There is a growing need for assays capable of assessing lipid signaling in colorectal tumors from disease models and patients. This need has been created by the advent of molecularly targeted therapies aimed at aberrant signal transduction pathways, particularly those involving phosphoinositide-3 kinase (PI3K), now known to play important roles in cancer initiation and progression. The ability to measure the enzymatic activity of dysregulated lipid signaling proteins would be extremely advantageous for directing treatment options as well as assessing treatment efficacy in individual patients. A collaborative interdisciplinary research program is proposed to create the instrumentation and chemical tools needed to directly assess the metabolism of phosphatidyl-inositol 4,5-bisphosphate (PI(4,5)P2) by PI3K and related enzymes in living cells from colorectal cancer cell lines and patient-derived metastatic colorectal cancer. The investigators will develop and optimize a microelectrophoresis platform for the biochemical analysis of single-cell samples using fluorescently labeled lipid substrates. A variety of fluorescent PI(4,5)P2 analogs will be designed, synthesized and screened for their suitability to report intracellular activity of PI3K i cells with and without pharmacologic inhibition. Initial screens will be performed in vitro using purified enzymes and cell lysates. The most promising compounds will then be tested in model colorectal carcinoma cell lines. The optimal reporter(s) will be employed to test clinically relevat hypotheses such as whether the PI3K pathway is activated in cells in a bimodal fashion and whether the PI3K pathway is used for dynamic reprogramming of cell signaling in response to targeted inhibitors. Finally, the biochemical activity of PI3K and other PI(4,5)P2-metabolizing enzymes will be profiled before and after pharmacologic therapy in single primary cells from metastatic tumors from colorectal cancer patients. The proposed technology will provide a new approach for clinical assays relevant to the emerging field of personalized medicine.
The purpose of the proposed research is to develop and implement an assay system for the study of active lipid signal transduction pathways in human colorectal cancer cells. The goal of this work is to enhance our understanding of this important aspect of the molecular basis of cancer and to lead to improved therapeutic interventions.