Role of Stat3 in regulating HIF-1alpha expression and tumor angiogenesis

Increased vascularization (angiogenesis) is a required adaptation for sustained tumor growth, and the primary mediator of de novo blood vessel formation is vascular endothelial growth factor (VEGF). The central transcriptional activator of VEGF is hypoxia inducible factor-1 (HIF-1), a heterodimeric transcription factor composed of an inducible HIF-1alpha subunit and a constitutively expressed HIF-1beta subunit. In addition to HIF-1, it has recently been reported that signal transducer and activator of transcription 3 (Stat3) is required for VEGF production and angiogenesis. Although it is known that Stat3 is an important mediator of many of the oncogenic signaling pathways that regulate HIF-1alpha, it was not known if Stat3 regulates HIF-1alpha. To answer this important question, the effect of blocking Stat3 signaling on both HIF-1alpha and VEGF expression was examined. Treatment of cells with IL-6, a potent activator of Stat3, resulted in HIF-1alpha and VEGF induction during normoxia. By blocking protein synthesis with cycloheximide, it was determined that IL-6 induction of HIF-1alpha resulted from increased translation. When Stat3 was silenced with siRNA, both basal level expression and IL-6 induction of HIF-1alpha and VEGF were significantly reduced. Furthermore, it is likely that Stat3 is required for HIF-1alpha induction by a variety of growth signals, as both HIF-1alpha and VEGF expression resulting from EGF and heregulin were abolished when Stat3 signaling was blocked. Because we had observed that Stat3 was required for induction of HIF-1alpha by growth signals, we wanted to determine if Stat3 was also required for HIF-1alpha induction by hypoxia. When stat3 was silenced and cells exposed to hypoxia, HIF-1alpha expression was again abolished. Furthermore, the hypoxic induction of VEGF and MMP-2 was also prevented. These results, along with loss of basal expression of HIF-1alpha, suggest that Stat3 is fundamentally involved with the cellular reponse to hypoxia. This idea is reinforced by our observation that Stat3 is activated by hypoxia in several cell lines. These findings position Stat3 as an important target for antiangiogenic therapy, not only through its direct regulation of the VEGF gene, but also because it is required for expression of the other most important transcriptional activator of VEGF.