UVM Theses and Dissertations
Format:
Print
Author:
Cassavaugh, Jessica M.
Dept./Program:
Pharmacology
Year:
2012
Degree:
PhD
Abstract:
Angiogenesis. the growth of new blood vessels from existing ones, is a primary cell response to hypoxia and significantly contributes to progression of cancer and vascular disorders. Current approaches to reduce angiogenic signaling in cancer have not been as successful as originally hoped, and the development of more effective therapies for regulation of angiogenesis is needed. Angiogenesis is primarily mediated through hypoxic stabilization of the transcription factor, hypoxia-inducible factor-1[alpha] (HIF-1[alpha]). Once stabilized, HIF1[alpha] initiates the transcription of multiple genes necessary for survival in hypoxic conditions. including activators that stimulate progression of angiogenesis. The overall goal of this research is to betterunderstand the mechanisms that promote and inhibit HIF-1[alpha] signaling to create more effective angiogenic treatments.
The primary mechanism of HIF-1[alpha] regulation in normal oxygen tension is mediated through interactions with the E3 ubiquitin ligase, von Hippel-Lindau, and is well understood. However, the mechanism of HIF-1[alpha] regulation in hypoxia remains unclear. HIF-1[alpha] was shown to be phosphorylated by glycogen synthase kinase 3 (GSK3) in hypoxia, and it was suggested that this phosphorylation leads to decreased HIF-1[alpha] stability; yet, no mechanism was established. We therefore tested the hypothesis that phosphorylation of HIF-1[alpha] by GSK3 promotes the interaction of HIF-1[alpha] with a novel E3 ubiquitin ligase, FBW7, leading to regulation of HIF-1[alpha] stabilization during hypoxia.
Using biochemical, cellular and in vivo techniques in an ovarian cancer model, we found that during hypoxia, HIF-1[alpha] is negatively regulated through phosphorylation by glycogen synthase kinase 3 (GSK3) and through interactions with FBW7. Inhibition of both GSK3 and FBW7 reduced ubiquitination of HIF-1[alpha] in hypoxia and mutation of GSK3 phosphorylation sites within HIF-1[alpha] greatly reduced the FBW7-HIF-1[alpha] interaction. GSK3 and FBW7 were also demonstrated to negatively affect endothelial cell tube formation and vessel growth in the chorioallantoic membrane assay, a model of in vivo angiogenesis. The 'findings within this dissertation are the first to identify both a mechanism of ubiquitin-dependent degradation of HIF-1[alpha] in hypoxia and two new potential targets for future therapeutic development. This novel regulatory mechanism of HIF-1alpha] will hopefully provide a starting point for the creation of new therapeutics to combat dysregulated angiogenesis and improve patient outcomes.
The primary mechanism of HIF-1[alpha] regulation in normal oxygen tension is mediated through interactions with the E3 ubiquitin ligase, von Hippel-Lindau, and is well understood. However, the mechanism of HIF-1[alpha] regulation in hypoxia remains unclear. HIF-1[alpha] was shown to be phosphorylated by glycogen synthase kinase 3 (GSK3) in hypoxia, and it was suggested that this phosphorylation leads to decreased HIF-1[alpha] stability; yet, no mechanism was established. We therefore tested the hypothesis that phosphorylation of HIF-1[alpha] by GSK3 promotes the interaction of HIF-1[alpha] with a novel E3 ubiquitin ligase, FBW7, leading to regulation of HIF-1[alpha] stabilization during hypoxia.
Using biochemical, cellular and in vivo techniques in an ovarian cancer model, we found that during hypoxia, HIF-1[alpha] is negatively regulated through phosphorylation by glycogen synthase kinase 3 (GSK3) and through interactions with FBW7. Inhibition of both GSK3 and FBW7 reduced ubiquitination of HIF-1[alpha] in hypoxia and mutation of GSK3 phosphorylation sites within HIF-1[alpha] greatly reduced the FBW7-HIF-1[alpha] interaction. GSK3 and FBW7 were also demonstrated to negatively affect endothelial cell tube formation and vessel growth in the chorioallantoic membrane assay, a model of in vivo angiogenesis. The 'findings within this dissertation are the first to identify both a mechanism of ubiquitin-dependent degradation of HIF-1[alpha] in hypoxia and two new potential targets for future therapeutic development. This novel regulatory mechanism of HIF-1alpha] will hopefully provide a starting point for the creation of new therapeutics to combat dysregulated angiogenesis and improve patient outcomes.