Potential Of Human Melanoma Cells Research Articles

Inadvertent Transfer of Murine VL30 Retrotransposons to CAR-T Cells.

For more than a decade, genetically engineered autologous T-cells have been successfully employed as immunotherapy drugs for patients with incurable blood cancers. The active components in some of these game-changing medicines are autologous T-cells that express viral vector-delivered chimeric antigen receptors (CARs), which specifically target proteins that are preferentially expressed on cancer cells. Some of these therapeutic CAR expressing T-cells (CAR-Ts) are engineered via transduction with γ-retroviral vectors (γ-RVVs) produced in a stable producer cell line that was derived from murine PG13 packaging cells (ATCC CRL-10686). Earlier studies reported on the copackaging of murine virus-like 30S RNA (VL30) genomes with γ-retroviral vectors generated in murine stable packaging cells. In an earlier study, VL30 mRNA was found to enhance the metastatic potential of human melanoma cells. These findings raise biosafety concerns regarding the possibility that therapeutic CAR-Ts have been inadvertently contaminated with potentially oncogenic VL30 retrotransposons. In this study, we demonstrated the presence of infectious VL30 particles in PG13 cell-conditioned media and observed the ability of these particles to deliver transcriptionally active VL30 genomes to human cells. Notably, VL30 genomes packaged by HIV-1-based vector particles transduced naïve human cells in culture. Furthermore, we detected the transfer and expression of VL30 genomes in clinical-grade CAR-T cells generated by transduction with PG13 cell-derived γ-retroviral vectors. Our findings raise biosafety concerns regarding the use of murine packaging cell lines in ongoing clinical applications.

Retraction: Green Tea Catechins Reduce Invasive Potential of Human Melanoma Cells by Targeting COX-2, PGE2 Receptors and Epithelial-to-Mesenchymal Transition.

Retraction: Green Tea Catechins Reduce Invasive Potential of Human Melanoma Cells by Targeting COX-2, PGE2 Receptors and Epithelial-to-Mesenchymal Transition.

YB-1 Expression and Phosphorylation Regulate Tumorigenicity and Invasiveness in Melanoma by Influencing EMT.

Cutaneous melanoma represents one of the most aggressive human tumor entities possessing a high tendency to metastasize. Cancer cells frequently exploit a highly conserved developmental program, the epithelial-to-mesenchymal transition (EMT), to gain migratory and invasive properties promoting their metastatic spread. Cytoplasmic localization of the oncogenic transcription and translation factor Y-box binding protein 1 (YB-1) is a powerful inducer of EMT in breast carcinoma cells. Interestingly, EMT-like processes have also been observed in cutaneous melanoma despite its neural crest origin. Here, increased expression of YB-1 negatively affects patient survival in malignant melanoma and promotes melanoma cell tumorigenicity both in vitro and in vivo Intriguingly, this effect seems to be mainly mediated by cytoplasmic YB-1 that does not exhibit phosphorylation at serine-102 (S102). Moreover, S102 unphosphorylated YB-1 enhances the migratory and invasive potential of human melanoma cells in two-dimensional (2D) and three-dimensional (3D) culture systems and facilitates acquisition of a mesenchymal-like invasive phenotype in the chick embryo model. Collectively, these data demonstrate that the cytoplasmic activity of YB-1 stimulates tumorigenicity and metastatic potential of melanoma cells by promoting EMT-like properties.Implications: This study reveals for the first time that YB-1 efficiently drives tumorigenicity and invasiveness of melanoma cells in its S102 unphosphorylated cytoplasmic state and that YB-1 expression represents a negative prognostic factor in primary melanoma patients. Mol Cancer Res; 16(7); 1149-60. ©2018 AACR.

Metabolic Rewiring by Oncogenic BRAF V600E Links Ketogenesis Pathway to BRAF-MEK1 Signaling

Many human cancers share similar metabolic alterations, including the Warburg effect. However, it remains unclear whether oncogene-specific metabolic alterations are required for tumor development. Here we demonstrate a "synthetic lethal" interaction between oncogenic BRAF V600E and a ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL). HMGCL expression is upregulated in BRAF V600E-expressing human primary melanoma and hairy cell leukemia cells. Suppression of HMGCL specifically attenuates proliferation and tumor growth potential of human melanoma cells expressing BRAF V600E. Mechanistically, active BRAF upregulates HMGCL through an octamer transcription factor Oct-1, leading to increased intracellular levels of HMGCL product, acetoacetate, which selectively enhances binding of BRAF V600E but not BRAF wild-type to MEK1 in V600E-positive cancer cells to promote activation of MEK-ERK signaling. These findings reveal a mutation-specific mechanism by which oncogenic BRAF V600E "rewires" metabolic and cell signaling networks and signals through the Oct-1-HMGCL-acetoacetate axis to selectively promote BRAF V600E-dependent tumor development.

FBXW7 modulates cellular stress response and metastatic potential through ​HSF1 post-translational modification.

Heat-shock factor 1 (HSF1) orchestrates the heat-shock response in eukaryotes. Although this pathway has been evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy. However, the mechanisms that regulate HSF1 and thus cellular stress response are poorly understood. Here we show that the ubiquitin ligase FBXW7 α interacts with HSF1 through a conserved motif phosphorylated by GSK3β and ERK1. FBXW7α ubiquitylates HSF1 and loss of FBXW7α results in impaired degradation of nuclear HSF1 and defective heat-shock response attenuation. FBXW7α is either mutated or transcriptionally downregulated in melanoma and HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. FBXW7α deficiency and subsequent HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells. These findings identify a post-translational mechanism of regulation of the HSF1 transcriptional program both in the presence of exogenous stress and in cancer.

Abstract 3683: Bioactive phytochemical proanthocyanidins target β-catenin signaling in preventing invasive potential of human melanoma cells.

Abstract Melanoma is the leading cause of death from skin diseases due to its propensity to metastasize. The overall incidence of melanoma is increasing in US, and is increasing rapidly in children. Since, melanoma is a highly malignant cancer with a potent capacity to metastasize distantly, an approach that decreases its metastatic potential may facilitate the development of an effective strategy for its prevention or treatment. Metastatic melanoma is often associated with activation of Wnt/β-catenin signaling pathway. To develop newer and more effective strategies for the prevention of melanoma metastasis, we have examined the inhibitory effect of proanthocyanidins, bioactive components of grape seeds, on cell migration of metastasis-specific human melanoma cell lines (A375 and Hs294t) and assessed whether Wnt/β-catenin signaling is the target of grape seed proanthocyanidins (GSPs). Using an in vitro invasion assay, we found that treatment of human melanoma cell lines with GSPs resulted in a dose-dependent inhibition of cell migration, which was associated with the degradation of cytosolic β-catenin, and subsequently reducing the nuclear accumulation of β-catenin (i.e., inactivation of β-catenin) and reducing the levels of matrix metalloproteinase (MMP) -2 and MMP-9 which are the down-stream targets of β-catenin and play a crucial role in cancer cell metastasis. GSPs increased the: (i) levels of casein kinase 1α, glycogen synthase kinase-3β and phosphorylated-β-catenin on critical serine residues (Ser45, Ser33/37 and Thr41), and (ii) binding of β-transducin repeat-containing proteins (β-TrCP) with phospho forms of β-catenin in melanoma cells. These molecular events lead to degradation and/or inactivation of β-catenin. To verify whether β-catenin is a potent molecular target of GSPs, the effect of GSPs was determined on β-catenin-activated (Mel1241) and β-catenin-inactivated (Mel1011) melanoma cells. Treatment of Mel1241 cells with GSPs or FH535, an inhibitor of Wnt/β-catenin pathway, significantly inhibited cell migration of Mel1241 cells, which was associated with the elevated levels of casein kinase 1α and glycogen synthase kinase-3β, and decreased accumulation of nuclear β-catenin and inhibition of MMP-2 and MMP-9 levels. However, this effect of GSPs and FH535 was not found in Mel1011 melanoma cells. These results indicate for the first time that grape seed proanthocyanidins inhibit the invasive potential of melanoma cells by targeting β-catenin signaling. This new insight into the anti-melanoma cell migration activity of GSPs could serve as the basis for chemoprevention or therapy of malignant melanoma in high risk human population. Citation Format: Santosh K. Katiyar, Mudit Vaid. Bioactive phytochemical proanthocyanidins target β-catenin signaling in preventing invasive potential of human melanoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3683. doi:10.1158/1538-7445.AM2013-3683

Assessment of the tumourigenic and metastatic properties of SK-MEL28 melanoma cells surviving electrochemotherapy with bleomycin

BackgroundElectrochemotherapy is a local treatment combining chemotherapy and electroporation and is highly effective treatment approach for subcutaneous tumours of various histologies. Contrary to surgery and radiation, the effect of electrochemotherapy on metastatic potential of tumour cells has not been extensively studied. The aim of the study was to evaluate the effect of electrochemotherapy with bleomycin on the metastatic potential of human melanoma cells in vitro.Materials and methodsViable cells 48 hours after electrochemotherapy were tested for their ability to migrate and invade through Matrigel coated porous membrane. In addition, microarray analysis and quantitative Real-Time PCR were used to detect changes in gene expression after electrochemotherapy.ResultsCell migration and invasion were not changed in melanoma cells surviving electrochemotherapy. Interestingly, only a low number of tumourigenesis related genes was differentially expressed after electrochemotherapy.ConclusionsOur data suggest that metastatic potential of human melanoma cells is not affected by electrochemotherapy with bleomycin, confirming safe role of electrochemotherapy in the clinics.

Green Tea Catechins Reduce Invasive Potential of Human Melanoma Cells by Targeting COX-2, PGE2 Receptors and Epithelial-to-Mesenchymal Transition

Melanoma is the most serious type of skin disease and a leading cause of death from skin disease due to its highly metastatic ability. To develop more effective chemopreventive agents for the prevention of melanoma, we have determined the effect of green tea catechins on the invasive potential of human melanoma cells and the molecular mechanisms underlying these effects using A375 (BRAF-mutated) and Hs294t (Non-BRAF-mutated) melanoma cell lines as an in vitro model. Employing cell invasion assays, we found that the inhibitory effects of green tea catechins on the cell migration were in the order of (-)-epigallocatechin-3-gallate (EGCG)>(-)-epigallocatechin>(-)-epicatechin-3-gallate>(-)-gallocatechin>(-)-epicatechin. Treatment of A375 and Hs294t cells with EGCG resulted in a dose-dependent inhibition of cell migration or invasion of these cells, which was associated with a reduction in the levels of cyclooxygenase (COX)-2, prostaglandin (PG) E2 and PGE2 receptors (EP2 and EP4). Treatment of cells with celecoxib, a COX-2 inhibitor, also inhibited melanoma cell migration. EGCG inhibits 12-O-tetradecanoylphorbol-13-acetate-, an inducer of COX-2, and PGE2-induced cell migration of cells. EGCG decreased EP2 agonist (butaprost)- and EP4 agonist (Cay10580)-induced cell migration ability. Moreover, EGCG inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in A375 melanoma cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited cell migration. Inhibition of melanoma cell migration by EGCG was associated with transition of mesenchymal stage to epithelial stage, which resulted in an increase in the levels of epithelial biomarkers (E-cadherin, cytokeratin and desmoglein 2) and a reduction in the levels of mesenchymal biomarkers (vimentin, fibronectin and N-cadherin) in A375 melanoma cells. Together, these results indicate that EGCG, a major green tea catechin, has the ability to inhibit melanoma cell invasion/migration, an essential step of metastasis, by targeting the endogenous expression of COX-2, PGE2 receptors and epithelial-to-mesenchymal transition.

Recognition of CD146 as an ERM-binding protein offers novel mechanisms for melanoma cell migration

Tumor cell migration is a well-orchestrated multistep process that drives cancer development and metastasis. Previous data indicated that CD146 expression correlates with malignant progression and metastatic potential of human melanoma cells. However, the exact molecular mechanism of how CD146 promotes melanoma cell migration still remains poorly understood. Here, we report that CD146 physically interacts with actin-linking ezrin-radixin-moesin (ERM) proteins and recruits ERM proteins to cell protrusions, promoting the formation and elongation of microvilli. Moreover, CD146-promoted melanoma cell migration is linked to RhoA activation and ERM phosphorylation. CD146 recruits Rho guanine nucleotide dissociation inhibitory factors 1 (RhoGDI1) through ERM proteins and thus sequesters RhoGDI1 from RhoA, which leads to upregulated RhoA activity and increased melanoma cell motility. CD146-activated RhoA also promotes further ERM phosphorylation and activation through Rho-phosphatidylinositol-4-phosphate-5-kinase-phosphatidylinositol 4,5-biphosphate pathway, which reinforces CD146/ERM association. Thus, our results provide a mechanistic basis to understand the role of CD146 in regulating human melanoma cell motility.

Abstract 4476: The effect of electrochemotherapy on metastatic potential of human melanoma cells SK-Mel28

Abstract Electrochemotherapy is a local treatment, combining chemotherapy and application of electric pulses to the tumour. Electrochemotherapy with bleomycin or cisplatin has demonstrated its effectiveness in controlling local tumour growth in the treatment of malignant melanoma and is proposed as one of the treatment options for in-transit melanoma metastases in Europe. An important aspect of successful treatment is prevention of metastasis, however, it is known that metastasis can be induced by different treatment modalities. Alterations in tumour microenvironment caused by surgery or radiation therapy, can lead to increased formation of metastasis. Furthermore, formation of metastases can be directly linked to migratory potential of the cells. Although electrochemotherapy is now routinely used for treatment of various cancers in many cancer centres, the effect of electrochemotherapy on the metastatic potential of tumour cells is not known. Therefore, the aim of this study was to evaluate the effect of electrochemotherapy with bleomycin and cisplatin on the metastatic potential of human malignant melanoma cells. Human malignant melanoma cells SK-MEL 28 were treated by electrochemotherapy with a range of bleomycin concentrations from 0.01 nM to 1μM and cisplatin concentrations from 2 to 80 μg/mL. SK-MEL 28 cells that were viable 48 h after electrochemotherapy were tested for their metastatic potential by ability to migrate and invade through Matrigel coated porous membrane. In addition, cell adhesion and proliferation using MTS assay were also tested. Furthemore, RNA was isolated from the cells 16 h after each treatment and differentially expressed genes were investigated by microarray analysis to evaluate the effect of electrochemotherapy with bleomycin or cisplatin on expression of genes involved in the development of cancer. The Gene Ontology Tree Machine program was used for gene enrichment analysis. Migration and invasion of melanoma cells that survived 48h after electrochemotherapy with bleomycin or cisplatin were not changed compared to control untreated cells. Also, there were no changes observed in cell adhesion on Matrigel. Gene expression analysis demonstrated that a very low number of genes were differentially expressed after electrochemotherapy with either bleomycin or cisplatin. Specifically, the expression of metastasis promoting genes was not increased after electrochemotherapy. Our data suggest that electrochemotherapy with bleomycin or cisplatin does not increase metastatic behaviour of human melanoma cells, having important clinical implication for safe use in treatment of melanoma metastases. 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 4476. doi:10.1158/1538-7445.AM2011-4476

Abstract OPS01-02: Inflammation and cancer: Interweaving microRNA, innate immune, and p53 pathways

Abstract Infection and chronic inflammation contribute to the etiology and pathogenesis of about one in four of all cancer cases. Mediators of the inflammatory response, e.g., cytokines, free radicals, prostaglandins, non-coding RNAs, and growth factors, can induce genetic and epigenetic changes including point mutations in tumor suppressor genes, DNA methylation and posttranslation modifications, causing alterations in critical pathways responsible for maintaining the normal cellular homeostasis and leading to the development and progression of cancer (1-6). IL-6 and IL-8 cooperate with microRNA in the induction of cellular senescence in benign tumors (7-9) and as autocrine growth factors in carcinoma (10-14). Cellular senescence is also a tumor suppressor mechanism of p53 (15,16). We are studying the molecular mechanisms of cellular senescence in normal and malignant human cells and the role of the telomeric multiprotein complex, shelterin, that includes TRF2 and POT1. Our ongoing studies have revealed that p53 and its endogenous isoforms regulate both specific microRNAs and TRF2 expression as mechanisms of replicative senescence. In addition, POT1 isoforms are functionally diverse in both maintaining telomeric integrity and preventing p53-dependent senescence induced by telomeric shortening (17,18). A switch in the expression patterns of p53 isoforms 133Np53 and p53 beta can cause cellular senescence in vitro and is also associated with the transition of benign to malignant human colon tumors in vivo (19). Expression of microRNAs and inflammatory genes are biomarkers of cancer risk, diagnosis, prognosis, and therapeutic outcome. We are especially interested in the interaction of the innate immune pathway with environmental tobacco smoking in lung cancer risk (20) and the interaction of inflammatory cytokines, p53 isoforms, and microRNAs as prognostic classifiers of early stage lung and colon cancer and their functional role in the development of micrometastases [reviewed in (3)] (21-25).

Cystatin E/M suppresses legumain activity and invasion of human melanoma

BackgroundHigh activity of cysteine proteases such as legumain and the cathepsins have been shown to facilitate growth and invasion of a variety of tumor types. In breast cancer, several recent studies have indicated that loss of the cysteine protease inhibitor cystatin E/M leads to increased growth and metastasis. Although cystatin E/M is normally expressed in the skin, its role in cysteine protease regulation and progression of malignant melanoma has not been studied.MethodsA panel of various non-melanoma and melanoma cell lines was used. Cystatin E/M and C were analyzed in cell media by immunoblotting and ELISA. Legumain, cathepsin B and L were analyzed in cell lysates by immunoblotting and their enzymatic activities were analyzed by peptide substrates. Two melanoma cell lines lacking detectable secretion of cystatin E/M were transfected with a cystatin E/M expression plasmid (pCST6), and migration and invasiveness were studied by a Matrigel invasion assay.ResultsCystatin E/M was undetectable in media from all established melanoma cell lines examined, whereas strong immunobands were detected in two of five primary melanoma lines and in two of six lines derived from patients with metastatic disease. Among the four melanoma lines secreting cystatin E/M, the glycosylated form (17 kD) was predominant compared to the non-glycosylated form (14 kD). Legumain, cathepsin B and L were expressed and active in most of the cell lines, although at low levels in the melanomas expressing cystatin E/M. In the melanoma lines where cystatin E/M was secreted, cystatin C was generally absent or expressed at a very low level. When melanoma cells lacking secretion of cystatin E/M were transfected with pCST6, their intracellular legumain activity was significantly inhibited. In contrast, cathepsin B activity was not affected. Furthermore, invasion was suppressed in cystatin E/M over-expressing melanoma cell lines as measured by the transwell Matrigel assay.ConclusionsThese results suggest that the level of cystatin E/M regulates legumain activity and hence the invasive potential of human melanoma cells.

Inflammation and melanoma growth and metastasis: The role of platelet-activating factor (PAF) and its receptor

An inflammatory tumor microenvironment fosters tumor growth, angiogenesis and metastatic progression. Platelet-activating factor (PAF) is an inflammatory biolipid produced from membrane glycerophospholipids. Through the activity of its G-protein coupled receptor, PAF triggers a variety of pathological reactions including tumor neo-angiogenesis. Several groups have demonstrated that inhibiting PAF-PAF receptor pathway at the level of a ligand or receptor results in an effective inhibition of experimental tumor growth and metastasis. In particular, our group has recently demonstrated that PAF receptor antagonists can effectively inhibit the metastatic potential of human melanoma cells in nude mice. Furthermore, we showed that PAF stimulated the phosphorylation of CREB and ATF-1 in metastatic melanoma cells, which resulted in overexpression of MMP-2 and MT1-MMP. Our data indicate that PAF acts as a promoter of melanoma metastasis in vivo. Since only metastatic melanoma cells overexpress CREB/ATF-1, we propose that these cells are better equipped to respond to PAF within the tumor microenvironment when compared to their non-metastatic counterparts.

Loss of Activator Protein-2α Results in Overexpression of Protease-activated Receptor-1 and Correlates with the Malignant Phenotype of Human Melanoma

Increasing evidence implicates the protease-activated receptor-1 (PAR-1) as a contributor to tumor invasion and metastasis of human melanoma. Here we demonstrate that the metastatic potential of human melanoma cells correlates with overexpression of PAR-1. We also provide evidence that an inverse correlation exists between the expression of activator protein-2alpha (AP-2) and the expression of PAR-1 in human melanoma cells. Reexpression of AP-2 in WM266-4 melanoma cells, which are AP-2-negative, resulted in decreased mRNA and protein expression of PAR-1. The promoter of the PAR-1 gene contains multiple putative consensus elements for the transcription factors AP-2 and specificity protein 1 (Sp1). Chromatin immunoprecipitation analysis of the PAR-1 promoter regions bp -365 to -329 (complex 1) and bp -206 to -180 (complex 2) demonstrated that Sp1 was predominantly bound to the PAR-1 promoter in metastatic cells, whereas AP-2 was bound to the PAR-1 promoter in nonmetastatic cells. In vitro analysis of complex 1 demonstrated that AP-2 and Sp1 bound to this region in a mutually exclusive manner. Transfection experiments with full-length and progressive deletions of the PAR-1 promoter luciferase constructs demonstrated that metastatic melanoma cells had increased PAR-1 promoter activity compared with low and nonmetastatic melanoma cells. Our data show that exogenous AP-2 expression decreased promoter activity, whereas transient expression of Sp1 further increased expression of the reporter gene. Mutational analysis of complex 1 within PAR-1 luciferase constructs further demonstrated that the regulation of PAR-1 was mediated through interactions with AP-2 and Sp1. Our data suggest that loss of AP-2 in metastatic cells alters the AP-2/Sp1 ratio, resulting in overexpression of PAR-1. Taken together, our results provide strong evidence that loss of AP-2 correlates with overexpression of PAR-1, which in turn contributes to the acquisition of the malignant phenotype of human melanoma.

A decrease in the expression of CD63 tetraspanin protein elevates invasive potential of human melanoma cells

CD63, which belongs to the tetraspanin membrane proteins, has been proposed to play an important role in inhibiting melanoma metastasis. To determine whether reduction of CD63 expression, which frequently occurs in the malignant progression of human melanoma, is responsible for metastasis promotion, we transfected the antisense CD63 cDNA into MelJuso melanoma cells having endogenous CD63 expression. The antisense CD63 transfectant clones showing decreased CD63 expression displayed increased cell motility, matrix- degrading activity, and invasiveness in vitro when compared with the control transfectant cells. The antisense CD63 cDNA-transfected cells also exhibited altered adhesiveness to extracellular matrix. The results suggest that reduced CD63 expression contributes to the invasive and metastatic ability of human melanoma cells.

Gene regulation in melanoma progression by the AP-2 transcription factor.

The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase [(VGP), metastatic phenotype] are not very well defined. We previously demonstrated that expression of the cell-surface adhesion molecule MCAM/MUC18 correlates directly with the metastatic potential of human melanoma cells. In addition, the progression of human melanoma towards the metastatic phenotype is associated with loss of expression of the tyrosine-kinase receptor c-KIT. In this review, I will summarize our recent studies demonstrating that the expression of both genes is regulated by the AP-2 transcription factor. Moreover, we have observed a loss of AP-2 expression in metastatic melanoma cells. Re-expression of AP-2 in the highly metastatic A375SM cells decreased their tumorigenicity and inhibited their metastatic potential in nude mice. MCAM/MUC18 mRNA and protein expression was significantly down-regulated while c-KIT expression was up-regulated in the AP-2-transfected cells. To further investigate the role of AP-2 in the progression of human melanoma, we attempted to inactivate AP-2 in primary cutaneous melanoma by using a dominant-negative AP-2, or the AP-2B gene. Expression of AP-2B in SB-2 cells augmented their tumorigenicity in nude mice, and upregulated MMP-2 expression and activity. As AP-2 also regulates other genes that are involved in the progression of human melanoma such as E-cadherin, p21/WAF-1, HER2/neu, Bcl-2, FAS/APO-1, IGF-R-1, VEGF and the thrombin receptor (PAR-1), we therefore propose that loss of AP-2 is a crucial event in the development of malignant melanoma. In addition, the transition of melanoma cells from RGP to VGP is also associated with over-expression of the transcription factors CREB and ATF-1. The notion that the balance between AP-2 and CREB/ATF-1 expression determines the progression of melanoma cells towards the metastatic phenotype will be discussed.

Role of AP-2 in tumor growth and metastasis of human melanoma.

We previously demonstrated that expression of the cell surface adhesion molecule MCAM/MUC18 correlates directly with the metastatic potential of human melanoma cells. In addition, the progression of human melanoma towards the metastatic phenotype is associated with loss of expression of the tyrosine-kinase receptor c-KIT. This review summarizes our recent data demonstrating that the expression of both genes is regulated by the AP-2 transcription factor. Moreover, we have observed a loss of AP-2 expression in metastatic melanoma cells. Re-expression of AP-2 in the highly metastatic A375SM cells decreased their tumorigenicity and inhibited their metastatic potential in nude mice. MCAM/MUC18 mRNA and protein expression was significantly downregulated while c-KIT expression was upregulated in the AP-2 transfected cells. Since AP-2 also regulates other genes that are involved in the progression of human melanoma such as E-cadherin, MMP-2, p21WAF-1, HER-2, BCL-2, and insulin like growth factor receptor-1, we propose that loss of AP-2 is a crucial event in the development of malignant melanoma.

Molecular changes in human melanoma metastasis.

The molecular changes associated with the transition of melanoma cells from radial growth phase to vertical growth phase (metastatic phenotype) are not well defined. Our recent studies have demonstrated that the two tumor suppressor genes, p53 and p16/CDKN2, do not play a major role in the acquisition of the metastatic phenotype in human melanoma. Mutations in p53 are infrequent and do not correlate with the metastatic potential of human melanoma cells while p16/CDKN2 abnormalities are frequent, but are not pre-requisite for the acquisition of the metastatic phenotype. On the other hand, the tyrosine-kinase receptor c-KIT and the cell adhesion molecule MCAM/MUC-18 play active roles in the progression of human melanoma. Metastatic melanoma cells overexpress MCAM and do not express the c-KIT receptor. Enforced c-KIT expression in metastatic cells significantly inhibited their growth and metastatic potential in nude mice. Furthermore, exposure of c-KIT-positive melanoma cells in vitro and in vivo to stem cell factor (SCF), the ligand for c-KIT, triggered apoptosis of these cells but not of normal melanocytes. Ectopic expression of MCAM into primary cutaneous melanoma cells enhanced their tumorigenicity and metastatic ability in vivo. We found that both genes, c-KIT and MCAM, are regulated by the transcription factor AP-2 and that metastatic melanoma cells do not express AP-2. We therefore propose that loss of AP-2 might be a crucial event in the progression of human melanoma.

Loss of AP-2 results in downregulation of c-KIT and enhancement of melanoma tumorigenicity and metastasis.

Expression of the tyrosine kinase receptor, c-KIT, progressively decreases during local tumor growth and invasion of human melanomas. We have previously shown that enforced c-KIT expression in highly metastatic cells inhibited tumor growth and metastasis in nude mice. Furthermore, the ligand for c-KIT, SCF, induces apoptosis in human melanoma cells expressing c-KIT under both in vitro and in vivo conditions. Here we show that loss of c-KIT expression in highly metastatic cells correlates with loss of expression of the transcription factor AP-2. The c-KIT promoter contains three binding sites for AP-2 and EMSA gels demonstrated that AP-2 protein binds directly to the c-KIT promoter. Transfection of wild-type AP-2 into c-KIT-negative A375SM melanoma cells activated a c-KIT promoter-driven luciferase reporter gene, while expression of a dominant-negative AP-2B in c-KIT-positive Mel-501 cells inhibited its activation. Endogenous c-KIT mRNA and expression of proteins were upregulated in AP-2-transfected cells, but not in control cells. In addition, re-expression of AP-2 in A375SM cells suppressed their tumorigenicity and metastatic potential in nude mice. These results indicate that the expression of c-KIT is highly regulated by AP-2 and that enforced AP-2 expression suppresses tumorigenicity and metastatic potential of human melanoma cells, possibly through c-KIT transactivation and SCF-induced apoptosis. Therefore, loss of AP-2 expression might be a crucial event in the development of malignant melanoma.

Loss of AP-2 Results in Up-regulation ofMCAM/MUC18 and an Increase in Tumor Growth and Metastasis of Human Melanoma Cells

MCAM/MUC18 is a cell-surface glycoprotein of 113 kDa, originally identified as a melanoma antigen, whose expression is associated with tumor progression and the development of metastatic potential. We have previously shown that enforced expression of MCAM/MUC18 in primary cutaneous melanoma led to increased tumor growth and metastatic potential in nude mice. The mechanism for up-regulation of MCAM/MUC18 during melanoma progression is unknown. Here we show that up-regulation of MCAM/MUC18 expression in highly metastatic cells correlates with loss of expression of the transcription factor AP-2. The MCAM/MUC18 promoter contains four binding sites for AP-2, and electrophoretic mobility shift assay gels demonstrated that the AP-2 protein bound directly to the MCAM/MUC18 promoter. Transfection of AP-2 into highly metastatic A375SM melanoma cells (AP-2-negative and MCAM/MUC18-positive) inhibited MCAM/MUC18 promoter-driven chloramphenicol acetyltransferase reporter gene in a dose-dependent manner. MCAM/MUC18 mRNA and protein expression were down-regulated in AP-2-transfected but not in control cells. In addition, re-expression of AP-2 in A375SM cells inhibited their tumorigenicity and metastatic potential in nude mice. These results indicate that the expression of MCAM/MUC18 is regulated by AP-2 and that enforced AP-2 expression suppresses tumorigenicity and metastatic potential of human melanoma cells, possibly by down-regulating MCAM/MUC18 gene expression. Since AP-2 also regulates other genes that are involved in the progression of human melanoma such as c-KIT, E-cadherin, MMP-2, and p21(WAF-1), we propose that loss of AP-2 is a crucial event in the development of malignant melanoma.