The Small Gtpase Rab35 Is A Novel Oncogenic Regulator Of Pi3K/Akt Signaling

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The phosphatidylinositol 4,5-bisphosphate 3'-OH kinase (PI3K) is a lipid kinase that regulates cell survival, proliferation and metabolism in response to external growth factors. PI3K signaling regulates a diverse set of effectors via the phosphoinositide dependent kinase 1 (PDK1), AKT/PKB, and mechanistic target of rapamycin complexes 1 and 2 (mTORC2). The components of this pathway are frequently mutated in human cancers, which give rise to oncogenic signals that drive tumorigenesis. As such, the proteins involved in PI3K/AKT signaling are attractive targets for targeted therapies. It is thought that most tumors upregulate PI3K/AKT signaling in some way. However, a large portion of tumors do not have any identifiable genetic lesions in the genes that code for proteins that regulate this pathway. Thus, we reasoned that there are likely to be many proteins that regulate PI3K/AKT signaling that are currently unappreciated. To identify novel regulators of the PI3K axis, we undertook an arrayed loss-of-function RNAi screen using a library of shRNA reagents that targeted all known human kinases and GTPases to identify proteins whose depletion altered AKT phosphorylation. Further, we triaged genes from this screen using oncogenomic databases to identify screen hit genes that were mutated in human tumor samples or cell lines. We reasoned that this approach could identify novel components of PI3K/AKT signaling that also play a role in tumorigenesis. This screen identified the small GTPase RAB35 as a novel positive regulator of PI3K/AKT signaling. Depletion of RAB35 in a variety of human and murine cell lines suppressed phosphorylation of PI3K-dependent proteins like AKT, FOXO1/3A and NDRG1. Moreover, stable expression of a GTPase-deficient, constitutively active allele of RAB35--but not wildtype RAB35--potently activated AKT signaling in the absence of growth factors. Further, we identified two RAB35 mutations in human tumor genome sequence databases that activated PI3K signaling and transformed NIH-3T3 cells in vitro in a PI3K-dependent manner. Finally, we find that RAB35 is likely regulating PI3K signaling by trafficking the platelet derived growth factor receptor (PDGFR) to a RAB7-positive compartment in the cell where the receptor actively signals PI3K. Thus, RAB35 is a novel oncogenic regulator of PI3K/AKT signaling.

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Cell & Developmental Biology

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Doctor of Philosophy

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