Regulation Of Melanoma Progression Research Articles

CD133-Dependent Activation of Phosphoinositide 3-Kinase /AKT/Mammalian Target of Rapamycin Signaling in Melanoma Progression and Drug Resistance.

Melanoma frequently harbors genetic alterations in key molecules leading to the aberrant activation of PI3K and its downstream pathways. Although the role of PI3K/AKT/mTOR in melanoma progression and drug resistance is well documented, targeting the PI3K/AKT/mTOR pathway showed less efficiency in clinical trials than might have been expected, since the suppression of the PI3K/mTOR signaling pathway-induced feedback loops is mostly associated with the activation of compensatory pathways such as MAPK/MEK/ERK. Consequently, the development of intrinsic and acquired resistance can occur. As a solid tumor, melanoma is notorious for its heterogeneity. This can be expressed in the form of genetically divergent subpopulations including a small fraction of cancer stem-like cells (CSCs) and non-cancer stem cells (non-CSCs) that make the most of the tumor mass. Like other CSCs, melanoma stem-like cells (MSCs) are characterized by their unique cell surface proteins/stemness markers and aberrant signaling pathways. In addition to its function as a robust marker for stemness properties, CD133 is crucial for the maintenance of stemness properties and drug resistance. Herein, the role of CD133-dependent activation of PI3K/mTOR in the regulation of melanoma progression, drug resistance, and recurrence is reviewed.

P32 promotes melanoma progression and metastasis by targeting EMT markers, Akt/PKB pathway, and tumor microenvironment

Melanoma originates from melanin-producing cells called melanocytes. Melanoma poses a great risk because of its rapid ability to spread and invade new organs. Cellular metastasis involves alteration in the gene expression profile and their transformation from epithelial to mesenchymal state. Despite of several advances, metastatic melanoma being a key cause of therapy failure and mortality remains poorly understood. p32 has been found to be involved in various physiological and pathophysiological conditions. However, the role of p32 in melanoma progression and metastasis remains underexplored. Here, we identify the role of p32 in the malignancy of both murine and human melanoma. p32 knockdown leads to reduced cell proliferation, migration, and invasion in murine and human melanoma cells. Furthermore, p32 promotes in vitro tumorigenesis, inducing oncogenes and EMT markers. Mechanistically, we show p32 regulates tumorigenic and metastatic properties through the Akt/PKB signaling pathway in both murine and human melanoma. Furthermore, p32 silencing attenuates melanoma tumor progression and lung metastasis in vivo, modulating the tumor microenvironment by inhibiting the angiogenesis, infiltration of macrophages, and leukocytes in mice. Taken together, our findings identify that p32 drives melanoma progression, metastasis, and regulates the tumor microenvironment. p32 can be a target of a novel therapeutic approach in the regulation of melanoma progression and metastasis.

Abstract 768: miR-378a-5p acts as a positive regulator of melanoma progression

Abstract Melanoma, the most aggressive form of skin cancer, is frequently associated with alterations in many genes, among which the Bcl-2 oncogene plays an important role in survival, progression, chemosensitivity and angiogenesis. Also microRNAs play an important role in melanoma development and progression affecting tumor proliferation, migration and invasion. HUVEC and a panel of human melanoma cells were used. Western Blot, qRT-PCR, miRNA Microarray, ELISA, vasculogenic mimicry, cell proliferation, clonogenic and invasion assays have been performed. MiR-378a-5p mimic and inhibitor have been used to study the biological and functional role of miR-378a-5p. In silico analysis have been carried out to find putative miRs targets using miRWalk, Diana Tools and Target Scan. To identify putative miRNAs, whose expression could be modulated by Bcl-2, M14 have been transfected with a smart-pool RNA targeting human Bcl-2 mRNA, and a miRNA Microarray has been performed. Four independent experiments indicate that, after Bcl-2 silencing, 13 miRs were significantly downregulated. Among these, miR-378a-5p, has been identified as significantly deregulated in different human melanoma cell lines. Based on the target prediction analysis, we identified and then confirmed, through qRT-PCR, several miR-378a-5p target genes, such as SUFU, STAMBP and KLF9. By performing in vitro experiments, we found that ectopic expression of miR-378a-5p does not significantly affect cell proliferation and clonogenic ability of melanoma cells, while it significantly increases invasion and the expression of MMP2 at protein level. It also facilitates the ability of tumor cells to form de novo vasculogenic structures. We have also evidences that conditioned medium from miR-378a-5p overexpressing melanoma increases the formation of capillary like structures in endothelial cells. To assess the molecular mechanism through which miR-378a-5p induce angiogenesis, ELISA showed that ectopic expression of miR-378a-5p significantly increases VEGF protein secretion, but not IL-8 mRNA expression. Experiments using an antiVEGF neutralizing antibody indicate VEGF implication on miR-378a-5p-induced vasculogenic mimicry. To identify other factors that may be implicated in miR-378a-5p proangiogenic/proinvasive functions, we focused our attention on Sp1/uPAR axis, a pathway we previously demonstrated to be regulated in cancer cells by Bcl-2 under hypoxic conditions. Our experiments indicate that ectopic expression of miR-378a-5p increases uPAR mRNA expression. Surprisingly, this treatment results in a Sp1 reduction, indicating that other transcription factor, other than Sp1, can be responsible for uPAR induction by miR-378a-5p. Based on these evidences, although a deeper understanding of the molecular mechanism involved in Bcl-2 modulation of miR-378a-5p is needed, our findings strongly suggest a proangiogenic and proinvasive role of miR-378a-5p in melanoma cells. Note: This abstract was not presented at the meeting. Citation Format: Maria Grazia Tupone, Marta Di Martile, Simona D'Aguanno, Elisabetta Valentini, Marianna Desideri, Sara Donzelli, Andrea Sacconi, Daniela Trisciuoglio, Giovanni Blandino, Donatella Del Bufalo. miR-378a-5p acts as a positive regulator of melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 768.

Abstract 1070: miR-579-3p is a novel master regulator of melanoma progression and drug resistance in metastatic melanoma

Abstract Introduction: Malignant melanoma is the most aggressive form of skin cancer presenting in approximately 50% of cases activating mutations in the BRAF oncogene. For these patients new therapeutic options are represented by the combination of BRAF inhibitors and MEK-inhibitors. However, a major limitation is the occurrence of resistance related in the majority of cases to mutations causing either reactivation of the MAPK/ERK pathway or of the PI3K/PTEN/AKT pathway. This complex scenario is worsened by the absence, in a significant proportion of cases, of new mutations. This suggests the involvement also of epigenetic or post-transcriptional changes at the basis of resistance. We have focused our attention over the past years to adaptive changes occurring in melanoma cells upon exposure to BRAF and/or MEK inhibitors in order to identify new therapeutic targets. Here we hypothesize that miRNAs deregulation upon cell exposure to kinase inhibitors may contribute to the development of drug resistance. Experimental procedures: The involvement of specific miRNAs in the establishment of drug resistance in melanoma was investigated through the online software miRò while target predictions were performed with TargetScan and microrna.org. Impact on melanoma cell growth, migration and establishment of drug resistance was assessed through enforced-transient miR overexpression. Tumor biopsies were obtained from patients before therapy with kinase inhibitors or after relapse and used for RNA extraction. Results: We identified by bioinformatics analysis a novel miRNA, miR-579-3p, relevant for BRAF mutated melanoma progression and found that it targets both BRAF, the relevant oncoprotein in these melanomas, and MDM2. miR-579-3p expression levels are high in nevi, heavily decline in stage III-IV melanomas and further decrease in melanomacells selected in vitro for resistance to BRAF and/or MEK inhibitors. Through in vitro studies we obtained evidence that mir-579-3p is able to inhibit melanoma cell growth and migration and to induce apoptosis alone or in combination with BRAF and/or MEKi. Moreover we found that miR-579-3p overexpression impairs the establishment of resistance to BRAFi in human melanoma cells. Finally we found this miR to be down-regulated in tumor samples obtained from patients who developed resistance to target therapies and its expression to be inversely correlated to the expression levels of its target genes, BRAF and MDM2. Conclusions: Our results strongly demonstrate that miR-579-3p is a novel oncosuppressor miRNA, controlling melanoma progression and development of drug resistance and which could be used as a new therapeutic target for intervention. Citation Format: Luigi Fattore, Mario Acunzo, Giulia Romano, Alessandro Laganà, Debora Malpicci, Maria Elena Pisanu, Antonio Maria Grimaldi, Franco Fulciniti, Carlo Maria Croce, Rita Mancini, Paolo Antonio Ascierto, Gennaro Ciliberto. miR-579-3p is a novel master regulator of melanoma progression and drug resistance in metastatic melanoma. [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 1070.

Dual, enzymatic and non-enzymatic, function of ecto-5'-nucleotidase (eN, CD73) in migration and invasion of A375 melanoma cells.

Ecto-5'-nucleotidase (eN, CD73) mediates extracellular adenosine production from 5'-AMP. Non-enzymatic functions of eN have also been reported. The aim of the study was to investigate the role of ecto-5'-nucleotidase in aggressive melanoma behaviour. Analysis of the involvement of eN in adhesion, migration and invasion revealed eN functions unknown to date. We found that following eN blockade by concanavalin A, the strength of adhesion to different ECM proteins was not altered, but at the same time the invasion ability of the cells was decreased. On the other hand, knocking down eN in melanoma cells did not influence cell invasion but abolished their migration on tenascin C (TnC). Ecto-5'-nucleotidase seems to fulfil a more distinct role as a receptor than as an enzyme in the cell interaction and mobility on TnC. Ecto-5'-nucleotidase activates also focal adhesion kinase and enhances the formation of complexes upon cell adhesion to TnC. All these observations prove that an eN-TnC complex is involved in cell migration and invasion and thus in the regulation of melanoma progression.

Abstract 314: The interaction between chronic alcohol consumption and melanoma skews the iNKT cell cytokine profile towards IL-4 dominance

Abstract Despite the growing evidence that alcohol consumption is associated with the incidence of many different types of cancer, there is little research regarding the effect of continued alcohol consumption on growth, progression and subsequent survival of cancer patients. Using a well-established mouse model we previously found that chronic alcohol consumption impairs the anti-melanoma functions of CD8+T cells and NK cells, and this leads to decreased survival of melanoma-bearing mice. The underlying mechanism for these effects is not known. Herein we examined the relationship of NKT cells to regulation of anti-tumor responses in alcohol consuming mice since NKT cells play important roles in the regulation of the anti-tumor immune response. The phenotype and function of invariant (i)NKT cells were examined in female B16BL6-bearing C57BL6 mice given 20% alcohol for three months. We found that alcohol consumption increased iNKT cells in the liver of mice not inoculated with melanoma, but did not alter iNKT cells in other organs. iNKT cells were dramatically increased in the blood and liver of chronic alcohol consuming, melanoma-bearing mice; however, these cells were not increased in the spleen or draining lymph nodes. Immature NK1.1- iNKT cells decreased 2-4 fold in the blood, spleen and liver of the alcohol-consuming, melanoma-bearing mice, suggesting that alcohol interacting with the melanoma tumors stimulates iNKT cells maturation. Upon activation, fewer iNKT cells produced IFN-gamma in these mice compared to water-drinking, melanoma-bearing mice. The ratio of IL-4:IFN-gamma-producing iNKT cells increased 2-fold as a result of the alcohol/tumor interaction, thus skewing the cytokine profile toward Th2 dominance. The Th2 dominant cytokine profile could negatively regulate the anti-tumor activities of CD8+ T cell and NK cells as well as increase myeloid derived suppressor cells, which we previously reported. Collectively, these data support an important role for iNKT cells in regulation of melanoma progression. (Supported by NIH grants R01AA07293 and K05AA017149, and WSU Alcohol and Drug Abuse Research Program grant 13B-2957-1246). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 314. doi:1538-7445.AM2012-314

Semaphorin 3A Suppresses Tumor Growth and Metastasis in Mice Melanoma Model

BackgroundRecent understanding on cancer therapy indicated that targeting metastatic signature or angiogenic switch could be a promising and rational approach to combat cancer. Advancement in cancer research has demonstrated the potential role of various tumor suppressor proteins in inhibition of cancer progression. Current studies have shown that axonal sprouting inhibitor, semaphorin 3A (Sema 3A) acts as a potent suppressor of tumor angiogenesis in various cancer models. However, the function of Sema 3A in regulation of melanoma progression is not well studied, and yet to be the subject of intense investigation.Methodology/Principal FindingsIn this study, using multiple in vitro and in vivo approaches we have demonstrated that Sema 3A acts as a potent tumor suppressor in vitro and in vivo mice (C57BL/6) models. Mouse melanoma (B16F10) cells overexpressed with Sema 3A resulted in significant inhibition of cell motility, invasiveness and proliferation as well as suppression of in vivo tumor growth, angiogenesis and metastasis in mice models. Moreover, we have observed that Sema 3A overexpressed melanoma clone showed increased sensitivity towards curcumin and Dacarbazine, anti-cancer agents.ConclusionsOur results demonstrate, at least in part, the functional approach underlying Sema 3A mediated inhibition of tumorigenesis and angiogenesis and a clear understanding of such a process may facilitate the development of novel therapeutic strategy for the treatment of cancer.

Abstract 1457: Raf kinase inhibitor protein (RKIP) negatively regulates melanoma differentiation associated gene-9/syntenin-mediated melanoma metastasis

Abstract Melanoma differentiation associated gene-9 (mda-9/syntenin) functions as a positive regulator of melanoma progression and metastasis by physically interacting with c-Src and promoting the formation of an active FAK/c-Src signaling complex leading to NFκB and matrix metalloproteinase (MMP) activation. In contrast, Raf Kinase Inhibitor Protein (RKIP), an inhibitor of RAF-mediated activation of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEKK), is strongly downregulated in metastatic melanoma cells suggesting a possible inverse relationship between expression of mda-9/syntenin and RKIP. We have now genetically modulated expression of mda-9/syntenin and RKIP in melanocytes and melanoma cells and evaluated biological consequences using invasion, anchorage-independent growth, Western blotting, immunoprecipitation and in vivo confocal imaging assays. These studies confirm an inverse correlation between mda-9/syntenin and RKIP expression. This reciprocal relationship was also documented in vivo using a chick embryo chorioallantoic membrane (CAM) model, which monitors spontaneous intravasation of cells from the upper to the lower layer by polymerase chain reaction (PCR) amplification of human Alu genomic DNA. Using tissue microarrays and a series of cell lines we confirmed a higher expression of MDA-9/syntenin and absence of RKIP in both malignant melanoma tumor sections and cell lines. We also established through gain-of-function and loss-of-function experiments that mda-9/syntenin transcriptionally downregulates RKIP, providing evidence for crosstalk between these two proteins. Moreover, transient overexpression of RKIP in metastatic melanoma cells overrides mda-9/syntenin-mediated signaling resulting in decreased cell invasion, anchorage-independent growth and spontaneous tumor cell dissemination in the CAM assay. RKIP through its RAF1 binding site (amino acids 77-108) physically interacts with MDA-9/syntenin and this interaction inversely correlates with downregulation of FAK and c-Src phosphorylation, crucial steps necessary for forming a stable FAK/c-Src complex and initiating a signaling cascade leading to an MDA-9/syntenin-mediated metastatic phenotype in melanoma cells. These provocative findings highlight a potential therapeutic role of RKIP in melanoma metastasis through its interaction with MDA-9/syntenin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1457. doi:10.1158/1538-7445.AM2011-1457

Tumour-associated macrophage infiltration, neovascularization and aggressiveness in malignant melanoma: role of monocyte chemotactic protein-1 and vascular endothelial growth factor-A

The objective of this study was to evaluate the role of tumour-associated macrophages (TAMs) in malignant melanoma progression, invasion and angiogenesis. We examined the levels of macrophage infiltration and monocyte chemotactic protein-1 (MCP-1), neovascularization and vascular endothelial growth factor-A (VEGF-A) in different Clark's level melanomas with varying thicknesses and metastases. The level of TAM density was significantly higher in thick (>0.75 mm) than thin (<or=0.75 mm) melanomas, and positively correlated with melanoma invasiveness and metastasis. In contrast, MCP-1 expression was significantly lower in thick (>0.75 mm) than thin (<or=0.75 mm) melanomas and negatively correlated with melanoma aggressiveness and invasion. We did not observe any significant difference in the levels of neovascularization between thin and thick melanomas, and no correlation with VEGF-A expression, TAM density or melanoma aggressiveness and invasion. Interestingly, levels of VEGF-A were significantly higher in metastatic melanoma than in thick melanoma. In addition, we observed lower levels of MCP-1 messenger RNA (mRNA) expression in more aggressive melanomas and in cell lines with higher metastatic potential. In summary, our data suggest a distinct pattern of TAM infiltration, MCP-1 expression, neovascularization and VEGF-A expression during human melanoma progression, and a complex interaction between TAMs and melanoma cells in the regulation of melanoma progression, angiogenesis and metastasis.

HGF induces fibronectin matrix synthesis in melanoma cells through MAP kinase-dependent signaling pathway and induction of Egr-1

The matrix fibronectin protein is a multifunctional adhesive molecule that promotes migration and invasiveness of many tumors including melanomas. Increased fibronectin synthesis has been associated with the metastatic potential of melanoma cells; however, the molecular mechanisms underlying fibronectin overexpression during melanoma development are poorly understood. We report that hepatocyte growth factor/scatter factor (HGF) induces fibronectin expression and its extracellular assembly on the surface of melanoma cells through activation of mitogen-activated protein (MAP) kinase pathway, and induction and transcriptional activation of Early growth response-1 (Egr-1). Inhibition of B-RAF/MAP kinase pathway by dominant-negative mutants and by U0126-abrogated HGF-induced Egr-1, and chromatin immunoprecipitation showed that Egr-1 is bound to the fibronectin promoter in response to HGF. Exogenously expressed Egr-1 increased fibronectin levels, while blockage of Egr-1 activation by expression of the Egr-1 corepressor NAB2 interfered with the upregulation of fibronectin synthesis induced by HGF, indicating that Egr-1 exerts a significant role in fibronectin expression in response to HGF. Finally, analysis of the expression pattern of fibronectin in melanoma cells demonstrated that fibronectin levels are correlated with constitutive MAP kinase signaling. Our data define a novel mechanism that might have important implications in regulation of melanoma progression by autocrine HGF signaling or by constitutive activation of MAP kinase pathway.

CD63 Associates with Transmembrane 4 Superfamily Members, CD9 and CD81, and with β1 Integrins in Human Melanoma

CD63 belongs to the Transmembrane 4 superfamily (TM4SF) of membrane proteins whose functions are largely unknown. Previous results have suggested that CD63 may play an important role in the regulation of melanoma progression. To explore the role of CD63 in melanoma we have examined its association with other molecules by immunoprecipitation of CD63 from detergent induced lysates of melanoma cells. These results are the first to demonstrate an association between CD63 and two other TM4SF members, CD9 and CD81 in 2 human melanoma cell lines. We were also able to identify an association between CD9 and CD63 with β1 integrins in melanoma. The results suggest that CD63 is capable of forming multicomponent complexes with TM4SF members and β1 integrins on the surface of melanoma. These findings provide further insights into the function of CD63.