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

Abstract

We previously reported that normoxic, serum-deprived prostate cancer (PCa) cells upregulate hypoxia-inducible factor-1alpha (HIF-1alpha) 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-1alpha, IGF-2 and cell survival in serum-deprived PC-3 and LNCaP PCa cells. 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-1alpha, phospho-Akt, total-Akt and IGF-IR protein was assessed by immunoblots. HIF-1alpha was suppressed with siRNA. 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-1alpha 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-1alpha expression, and also its own transcription, suggesting autocrine regulation of HIF-1alpha 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-1alpha suppression attenuated the serum deprivation-mediated increase in Akt and IGF-IR activation. Collectively, our study demonstrates existence of a pro-survival HIF-1alpha-dependent autocrine feedback loop in normoxic, serum-deprived PCa cells.