Signaling through the insulin-like growth factor receptor (IGF-1R) is associated with the aberrant growth of several cancer types, including breast cancer, Members of the Forkhead (Fl-I) family of transcription factors were recently discovered to act downstream of IGF-1R, phosphatidylinositide-3-kinase (Ptdlns-3-kinase) and Akt to mediate cell survival, hi vitro studies have demonstrated that when survival signaling is blocked, dephosphorylated Fl-I proteins accumulate in the nucleus where they act as transcriptional activators to induce apoptosis. The proposed pilot project will test the hypothesis that activation of FH transcriptional activity can be used to monitor the effectiveness of cancer therapies that disrupt survival signaling. Breast cancer cell lines will be used as the model system; the cells will be treated with wortmannin, DPIEL, 17AAG or chloronovobiocin to block survival signaling.
The specific aims are: 1. To assess the effect of anti-survival signaling drugs on Fl-i activity. Western blots will be used to monitor the level of phosphorylated PH proteins, laser scanning cytometry will be used to determine the cellular localization of phosphorylated vs. total EN proteins and transient transfections with a FE reporter construct will be used to measure transcriptional activity. 2. To determine whether FH transcriptional activity is essential for induction of apoptosis. Sensitivity to the anti-survival signaling drugs will be tested after transient transfection of cells with a dominant negative FH gene. 3. To develop assays for monitoring PH activation in small numbers of cells. Real time TaqMan PCR will be used to monitor expression of FasL, a FH-responsive gene, in a population of cells and Immunohistochemistry will be used to detect FasL protein levels in individual cells. The long-term objective of the pilot project is identify downstream transcriptional targets for monitoring the effectiveness ?of therapies directed against survival signaling. The ability to monitor such targets is expected to provide definitive evidence, in a clinical setting that a drug has induced apoptosis of the cancer cells.
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