Date of Award

2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell and Molecular Biology

Abstract

Cancerous tumors adjust their signaling pathways to facilitate survival in a hypoxic environment. The pro-angiogenic transcription factor hypoxia inducible factor -1α (HIF-1α) is stabilized in hypoxia and promotes tumor cell survival, proliferation, and metastasis by inducing transcription of many targets including vascular endothelial growth factor (VEGF). High expression levels of HIF-1α have been correlated with aggressive tumor types. As such, HIF-1α is an attractive candidate for targeted therapies in ovarian cancer. Though there have been attempts at developing chemotherapies that inhibit HIF-1α, to this date there remains no approved anti-angiogenic drug that directly targets HIF-1α. Because the cytokine EPO (erythropoietin) is transcriptionally regulated by HIF-1α, we hypothesized that negative feedback inhibition from EPO to HIF-1α would decrease HIF-1α protein levels. In fact, exogenous EPO significantly inhibited hypoxia-induced HIF-1α expression in cultured human ovarian cancer cells in a dose-dependent fashion and significantly reduced hypoxia-induced VEGF transcription to near basal levels. Furthermore, although EPO is a growth factor, there was no difference in cell growth with EPO treatment in either the normoxic or hypoxic condition at any time point tested. The negative feedback of EPO on HIF-1α was not limited to ovarian cancer cells as EPO efficiently inhibited hypoxia-induced HIF-1α stabilization in MCF-7 breast cancer cells. Initially, we hypothesized that EPO inhibition of HIF-1α was through activation of GSK3 (glycogen synthase kinase). Though EPO likely does not signal through activation of GSK3, initial experiments investigating this pathway generated promising results and prompted the continued exploration into the regulation of HIF-1α by GSK3. Therefore, the second goal of this dissertation was to investigate the role of GSK3 phosphorylation on HIF-1α protein levels and proteosomal degradation. Recently, GSK3 has been implicated in the degradation of HIF-1α, the role of GSK3 in the regulation of HIF-1α in hypoxia has yet to be studied. We hypothesized that phosphorylation by GSK3 would target HIF-1α for proteosomal degradation in an oxygen-independent manner. In support of this hypothesis, we show that GSK3 inhibition in hypoxia lead to an increase in stabilization of HIF-1α that was echoed by an increase in VEGF transcription, suggesting an augmentation of HIF-1α transcriptional activity. Overexpression of GSK3 significantly decreased both HIF protein and VEGF transcription. Further, these studies identified five putative GSK3 sites within the HIF-1α protein that were necessary for phosphorylation by GSK3. Mutation of these sites also induced HIF-1α stabilization in normoxia and hypoxia beyond that of wild-type HIF-1α. From these results, we propose a model whereby GSK3 phosphorylates and subsequently destabilizes HIF-1α thus halting HIF-1α−dependent VEGF transcription. The work presented in this dissertation illustrates two ways in which the HIF-1α pathway can be modulated. Further exploration and exploitation of these pathways could prove to be therapeutically advantageous in the treatment of cancer.

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