Roles of BRCA1 in sporadic breast cancer

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The breast and tumor suppressor BRCA1 is a hub protein that integrates multiple cellular pathways, and its functional failure is implicated in the carcinogenesis of sporadic breast cancer. I have investigated the possible importance of factors act in the BRCA1 ubiquitin ligase cascade as biological indicators for sporadic breast cancer. I focused in BRCA1 inhibition by CDK2-Cyclin E in 2004. CDK2-Cyclin E destabilized BRCA1 at protein level in vivo and dramatically inhibited BRCA1's ubiquitin ligase activity. Therefore I analyzed expression pattern of BRCA1 and Cyclin E in primary breast cancer tissues by immunohistochemistry. The BRCA1 expression was reversely correlated with Cyclin E. In 2005 I mainly investigated RPB8, a subunit of RNA polymerases, and spindle-associated protein SPIN, that I have identified as substrates of BRCA1 by proteomics screens. RPB8 was ubiquitinated by BRCA1-BARD1 in vivo and in vitro, and the ubiquitination was abolished by substitution of five Lys residues of RPB8 with Arg. HeLa cell line stably expressing this ubiquitin resistant mutant of RPB8 exhibited hypersensitivity to DNA damage, the phenotype known in cells with BRCA1 deficiency. Interestingly two of the five Lys residues were substituted in human as single nucleotide polymorphisms (SNPs), that could be implicated in breast cancer susceptibility or sensitivity to anti-cancer reagents such as anthracyclin. A part of the results has been submitted for publication. I also found that endogenous or overexpressed SPIN interacted with BRCA1, and BRCA1-BARD1 dependent polyubiquitination of SPIN in vivo. I established antibodies to RPB8 and SPIN and tested its availability for immunohistochemistry. However, we have not determined appropriate condition to detect the proteins in breast cancer specimens yet.