A HIF-1α-Dependent Autocrine Feedback Loop Promotes Survival of Serum-Deprived Prostate Cancer Cells

BACKGROUND. We previously reported that normoxic, serum-deprived Prostate cancer (PCa) cells upregulate hypoxia-inducible factor-1 alpha (HIF-1 alpha) protein, which Promotes survival during serum deprivation via insulin-like growth factor-2 (IGF-2) upregulation. This study investigated the molecular mechanism of autocrine regulation of HIF-1 alpha, IGF-2 and cell Survival in serum-deprived PC-3 and LNCaP PCa cells. METHODS. Cell viability was assessed by trypan blue assay. PI3K activity was inhibited with LY294002, and PTEN overexpression. mRNA was assessed by RT-PCR, and IGF-2 protein by ELISA. Activated insulin-like growth factor-I receptor (IGF-IR) was detected by probing immunoprecipitated IGF-IR for phospho-tyrosine. IGF-IR activity was inhibited with IGF-2 neutralizing antibody and IGF-IR-specific siRNA. HIF-1 alpha, phospho-Akt, total-Akt and IGF-IR protein was assessed by immunoblots. HIF-1 alpha was suppressed with siRNA. RESULTS. 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-1 alpha and cell survival. Importantly, IGF-2 secretion significantly increased during serum deprivation, and was accompanied by increased activation of its receptor, IGF-IR. Additionally, inhibition of IGF-2 activity markedly attenuated the serum deprivation-mediated increase in IGF-IR and Akt activation, HIF-1 alpha expression, and also its own transcription, suggesting autocrine regulation of: HIF-1 alpha expression via IGF-2. Cross-talk between IGF-2/IGF-IR system and PI3K-Akt pathway was further demonstrated by findings wherein IGF-IR suppression inhibited Akt activation, and IGF-IR activation was inhibited following PI3K inhibition. Furthermore, HIF-1 alpha suppression attenuated the serum deprivation-mediated increase in Akt and IGF-IR activation. CONCLUSION. Collectively, our study demonstrates existence of a pro-survival HIF-1 alpha-dependent autocrine feedback loop in normoxic, serum-deprived PCa cells. Prostate 69: 263275,2009 (C) 2008 Wiley-Liss, Inc.