Our laboratory investigations are focused on studying genes and pathways that regulate growth, differentiation, and programmed cell death of epithelial cancer cells with a focus on breast and ovarian cancer. These investigations will be used as the basis for preclinical development of targeted therapeutics to breast and ovarian cancers including the characterization of the mechanisms that regulate sensitivity and resistance of cancer cells to targeted therapeutics. Ongoing projects include: (1) the regulation of growth factor receptor function by cbl proteins; (2) the function of death receptors in epithelial cancer cells; and (3) the assessment of molecular effects of EGFR inhibitors in cancer patients. (1) The regulation of growth factor receptor function by cbl proteins. Receptor tyrosine kinases, such as the EGFR, are often inappropriately active (due to mutation or overexpression) in a wide array of epithelial malignancies. Understanding the mechanisms that regulate signaling by these receptors should uncover new ways to inhibit cancer cell growth. cbl proteins were first implicated as negative regulators of the EGFR in genetic studies from C. elegans. Our lab has cloned two of the three members of the mammalian cbl protein family. We have shown that all mammalian cbl proteins act as ubiquitin protein ligases for the activated EGFR resulting in the ubiquitination and degradation of the activated EGFR. We have further shown that cbl proteins, as well as other members of the signaling complex, are degraded upon activation of the EGFR. This coordinated degradation is dependent on the cbl protein ubiquitin ligase activity. Thus, cbl proteins negatively regulate EGFR function by degrading the active receptor signaling complex. In addition, the cbl proteins negatively autoregulate their own activity as they are degraded along with their targets. Ongoing work is focused on understanding the biochemical and physiologic functions of the three mammalian cbl proteins and elucidating the differences in the specificity and/or function of the three mammalian cbl proteins. We are also collaborating with Josef Penninger at the Amgen Institute in Toronto to knock out cbl-b and cbl-3 in order to study the role of the different cbl proteins in vivo. cbl-b null mice have been generated and the cbl-3 knockout is in progress. (2) The function of death receptors in epithelial cancer cells. Cancer cells avoid apoptosis by a variety of genetic and epigenetic mechanisms. We are investigating the expression and function of death receptors of the TNFR family (e.g. TNFR, Fas, Dr3, Dr4, and Dr5) and their ligands (e.g. TNF, Fas Ligand, and TRAIL) in normal and malignant breast and ovarian epithelial cells in order to selectively trigger apoptosis in the cancer cells. In our initial studies of the Fas receptor, we demonstrated that most breast cancer cells are resistant to Fas-induced apoptosis while the normal breast epithelial cells are sensitive. Treatment of resistant breast cancer cells with interferon- increased the expression of the Fas receptor and of downstream caspases. This reversed the resistance of breast cancer cells to Fas-induced apoptosis. We have shown that most normal and malignant breast cancer cell lines are resistant to the induction of apoptosis by TRAIL, the ligand for Fas-related death receptors DR4 and DR5. This resistance can be overcome by co-incubation of the cells with sub-toxic doses of drugs such as adriamycin. More recently, we have shown that many ovarian cancer cell lines are also resistant to TRAIL-induced apoptosis and again that chemotherapeutic agents, commonly used to treat ovarian cancer, synergistically enhance TRAIL-induced apoptosis. We have also shown that trastuzumab, the monoclonal antibody to the erbB2 receptor, specifically enhanced TRAIL-induced apoptosis in cancer cells which overexpress the erbB2 receptor but not in cells with normal or low levels of erbB2. Thus, this may allow us to target TRAIL-induced apoptosis to a subset of cancer cells without affecting normal cells. Mechanistically, we have shown that trastuzumab enhances TRAIL-induced apoptosis by downregulating the erbB2 receptor and that this results in decreased AKT activity. Our current work is focused on understanding the mechanisms that underlie the resistance to death mediated by TRAIL ligand in breast and ovarian cancer cells. Our work includes screening death receptor pathway components for evidence of mutations that could account for the resistance of tumor cells to apoptosis. In addition, we are investigating mechanisms to overcome this resistance. (3) The assessment of molecular effects of EGFR inhibitors in cancer patients. In collaboration with Sandra Swain, we are investigating the biochemical consequences of EGFR inhibition in a pilot study of breast cancer patients treated with the small molecule EGFR inhibitor OSI 774. In this pilot study, tumors are biopsied before and after treatment with OSI-774. The function of the EGFR and of downstream MAP and AKT kinases are being assessed.