In this revised renewal application, we propose to investigate how cell survival mechanisms contribute to the invasive properties of endothelial cells (ECs) and tumor cells during angiogenesis and metastasis, respectively. In the previous funding cycle, we showed that bFGF and VEGF suppress apoptosis of angiogenic ECs induced by intrinsic and extrinsic death pathways, respectively. These growth factors differed in their pro-survival functions based on their ability to differentially activate Raf-1 kinase. In recent studies, we have identified apoptosis signal regulating kinase 1 (ASK1) as a target of pro-survival signaling by bFGF, but not VEGF, in ECs.
In Aim 1 of this proposal, we outline studies to characterize the mechanism by which Raf-1/ASK1 interact and how this interaction regulates the pro-apoptotic activity of ASK1, thereby facilitating EC invasion during angiogenesis and resistance to anti-angiogenic chemotherapeutics. The role of ASK1 in regulation of EC survival during angiogenesis and chemoresistance of tumor-associated vasculature will be examined using ASK1 knockout animals that we have recently obtained. Elevated EGFR and Src kinase activities among invasive tumor cells has been shown to promote anchorage-independent growth and survival. We have shown that Src activity leading to Raf-1 phosphorylation within its activation domain on tyrosines 340/341 promotes cellular protection from death receptor-mediated apoptosis. Thus, in Aim 2 we will test the hypothesis that the EGFR/Src/Raf signaling module and its ability to promote cell survival is critical for metastasis of invasive carcinoma cells. We contend that suppression of this survival pathway will interfere with metastatic disease within tissue microenvironments where otherwise death receptor-mediated apoptosis is limiting. Finally, in Aim 3 we propose to establish how the extracellular matrix (ECM) influences the metastatic properties of invasive tumor cells. During the previous cycle, we identified a novel apoptotic process termed integrin-mediated death (IMD) in which caspase 8 recruitment to unligated integrins on inappropriately placed cells leads to apoptosis. We recently determined that loss of IMD by suppression of caspase 8 expression was sufficient to promote invasion and metastasis in vivo. Therefore, we will establish how caspase 8 becomes activated during IMD and test the hypothesis that rescue of caspase 8 in metastatic tumor cells renders them non-metastatic. The studies outlined in this proposal will established a molecular basis to support our hypothesis that invasive cell behavior depends on the ability of cells to suppress one or more of the above mentioned apoptotic pathways. These studies may reveal new therapeutic strategies to regulate angiogenesis or the invasive properties of cancer cells. ? ? ?
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