Hypoxia-inducible factor 1α promotes primary tumor growth and tumor-initiating cell activity in breast cancer

Introduction: Overexpression of the oxygen-responsive transcription factor hypoxia-inducible factor 1 alpha (HIF-1 alpha) correlates with poor prognosis in breast cancer patients. The mouse mammary tumor virus polyoma virus middle T (MMTV-PyMT) mouse is a widely utilized preclinical mouse model that resembles human luminal breast cancer and is highly metastatic. Prior studies in which the PyMT model was used demonstrated that HIF-1 alpha is essential to promoting carcinoma onset and lung metastasis, although no differences in primary tumor end point size were observed. Using a refined model system, we investigated whether HIF-1 alpha is directly implicated in the regulation of tumor-initiating cells (TICs) in breast cancer. Methods: Mammary tumor epithelial cells were created from MMTV-PyMT mice harboring conditional alleles of Hif1a, followed by transduction ex vivo with either adenovirus beta-galactosidase or adenovirus Cre to generate wildtype (WT) and HIF-1 alpha-null (KO) cells, respectively. The impact of HIF-1 alpha deletion on tumor-initiating potential was investigated using tumorsphere assays, limiting dilution transplantation and gene expression analysis. Results: Efficient deletion of HIF-1 alpha reduced primary tumor growth and suppressed lung metastases, prolonging survival. Loss of HIF-1 alpha led to reduced expression of markers of the basal lineage (K5/K14) in cells and tumors and of multiple genes involved in the epithelial-to-mesenchymal transition. HIF-1 alpha also enhanced tumorsphere formation at normoxia and hypoxia. Decreased expression of several genes in the Notch pathway as well as Vegf and Prominin-1 (CD133) was observed in response to Hif1a deletion. Immunohistochemistry confirmed that CD133 expression was reduced in KO cells and in tumorspheres. Tumorsphere formation was enhanced in CD133(hi) versus CD133(neg) cells sorted from PyMT tumors. Limiting dilution transplantation of WT and KO tumor cells into immunocompetent recipients revealed > 30-fold enrichment of TICs in WT cells. Conclusion: These results demonstrate that HIF-1 alpha plays a key role in promoting primary mammary tumor growth and metastasis, in part through regulation of TICs. HIF-1 alpha regulates expression of several members of the Notch pathway, CD133 and markers of the basal lineage in mammary tumors. Our results suggest that CD133, which has not been profiled extensively in breast cancer, may be a useful marker of TICs in the PyMT mouse model. These data reveal for the first time that HIF-1 alpha directly regulates breast TIC activity in vivo.