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(V600E)BRAF is associated with disabled feedback inhibition of RAF-MEK signaling and elevated transcriptional output of the pathway.

ABSTRACT: Tumors with mutant BRAF and those with receptor tyrosine kinase (RTK) activation have similar levels of phosphorylated ERK, but only the former depend on ERK signaling for proliferation. The mitogen-activated protein kinase, extracellular signal-regulated kinase kinase (MEK)/ERK-dependent transcriptional output was defined as the genes whose expression changes significantly 8 h after MEK inhibition. In (V600E)BRAF cells, this output is comprised of 52 genes, including transcription factors that regulate transformation and members of the dual specificity phosphatase and Sprouty gene families, feedback inhibitors of ERK signaling. No such genes were identified in RTK tumor cells, suggesting that ERK pathway signaling output is selectively activated in BRAF mutant tumors. We find that RAF signaling is feedback down-regulated in RTK cells, but is insensitive to this feedback in BRAF mutant tumors. Physiologic feedback inhibition of RAF/MEK signaling down-regulates ERK output in RTK cells; evasion of this feedback in mutant BRAF cells is associated with increased transcriptional output and MEK/ERK-dependent transformation.

SUBMITTER: Pratilas CA

PROVIDER: S-EPMC2649208 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background Main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF- mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to define in this context the specific role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit and regarded as an oncogenic driver in several tumor types, including MM. Methods After analyzing the TCGA MM patients’ database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAF V600E MM cell lines on their response to BRAF/MEKi in vitro. We performed a proteomic screening to identify proteins modulated by changes in RICTOR expression and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. Results We found that low RICTOR levels in MM correlate with an overall worse clinical outcome. GSEA of low- RICTOR tumors revealed a gene expression signature suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy producing pathway. RICTOR-deficient BRAF V600E cells are intrinsically tolerant to BRAFi/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. In RICTOR- depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, while their pharmacological inhibition restores sensitivity to BRAFi. Conclusions Our work unveils a novel tumor suppressing role for mTORC2 in the responses of BRAF V600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low- RICTOR tumors. Importantly, our findings support the concept that the evaluation of intra-tumor RICTOR levels in MM has a prognostic value, and may help predicting the response of patients to targeted therapy.

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(V600E)BRAF is associated with disabled feedback inhibition of RAF-MEK signaling and elevated transcriptional output of the pathway.

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