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, and regulates the ubiquitin E3 ligase activity of BRCC. Importantly, BRCC36 is aberrantly expressed in the vast majority of breast tumors, indicating a potential role in the pathogenesis of this disease. To further elucidate the functional consequence of abnormal BRCC36 expression in breast cancer, we have done in vivo silencing studies using small interfering RNAs targeting BRCC36 in breast cancer cell lines, i.e., MCF-7, ZR-75-1, and T47D. Knock-down of BRCC36 alone does not affect cell growth, but when combined with ionizing radiation (IR) exposure, it leads to an increase in the percentage of cells undergoing apoptosis when compared with the small interfering RNA control group in breast cancer cells. Immunoblot analysis shows that inhibition of BRCC36 has no effect on the activation of ATM, expression of p21 and p53, or BRCA1-BARD1 interaction following IR exposure. Importantly, BRCC36 depletion disrupts IR-induced phosphorylation of BRCA1. Immunofluorescent staining of BRCA1 and γ-H2AX indicates that BRCC36 depletion prevents the formation of BRCA1 nuclear foci in response to DNA damage in breast cancer cells. These results show that down-regulation of BRCC36 expression impairs the DNA repair pathway activated in response to IR by inhibiting BRCA1 activation, thereby sensitizing breast cancer cells to IR-induced apoptosis. (Cancer Res 2006; 66(10): 5039-46)
- breast cancer
- ionizing radiation (IR)
- DNA repair and apoptosis
Breast cancer is the most common cancer affecting women, with a woman's lifetime risk of breast cancer at ∼10% by the age of 80 years. In the U.S., it was estimated that in 2005, ∼211,000 new cases of breast cancer were diagnosed, and >40,000 deaths resulted from this disease ( 1). Breast cancer is a genetically heterogeneous disease, and germ line mutations in BRCA1 and BRCA2 genes predispose women to early onset breast cancer and/or ovarian cancer ( 2, 3). Since their cloning and characterization in the mid-1990s ( 4, 5), BRCA1 and BRCA2 proteins have been implicated in many cellular processes, including DNA repair and cell cycle-checkpoint control ( 6– 10). BRCA1 has also been reported to be involved in protein ubiquitylation and chromatin remodeling ( 11, 12). Despite the fact that BRCA1 and BRCA2 mutations contribute to hereditary breast/ovarian cancer predisposition, somatic mutations are rarely found in sporadic breast cancers ( 13– 15). Nevertheless, evidence is accumulating that dysfunction of other genes, coding for proteins in similar or redundant pathways as BRCA1 and BRCA2, might be important in the pathogenesis of a significant fraction of nonfamilial breast cancers. This speculation comes from several lines of evidence, including both phenotypic analyses of breast and ovarian tumors, as well as mechanistic studies of BRCA1- and BRCA2-associated pathways ( 16– 18).
We have previously reported a novel multiprotein complex, termed BRCC, containing seven polypeptides including BRCA1, BRCA2, BARD1, and RAD51 ( 19). 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, has been found to be associated with BRCA1 and BRCA2, and has been shown to play an important role in the regulation of the ubiquitin E3 ligase activity of BRCC. The BRCC36 gene is located at the Xq28 locus, a chromosomal break point in patients with prolymphocytic T cell leukemia ( 20). BRCC36 displays sequence homology with the human Poh1/Pad1 subunit of the 26S proteasome and with subunit 5 (Jab1) of the COP9 signalosome ( 19). We have shown that cancer-associated mutations in BRCA1 abrogated the association of BRCC36 with BRCC and BRCA1 ( 19). Furthermore, reconstitution of a recombinant four-subunit BRCC complex containing BRCA1/BARD1/BRCC45/BRCC36 reveals an enhanced E3 ligase activity compared with that of BRCA1/BARD1 heterodimer ( 19). In addition, we have reported aberrant expression of BRCC36 in the majority of breast cancer cell lines and invasive ductal carcinomas ( 19). The mechanism and consequences of abnormal BRCC36 expression in breast cancer are presently unknown.
Previous studies have shown that BRCA1 is activated via the ATM/CHEK2 (CHK2) signaling pathway following the exposure of cells to DNA-damaging agents such as ionizing radiation (IR; refs. 21, 22). Following IR, BRCA1 is phosphorylated and forms discrete nuclear foci (dots) in response to DNA damage ( 23). Because BRCC36 directly interacts with BRCA1, we investigated the effects of knocking down BRCC36 expression, using small interfering RNAs (siRNA) on the growth and apoptosis of breast cancer cells. We further determined the role of BRCC36 in the BRCA1-associated DNA repair pathway activation following DNA damage. Here, our studies show that BRCC36 is a direct regulator of BRCA1 activation in response to IR.
Materials and Methods
Cell culture, siRNA transfection, and IR. Nontumorigenic epithelial cell lines, MCF-10F and 12A were purchased from American Type Culture Collection (Manassas, VA) and cultured in DMEM/F12 with reduced Ca2+ (0.04 mmol/L final), 20 ng/mL epidermal growth factor, 100 ng/mL cholera toxin, 0.01 mg/mL insulin, 500 ng/mL hydrocortisone, and 5% Chelex-treated horse serum. The human breast cancer cell lines, MCF-7, T47D, and ZR-75-1, were also obtained from the American Type Culture Collection. MCF-7 cells were maintained in DMEM medium, supplemented with 10% fetal bovine serum, penicillin, and streptomycin. T47D and ZR-75-1 cells were maintained in RPMI 1640, supplemented with 10% fetal bovine serum, penicillin, and streptomycin. 293-BARD1 cells were generously provided by Dr. R. Shiekhattar (Wistar Institute, Philadelphia, PA) and were maintained in MEM (Eagle) with 10% heat-inactivated horse serum.
For the BRCC36 depletion studies, breast cancer cells were plated at a density of 5 × 103 cells/cm2. After reaching 30% to 40% confluence, cells were transfected with siRNA using OligofectAMINE and OPTI-MEM I reduced serum medium (Invitrogen/Life Technologies, Inc., Carlsbad, CA) according to the manufacturer's protocol. The siRNA sequences targeting BRCC36 corresponded to the coding region 253 to 273 bp (5′-AAGAGGAAGGACCGAGTAGAA-3′) relative to the start codon. The corresponding siRNA duplexes with the following sense and antisense sequences were used: 5′-GAGGAAGGACCGAGUAGAAdTdT (sense) and 5′-UUCUACUCGGUCCUUCCUCdTdT (antisense). This siRNA has been used in a previous study ( 19), as well as another siRNA targeting BRCC36 (corresponding to the coding region 120-138 bp). Both resulted in similar levels of transcript depletion. Green fluorescent protein siRNA was used as the negative control. All of the siRNA duplexes were synthesized by Dharmacon Research, Inc. (Lafayette, CO) using 2′-ACE protection chemistry. Twenty-four hours after the initial transfection, cells were subcultured and replated at 5 × 103 cells/cm2. The cells were then retransfected under similar conditions 24 hours after replating. Seventy-two hours after the first transfection, the cells were irradiated using a Cesium 137 irradiator (model 81-14R). The cells received 4 Gy total IR (1.132 Gy/min for 3.53 minutes) for a targeted 50% induction of apoptosis ( 24). Cells were then grown for an additional 72 hours prior to harvesting and further analyses.
RNA isolation, reverse transcription, and quantitative PCR. Total cellular RNA was isolated from cells using TRIzol reagent (Invitrogen) according to the protocols provided by the manufacturer. Total RNA (2 μg) was used as a template to be reverse-transcribed in a 20 μL reaction containing 5 μmol/L random hexamers, 500 μmol/L of deoxynucleoside triphosphate mix, 1× reverse transcriptase buffer, 5 mmol/L MgCl2, 1.5 units of RNase inhibitor, and 7.5 units of MuLV reverse transcriptase. All reagents were obtained from Applied Biosystems (Branchburg, NJ). The reaction conditions were as follows: 10 minutes at 25°C, 1 hour at 42°C, and 5 minutes at 94°C. The cDNA mixture (0.625 μL) was used in a real-time PCR reaction (25 μL total volume) done with ABI 7900HT (Applied Biosystems) following protocols recommended by the manufacturer. Optimal conditions were defined as: step 1, 95°C for 10 minutes; step 2, 95°C for 15 seconds, 60°C for 60 seconds with Optics, repeated for 40 cycles. The relative mRNA expressions of BRCC36 were adjusted with β-actin (ACTB). The primer and probe sets used for real-time PCR were as follows: BRCC36, forward primer, 5′-AATTTCTCCAGAGCAGCTGTCTG; reverse primer, 5′-CATGGCTTGTGTGCGAACAT; TaqMan probe, (FAM) 5′-AACTGACAGGCCGCC-CCATGAG-(BHQ1); β-actin, forward primer, 5′-GCCAGGTCATCACCATTGG; reverse primer, 5′-GCGTACAGGTCTTTGCGGAT; TaqMan probe, (Cal red) 5′-CGGTTCCGCTGCCCTGAGGC-(BHQ2).
Coimmunoprecipitation. 293 cells that stably express FLAG-BARD1 ( 19) at 70% to 80% confluence were washed twice with ice-cold D-PBS before scraping on ice with lysis buffer [50 mmol/L Tris-HCl (pH 7.4), with 150 mmol/L NaCl, 1 mmol/L EDTA, and 1% Triton X-100 and one tablet of protease inhibitor mixture per 40 mL of lysis buffer (Roche Molecular Biochemicals, Indianapolis, IN)]. Cellular debris was removed by centrifugation (14,000 × g for 15 minutes at 4°C), and protein concentrations were determined using the Bio-Rad detergent-compatible protein assay reagent. Cell lysate (1.5 mg) was added to the anti-FLAG M2-agarose affinity gel (Sigma, St. Louis, MO). All samples were placed on a roller shaker overnight at 4°C. After centrifugation, the supernatants were removed and the gel beads were washed thrice with 0.5 mL of wash buffer [50 mmol/L Tris-HCl (pH 7.4), with 150 mmol/L NaCl]. The beads were washed an additional four times with the wash buffer, resuspended in 20 μL of 2× SDS sample buffer before boiling for 5 minutes. Fifteen microliters of immunoprecipitate were separated by SDS-PAGE electrophoresis on 4% to 20% and 5% linear gradient Tris-HCl ready gels (Bio-Rad, Richmond, CA).
Western blot and antibodies. Cells were homogenized in lysis buffer [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 2.5 mmol/L Na-pyrophosphate, 1 mmol/L Na-β-glycerophosphate, 20 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, 1% Triton X-100, one tablet of protease inhibitor mixture (Roche Molecular Biochemicals)]. Cellular debris was removed by centrifugation as above. Protein concentrations were determined with Bio-Rad detergent-compatible protein assay (Bio-Rad). For BRCA1 and pATM, cells were lysed directly in Laemmli sample buffer (Bio-Rad). Proteins were resolved on 5% (BRCA1 and pATM) or 4% to 20% linear gradient (β-actin, p53, p21, and RAD51) SDS-PAGE ready gels at 120 V for 1.5 to 3 hours with 1× SDS running buffer. SDS-PAGE gels were transferred onto an Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA). Primary antibodies used for Western analysis were mouse anti-BRCA1 (1:100; EMD Biosciences, San Diego, CA), mouse anti-phosphorylated ATM-S1981 (1:500; Rockland, Gilbertsville, PA), mouse anti-p53 (1:1,000, Cell Signaling, Beverly, MA), mouse anti-p21WAF (1:100, EMD Biosciences), mouse anti-RAD51 (1:500; Upstate Biotechnology, Lake Placid, NY), mouse anti-FLAG M2 (1:1,000; Sigma), and mouse anti-β-actin (1:5,000; Sigma). Secondary antibodies were mouse and rabbit IgG, horseradish peroxidase–linked (1:10,000; Amersham, Piscataway, NJ). Perkin-Elmer Life Sciences renaissance enhanced luminol reagents (Boston, MA) were used as substrates for detection. To reprobe immunoblot membranes, Restore Western blot stripping buffer (Pierce, Rockford, IL) was used to strip the membrane.
Apoptosis assay. Breast cancer cells were collected by trypsinization and pelleted by centrifuging for 5 minutes at 800 × g at 4°C. After washing with 1 mL of ice-cold 1× Nexin buffer (Guava Technologies, Hayward, CA), the cells were resuspended in 100 μL of the same buffer. After labeling with Annexin V and 7-amino actinomycin D, the proportion of apoptotic cells was determined using a Guava personal cytometer (Guava Technologies) according to the manufacturer's instruction. Cell apoptosis was also analyzed using a terminal nucleotidyl transferase–mediated nick end labeling (TUNEL) assay. In brief, breast cancer cells were grown in four-well chamber slides (Nalge Nunc International, Rochester, NY). After fixing with 4% paraformaldehyde in PBS and permeabilizing with 0.1% Triton X-100 in 0.1% sodium citrate solution, apoptotic cells were detected using an in situ cell death detection kit (Roche, Germany) according to the manufacturer's instructions. At least 1,200 cells from eight independent fields were counted to evaluate the percentage of apoptotic cells.
Immunofluorescence and antibodies. MCF-7, ZR-75-1, and T47D cells were grown in four-well chamber slides (Nalge Nunc International) and processed for immunofluorescent analysis as described previously ( 25). For nuclear foci formation and colocalization of the BRCA1 and γ-H2AX, cells were preextracted in protein extraction solution (20 mmol/L HEPES, 50 mmol/L NaCl, 3 mmol/L MgCl2, 300 mmol/L sucrose, and 0.5% Triton X-100), fixed in 3.7% formaldehyde (Fisher, Pittsburgh, PA) in PBS for 10 minutes and permeabilized in 0.5% NP40 in PBS prior to incubation with the following antibodies: rabbit anti-BRCA1 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-γ-H2AX (1:200; Upstate Biotechnology). Primary antibodies were detected with tetramethyl rhodamine isothiocyanate–conjugated donkey anti-rabbit IgG and FITC-conjugated donkey anti-mouse IgG (1:100; Jackson ImmunoResearch, West Grove, PA). DNA was counterstained with 0.1 μg/mL of 4′,6′-diamidino-2-phenylindole (Sigma) and mounted in embedding medium (0.1% p-phenylene diamine in 90% glycerol, 1× PBS). Microscopic analysis was carried out using the Eclipse TE2000 (Nikon, Melville, NY) and images captured using a Cascade 650 monochrome camera (PhotoMetrics, Huntington Beach, CA). A series of 0.5-μm sections were collected for seven fields of each treatment group. Image acquisition from a Cascade 650 monochrome camera (PhotoMetrics) was controlled by MetaVue (v6.2r6, Universal Imaging/Molecular Devices, Downingtown, PA). An automated Ludl MAC2000 x-y stage and z-axis motor were also controlled using the MetaVue software (v6.2r6).
Image analysis. Quantification of BRCA1 and γ-H2AX nuclear foci formation was done with Metamorph software (v6.1; Universal Imaging/Molecular Devices). In brief, a series of Z-sections for each channel was reassembled using the “maximum” type option within the “3-D reconstruction” function. Nuclei were defined in the 4′,6′-diamidino-2-phenylindole channel using the functions of “threshold for light objects” and “create regions from objects.” Adjacent nuclei were separated into independent regions using the “cut-drawing” tool. Regions were then transposed onto the reassembled image for each digital channel, after background was removed using the “flatten background” function, and positive signals were identified by manual thresholding (high, 2,836; low, 1,758). For each nucleus, the number of the BRCA1 or γ-H2AX foci was calculated using the “foci measure” function. Approximately 70 cells of each treatment group from seven independent fields were analyzed to evaluate the number of BRCA1 foci.
Statistical analysis. Student's t test was employed using SAS software 8.0 (SAS Institute, Cary, NC). P < 0.05 was considered significant and results were presented as the mean ± SD.
Inhibition of BRCC36 gene expression by siRNA. To further elucidate the functional consequence of BRCC36 aberrant expression in the pathogenesis of breast cancer, we did in vivo silencing studies targeting BRCC36 in MCF-7, T47D, and ZR-75-1 breast cancer cell lines, which constitutively expresses high levels of BRCC36 transcript relative to nontumorigenic breast epithelial cells, i.e., MCF-10F and MCF12A ( Fig. 1A ). Because antibodies specific to BRCC36 protein are not available, quantitative PCR was used to establish constitutive and attenuated BRCC36 mRNA levels. We used siRNA targeting BRCC36 or green fluorescent protein (negative control) to assess the response of BRCC36 depletion in breast cancer cells. Various siRNAs to BRCC36 were previously evaluated ( 19) and shown to be effective alone or in combination; however, for the purpose of these studies, BRCC36-siRNA1 was used. Treatment with this siRNA resulted in a >50% to 80% decrease in BRCC36 mRNA levels in comparison to mock-treated or siRNA control–transfected cells (P < 0.05) by 72 hours. The greatest level of suppression was observed in MCF-7 cells ( Fig. 1B).
Abrogation of BRCC36-enhanced IR-induced breast cancer cell apoptosis. Following depletion of BRCC36 via siRNA, MCF-7, ZR-75-1, or T47D were treated with and without IR (4 Gy). The cells were cultured for an additional 72 hours prior to harvesting and were examined for DNA damage–induced cell apoptosis via Annexin V and 7-amino actinomycin D staining. No significant difference in the fraction of cells undergoing apoptosis in mock-treated, siRNA control-transfected, or siRNA-BRCC36–transfected cells was observed in the absence of IR, indicating that depletion of BRCC36 alone is not lethal ( Fig. 2A ; data not shown). However, when combined with BRCC36 knock-down, IR exposure led to a significant increase in the percentage of MCF-7 cells that undergo apoptosis (45.9 ± 4.3%) when compared with the siRNA control group (34.9 ± 1.9%, P < 0.05; Fig. 2A; Table 1 ). Consistent with these results, the overall cell viability was substantially lower in siRNA-BRCC36–treated cells following IR as compared with control cells (50.9 ± 5.8% versus 58.4 ± 5.7%; Table 1). Similar results were observed in T47D cells treated with siRNA-BRCC36 and IR versus controls (42.2 ± 4.5% versus 23.3 ± 1.9%, P < 0.05; Fig. 2A; Table 1). Although the trend was evident for ZR-75-1 cells, the fraction of cells undergoing apoptosis following depletion of BRCC36 and IR were not statistically significantly different (data not shown). Induction of apoptosis was confirmed using a TUNEL assay and MCF-7 cells ( Fig. 2B). The combination of BRCC36 siRNA abrogation and IR exposure again resulted in a significant increase in the fraction of cells undergoing apoptosis when compared with the siRNA control–treated cells (40.9 ± 2.7% versus 24.9 ± 3.3%, P < 0.05; Fig. 2C).
Inhibition of BRCC36 disrupted BRCA1 phosphorylation in breast cancer cells exposed to IR. Previous studies have indicated that the BRCA1 protein is phosphorylated in response to DNA-damaging agents ( 23). Because BRCC36 directly interacts with BRCA1 ( 19), we examined the effect of BRCC36 depletion on BRCA1-associated DNA repair/damage pathways. MCF-7 cells were treated with siRNA targeting BRCC36, and then exposed to 4 Gy of IR to induce DNA damage. MCF-7 cells were harvested 2 hours after IR. Western blot analysis was carried out to examine the expression and modification of BRCA1, p21, p53, and ATM. Western analysis clearly shows that DNA damage induced by IR resulted in increased expression of p21, stabilization of p53, and phosphorylation of BRCA1 and ATM (S1981) as expected ( Fig. 3 ). This same pattern was observed in siRNA control–treated cells. Importantly, the reduction of BRCC36 blocked IR-induced phosphorylation of BRCA1. In comparison, BRCC36 knock-down had no effect on IR-induced expression of p21, stabilization of p53, and phosphorylation of ATM.
Effects of inhibition of BRCC36 on integrity of BRCA1-BARD1 heterodimer. To determine if depletion of BRCC36 affects the integrity of the BRCA1-BARD1 heterodimer, we targeted BRCC36 mRNA in a 293-derived cell line expressing FLAG-BARD1 ( 19). Immunoprecipitation was done using anti-FLAG on lysates prepared from cells transfected with the BRCC36 or control siRNAs. BRCA1 and BARD1 were examined with SDS-PAGE and Western blot. Reduction of BRCC36 did not seem to alter the BRCA1-BARD1 interaction in either untreated or IR-treated cells ( Fig. 4 ).
Inhibition of BRCC36 disrupts BRCA1 nuclear foci formation in breast cancer cells exposed to IR. It is well characterized that BRCA1 localizes to discrete nuclear foci (dots) during S phase or in response to DNA damage. In our previous report, we showed that BRCC36 directly interacts with BRCA1 at the region encompassing amino acids 502 to 1,054 ( 19). This region falls within the BRCA1 DNA-binding domain (amino acids 452-1079). Because the DNA-binding domain has been shown to contribute to the BRCA1 relocalization after DNA damage ( 23, 26), we sought to evaluate the role of BRCC36 in the formation of BRCA1 nuclear foci in response to DNA damage. MCF-7, T47D, and ZR-75-1 cells, mock-treated or transfected with siRNA-control or siRNA-BRCC36, were exposed to IR (4 Gy), and were evaluated for BRCA1 and γ-H2AX subcellular location. Recent studies have shown that the three breast cancer cell lines used in this study possess wild-type BRCA1 ( 27). As shown in Fig. 5 , BRCC36 deficiency inhibits BRCA1 focus formation as compared with mock-treated and siRNA control–transfected cells. Importantly, γ-H2AX response to IR was unaffected in the cells transfected with BRCC36 siRNA ( Fig. 5A-C). Quantification of BRCA1 nuclear foci showed that siRNA-BRCC36 transfection in MCF-7 cells resulted in 63% and 52% decrease compared with siRNA-control cells at 2 and 4 hours post-IR, respectively (P < 0.05; Fig. 5D and E). Similar results were observed in T47D (49% and 36%) and ZR-75-1 (59% and 71%) cells ( Fig. 5D and E). Collectively, these results show that down-regulation of BRCC36 expression impairs the DNA repair pathway activated in response to IR by inhibiting BRCA1 activation.
In this study, we have evaluated the role of BRCC36 in the ATM/BRCA1 DNA repair pathway in breast cancer cells in response to IR. The key findings of this work lie in the following: first, we have shown that the depletion of BRCC36 mRNA enhances IR-induced apoptosis of breast cancer cells ( Fig. 2). Second, the reduction of BRCC36 prevents the IR-induced activation of BRCA1 whereas other IR response proteins, such as ATM, p53 and p21, are unaffected ( Fig. 3). Third, BRCC36 abrogation inhibits the formation of BRCA1 nuclear foci following IR, without preventing the interaction of BRCA1 with its well-characterized binding partner, BARD1 ( Figs. 4 and 5; data not shown).
The damage caused by IR activates various DNA repair pathways, including the ATM/ATR/CHEK2 pathways ( 21, 28). The central component of these DNA repair pathways is ATM kinase ( 29). ATM is activated by DNA damage and phosphorylates multiple factors, including BRCA1 and p53, which are involved in DNA repair, apoptosis and cell cycle arrest ( 21, 30, 31). As our results indicate, depletion of BRCC36 expression by siRNAi blocks BRCA1 activation, i.e., phosphorylation and nuclear foci formation in breast cancer cells following IR exposure, but has no direct effect on IR-induced apoptosis. Because of the role of BRCA1 in DNA repair, we propose that disrupting BRCA1 activation by BRCC36 depletion creates an imbalance between the DNA repair/cell survival and DNA damage/cell apoptosis pathways in cells following IR exposure ( Fig. 6 ). As a result, BRCC36 depletion seems to substantially sensitize breast cancer cells to IR-induced apoptosis. However, it should be noted that these studies were done in a limited number of breast cancer cell lines, the caveat being that the DNA damage response may be altered in any or all cancer cell lines.
BRCA1 has been examined for a possible role in the development of radioresistant breast tumors. In fact, researchers have reported that BRCA1-deficient breast cancer cells have an increased sensitivity to IR ( 32). More recent studies have focused on the genes that code for proteins with equivalent/complementary functions to BRCA1 or function in the same pathway as BRCA1. A number of studies ( 33– 37) have reported that manipulation of BRCA1-associated proteins affects cellular resistance or sensitivity to IR. The abnormal change (loss or gain) of any component in these BRCA1-related protein complexes may lead to their functional defects, which would result in a “BRCA1 null” phenotype. This may begin to explain why BRCA1 itself is rarely mutated and only occasionally (∼10%) epigenetically down-regulated in sporadic diseases ( 18, 38). Therefore, BRCA1-associated proteins, including BRCC proteins, may serve as potential targets for the treatment of breast cancer, including radiation therapy.
In a previous report, we have shown that BRCC36 directly interacts with amino acids 502 to 1,054 of BRCA1 ( 19). In our current study, we have found that IR induced-BRCA1 nuclear foci formation is disrupted in BRCC36-depleted breast cancer cells ( Fig. 5). The mechanism by which BRCC36 interferes with IR induced-BRCA1 localization is not clear. Previous studies have shown that BRCA1 consists of a DNA-binding domain region encompassing amino acids 452 to 1,079, and this BRCA1 DNA-binding domain contributes to the DNA repair–related functions of BRCA1, including the BRCA1 relocalization after DNA damage ( 23). The function of BRCA1 DNA-binding domain has been reported to be partially mediated through a protein complex, termed as BRCA1-associated surveillance complex (BASC; ref. 26). Interestingly, the location of BRCA1 DNA-binding domain coincides with the region that BRCC36 binds to, i.e., amino acids 452 to 1,079 versus amino acids 502 to 1,054 of BRCA1, respectively. In this study, we have found that depletion of BRCC36 by siRNA knock-down prevents the phosphorylation of BRCA1 following IR ( Fig. 3). Previous studies have shown that BRCA1 is bound and phosphorylated by the ATM kinase and the G2-M control kinase (CHEK2) after IR ( 21, 39, 40). Coincidentally, a host of studies have suggested that ATM and CHEK2 also bind to this central region of BRCA1 [reviewed by Narod and Foulkes ( 38)]. These findings may provide insight as to why depletion of BRCC36 in our studies inhibits BRCA1 activation, e.g., BRCC36 could help recruit BRCA1 to ATM and CHEK2 or stabilize their interactions following activation of the DNA damage response pathway. Our future studies are geared towards determining if BRCC36 remains associated with activated BRCA1 or whether BRCC36 must be displaced prior to phosphorylation by ATM and CHEK2. We have begun to explore these questions and have found that BARD1 and RAD51 remain associated with BRCA1 following BRCC36 depletion ( Fig. 4; data not shown). However, we have yet to determine if BRCC36 depletion affects the interaction between BRCA1 and ATM and/or CHEK2 (data not shown).
Overall, our studies define BRCC36 as a direct regulator of BRCA1 activation and nuclear foci formation in response to IR in a number of breast cancer cell lines. Our results suggest that down-regulation of BRCC36 expression impairs the DNA repair pathway activated in response to IR and seems to sensitize breast cancer cells to IR-induced apoptosis. Therefore, it is intriguing to speculate that targeting BRCC36 may aid in the treatment of radiation-resistant breast tumors.
Grant support: AACR-Anna Barker Award for basic research (X. Chen), Cheryl Herman and the Eileen Stein-Jacoby Fund, and grants from the Department of Defense, DAMD17-03-1-0707 (A.K. Godwin), and W81XWH-04-1-0573 (X. Chen). This project was also funded, in part, under a grant from the Pennsylvania Department of Health. The Department specifically disclaims responsibility for any analyses, interpretations, or conclusions.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. A. Knudson for his thoughtful comments, Dr. R. Shiekhattar for generously providing the 293-BARD1 cell line, Dr. J. Johnson for the help in Metamorph image analysis, and Z-Y. Song for help with the graphics.
- Received November 23, 2005.
- Revision received March 7, 2006.
- Accepted March 10, 2006.
- ©2006 American Association for Cancer Research.