ABSTRACT
Activating mutations in BRAF are the most common genetic alterations in melanoma. Inhibition of BRAF by small molecules leads to cell-cycle arrest and apoptosis. We show here that BRAF inhibition also induces an oxidative phosphorylation gene program, mitochondrial biogenesis, and the increased expression of the mitochondrial master regulator, PGC1α. We further show that a target of BRAF, the melanocyte lineage factor MITF, directly regulates the expression of PGC1α. Melanomas with activation of the BRAF/MAPK pathway have suppressed levels of MITF and PGC1α and decreased oxidative metabolism. Conversely, treatment of BRAF-mutated melanomas with BRAF inhibitors renders them addicted to oxidative phosphorylation. Our data thus identify an adaptive metabolic program that limits the efficacy of BRAF inhibitors.
Subject(s)
Heat-Shock Proteins/metabolism , Melanoma/metabolism , Microphthalmia-Associated Transcription Factor/metabolism , Proto-Oncogene Proteins B-raf/metabolism , Transcription Factors/metabolism , Apoptosis , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Heat-Shock Proteins/genetics , Humans , Indoles/pharmacology , Melanocytes/metabolism , Melanoma/genetics , Mitochondria/metabolism , Mitogen-Activated Protein Kinases/metabolism , Mutation , Oxidative Phosphorylation , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , RNA, Messenger/biosynthesis , Signal Transduction , Sulfonamides/pharmacology , Transcription Factors/genetics , VemurafenibABSTRACT
The RAS to extracellular signal-regulated kinase (ERK) signal transduction cascade is crucial to cell proliferation, differentiation, and survival. Although numerous growth factors activate the RAS-ERK pathway, they can have different effects on the amplitude and duration of the ERK signal and, therefore, on the biological consequences. For instance, nerve growth factor, which elicits a larger and more sustained increase in ERK phosphorylation in PC12 cells than does epidermal growth factor (EGF), stimulates PC12 cell differentiation, whereas EGF stimulates PC12 cell proliferation. Here, we show that protein arginine methylation limits the ERK1/2 signal elicited by particular growth factors in different cell types from various species. We found that this restriction in ERK1/2 phosphorylation depended on methylation of RAF proteins by protein arginine methyltransferase 5 (PRMT5). PRMT5-dependent methylation enhanced the degradation of activated CRAF and BRAF, thereby reducing their catalytic activity. Inhibition of PRMT5 activity or expression of RAF mutants that could not be methylated not only affected the amplitude and duration of ERK phosphorylation in response to growth factors but also redirected the response of PC12 cells to EGF from proliferation to differentiation. This additional level of regulation within the RAS pathway may lead to the identification of new targets for therapeutic intervention.
Subject(s)
Cell Differentiation/physiology , Cell Proliferation , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Protein Methyltransferases/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Animals , COS Cells , Cell Differentiation/drug effects , Chlorocebus aethiops , Epidermal Growth Factor/metabolism , Epidermal Growth Factor/pharmacology , HEK293 Cells , Humans , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Nerve Growth Factor/metabolism , Nerve Growth Factor/pharmacology , PC12 Cells , Phosphorylation/drug effects , Phosphorylation/physiology , Protein Methyltransferases/genetics , Protein-Arginine N-Methyltransferases , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins c-raf/genetics , RatsABSTRACT
BACKGROUND: Melanoma is the most deadly form of skin cancer without effective treatment. Methylthioadenosine (MTA) is a naturally occurring nucleoside with differential effects on normal and transformed cells. MTA has been widely demonstrated to promote anti-proliferative and pro-apoptotic responses in different cell types. In this study we have assessed the therapeutic potential of MTA in melanoma treatment. METHODS: To investigate the therapeutic potential of MTA we performed in vitro proliferation and viability assays using six different mouse and human melanoma cell lines wild type for RAS and BRAF or harboring different mutations in RAS pathway. We also have tested its therapeutic capabilities in vivo in a xenograft mouse melanoma model and using variety of molecular techniques and tissue culture we investigated its anti-proliferative and pro-apoptotic properties. RESULTS: In vitro experiments showed that MTA treatment inhibited melanoma cell proliferation and viability in a dose dependent manner, where BRAF mutant melanoma cell lines appear to be more sensitive. Importantly, MTA was effective inhibiting in vivo tumor growth. The molecular analysis of tumor samples and in vitro experiments indicated that MTA induces cytostatic rather than pro-apoptotic effects inhibiting the phosphorylation of Akt and S6 ribosomal protein and inducing the down-regulation of cyclin D1. CONCLUSIONS: MTA inhibits melanoma cell proliferation and in vivo tumor growth particularly in BRAF mutant melanoma cells. These data reveal a naturally occurring drug potentially useful for melanoma treatment.
