REGULATION OF THE GABPβ PROMOTER AND THE ROLE OF NRF-1 IN MAMMARY EPITHELIAL MORPHOGENESIS
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Decreased expression of the tumor suppressor gene BRCA1 (breast cancer 1, early onset) is frequently observed in sporadic breast tumors, with the decrease not attributed to mutations or hypermethylation of the promoter. A critical regulator of the BRCA1 promoter is the GA-Binding Protein (GABP), a heterotetramer of GABPα and GABPβ. Previous analysis of the GABPβ promoter revealed a regulatory multi-protein complex containing Nuclear Respiratory Factor 1 (NRF-1), which was aberrant in SK-BR-3 cells, resulting in decreased GABPβ and BRCA1 expression. To identify the unknown co-regulators of the NRF-1-containing complex at the GABPβ promoter, an immobilized-template assay containing the complex binding site was utilized. Despite optimization of binding and elution conditions through variations in salt content, Mg2+ concentration and pH, non-specific DNA-binding proteins were present in the column eluate. Further experimentation is therefore required to distinguish between non-specific proteins and complex co-activators. As this complex containing NRF-1 is also able to modulate BRCA1, a regulator of luminal progenitor differentiation, a defect in NRF-1 or complex co-activators could contribute to the abnormal morphogenesis and differentiation of BRCA1-deficient tumors. We therefore investigated the role of NRF-1 in mammary cell morphogenesis and its link to the GABP/BRCA1 pathway. As both GABPβ and NRF-1 are known regulators of nuclear-encoded mitochondrial proteins, this association also provides a link between mitochondrial metabolism and breast differentiation, both of which are frequently disrupted in breast cancer. An inducible lentiviral system was used to generate NRF-1 knockdown cell lines to examine its effect on morphogenesis. Growth in 3D culture resulted in abnormal cell structures having impaired cell polarization and lumen formation. In monolayer culture, prolonged NRF-1 knockdown did not result in decreased BRCA1 or GABP expression. However, these cells did display notable mitochondrial dysfunction, accompanied by the downregulation of several NRF-1 target genes involved in mitochondrial biogenesis including Tfam and cytochrome c. These results suggest a role for NRF-1 in mediating mammary morphogenesis through maintenance of functional mitochondria. Further investigation into the role of the NRF-1-containing complex at the GABPβ promoter during differentiation might also provide insight into the altered cell metabolism and differentiation observed in cancer cells.