ABSTRACT
Several studies have highlighted the importance of the PI3K pathway in melanocytes and its frequent over-activation in melanoma. However, little is known about regulation of the PI3K pathway in melanocytic cells. We showed that normal human melanocytes are less sensitive to selective PI3K or mTOR inhibitors than to dual PI3K/mTOR inhibitors. The resistance to PI3K inhibitor was due to a rapid AKT reactivation limiting the inhibitor effect on proliferation. Reactivation of AKT was linked to a feedback mechanism involving the mTORC2 complex and in particular its scaffold protein RICTOR. RICTOR overexpression in melanocytes disrupted the negative feedback, activated the AKT pathway and stimulated clonogenicity highlighting the importance of this feedback to restrict melanocyte proliferation. We found that the RICTOR locus is frequently amplified and overexpressed in melanoma and that RICTOR over-expression in NRAS-transformed melanocytes stimulates their clonogenicity, demonstrating that RICTOR amplification can cooperate with NRAS mutation to stimulate melanoma proliferation. These results show that RICTOR plays a central role in PI3K pathway negative feedback in melanocytes and that its deregulation could be involved in melanoma development.
Subject(s)
Carrier Proteins/metabolism , Melanocytes/metabolism , Melanoma/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Blotting, Western , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Carrier Proteins/genetics , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Cells, Cultured , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Humans , Imidazoles/pharmacology , Melanocytes/drug effects , Melanoma/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mutation , Phosphoinositide-3 Kinase Inhibitors , Pyrimidines/pharmacology , Rapamycin-Insensitive Companion of mTOR Protein , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Triazines/pharmacologyABSTRACT
There has been a great deal of interest in understanding the role of KIT in melanoma since the discovery of KIT mutations in a subset of melanoma. Although a significant proportion of these melanomas respond to KIT inhibitors, the presence of a KIT mutation does not guarantee a response to KIT inhibitors. Because recent data seem to indicate that only melanoma with specific KIT mutations respond to KIT inhibitors, we investigated which KIT mutations are driver mutations in melanoma and are therefore therapeutically relevant. We established that 70% of KIT mutations in melanoma are located in four hotspots (L576, K642, W557-V560, and D816-A829) and that these mutations are oncogenic in melanocytes and are bona-fide driver mutations. Testing for KIT mutations should therefore concentrate on these four hotspots, which can be targeted therapeutically.
