To identify protein kinases with functional somatic mutations that contribute to the process of tumorigenesis, protein kinases newly implicated in cancer through the presence of putative driver mutations identified in recent cancer genome sequencing efforts will be characterized. Initially, the focus will be on four such kinases, DAPK3, SGK085 and MLK4, which score highly in the devised ranking system as being the most likely to play a role in cancer, and protein kinase C family members. In vitro and in vivo analysis of kinase activity will be carried out in order to determine whether individual mutations in DAPK3, SGK085 and MLK4 increase or decrease activity, attempt to identify substrates for the mutant and WT kinases and define in what signaling pathway(s) they act, and determine the effects of expressing mutant and WT kinases in normal and cancer cells, assaying for changes in proliferation, cell cycle progression, apoptosis and autophagy, morphological transformation, anchorage independent growth in soft agar, and tumorigenesis in nude mice. This will define whether each mutant kinase has gain- or loss-of-function mutations, whether it acts as an oncoprotein or opposes the effects of its normal tumor suppressor kinase counterpart, and whether the kinase might serve as a new drug target for cancer therapy. The protein kinase C (PKC) family of kinases has been extensively studied in cancer, through their role as receptors for tumor promoting chemicals, but few cancer mutations have been identified, and there is a debate regarding whether activation or inactivation of PKC family of kinases is important for cancer. The discovery of nonsynonymous point mutations in many PKCs, mainly in colorectal cancer (CRC) and glioblastoma multiforme (GBM), provides an opportunity to determine if PKCs are activated or inactivated in cancer and determine how these mutations contribute to tumorigenesis. Mutations observed in the PKC family of isozymes in cancer utilizing live-cell imaging techniques will be characterized. Additionally, all the PKC mutations we are studying in CRC occur in the context of activating K-Ras mutations. K-Ras is a substrate for PKC, and phosphorylation of K-Ras by PKC alters its subcellular targeting and causes K-Ras to promote apoptosis. The intention is to determine if loss of function mutations in PKC can promote the survival of colon cancer cells harboring K-Ras mutations, by suppressing this K-Ras-induced apoptotic feedback loop, thereby fine-tuning oncogenic K-Ras addiction. An investigation of the mutations in the atypical PKC, aPKC6, in GBM will be carried out to determine their effect on the activity of aPKC6 using similar approaches and their transforming potential will be tested in a new mouse model for GBM.
The studies proposed in this application seek to elucidate how protein kinases that have been newly implicated in cancer through cancer genome sequencing act in the initiation and maintenance of cancer phenotypes, with the ultimate goal of identifying protein kinases and pathways that may offer the possibility of defining new cancer therapeutic targets.
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