Rapid non-genomic signalling by 17β-oestradiol through c-Src involves mTOR-dependent expression of HIF-1α in breast cancer cells

Background: Hypoxia-inducible factor 1 (HIF1) has been implicated in regulating many of the genes responsible for angiogenesis, erythropoiesis, glucose metabolism and cancer pathogenesis. In this study, we demonstrate that exposure of human breast cancer lines to 17 β -oestradiol (E2) rapidly induced the expression of HIF-1 α , the regulated subunit of HIF1, in normoxic condition. Hypoxia-inducible factor-1 α is normally degraded in normoxia through ubiquitination-mediated proteolysis, whereas hypoxia modulates HIF-1 α level by inhibiting ubiquitination-mediated degradation. Methods: Oestradiol-induced accumulation of HIF-1 α in breast cancer lines was detected by western blot analysis and its promoter activity was measured by HIF1 reporter assay. Molecular signalling of oestradiol-mediated HIF-1 α expression was studied using specific pharmacological inhibitors and small interference RNA by co-immunoprecipitation and western blotting analysis. Results: Oestradiol has been observed to rapidly activate the nongenomic signalling cascade leading to HIF-1 α protein synthesis. The results define a signalling pathway in breast cancer cells whereby oestradiol induces a rapid protein–protein interaction of ER α -c-Src-PI3K, resulting in the activation of PI3K/AKT pathway leading to mammalian target of rapamycin (mTOR) phosphorylation. The mTOR then stimulates translation by phosphorylating p70 S6 kinase and 4EB-P1, modulating HIF-1 α protein synthesis. Oestradiol-stimulated HIF-1 α activity was inhibited by either siRNA or pharmacological inhibitors to ER α , c-Src, PI3K and mTOR, providing a mechanism for the modulation of HIF-1 α protein synthesis. Conclusion: These results show oestradiol-induced expression of HIF-1 α , downstream of the ER α /c-Src/PI3K/AKT/mTOR pathway in human breast cancer cells.