TGF‐β induces SOX2 expression in a time‐dependent manner in human melanoma cells

The sry‐related high‐mobility box (SOX)‐2 protein has recently been proven to play a significant role in progression, metastasis, and clinical prognosis spanning several cancer types. Research on the role of SOX2 in melanoma is limited and currently little is known about the mechanistic function of this gene in this context. Here, we observed high expression of SOX2 in both human melanoma cell lines and primary melanomas in contrast to melanocytic nevi. This overexpression in melanoma can, in part, be explained by extra gene copy numbers of SOX2 in primary samples. Interestingly, we were able to induce SOX2 expression, mediated by SOX4, via TGF‐β1 stimulation in a time‐dependent manner. Moreover, the knockdown of SOX2 impaired TGF‐β‐induced invasiveness. This phenotype switch can be explained by SOX2‐mediated cross talk between TGF‐β and non‐canonical Wnt signaling. Thus, we propose that SOX2 is involved in the critical TGF‐β signaling pathway, which has been shown to correlate with melanoma aggressiveness and metastasis. In conclusion, we have identified a novel downstream factor of TGF‐β signaling in melanoma, which may have further implications in the clinic.     

Co‐localization of β1,6‐branched Oligosaccharides and Coarse Melanin in Macrophage–Melanoma Fusion Hybrids and Human Melanoma Cells In Vitro

Fusion hybrids between normal macrophages and Cloudman S91 melanoma cells were shown earlier to have increased metastatic potential, along with high expression of β1,6‐N‐acetylglucosaminyltransferase  V and β1,6‐branched oligosaccharides. Curiously, hybrids, but not parental melanoma cells, also produced ‘coarse melanin’– autophagic vesicles with multiple melanosomes. As β1,6‐branched oligosaccharides were known to be associated with metastasis, and coarse melanin had been described in invasive human melanomas, we looked for potential relationships between the two. Using lectin‐ and immunohistochemistry, we analyzed cell lines producing coarse melanin for β1,6‐branched oligosaccharides: gp100/pmel‐17 (a melanosomal structural component) and CD63 (a late endosome/lysosome component associated with melanoma and certain other human cancers). Cell lines used in this study were (i) hybrid 94‐H48, a highly metastatic, macrophage–melanoma experimental fusion hybrid; (ii) 6^neo mouse melanoma cells, the weakly metastatic, parental fusion partner; and (iii) SKmel‐23, a human melanoma cell line derived from a metastasis. Coarse melanin granules were prominent both in hybrids and in SKmel‐23 cells, and co‐localized with stains for β1,6‐branched oligosaccharides, gp100/pmel 17, and CD63. This is the first report of this phenotype being expressed in vitro, although co‐expression of β1,6‐branched oligosaccharides and coarse melanin was recently shown to be a common and pervasive characteristic in archival specimens of human melanomas, and was most prominent in metastases. The results suggest that pathways of melanogenesis in melanoma may differ significantly from those in normal melanocytes. In vitro expression of this phenotype provides new biological systems for more detailed analyses of its genesis and regulation at the molecular genetic level.     

Melanoma‐associated GRM3 variants dysregulate melanosome trafficking and cAMP signaling

Large‐scale sequencing studies have revealed several genes that are recurrently mutated in melanomas. To annotate the melanoma genome, we have expressed tumor‐associated variants of these genes in zebrafish and characterized their effects on melanocyte development and function. Here, we describe expression of tumor‐associated variants of the recurrently mutated metabotropic glutamate receptor 3 (GRM3) gene. Unlike wild‐type GRM3, tumor‐associated GRM3 variants disrupted trafficking of melanosomes, causing their aggregation in the cell body. Melanosomes are trafficked in a cAMP‐dependent manner, and drugs that directly or indirectly increased cAMP levels were able to suppress melanosome aggregation in mutant GRM3‐expressing melanocytes. Our data show that oncogenic GRM3 variants dysregulate cAMP signaling, a heretofore unknown role for these oncogenes. cAMP signaling has been implicated in melanoma progression and drug resistance, and our data show that oncogenic properties of GRM3 could be mediated, at least in part, by alterations in cAMP signaling.     

Identification of glycogen synthase kinase 3α as a therapeutic target in melanoma

Deregulated expression or activity of kinases can lead to melanomas, but often the particular kinase isoform causing the effect is not well established, making identification and validation of different isoforms regulating disease development especially important. To accomplish this objective, an siRNA screen was undertaken that which identified glycogen synthase kinase 3α (GSK3α) as an important melanoma growth regulator. Melanocytes and melanoma cell lines representing various stages of melanoma tumor progression expressed both GSK3α and GSK3β, but analysis of tumors in patients with melanoma showed elevated expression of GSK3α in 72% of samples, which was not observed for GSK3β. Furthermore, 80% of tumors in patients with melanoma expressed elevated levels of catalytically active phosphorylated GSK3α (pGSK3αY279), but not phosphorylated GSK3β (pGSK3βY216). siRNA‐mediated reduction in GSK3α protein levels reduced melanoma cell survival and proliferation, sensitized cells to apoptosis‐inducing agents and decreased xenografted tumor development by up to 56%. Mechanistically, inhibiting GSK3α expression using siRNA or the pharmacological agent AR‐A014418 arrested melanoma cells in the G0/G1 phase of the cell cycle and induced apoptotic death to retard tumorigenesis. Therefore, GSK3α is a key therapeutic target in melanoma.     

Principal expression of two mRNA isoforms (ABCB 5α and ABCB 5β ) of the ATP‐binding cassette transporter gene ABCB 5 in melanoma cells and melanocytes

ATP‐binding cassette (ABC) transporters play a pivotal role in physiology and pathology. We identified and cloned two novel mRNA isoforms (ABCB 5α and ABCB 5β) of the ABC transporter ABCB 5 in human melanoma cells. The deduced ABCB 5α protein appears to be an altered splice variant containing only a putative ABC, whereas the ABCB 5β isoform shares approximately 70% similarity with ABCB1 (MDR1) and has a deduced topological arrangement similar to that of the whole carboxyl terminal half of the ABCB1 gene product, P‐glycoprotein, including an intact ABC. Northern blot, real‐time PCR, and conventional RT‐PCR were used to verify the expression profiles of ABCB 5α/β. We found that the melanomas included among the NCI‐60 panel of cell lines preferentially expressed both ABCB 5α and ABCB 5β. However, ABCB 5α/β expression was undetectable in two amelanotic melanomas (M14 and LOX‐IMVI). The expression profile of ABCB 5α/β in all of the other melanomas of the panel was confirmed both by RT‐PCR and by sequencing. Neither ABCB 5α nor ABCB 5β expression was found in normal tissues such as liver, spleen, thymus, kidney, lung, colon, small intestines or placenta. ABCB 5α/β mRNAs were also expressed in normal melanocytes and in retinal pigment epithelial cells, suggesting that ABCB 5α/β expression is pigment cell‐specific and might be involved in melanogenesis. Our findings indicate that expression of ABCB 5α/β might possibly provide two novel molecular markers for differential diagnosis of melanomas and constitute potential molecular targets for therapy of melanomas.     

RAC1mutation is not a predictive biomarker for PI3’‐kinase‐β‐selective pathway‐targeted therapy

Mutational activation of RAC1 is detected in ~7% of cutaneous melanoma, with the most frequent mutation (RAC1^C85T) encoding for RAC1^P29S. RAC1^P29S is a fast‐cycling GTPase that leads to accumulation of RAC1^P29S‐GTP, which has potentially pleiotropic regulatory functions in melanoma cell signaling and biology. However, the precise mechanism by which mutationally activated RAC1^P29S propagates its pro‐tumorigenic effects remains unclear. RAC1‐GTP is reported to activate the beta isoform of PI3’‐kinase (PIK3CB/PI3Kβ) leading to downstream activation of PI3’‐lipid signaling. Hence, we employed both genetic and isoform‐selective pharmacological inhibitors to test if RAC1^P29S propagates its oncogenic signaling in melanoma through PI3Kβ. We observed that RAC1^P29S‐expressing melanoma cells were largely insensitive to inhibitors of PI3Kβ. Furthermore, RAC1^P29S melanoma cell lines showed variable sensitivity to pan‐class 1 (α/β/γ/δ) PI3’‐kinase inhibitors, suggesting that RAC1‐mutated melanoma cells may not rely on PI3’‐lipid signaling for their proliferation. Lastly, we observed that RAC1^P29S‐expressing cell lines also showed variable sensitivity to pharmacological inhibition of the RAC1 → PAK1 signaling pathway, questioning the relevance of inhibitors of this pathway for the treatment of patients with RAC1‐mutated melanoma.     

Syndecan‐2 regulates melanin synthesis via protein kinase C βII‐mediated tyrosinase activation

Syndecan‐2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan‐2 in melanogenesis. Syndecan‐2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA‐mediated knockdown of syndecan‐2 was associated with reduced melanin synthesis, whereas overexpression of syndecan‐2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan‐2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan‐2 expression, and this up‐regulation of syndecan‐2 was required for UVB‐induced melanin synthesis. Taken together, these data suggest that syndecan‐2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin‐associated diseases.     

Role of melanoma inhibitory activity in melanocyte senescence

The protein melanoma inhibitory activity (MIA) is known to be expressed in melanoma and to support melanoma progression. Interestingly, previous studies also observed the expression of MIA in nevi. Concentrating on these findings, we revealed that MIA expression is correlated with a senescent state in melanocytes. Induction of replicative or oncogene‐induced senescence resulted in increased MIA expression in vitro. Notably, MIA knockdown in senescent melanocytes reduced the percentage of senescence‐associated beta‐Gal‐positive cells and enhanced proliferation. Using the melanoma mouse model Tg(Grm1), MIA‐deficient mice supported the impact of MIA on senescence by showing a significantly earlier tumor onset compared to controls. In melanocytes, MIA knockdown led to a downregulation of the cell cycle inhibitor p21 in vitro and in vivo. In contrast, after induction of hTERT in human melanoma cells, p21 regulation by MIA was lost. In summary, our data show for the first time that MIA is a regulator of cellular senescence in human and murine melanocytes.     

Senescence evasion in melanoma progression: uncoupling of DNA‐damage signaling from p53 activation and p21 expression

The best‐established function of the melanoma‐suppressor p16 is mediation of cell senescence, a permanent arrest following cell proliferation or certain stresses. The importance of p16 in melanoma suggests indolence of the other major senescence pathway through p53. Little or no p53 is expressed in senescent normal human melanocytes, but p16‐deficient melanocytes can undergo p53‐mediated senescence. As p16 expression occurs in nevi but falls with progression toward melanoma, we here investigated whether p53‐dependent senescence occurs at some stage and, if not, what defects were detectable in this pathway, using immunohistochemistry. Phosphorylated checkpoint kinase 2 (CHEK2) can mediate DNA‐damage signaling, and under some conditions senescence, by phosphorylating and activating p53. Remarkably, we detected no prevalent p53‐mediated senescence in any of six classes of lesions. Two separate defects in p53 signaling appeared common: in nevi, lack of p53 phosphorylation by activated CHEK2, and in melanomas, defective p21 upregulation by p53 even when phosphorylated.     

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.     

Near‐genomewide RNAi screening for regulators of BRAFV600E‐induced senescence identifies RASEF,a gene epigenetically silenced in melanoma

The activation of oncogenes in primary cells blocks proliferation by inducing oncogene‐induced senescence (OIS), a highly potent in vivo tumor‐suppressing program. A prime example is mutant BRAF, which drives OIS in melanocytic nevi. Progression to melanoma occurs only in the context of additional alteration(s) like the suppression of PTEN, which abrogates OIS. Here, we performed a near‐genomewide short hairpin (sh)RNA screen for novel OIS regulators and identified by next generation sequencing and functional validation seven genes. While all but one were upregulated in OIS, depletion of each of them abrogated BRAF^V600E‐induced arrest. With genome‐wide DNA methylation analysis, we found one of these genes, RASEF, to be hypermethylated in primary cutaneous melanomas but not nevi. Bypass of OIS by depletion of RASEF was associated with suppression of several senescence biomarkers including senescence‐associated (SA)‐β‐galactosidase activity, interleukins, and tumor suppressor p15^INK4B. Restoration of RASEF expression inhibited proliferation. These results illustrate the power of shRNA OIS bypass screens and identify a potential novel melanoma suppressor gene.     

Thrombospondin‐1 is part of a Slug‐independent motility and metastatic program in cutaneous melanoma,in association with VEGFR‐1 and FGF‐2

Differently from most transformed cells, cutaneous melanoma expresses the pleiotropic factor thrombospondin‐1 (TSP‐1). Herein, we show that TSP‐1 (RNA and protein), undetectable in four cultures of melanocytes and a RGP melanoma, was variously present in 13 cell lines from advanced melanomas or metastases. Moreover, microarray analysis of 55 human lesions showed higher TSP‐1 expression in primary melanomas and metastases than in common and dysplastic nevi. In a functional enrichment analysis, the expression of TSP‐1 correlated with motility‐related genes. Accordingly, TSP‐1 production was associated with melanoma cell motility in vitro and lung colonization potential in vivo. VEGF/VEGFR‐1 and FGF‐2, involved in melanoma progression, regulated TSP‐1 production. These factors were coexpressed with TSP‐1 and correlated negatively with Slug (SNAI2), a cell migration master gene implicated in melanoma metastasis. We conclude that TSP‐1 cooperates with FGF‐2 and VEGF/VEGFR‐1 in determining melanoma invasion and metastasis, as part of a Slug‐independent motility program.     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.