Metastatic melanoma is a deadly cancer that fails to respond to conventional chemotherapy and is poorly understood at the molecular level. p53 mutations often occur in aggressive and chemoresistant cancers but are rarely observed in melanoma. Here we show that metastatic melanomas often lose Apaf-1, a cell-death effector that acts with cytochrome c and caspase-9 to mediate p53-dependent apoptosis. Loss of Apaf-1 expression is accompanied by allelic loss in metastatic melanomas, but can be recovered in melanoma cell lines by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (5aza2dC). Apaf-1-negative melanomas are invariably chemoresistant and are unable to execute a typical apoptotic programme in response to p53 activation. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment markedly enhances chemosensitivity and rescues the apoptotic defects associated with Apaf-1 loss. We conclude that Apaf-1 is inactivated in metastatic melanomas, which leads to defects in the execution of apoptotic cell death. Apaf-1 loss may contribute to the low frequency of p53 mutations observed in this highly chemoresistant tumour type.

The highly aggressive character of melanoma makes it an excellent model for probing the mechanisms underlying metastasis, which remains one of the most difficult challenges in treating cancer. We find that miR-182, member of a miRNA cluster in a chromosomal locus (7q31-34) frequently amplified in melanoma, is commonly upregulated in human melanoma cell lines and tissue samples; this up-regulation correlates with gene copy number in a subset of melanoma cell lines. Moreover, miR-182 ectopic expression stimulates migration of melanoma cells in vitro and their metastatic potential in vivo, whereas miR-182 down-regulation impedes invasion and triggers apoptosis. We further show that miR-182 over-expression promotes migration and survival by directly repressing microphthalmiaassociated transcription factor-M and FOXO3, whereas enhanced expression of either microphthalmia-associated transcription factor-M or FOXO3 blocks miR-182's proinvasive effects. In human tissues, expression of miR-182 increases with progression from primary to metastatic melanoma and inversely correlates with FOXO3 and microphthalmia-associated transcription factor levels. Our data provide a mechanism for invasion and survival in melanoma that could prove applicable to metastasis of other cancers and suggest that miRNA silencing may be a worthwhile therapeutic strategy.microRNA ͉ cancer ͉ invasion M etastasis is a central problem in cancer, yet the mechanisms underlying a cell's ability to extravasate from the primary tumor, circulate, and invade new tissue remain poorly understood. We reasoned that melanoma, one of the most notoriously invasive neoplasia, would provide an excellent model for investigating the alterations that contribute to metastasis. Melanomas are characterized by certain well-defined genetic alterations (reviewed in ref. 1) as well as frequent chromosomal aberrations associated with tumor progression (2). Recent work has also shown that melanomas display genomic alterations involving numerous microRNA genes (3). MicroRNAs (miRNAs) are endogenous noncoding small RNAs that interfere with the translation of coding messenger RNAs (mRNAs) in a sequence-specific manner (4), often to regulate processes involved in development or tissue homeostasis (5-7). Intriguingly, dysregulation of miRNAs has been found to contribute to neoplasia (8). We decided to investigate the possible contributions of miRNA dysregulation to melanoma extravasation, migration, and invasion.We compared the expression of miRNAs in a large cohort of melanoma cell lines with that of normal melanocytes. We found that miR-182, flanked by the c-MET and BRAF oncogenes in the 7q31-34 region that is frequently amplified in melanoma (9, 10), is highly expressed in metastatic melanoma cell lines and tumors, often in association with increased copy number. Moreover, we demonstrate that antisense-mediated repression of miR-182 inhibited invasion and induced melanoma cell death, whereas ectopic miR-182 up-regulation enhanced the oncogenic activity of melanoma cells in vitro ...

Cancer is a disease consisting of both genetic and epigenetic changes. While increasing evidence demonstrates that tumour progression entails chromatin-mediated changes such as DNA methylation, the role of histone variants in cancer initiation and progression currently remains unclear. Here, we report that the histone variant macroH2A (mH2A) suppresses tumour progression of malignant melanoma. Loss of mH2A isoforms, histone variants generally associated with condensed chromatin and fine-tuning of developmental gene expression programs1-4, is positively correlated with increasing malignant phenotype of melanoma cells in culture and human tissue samples. Knockdown of mH2A isoforms in melanoma cells of low malignancy results in significantly increased proliferation and migration in vitro and growth and metastasis in vivo. Restored expression of mH2A isoforms rescues these malignant phenotypes in vitro and in vivo. We demonstrate that the tumour promoting function of mH2A loss is mediated, at least in part, through direct transcriptional up-regulation of CDK8. Suppression of CDK8, a colorectal cancer oncogene5, 6, inhibits proliferation of melanoma cells, and knockdown of CDK8 in cells depleted of mH2A suppresses the proliferative advantage induced by mH2A loss. Moreover, a significant inverse correlation between mH2A and CDK8 expression levels exists in melanoma patient samples. Taken together, our results demonstrate that mH2A is a critical component of chromatin that suppresses the development of malignant melanoma, a highly intractable cutaneous neoplasm.

The familial melanoma gene (INK4a/MTS1/CDKN2) encodes potent tumor suppressor activity. Although mice null for the ink4a homolog develop a cancer-prone condition, a pathogenetic link to melanoma susceptibility has yet to be established. Here we report that mice with melanocyte-specific expression of activated H-ras G12V on an ink4a-deficient background develop spontaneous cutaneous melanomas after a short latency and with high penetrance. Consistent loss of the wild-type ink4a allele was observed in tumors arising in ink4a heterozygous transgenic mice. No homozygous deletion of the neighboring ink4b gene was detected. Moreover, as in human melanomas, the p53 gene remained in a wild-type configuration with no observed mutation or allelic loss. These results show that loss of ink4a and activation of Ras can cooperate to accelerate the development of melanoma and provide the first in vivo experimental evidence for a causal relationship between ink4a deficiency and the pathogenesis of melanoma. In addition, this mouse model affords a system in which to identify and analyze pathways involved in tumor progression against the backdrop of genetic alterations encountered in human melanomas.

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