Hypoxia-inducible factor 1α promotes primary tumor growth and tumor-initiating cell activity in breast cancer.
BACKGROUND
Overexpression of the oxygen-responsive transcription factor hypoxia-inducible factor 1α (HIF-1α) 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-1α 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-1α 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 β-galactosidase or adenovirus Cre to generate wild-type (WT) and HIF-1α-null (KO) cells, respectively. The impact of HIF-1α deletion on tumor-initiating potential was investigated using tumorsphere assays, limiting dilution transplantation and gene expression analysis.


RESULTS
Efficient deletion of HIF-1α reduced primary tumor growth and suppressed lung metastases, prolonging survival. Loss of HIF-1α 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-1α 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 CD133hi versus CD133neg 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.


CONCLUSIONS
These results demonstrate that HIF-1α plays a key role in promoting primary mammary tumor growth and metastasis, in part through regulation of TICs. HIF-1α 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-1α directly regulates breast TIC activity in vivo.
Center for Cancer Research and the Department of Pathology and Laboratory Medicine, The University of Tennessee Health Science Center, Cancer Research Building, 19 South Manassas Street, Memphis, TN 38163, USA. tseagro1@uthsc.edu
Luciana P
LP
Schwab
Danielle L
DL
Peacock
Debeshi
D
Majumdar
Jesse F
JF
Ingels
Laura C
LC
Jensen
Keisha D
KD
Smith
Richard C
RC
Cushing
Tiffany N
TN
Seagroves
D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation; D002469:Cell Separation; D004815:Epidermal Growth Factor / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology / Q000502:physiology; D047628:Estrogen Receptor alpha / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D019394:Ki-67 Antigen / Q000378:metabolism; D008175:Lung Neoplasms / Q000378:metabolism* / Q000556:secondary; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009361:Neoplasm Invasiveness; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology; D010750:Phosphoproteins / Q000378:metabolism; D018874:Spheroids, Cellular / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D047368:Tumor Burden; D014407:Tumor Cells, Cultured
D047628:Estrogen Receptor alpha; C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D019394:Ki-67 Antigen; D010750:Phosphoproteins; D015534:Trans-Activators; C486419:Trp63 protein, mouse; D004815:Epidermal Growth Factor; C505409:Casp3 protein, mouse; D053148:Caspase 3