Sustained attenuation of RICTOR/mTORC2 functions promotes melanoma aggressiveness by paradoxical reactivation of Akt signaling.

ABSTRACT Melanoma is the most severe type of skin cancer, whose incidence over the past several years has dramatically increased, becoming one of the most frequent tumor in fair-skinned populations. About 50% of melanomas are driven by activating mutations of the BRAF oncogene (V600E). Bioinformatic analysis carried out in this work on the entire dataset of skin melanoma patients in the TCGA (The Cancer Genome Atlas) evidenced a significative negative correlation between patients’ survival and levels of RICTOR expression in tumor cells. Rictor is a core component of mTOR complex 2, indispensable for its formation and function. Our precedent work demonstrated that lack of mTORC2 signaling in normal keratinocytes leads to increased survival of these cells under stressful conditions. Considering the results of the bioinformatic analysis on TCGA melanoma patients, we aimed to determine whether Rictor deficiency may provide a growth advantage also to BRAF-mutated melanoma cells. In this setting, Rictor deficient A375 BRAFV600E melanoma cells were generated, and these displayed an impaired mTORC2 signaling. These cells were injected in NSG mice to generate tumor xenografts. Remarkably, Rictor deficient tumors were found endowed with greatly increased growth capacity if compared to Rictor proficient tumors. Surprisingly, downregulation of Rictor in these tumors was paralleled by “paradoxical” hyperphosphorylation of Akt at mTORC2 target site within the hydrophobic regulatory motif, a modification required for full Akt kinase activation. Analysis of Rictor deficient cells after prolonged culture evidenced that the Akt Ser473 was robustly phosphorylated even in the absence of growth factors, and this provides a growth advantage to these cells under reduced serum conditions. Pharmacological inhibition of Akt was able to eliminate such growth advantage in vitro, indicating that Akt signaling activity plays a major role in this context. Overall, our work demonstrates that a sustained Rictor/mTORC2 downregulation promotes tumor progression in the A375 melanoma model, which is consistent with the results obtained in our bioinformatic analysis. Our study also suggests a molecular strategy to overcome the observed aggressive behaviour resulting from Rictor downregulation in melanoma cells.