Hypoxia and metabolic phenotypes during breast carcinogenesis: expression of HIF-1α, GLUT1, and CAIX

Hypoxia and acidosis are microenvironmental selection forces during somatic evolution in breast carcinogenesis. The effect of cobalt chloride (CoCl₂)-induced hypoxia on the expression of hypoxia-inducible factor (HIF)-1α, glucose transporter 1 (GLUT1), and carbonic anhydrase IX (CAIX) was assessed in breast cancer cells derived from primary sites (HCC1395 and HCC1937) and metastatic sites (MCF-7 and MDA-MB-231) by reverse transcriptase-polymerase chain reaction and immunoblotting. We analyzed these proteins' expression in tissue samples from normal breast tissue, usual ductal hyperplasia (DH), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) using immunohistochemistry. CAIX mRNA was expressed constitutively in MDA-MB-231 cells but not in the other three cell lines. CAIX mRNA expression was increased after CoCl₂-induced hypoxia in all four breast cancer cell lines. The expression of HIF-1α and GLUT1 proteins was increased after CoCl₂-induced hypoxia in all breast cancer cell lines tested. Hypoxia significantly increased CAIX protein expression in primary cancer cells but not in metastatic ones. HIF-1α was not expressed in benign breast tissue, whereas it was significantly expressed in DH, ADH, DCIS, and IDC (p < 0.001). GLUT1 and CAIX were expressed only in DCIS (56.8% and 25.0%) and IDC (44.1% and 30.5%), with higher expression in high grade DCIS than low/intermediate grade DCIS (79.2% vs. 30.0%, p = 0.001 and 37.5% vs. 10.0%, p = 0.036, respectively). High CAIX expression was significantly associated with poor histological grade of IDC (p = 0.005). During breast carcinogenesis, the role of HIF-1α changes from response to proliferation to tumor progression. GLUT1 expression (glycolytic phenotype) and CAIX expression (acid-resistant phenotype) may result in a powerful adaptive advantage and represent an aggressive phenotype.