Interferon-beta from melanoma cells suppresses the proliferations of melanoma cells in an autocrine manner

Interferon (IFN)-alpha and IFN-beta have been utilized in the treatment of melanoma as a form of cytokine therapy. While previous studies have demonstrated that melanocytes and melanoma cells produce a number of cytokines, it remains unclear whether or not melanocytes and melanoma cells per se produce IFN-alpha or IFN-beta. In the present study, we investigated the expression of IFN-alpha or IFN-beta in human melanocytes and five melanoma cell lines: G-361, C32TG, MMAc, MEWO and VMRC-MELG at both mRNA and protein levels. Both IFN-alpha and IFN-beta mRNA were detected in normal human melanocytes. Likewise, IFN-alpha mRNA was detected in all five melanoma cell lines. However, IFN-beta mRNA was only detected in one melanoma cell line, VMRC-MELG. When melanocytes and melanoma cells were treated with a potent IFN inducer, polyinosinic:polycytidylic acid (poly I:C), the mRNA expression of both IFN-alpha and IFN-beta was significantly upregulated. Poly I:C was not able to induce melanocytes or melanoma cells to produce detectable amounts of IFN-alpha protein, but able to induce a significant amount of IFN-beta in melanocytes and two of the melanoma cell lines: MMAc and VMRC-MELG. Moreover, similar to exogenous IFN-alpha and IFN-beta, poly I:C significantly inhibited the proliferation of all five melanoma cell lines. This suppressive effect was partially blocked by anti-IFN-beta antibody treatment in the IFN-beta-producing melanoma cell lines: MMAc and VMRC-MELG, but not in the non-IFN-beta-producing cell lines: G-361, C32TG and MEWO. Collectively, these studies have demonstrated for the first time that human melanocytes and melanoma cells produce IFN-beta. Furthermore, melanoma cells are capable of suppressing their own proliferation via secretion of endogenous IFN-beta. This finding may have important implications for melanoma therapy.     

A secreted form of P-cadherin is expressed in malignant melanoma

Cadherins are Ca-dependent homophilic cell-cell adhesion molecules which are responsible for correct location of cells and tissue integrity. They are critical for development and maintenance of epithelial architecture. Aberrantly expressed cadherins are known to be involved in malignant transformation of different types of tissues. In this study, we show the expression of a short truncated 50 kDa form of the N-terminal part of P-cadherin in seven melanoma cell lines compared to melanocytes and keratinocytes. In vitro protein analysis on cell culture supernatant as well as immunohistochemistry of primary and metastatic melanoma tissue revealed the expression of this short form of P-cadherin. Furthermore, analysis showed that this short 50 kDa form of P-cadherin is secreted by melanoma cells in contrast to the membrane bound form in melanocytes. Analysis on mRNA level detected only exon 1 to 10 of P-cadherin resulting in the 50 kDa form missing the transmembrane and cytoplasmatic region. Genomic sequence analysis did not show any mutations in melanoma cells neither in the exons nor in the exon-intron boundaries. Furthermore, there was no loss of exons 11-16 on the genomic level. Functionally, the secreted form of P-cadherin could play a role as regulator of the homophilic interaction between P-cadherin molecules by antagonizing their biological role acting as a dominant negative form to interrupt cell-cell attachment.     

Stimulation of cyclic GMP efflux in human melanocytes by hypergravity generated by centrifugal acceleration

Gravity alteration (micro- and hypergravity) is known to influence cell functions. As guanosine 3',5'-cyclic monophosphate (cGMP) plays an important role in human melanocyte functions and different guanylyl cyclase isoforms are responsible for cGMP synthesis in human non-metastatic and metastatic melanoma cells, we investigated the effects of hypergravity on the regulation of cGMP levels in cultured human melanocytes and in melanoma cell lines with different metastatic potentials. Hypergravity was produced by horizontal centrifugal acceleration. Here we report that long-term application of hypergravity (up to 5 g for 24 h) stimulated cGMP efflux in cultured melanocytes and in non-metastatic melanoma cells in the presence of 0.1 mM 3-isobutyl-1-methylxanthine (IBMX), a non-selective phosphodiesterase (PDE) inhibitor. Under these conditions, cAMP synthesis and melanin production were up-regulated in pigmented melanocytes and non-metastatic melanoma cells. Hypergravity also stimulated cGMP transport in the presence of 1 microM trequinsin, an inhibitor of cGMP-binding PDE (PDE5) and of transport by multidrug resistance proteins MRP4/5, whereas 50 microM trequinsin partially inhibited cGMP transport. Transport was further inhibited by probenecid, an inhibitor of endogenous non-selective transporters as well as of MRP4/5 and by cycloheximide as an inhibitor of de novo protein synthesis. In contrast, hypergravity did not affect cGMP efflux in metastatic melanoma cells, which might be related to an up-regulated cGMP efflux at 1 g. The results of the present study indicate that hypergravity may stimulate cGMP efflux in melanocytes and in non-metastatic melanoma cells most probably by an enhanced expression of endogenous transporters and/or MRP4/5. Thus, an altered acceleration vector may induce signaling events in melanocytic cells.     

RelA, p50 and inhibitor of kappa B alpha are elevated in human metastatic melanoma cells and respond aberrantly to ultraviolet light B.

Metastatic melanomas are typically resistant to radiation and chemotherapy. The underlying basis for this phenomenon may result in part from defects in apoptotic pathways. Nuclear factor kappa B (NFkappaB) has been shown to control apoptosis in many cell types and normally functions as an immediate stress response mechanism that is rigorously controlled by multiple inhibitory complexes. We have previously shown that NFkappaB binding is elevated in metastatic melanoma cells relative to normal melanocytes. In the current study, Western blot analysis showed that, compared with normal melanocytes, melanoma cell lines have higher nuclear levels of the NFkappaB subunits p50 (7-fold) and RelA (5-10-fold). In response to tumor necrosis factor-alpha (TNFalpha), both melanocytes and melanoma cells showed increased nuclear p50 and RelA levels, but levels in melanoma cells remained higher than in melanocytes. We also found that melanoma cells expressed higher cytoplasmic levels of RelA, p105/p50 and the inhibitory protein, inhibitor of kappa B alpha (IkappaBalpha) than melanocytes. To directly test whether RelA expression has an impact on melanoma cell survival, we used antisense RelA phosphorothioate oligonucleotides and found that melanoma cell viability was significantly decreased compared with untreated or control cultures. The constitutive activation of NFkappaB in metastatic melanoma cell cultures may, therefore, support an inappropriate cell survival pathway that can be therapeutically manipulated.     

Deletion of specific protein kinase C subspecies in human melanoma cells

It has been shown that tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulates the proliferation of normal human melanocytes, whereas it inhibits the growth of human melanoma cell lines. The expression of protein kinase C (PKC) subspecies, the major intracellular receptors for TPA, was examined in normal melanocytes and the four melanoma cell lines HM3KO, MeWo, HMV-1, and G361. PKC was partially purified and then separated into subspecies by column chromatography on Mono Q and hydroxyapatite successively, and finally subjected to immunoblot analysis using antibodies specific for the PKC subspecies. Of the PKC subspecies examined, δ-, ϵ-, and ζ-PKC were detected in both normal melanocytes and the four melanoma cell lines. In contrast, both α-PKC and β-PKC were expressed in normal melanocytes, whereas either α-PKC or β-PKC was detected in melanoma cells. Specifically, HM3KO, MeWo, and HMV-1 cells were shown to contain α-PKC but not β-PKC, while G361 cells expressed β-PKC but not α-PKC. The growth of these melanoma cells was suppressed by TPA treatment, and the growth of the G361 cells lacking α-PKC was inhibited more efficiently than the other melanoma cell lines which lacked β-PKC. It was further shown that β-PKC was not detected in freshly isolated human primary or metastatic melanoma tissues. These results suggest that the expression of α-PKC or β-PKC may be altered during the malignant transformation of normal melanocytes and that loss of α-PKC or β-PKC may be related to the inhibitory effect of TPA on the growth of melanoma cells. © 1996 Wiley-Liss, Inc.     

Lumican, a small leucine-rich proteoglycan substituted with keratan sulfate chains is expressed and secreted by human melanoma cells and not normal melanocytes

Melanoma is a frequent and therapy-resistant human disease. Malignant melanocytes modulate their microenvironment in order to penetrate the dermal/epidermal junction and eventually invade the dermis. The small leucine-rich proteoglycans (SLRPs) constitute important constituents of the dermis extracellular matrix (ECM), participating in both the structural and the functional organization of the skin. The role of a keratan sulphate SLRP lumican, has recently been investigated in the growth and metastasis of several cancers. In this study, the expression of lumican was studied in two human melanoma cell lines (WM9, M5) as well as in normal neonatal human melanocytes (HEMN) using real time PCR, western blotting with antibodies against the protein core and keratan sulfate, and treatments with specific enzymes. Both human metastatic melanoma cell lines were found to express lumican mRNA and effectively secrete lumican in a proteoglycan form, characterized to be substituted mostly with keratan sulfate chains. Lumican mRNA was not detected in normal melanocytes. This is the first time that the synthesis and secretion of lumican in human melanoma cell lines is reported. The role of this proteoglycan in the development and progression of malignant melanoma has to be further investigated.     

Suppression of human melanoma metastasis by the metastasis suppressor gene, BRMS1

We recently identified a novel metastasis suppressor gene, BRMS1, in breast cancer. Since the BRMS1 gene maps to chromosome 11q13.1-q13.2 and since chromosome 11q defects have been described in various stages of human melanoma progression, we hypothesized that BRMS1 may function as a tumor or metastasis suppressor in melanomas as well. Quantitative real-time RT-PCR revealed that BRMS1 mRNA expression was high in melanocytes, considerably reduced in early melanoma-derived cell lines, and barely detectable in advanced/metastatic cell lines. Stable transfectants of BRMS1 in the human melanoma cell lines MelJuSo and C8161.9 did not alter the tumorigenicity of either cell line, but significantly suppressed metastasis compared to vector-only transfectants. Orthotopic tumors continued to express BRMS1, but expression was lost in lung metastases. In vitro morphology, growth rate, and histology of BRMS1 transfectants were similar to controls. BRMS1 transfectants were less invasive in a collagen sandwich assay and had restored homotypic gap junctional intercellular communication (GJIC). Thus, BRMS1 functions as a metastasis suppressor in more than one tumor type (i.e., breast carcinoma and cutaneous melanoma) by modifying several metastasis-associated phenotypes.     

Differential biological effects of 1,25-dihydroxyVitamin D3 on melanoma cell lines in vitro

1,25-DihydroxyVitamin D(3) and analogs have been shown to inhibit proliferation and to induce differentiation in different cell types, including human melanocytes. However, various tumor cell lines that fail to respond to the antiproliferative effects of Vitamin D analogs have also been reported. Using real-time PCR (LightCycler), we have compared mRNA expression of Vitamin D receptor (VDR), Vitamin D-25-hydroxylase (25-OHase), 25-hydroxyVitamin D-1alpha-hydroxylase (1alpha-OHase), and 1,25-dihydroxyVitamin D-24-hydroxylase (24-OHase) in a melanoma cell line that responds to antiproliferative effects of Vitamin D (MeWo) with a non-responsive melanoma cell line (SkMel5). Additionally, modulation of cell proliferation by calpain inhibitors, as well as regulation of mRNA expression of VDR, 1alpha-OHase, and 24-OHase genes by Vitamin D analogs were assessed in melanoma cell lines in vitro using a WST-1 based colorimetric assay and real-time PCR, respectively. RNA for VDR, 25-OHase, 1alpha-OHase, and 24-OHase was detected in melanoma cell lines. In contrast to SkMel5 cells, treatment of MeWo cells with calcitriol resulted in a dose-dependent increase in mRNA for VDR and 24-OHase as well as in a suppression of cell proliferation (up to approximately 50%). Our findings demonstrate that local synthesis or metabolism of Vitamin D metabolites may be of importance for growth regulation of MM and melanoma cell lines. Additionally, metastasizing MM represents a promising target for palliative treatment with new Vitamin D analogs that exert little calcemic side effects or for pharmacological modulation of calcitriol synthesis/metabolism in these tumors.     

Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells

To identify C-MYC targets rate-limiting for proliferation of malignant melanoma, we stably inhibited C-MYC in several human metastatic melanoma lines via lentivirus-based shRNAs approximately to the levels detected in normal melanocytes. C-MYC depletion did not significantly affect levels of E2F1 protein reported to regulate expression of many S-phase specific genes, but resulted in the repression of several genes encoding enzymes rate-limiting for dNTP metabolism. These included thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). C-MYC depletion also resulted in reduction in the amounts of deoxyribonucleoside triphosphates (dNTPs) and inhibition of proliferation. shRNA-mediated suppression of TS, IMPDH2 or PRPS2 resulted in the decrease of dNTP pools and retardation of the cell cycle progression of melanoma cells in a manner similar to that of C-MYC-depletion in those cells. Reciprocally, concurrent overexpression of cDNAs for TS, IMPDH2 and PRPS2 delayed proliferative arrest caused by inhibition of C-MYC in melanoma cells. Overexpression of C-MYC in normal melanocytes enhanced expression of the above enzymes and increased individual dNTP pools. Analysis of in vivo C-MYC interactions with TS, IMPDH2 and PRPS2 genes confirmed that they are direct C-MYC targets. Moreover, all three proteins express at higher levels in cells from several metastatic melanoma lines compared to normal melanocytes. Our data establish a novel functional link between C-MYC and dNTP metabolism and identify its role in proliferation of tumor cells.     

A melanoma octamer binding protein is responsive to differentiating agents.

Analysis of human melanocytes and melanoma cell lines for proteins interacting with the octamer control sequence (ATGCAAAT) has revealed two distinct melanoma octamer binding proteins, Oct-M1 and Oct-M2. The latter was restricted to cell lines derived from tumor metastases. The level of Oct-M1 activity in a pigmented melanoma line was enhanced in comparison to the general octamer binding protein Oct-1 when cells were cultured in the presence of the depigmenting agent dithiothreitol and conversely was reduced by the differentiating and pigment inducing agents butyric acid and dimethyl sulfoxide.     

Status of RASSF1A in uveal melanocytes and melanoma cells

RASSF1A gene, found at the 3p21.3 locus, is a tumor suppressor gene frequently hypermethylated in human cancers. In this study, we report that compared with melanocytes in normal choroid, RASSF1A is downregulated in uveal melanoma samples and in uveal melanoma cell lines. LOH at 3p21.3 was detected in 50% of uveal melanoma. Moreover, methylation of the RASSF1A promoter was detected in 35 of 42 tumors (83%) and RASSF1A was also weakly expressed at the mRNA level. These data indicate that LOH at the RASSF1A locus or RASSF1A promoter methylation may partly account for the suppression of RASSF1A expression observed in uveal melanoma. Furthermore, following ectopic expression in three RASSF1A-deficient melanoma cell lines (OCM-1, Mel270, and 92.1), RASSF1A weakly reduces cell proliferation and anchorage-independent growth of uveal melanoma cells without effect on ERK1/2 activation, cyclin D1 and p27(Kip1) expression. This study explored biological functions and underlying mechanisms of RASSF1A in the ERK1/2 pathway in normal uveal melanocytes. We showed that siRNA-mediated depletion of RASSF1A increased ERK1/2 activation, cyclin D1 expression, and also decreased p27(Kip1) expression in normal uveal melanocytes. Moreover, that the depletion of RASSF1A induced senescence-associated β-galactosidase activity and increased p21(Cip1) expression suggests that RASSF1A plays a role in the escape of cellular senescence in normal uveal melanocytes. Interestingly, we found that RASSF1A was epigenetically inactivated in long-term culture of uveal melanocytes. Taken together, these data show that depletion of RASSF1A could be an early event observed during senescence of normal uveal melanocytes and that additional alterations are acquired during malignant transformation to uveal melanoma.     

Roscovitine inhibits differentiation and invasion in a three-dimensional skin reconstruction model of metastatic melanoma

The aim of this study was to investigate the therapeutic potential of a cyclin-dependent kinase inhibitor, roscovitine, in cultured melanoma cells and a three-dimensional skin reconstruction model of metastatic melanoma. The modulatory effects of roscovitine on the growth and survival of normal melanocytes and cultured melanoma cell lines were tested. Additionally, we investigated the potential of roscovitine to regulate the growth and differentiation of a metastatic melanoma cell line (A375) in a three-dimensional skin reconstruction culture consisting of A375 cells admixed with normal human keratinocytes embedded within a collagen-constricted fibroblast matrix. We show that roscovitine is able to induce apoptosis in the melanoma cell lines A375, 888, and 624 but not in normal human cultured epithelial melanocytes. The degree of apoptosis within these cell lines correlated with the accumulation of p53 protein and concomitant reduction of X-linked inhibitor of apoptosis protein, with no change in the proteins Bcl-2 and survivin. We also found that roscovitine inhibited the growth and differentiation of A375 melanoma cells within the dermal layer of the skin. The results of this study show that roscovitine has the potential to inhibit the differentiation and invasion of metastatic melanoma and may be useful as a therapy for the treatment of patients with metastatic melanoma.     

During human melanoma progression AP-1 binding pairs are altered with loss of c-Jun in vitro

We demonstrated previously that c-Jun, JunB and c-Fos RNA were dysregulated in metastatic melanoma cells compared with normal human melanocytes. The purpose of this study was to evaluate the distribution in composition of AP-1 dimers in human melanoma pathogenesis. We investigated AP-1 dimer pairing in radial growth phase-like (RGP) (w3211) and vertical growth phase-like (VGP) (w1205) human melanoma cells and metastatic cell lines (cloned from patients, c83-2c, c81-46A, A375, respectively) compared with melanocytes using electrophoretic mobility shift assay (EMSA), Western blot and transfection analyses. There are progressive variations in AP-1 composition in different melanoma cell lines compared with normal melanocytes, in which c-Jun, JunD and FosB were involved in AP-1 complexes. In w3211, c-Jun, JunD and Fra-1 were involved in AP-1 binding, while in w1205, overall AP-1 binding activity was decreased significantly and supershift binding was detected only with JunD antibodies. In metastatic c81-46A and A375 cells, only JunD was involved in AP-1 binding activity, but in a third (c83-2c) c-Jun, JunD and Fra-1 were present. Western blot evaluation detected c-Jun in melanocytes and w3211, but this component was decreased significantly or was not detectable in w1205, c81-46A and A375 cells. In contrast, JunD protein was elevated in c81-46A and c83-2c cells compared with melanocytes and RGP and VGP cell lines. Normal melanocytes and c83-2c cells (which have c-Jun involved in AP-1 binding), transfected with c-Jun antisense and treated with cisplatin, showed higher viability compared with untransfected cells, while in c81-46A cells (in which only JunD is detectable) no change in cell viability was observed following treatment with cisplatin and c-jun antisense transfection. A dominant-negative c-Jun mutant (TAM67) significantly increased the soft agar colony formation of w3211 and c83-2c cells. These results suggest that components of AP-1, especially c-Jun, may offer a new target for the prevention or treatment of human melanoma progression.