HIF: A Key Survival Factor for Serum-Deprived Prostrate Cancer Cells

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.