BRCA1 phosphorylation and nuclear foci formation following IR exposure

We have previously reported a novel multiprotein complex, termed BRCC, containing seven polypeptides including BRCA1, BRCA2, BARD1, and RAD5. BRCC is an ubiquitin E3 ligase complex exhibiting an E2-dependent ubiquitination of the tumor suppressor p53. In this multiprotein complex, one of these proteins, referred to as BRCC36, was found to be associated with BRCA1 and BRCA2 and was shown to play an important role in the regulation the ubiquitin E3 ligase activity of BRCC. We have shown that cancer-associated mutations in BRCA1 abrogated the association of BRCC36 with BRCC and BRCA1. Furthermore, reconstitution of a recombinant four-subunit BRCC complex containing BRCA1/BARD1/-BRCC45/BRCC36 revealed an enhanced E3 ligase activity compared to that of BRCA1/BARD1 heterodimer. In addition, we have reported over-expression of BRCC36 in the majority of breast cancer cell lines and invasive ductal carcinomas. The mechanism and consequences of abnormal BRCC36 expression in breast cancer are presently unknown. 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 the MCF-7 breast cancer cell line. Knock-down of BRCC36 alone did not affect cell growth, but when combined with ionizing radiation (IR) exposure, led to an increase in apoptotic cells when compared to the siRNA control group. Immunoblot analysis showed that inhibition of BRCC36 had no effect on activation of ATM, expression of p21 and p53, or BRCA1- BARD1 interaction following IR exposure (Figure (left):BRCA1 Phosphorylation and Nuclear Foci Formation in Breast Cancer Cells Following IR Exposure). Importantly, BRCC36 depletion disrupted IR-induced phosphorylation of BRCA1. Immunofluorescent staining of BRCA1 and γ-H2AX indicated that BRCC36 depletion prevented the formation of BRCA1 nuclear foci in response to DNA damage (Figure (right):BRCA1 Phosphorylation and Nuclear Foci Formation in Breast Cancer Cells Following IR Exposure). These results imply that down-regulation of BRCC36 expression impairs the DNA repair pathway activated in response to IR via abolishing BRCA1 activation and thereby to sensitizes breast cancer cells to IR-induced apoptosis.

Allelic imbalance in BRCA1 expression

The contribution of BRCA1 to familial and non-familial forms of breast cancer has been difficult to accurately estimated because the myriad of a potential genetic and epigenetic mechanisms that can ultimately influence their expression and cellular activities. Therefore, we investigated if allelic loss of BRCA1 expression was associated with an increased risk of developing breast cancer. By designing allele specific real-time PCR to target a common polymorphism, BRCA1-c.4308T/C, we were able to evaluate quantitatively the allelic imbalance (AI) in BRCA1 gene expression. By using mRNA from BRCA1 frameshift mutation carriers, we first demonstrated, that the transcript ratios of between the wild-type alleles to the corresponding mutant alleles (BRCA1-c.3671ins4) were significantly increased (Chen et al, 2008). The observed AI is the result of non-sense mediated mRNA decay and leads to subsequent loss of protein expression. We next measured AI for BRCA1 in heterozygous lymphocytes from three groups, familial breast cancer patients, non-familial breast cancer patients, and age-matched cancer-free females. The AI ratios of BRCA1 in the lymphocytes from familial breast cancer patients were found to be significantly increased as compared to cancer-free women [BRCA1: 0.424 ± 0.157 (n=32) vs. 0.211 ± 0.169 (n=40), p=0.00001] (Figure: Allelic Imbalance in BRCA1 Expression). Similarly, the AI ratios were greater for BRCA1 in the lymphocytes of non-familial breast cancer cases versus controls [BRCA1: 0.353 ± 0.209 (n=21), p=0.002] (Figure: Allelic Imbalance in BRCA1 Expression). Furthermore, we have demonstrated that the AI patterns for BRCA1 expression could be transmitted by Mendelian inheritance (Chen et al, 2008). These findings indicate that AI in BRCA1 appears to be a common event in breast cancer development and point towards the importance of identifying other mechanisms that regulate/suppress BRCA1 activity as contributors to breast tumorigenesis.

siRNA targeting mediated by diabody-protamine

Since tumor cells in general are genomically unstable and have defects in DNA damage responses, it has been proposed that targeting DNA repair pathways may lead to a therapeutic index in tumor cells over “normal” cells. Our previous studies have demonstrated that BRCC36 is over-expressed in the vast majority of invasive breast cancers and that depletion of BRCC36 sensitizes breast cancer cells to IR via the BRCA1 DNA repair pathway. Therefore, we hypothesize that abrogation of BRCC36 will sensitize breast tumors to the DNA-damage based therapies. To test this hypothesis, we will utilize a novel diabody (anti-HER2)-protamine (P) based siRNA delivery system to selectively deplete BRCC36 in breast tumor xenografts (Figure: siRNA targeting mediated by diabody-protamine). The diabody (C6.5) used in the proposed studies displays approximately 40-fold greater affinity for HER2 and significantly prolongs association with HER2 as compared with scFv. Therefore, this C6.5 diabody provides an in vivo targeting advantage and has been effectively used as a delivery vehicle for therapeutic radioisotopes.