Project Abstract Our long-term goal is to identify protein kinase C (PKC) signaling mechanisms that contribute to cancer and translate these mechanistic insights into better prognostic and therapeutic intervention strategies. In previous funding periods, we: 1) identified atypical PKC isozyme PKCι as an oncogene in non-small cell lung cancer (NSCLC), the leading cause of cancer death in the U.S.; 2) established that PKCι enforces a highly aggressive tumor-initiating cell (TIC) phenotype in two major NSCLC subtypes, lung adenocarcinoma (LADC) and lung squamous cell carcinoma (LSCC); and 3) identified a small molecule PKCι inhibitor that shows clinical promise for lung and ovarian cancer treatment. During the current funding period we showed that: 1) PKCι drives a LSCC TIC phenotype by directly phosphorylating and regulating the transcriptional activity of Sox2, a LSCC lineage-specific pluripotent stem cell factor; 2) PKCι establishes a LADC TIC phenotype by phosphorylating and regulating Elf3, a key transcription factor in LADC; 3) PKCι directly phosphorylates and activates the Rho family GTPase GEF Ect2 to regulate its oncogenic activity; 4) Ect2 regulates ribosomal DNA transcription by binding the Upstream binding factor 1 (Ubf1), a major rDNA transcription factor, and recruiting Rac1 and Npm to rDNA to drive transformed growth and lung tumor formation in vivo; 5) PKCι-mediated Ect2 phosphorylation is required for Ect2-driven rDNA transcription. Our preliminary studies indicate that: 1) PKCι phosphorylates Ubf1 at a unique site required for Ect2 binding and rDNA transcription; 2) PKCι-mediated Sox2 phosphorylation regulates Sox2 binding to direct transcriptional targets implicated in LSCC transformation; 3) PKCι and Ect2 are overexpressed in a genetically-tractable mouse model of Sox2-dependent LSCC that faithfully recapitulates many aspects of human LSCC. Based on these data, we hypothesize that: 1) PKCι regulates Ubf1-, Ect2-dependent rRNA transcription and LSCC transformation through phosphorylation- dependent regulation of Ubf1-Ect2 binding interactions; 2) PKCι-mediated Sox2 phosphorylation controls Sox2 transcriptional programming of LSCC TICs; and 3) Prkci and Ect2 are required for Sox2-driven mouse LSCC tumorigenesis in vivo. These hypotheses will be tested by completing three interrelated specific aims designed to: 1) determine the mechanism by which PKCι regulates ribosomal RNA transcription in LSCC cells; 2) identify and functionally characterize direct PKCι-Sox2 transcriptional targets involved in LSCC tumorigenesis; and 3) determine the role of PKCι and Ect2 in Sox2-dependent LSCC tumorigenesis in vivo. Successful completion of these aims will provide significant new insight into how three oncogenes that are coordinately amplified and overexpressed in the vast majority of human LSCCs, PRKCI, ECT2 and SOX2, cooperate to drive LSCC tumorigenesis. Mechanistic insights gained through these studies will provide new therapeutic opportunit...