The kinase pathway comprising RAS, RAF, mitogen-activated protein kinase kinase (MEK) and extracellular signal regulated kinase (ERK) is activated in most human tumours, often through gain-of-function mutations of RAS and RAF family members 1 . Using small-molecule inhibitors of MEKand an integrated genetic and pharmacologic analysis, we find that mutation of BRAF is associated with enhanced and selective sensitivity to MEK inhibition when compared to either 'wild-type' cells or cells harbouring a RAS mutation. This MEK dependency was observed in BRAF mutant cells regardless of tissue lineage, and correlated with both downregulation of cyclin D1 protein expression and the induction of G1 arrest. Pharmacological MEK inhibition completely abrogated tumour growth in BRAF mutant xenografts, whereas RAS mutant tumours were only partially inhibited. These data suggest an exquisite dependency on MEK activity in BRAF mutant tumours, and offer a rational therapeutic strategy for this genetically defined tumour subtype.Activating RAS and BRAF mutations typically demonstrate mutual exclusivity in tumours 1-3 . This suggests an epistatic relationship whereby either mutation is sufficient to deregulate a common effector pathway such as the MEK-ERK kinase cascade. If so,

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 ...

Resistance to RAF- and MEK-targeted therapy is a major clinical challenge1–4. RAF and MEK inhibitors are initially but only transiently effective in some but not all patients with BRAF gene mutation and are largely ineffective in those with RAS gene mutation because of resistance5–14. Through a genetic screen in BRAF-mutant tumor cells, we show that the Hippo pathway effector YAP (encoded by YAP1) acts as a parallel survival input to promote resistance to RAF and MEK inhibitor therapy. Combined YAP and RAF or MEK inhibition was synthetically lethal not only in several BRAF-mutant tumor types but also in RAS-mutant tumors. Increased YAP in tumors harboring BRAF V600E was a biomarker of worse initial response to RAF and MEK inhibition in patients, establishing the clinical relevance of our findings. Our data identify YAP as a new mechanism of resistance to RAF- and MEK-targeted therapy. The findings unveil the synthetic lethality of combined suppression of YAP and RAF or MEK as a promising strategy to enhance treatment response and patient survival.

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.

Although remission rates for metastatic melanoma are generally very poor, some patients can survive for prolonged periods following metastasis. We used gene expression profiling, mitotic index (MI), and quantification of tumor infiltrating leukocytes (TILs) and CD3؉ cells in metastatic lesions to search for a molecular basis for this observation and to develop improved methods for predicting patient survival. We identified a group of 266 genes associated with postrecurrence survival. Genes positively associated with survival were predominantly immune response related (e.g., ICOS, CD3d, ZAP70, TRAT1, TARP, GZMK, LCK, CD2, CXCL13, CCL19, CCR7, VCAM1) while genes negatively associated with survival were cell proliferation related (e.g., PDE4D, CDK2, GREF1, NUSAP1, SPC24). Furthermore, any of the 4 parameters (prevalidated gene expression signature, TILs, CD3, and in particular MI) improved the ability of Tumor, Node, Metastasis (TNM) staging to predict postrecurrence survival; MI was the most significant contributor (HR ‫؍‬ 2.13, P ‫؍‬ 0.0008). An immune response gene expression signature and presence of TILs and CD3؉ cells signify immune surveillance as a mechanism for prolonged survival in these patients and indicate improved patient subcategorization beyond current TNM staging.gene expression analysis ͉ immune response ͉ TNM staging ͉ tumor infiltrating leukocytes

No abstract

Summary To metastasize, a tumor cell must acquire abilities such as the capacity to colonize new tissue and evade immune surveillance. Recent evidence suggests that microRNAs can promote the evolution of malignant behaviors by regulating multiple targets. We performed a microRNA analysis of human melanoma, a highly invasive cancer, and found that miR-30b/30d upregulation correlates with stage, metastatic potential, shorter time to recurrence and reduced overall survival. Ectopic expression of miR-30b/30d promoted the metastatic behavior of melanoma cells by directly targeting the GalNAc transferase GALNT7, resulted in increased synthesis of the immunosuppressive cytokine IL-10, and reduced immune cell activation and recruitment. These data support a key role of miR-30b/30d and GalNAc transferases in metastasis, by simultaneously promoting cellular invasion and immunosuppression.

The immunoregulatory protein T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) mediates T cell exhaustion and contributes to the suppression of immune responses in both viral infections and tumors. Tim-3 blockade reverses the exhausted phenotype of CD4+ and CD8+ T cells in several chronic diseases including melanoma. Interestingly, natural killer (NK) cells constitutively express Tim-3; however, its role in modulating the function of these innate effector cells remains unclear, particularly in human disease. In this study, we compared the function of Tim-3 in NK cells from healthy donors and patients with metastatic melanoma. NK cells from the latter were functionally impaired/exhausted and Tim-3 blockade reversed this exhausted phenotype. Moreover, Tim-3 expression levels correlated with the stage of the disease and poor prognostic factors. These data indicate that Tim-3 can function as an NK cell exhaustion marker in advanced melanoma and supports the development of Tim-3-targeted therapies to restore antitumor immunity.