Abstract Colorectal cancer (CRC) is the second-leading cause of death from cancer in the USA. Chronic inflammation in the gastrointestinal tract is a significant risk factor for developing CRC. Generally, CRC involves the upregulation of inflammation-induced cyclooxygenase-2 (COX-2) that is blocked by non-steroidal anti-inflammatory drugs (NSAIDS). Among the downstream prostaglandin synthases that produce bioactive prostaglandins (PGs), prostaglandin E2 (PGE2) synthase products are proinflammatory. After export from cells, PGE2 can bind to G-protein coupled PG cell surface receptors EP2/4 and activate adenylate cyclase and elevate cAMP. In turn, cAMP activates downstream effectors such as protein kinase A (PKA) and/or exchange protein directly activated by cAMP (EPAC). We studied effects of PGE2 on PKA activation in CRC cells and their relationship to lipid raft function. After isolating lipid rafts from PGE2 stimulated cells we performed shotgun proteomics. Among the lipid raft proteins identified were DNA-protein kinase (DNA-PK), and heterodimer complex proteins Ku70 and Ku80, which we verified by immunoprecipitation and immunofluorescence. Of these lipid raft proteins, Ku80 contained a PKA consensus phosphorylation sites and phosphorylation increased after stimulation of CRC cells by PGE2. Typically found in the nucleus, Ku70, Ku80 and DNA-PK complexes bind to DNA double-strand breaks and initiate non-homologous end joining DNA repair. These data are the first to demonstrate the Ku70, Ku80 and DNA-PK complexes in a new non-nuclear action, which involves lipid rafts and the DNA repair complex. Furthermore, PGE2 stimulated LS174T CRC cells exhibit an increase in non-nuclear pDNA-PK (S2056). Additionally, treatment of a number of colon cancer cell lines with the selective DNA-PK inhibitor Nu7026 significantly decreased cell viability in the absence of any overt DNA damaging stimuli, suggesting that non-nuclear, and in particular, lipid raft localized DNA-PK complex may play an important role in cell growth. Finally, treatment with both PGE2 and Nu7026 causes a profound change in cell morphology involving numerous spindle-like processes. These data suggest there are important downstream effects of proinflammatory PGE2 on CRC cells that influence lipid raft associated Ku70, Ku80 and DNA-PK complexes as well as cell proliferation. Support: T. Boone Pickens Distinguished Chair for Early Prevention of Cancer to E.T.H., R37 DK47297, NCI P01 CA77839, CPRIT-RP100960, Ellen F. Knisely Distinguished Chair in Colon Cancer Research, The National Colorectal Cancer Research Alliance to R.N.D. and 5U54CA151668-02 to D.G.M. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3251. doi:1538-7445.AM2012-3251