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Inhibition of mTORC2/RICTOR Impairs Melanoma Hepatic Metastasis.

ABSTRACT: Mammalian target of rapamycin complex 2 (mTORC2) with its pivotal component rapamycin-insensitive companion of mTOR (RICTOR) is the major regulator of AKT phosphorylation and is increasingly implicated in tumor growth and progression. In cutaneous melanoma, an extremely aggressive and highly metastatic disease, RICTOR overexpression is involved in tumor development and invasiveness. Therefore, we investigated the impact of RICTOR inhibition in melanoma cells in vitro and in vivo with special emphasis on hepatic metastasis. Moreover, our study focused on the interaction of tumor cells and hepatic stellate cells (HSC) which play a crucial role in the hepatic microenvironment. In silico analysis revealed increased RICTOR expression in melanoma cells and tissues and indicated higher expression in advanced melanoma stages and metastases. In vitro, transient RICTOR knock-down via siRNA caused a significant reduction of tumor cell motility. Using a syngeneic murine splenic injection model, a significant decrease in liver metastasis burden was detected in vivo. Moreover, stimulation of melanoma cells with conditioned medium (CM) from activated HSC or hepatocyte growth factor (HGF) led to a significant induction of AKT phosphorylation and tumor cell motility. Blocking of RICTOR expression in cancer cells diminished constitutive and HGF-induced AKT phosphorylation as well as cell motility. Interestingly, RICTOR blockade also led to an abrogation of CM-induced effects on AKT phosphorylation and motility in melanoma cells. In conclusion, these results provide first evidence for a critical role of mTORC2/RICTOR in melanoma liver metastasis via cancer cell/HSC interactions.

SUBMITTER: Schmidt KM

PROVIDER: S-EPMC6224335 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Inhibition of mTORC2/RICTOR Impairs Melanoma Hepatic Metastasis.

Schmidt Katharina M KM Dietrich Peter P Hackl Christina C Guenzle Jessica J Bronsert Peter P Wagner Christine C Fichtner-Feigl Stefan S Schlitt Hans J HJ Geissler Edward K EK Hellerbrand Claus C Lang Sven A SA

Neoplasia (New York, N.Y.) 20181104 12

Mammalian target of rapamycin complex 2 (mTORC2) with its pivotal component rapamycin-insensitive companion of mTOR (RICTOR) is the major regulator of AKT phosphorylation and is increasingly implicated in tumor growth and progression. In cutaneous melanoma, an extremely aggressive and highly metastatic disease, RICTOR overexpression is involved in tumor development and invasiveness. Therefore, we investigated the impact of RICTOR inhibition in melanoma cells in vitro and in vivo with special emp ...[more]

PMID: 30404068

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Project description:Nearly 50% of cutaneous melanomas carry activating mutations on the BRAF oncogene, and the combination of BRAF- and MEK-inhibitors (BRAF/MEKi) is frequently used for their clinical management. One major drawback of BRAF/MEKi targeted therapy is the rapid development of therapeutic resistance, which can occur via multiple mechanisms, including the metabolic rewiring of cancer cells that often involves the upregulation of mitochondrial bionergetics and NAD+ biosynthetic pathways. mTORC2 is a signaling complex that requires the presence of its essential RICTOR subunit to play its regulatory functions in cell growth and metabolism. mTORC2 is believed to play mostly pro-oncogenic roles in several tumor types, including melanoma. However, bioinformatics analysis of TCGA melanoma patients’ database revealed that low RICTOR levels in tumors correlate with an overall worse clinical outcome. GSEA analysis of low-RICTOR tumors evidenced also a gene expression signature suggestive of activation of mitochondrial energy producing pathways. On these bases, we have hypothesized that inhibition of mTORC2 activity may render BRAFV600E melanoma cells resistant to BRAFi/MEKi. We show here that RICTOR/mTORC2-deficient cells are intrinsically tolerant to BRAFi/MEKi, and anticipate the onset of resistance to BRAFi after sustained drug exposure both in vitro and in vivo, indicating that mTORC2 activity normally opposes the acquisition of targeted therapy resistance in BRAFV600E melanomas. Mechanistically, RICTOR-deficient cells show an enhanced mitochondrial respiratory potential and increased expression of nicotinamide phosphoribosyltransferase (NAMPT) protein, the rate-limiting enzyme of NAD+ salvage pathway, and pharmacological inhibition of these processes in RICTOR-deficient cells is sufficient to restore sensitivity to BRAFi. Thus, our work identifies a novel role for mTORC2 in favoring the responses of BRAF-mutated melanoma cells to targeted therapy, and suggest that the evaluation of the intratumor level of RICTOR may help to predict the responses of melanoma patients to these treatments.

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.

Dataset Information

Inhibition of mTORC2/RICTOR Impairs Melanoma Hepatic Metastasis.

Publications

Inhibition of mTORC2/RICTOR Impairs Melanoma Hepatic Metastasis.

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