Date of Award

5-1-2008

Degree Type

Restricted Access Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Microbiology and Immunology

Department

Graduate School of Biomedical Sciences

First Advisor

Jamboor Vishwanatha

Second Advisor

Harlan Jones

Third Advisor

Raghu Krishnamoorthy

Abstract

Thomas, Rusha, HIF: A key survival factor for serum-deprived prostate cancer cells. Doctor of Philosophy (Molecular Biology and Immunology), May 2008, 134 pages, 47 illustrations, reference list, 105 titles. The hypoxia-inducible factor (HIF) is central to hypoxic adaptation of tumors, and consists of an oxygen-labile HIF-1α and a constitutively expressed HIF-1β subunit. In specific aim 1, we report that prolonged serum deprivation is a potent inducer of HIF-1α in PC-3 and LNCaP prostate cancer (PCa) cells, despite normal oxygen conditions. In contrast, cells grown in the presence of serum did not upregulate HIF-1α protein levels. Moreover, HIF-1α protein increase during serum deprivation correlated with increased cell survival, while suppression of HIF-1α expression significantly decreased PCa cell viability. Our results further demonstrate that HIF-1α protein increase during serum deprivation is due to increased HIF-1α protein synthesis. First, there was no significant increase in HIF-1α mRNA. Secondly, cycloheximide, a protein synthesis inhibitor, prevented HIF-1α protein increase in serum-deprived PCa cells. Moreover, the expression of HIF-1α-target genes, VEGF and IGF-2, was concomitantly increased in serum-deprived PCa cells, while suppression of HIF-1α expression markedly inhibited their induction. Most interestingly, our study showed a significant decline in PCa cell survival following inhibitor of IGF-2 activity. Taken together, our study demonstrates for the first time that PCa cells counteract the stress of prolonged serum deprivation by upregulating HIF-1α protein which increases IGF-2 expression to promote cell survival. In specific aims 2 and 3, we investigated the molecular mechanism of autocrine regulation of HIF-1α, IGF-2 and cell survival in serum-deprived PC-3 and LNCaP PCa cells. We detected a time-dependent increase in Akt activation during serum deprivation, and inhibition of Akt activation attenuated the serum deprivation-mediated increase in HIF-1α and cell survival. Importantly, IGF-2 secretion significantly increased during serum deprivation, and was accompanied by increased activation of its receptor, insulin-like growth factor-I receptor (IGF-IR). Additionally, inhibition of IGF-2 activity markedly suppressed the serum deprivation-mediated increase in IGF-IR and Akt activation, HIF-1α expression, as well as its own transcription, suggesting autocrine regulation of HIF-1α expression via IGF-2. Reciprocal regulation of the IGF-2/IGF-IR system and P13K-Akt pathway was further demonstrated by findings wherein Akt activation was prevented following suppression of IGF-IR expression, and IGF-IR activation was inhibited following P13K inhibition. Lastly, HIF-1α suppression abolished the serum deprivation-mediated increase in Akt activation, and also resulted in higher IGF-IR protein levels indicating reduced IGF-IR activation. Collectively, our study demonstrates that a HIF-1α-dependent autocrine feedback loop upregulates HIF-1α, and thus promotes survival of normoxic, serum-deprived PCa cells.

Comments

Thomas, Rusha, HIF: A key survival factor for serum-deprived prostate cancer cells. Doctor of Philosophy (Molecular Biology and Immunology), May 2008, 134 pages, 47 illustrations, reference list, 105 titles. W 4 T461H 2008

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