BRCA1 has been shown to have a major role in the pathogenesis of hereditary, and to a lesser extent, sporadic forms of breast cancer. We hypothesized that additional proteins, which have equivalent or complementary functions as BRCA1, may also contribute to the development of breast cancer when aberrantly expressed or when inappropriately activated. We have previously reported the identification and characterization of a novel BRCA1/2 interacting protein complex, BRCC (BRCA1/2 Containing Complex). BRCC36, one of the proteins in BRCC, directly interacts with BRCA1, regulates the ubiquitin E3 ligase activity of BRCC, and is aberrantly expressed in the majority of invasive ductal carcinomas. To further elucidate the functional consequence of abnormal BRCC36 expression in breast cancer, we performed in vivo silencing studies using small interfering RNAs (siRNA) targeting BRCC36 in breast cancer cells. Since the BRCA1 protein is activated via the ATM/CHK2 signaling pathway following the exposure of cells to DNA damaging agents such as ionizing radiation (IR), we sought to determine the role, if any, of BRCC36 in this response. Knock-down of BRCC36 alone did not affect tumor cell growth, but when combined with ionizing radiation (IR) exposure led to an increase in apoptotic cells (45.9% ± 4.3% vs. 34.9% ± 1.9%) (p<0.05) and a decrease in cell viability (50.9% ± 5.8% vs. 58.4% ± 5.7%) when compared to the siRNA control group. Importantly, immunoblot analysis showed that BRCC36 depletion disrupted IR-induced phosphorylation of BRCA1, but had no apparent effects on the activation of ATM, p21 and p53 or the interaction between BRCA1 and its binding partners, e.g., BARD1, following IR exposure. Furthermore, we evaluated the role of BRCC36 in the formation of BRCA1 nuclear foci in response to DNA damage by immunofluorescent staining. The number of BRCA1-associated nuclear foci following IR exposure in siRNA-BRCC36 knock-down cells was dramatically decreased, i.e., >60% and >50%, as compared with siRNA-control cells at 2h and 4h post-IR, respectively (p<0.05). Importantly, γH2AX-associated nuclear foci in response to IR was unaffected. Therefore, our studies show that BRCC36 may be a key regulator of BRCA1’s DNA repair activity and that BRCC36 abrogation abolishes the activation of BRCA1 and prevents the formation of BRCA1 nuclear foci in response to IR. These results also suggest that BRCC36 may be a therapeutic target for the management of radiation resistant breast tumors. This work was supported in part by an AACR Anna Barker Award for basic research, the Eileen Stein-Jacoby Fund, and grants from the Department of Defense, DAMD17-03-1-0707 and W81XWH-04-1-0573.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]