PHOSPHOINOSITIDE 3-KINASE ASSAYS IN CANCER DIAGNOSIS

Phosphoinositides are key lipid second messengers in cellular signaling. No approved diagnostics evaluate lipids as markers for cancer, but analysis of lipids in addition to protein and DNA may enhance overall accuracy of diagnosis and simultaneously suggest a molecular target for treatment. The tumor promoter phosphatidylinositol 3-kinase (PI3-K) regulates protein trafficking, proliferation and differentiation, tumorigenesis, and apoptosis. Both PI 3-K and its lipid product PI(3,4,5)P3 are potential tumor markers for the early detection of cancer. PI(3,4,5)P3 accumulation is exacerbated by mutations in the tumor suppressor PTEN, a lipid 3-phosphatase. In Phase I, Echelon has demonstrated feasibility and developed reagents and assay platforms to measure PI 3-K activity and to measure lipid product PI(3,4,5)P3 in high-throughput microplate assays. In addition, we have developed and produced two monoclonal antibodies to PI(3,4,5)P3 that show striking results in immunodetection of PI(3,4,5)P3 in pathology samples from breast, ovarian, prostate, and colorectal cancers. In Phase II, we will validate our methods for identification of PI(3,4,5)P3 levels in drug discovery assays and in serum/cell extract detection formats. Anti- PI(3,4,5)p3, antibodies will be used for immunohistochemistry of cancer samples with known disease progression. The data will be analyzed quantitatively and used to establish protocols for human clinical trials to validate the prognostic value of these lipid markers for aggressive cancers. PROPOSED COMMERCIAL APPLICATIONS: Microplate assays for PI 3-K and PI(3,4,5)P3 quantification have value in research, clinical, and drug development settings. Systematic screening of cell extracts and pathology samples will allow rapid and early detection of an important molecular lesion in many cancers.