Enhanced H-Ras-mediated transformation of human mammary epithelial cells following BRCA1 knockdown is associated with elevated cyclin D1, c-myc and ER-alpha Free

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Recent success in genetic manipulation to transform normal human cells has provided a strong experimental approach to investigate the changes that occur and contribute to human mammary tumorigenesis. To gain more insight into the role of tumor suppressor BRCA1 in human breast tumorigenesis we transformed immortalized human mammary epithelial cells with or without retrovirally-mediated BRCA1 gene knockdown. Stable knock down of BRCA1 in MCF10A cells led to centrosome amplification and genomic instability even prior to experimental evidence for transformation. Knockdown of BRCA1 resulted in impaired p53 protein stability, increased sensitivity to DNA damaging agents such as adriamycin and defective chromosomal decatenation. BRCA1-depleted MCF10A cells also showed elevated levels of cyclin D1, c-Myc and interestingly ER-alpha. Overexpression of a mutated H-Ras oncogene in MCF10A cells resulted in enhanced downstream MAPK activity and tumor growth in xenografted tumors that were imaged using bioluminescence. While H-Ras overexpression was sufficient to transform MCF10A cells, depletion of BRCA1 in those H-Ras over expressing MCF10A cells greatly increased the in vivo tumorigenesis. Stable knockdown of p53 in the MCF10A cells did not enhance the in vivo H-Ras-induced tumorigenesis either in the presence or absence of BRCA1 knockdown. These results suggest that knockdown of BRCA1 may contribute to Ras-MAPK driven human mammary tumorigenesis with associated increases in cyclin D1, c-Myc and ER-alpha.