Tumor progression is intrinsically tied to the clonal selection of tumor cells with acquired phenotypes allowing to cope with a hostile microenvironment. Hypoxiainducible factors (HIFs) master the transcriptional response to local tissue hypoxia, a hallmark of solid tumors. Here, we report significantly longer patient survival in breast cancer with high levels of HIF-2a. Amphiregulin (AREG) and WNT1-inducible signaling pathway protein-2 (WISP2) expression was strongly HIF2a-dependent and their promoters were particularly responsive to HIF-2a. The endogenous AREG promoter recruited HIF-2a in the absence of a classical HIF-DNA interaction motif, revealing a novel mechanism of gene regulation. Loss of AREG expression in HIF-2a-depleted cells was accompanied by reduced activation of epidermal growth factor (EGF) receptor family members. Apparently opposing results from patient and in vitro data point to an HIF-2a-dependent auto-stimulatory tumor phenotype that, while promoting EGF signaling in cellular models, increased the survival of diagnosed and treated human patients. Our findings suggest a model where HIF2a-mediated autocrine growth signaling in breast cancer sustains a state of cellular self-sufficiency, thereby masking unfavorable microenvironmental growth conditions, limiting adverse selection and improving therapy efficacy. Importantly, HIF-2a/AREG/WISP2-expressing tumors were associated with luminal tumor differentiation, indicative of a better response to classical treatments. Shifting the HIF-1/2a balance toward an HIF-2-dominated phenotype could thus offer a novel approach in breast cancer therapy.

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Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O2 transport. It has been reported that this protein contributes to gas permeation (CO2, NO and O2) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5′ proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl2) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases.