A HIF‐1α‐dependent autocrine feedback loop promotes survival of serum‐deprived prostate cancer cells

Abstract

BACKGROUND: We previously reported that normoxic, serum‐deprived prostate cancer (PCa) cells upregulate hypoxia‐inducible factor‐1α (HIF‐1α) 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‐1α, 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‐1α, phospho‐Akt, total‐Akt and IGF‐IR protein was assessed by immunoblots. HIF‐1α 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‐1α 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‐1α expression, and also its own transcription, suggesting autocrine regulation of HIF‐1α 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‐1α suppression attenuated the serum deprivation‐mediated increase in Akt and IGF‐IR activation. CONCLUSION: Collectively, our study demonstrates existence of a pro‐survival HIF‐1α‐dependent autocrine feedback loop in normoxic, serum‐deprived PCa cells. Prostate 69:263–275, 2009. © 2008 Wiley‐Liss, Inc.