cells. In conclusion, we demonstrated that ανβ5 integrin is involved in the highly aggressive phenotype of melanoma cells expressing NRP-1. Moreover, we identified a novel mechanism that contributes to the antimelanoma activity of the αv integrin inhibitor cilengitide based on the inhibition of vasculogenic mimicry.