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The tumor suppressor BRCA1 is a nuclear shuttling protein. However, the role of BRCA1 localization in the control of its functions remains to be elucidated. Given the central role of BRCA1 in DNA damage repair, we hypothesized that depletion of nuclear BRCA1 will compromise its nuclear function in DNA repair and thereby result in enhanced cytotoxic response to DNA damage. In this study, we showed that repair of DNA double strand breaks (DSBs) required BRCA1 in the nucleus. In addition, sequestering BRCA1 in the cytosol enhanced the cytotoxic response to ionizing radiation (IR) or cisplatin in human breast and colon cancer cells. However, further genetic dissection of the mechanism of this enhanced cytotoxicity using BRCA1 mutants deficient in DSB repair unexpectedly revealed a dissociation of BRCA1’s function in DNA repair from its effects on cellular sensitivity to DNA damage. Interestingly, we observed a dependence of the DNA damage-induced cell killing on the translocation and accumulation of BRCA1 in the cytosol. Together, these data suggest a novel role of cytoplasmic translocation of BRCA1, not only in controlling its DNA repair functions, but also in the regulation of cell death processes following DNA damage. Further dissection of the mechanism of cytotoxicity induced by BRCA1 cytoplasmic translocation revealed involvement of the apoptotic pathway. We propose that the status of BRCA1 nuclear/cytoplasmic shuttling may provide a molecular marker to predict tumor response and a potential novel target to sensitize cancer cells to DNA damage-based therapy.
Mutations of the breast cancer susceptibility gene BRCA1 have been implicated in the development of familial breast cancers (1). The exact role of BRCA1 as a tumor suppressor, however, is not fully defined. BRCA1 functions in a number of cellular processes, including repair of chromosomal breaks, DNA replication, chromatin remodeling, cell cycle checkpoints, and apoptosis(2). Disruption of any or all of these processes may contribute to the increased risk for carcinogenesis seen in carriers of germline BRCA1 mutations.
In contrast, somatic BRCA1 mutations are rare in sporadic cancers (3). It has been hypothesized that deregulation of wild type BRCA1 function can contribute to the pathogenesis of non-familial cancers, which represent up to 90% of breast cancers. Consistent with this, reduced or absent BRCA1 expression has been found in high grade sporadic breast cancer and occurs at the transcriptional level (4). In addition to transcriptional regulation, BRCA1 function is also regulated by other mechanisms such as protein-protein interactions and post-translational modification (5). More recently, we and others have demonstrated that BRCA1 is a nuclear/cytoplasmic shuttling protein and its functions may be controlled via active shuttling between cellular compartments (5–7).
BRCA1 nuclear shuttling is a complex process. There are two nuclear localization signals (NLSs) within BRCA1, which target it to the nucleus via the importin α/β transport system (8). BRCA1 also contains two nuclear export sequences (NESs) at the N-terminus in its ring domain, which transports BRCA1 to the cytoplasm through the chromosome region maintenence 1 (CRM1)/exportin pathway (9, 10). BRCA1 shuttling can also be influenced by its association with other proteins. The BRCA1-associated ring domain protein (BARD1) has been shown to bind to the ring domain and mask the NES of BRCA1, subsequently preventing nuclear export of BRCA1 through CRM1 (6, 7). The tumor suppressor p53 can also bind BRCA1 and affect BRCA1 cellular localization (5, 11). In contrast to BARD1, however, p53 mediates BRCA1 nuclear export in response to IR-induced DNA damage (5). Consistent with a role of p53 in BRCA1 nuclear export, breast cancer cells with p53 mutations exhibit a predominantly nuclear BRCA1 staining pattern ((5), Wang et al., manuscript submitted). Thus, in vitro and in vivo evidence suggests that control of BRCA1 subcellular localization is a vital process in the temporal and spatial regulation of BRCA1 both for physiological activities such as DNA repair and for the cellular response to DNA damage.
Given these diverse functions of BRCA1 and the poorly defined mechanisms by which its localization regulates these functions, we assessed the repair of DNA DSBs and the DNA damage-induced cytotoxic response as a function of BRCA1 location. The data suggest that BRCA1 shuttling may not only control its nuclear function in DNA repair but may also facilitate additional cellular processes involved in the execution of DNA damage-induced cell death.
MCF7 cells were maintained as previously described (12). Isogenic lines derived from HCC1937 cells carrying wild type BRCA1 expression plasmid (HCCwt-BRCA1), mutated BRCA1 Chk2 phosphorylation site S988A (HCCBRCA1S988A), mutated BRCA1 RING domain C64G (HCCBRCA1C64G), or mutated BRCA1 BRCT domain P1749R (HCCBRCA1P1749R) were maintained as previously described (12). MCF7 and HCC1937 cells were purchased from ATCC (Manassas, VA). The genetic background including expression and function of BRCA1, p53, and caspase 3, 8, and 9, as well as the growth characteristics and their response to genotoxic agents, were tested most recently in May 2010 using western blot analysis, immunohistochemistry, and colony formation assays. All transfections were performed using FuGene6 according to the manufacturer’s recommendations (Roche).
Cells were cultured and mounted onto sterile glass slides and subjected to various treatments as indicated. Immunohistochemistry was performed as previously described (5). Primary antibodies were 1:500 rabbit anti-Rad51 antibody (Calbiochem, Cat#PC130) and 1:150 mouse anti-BRCA1 (Ab-1) (EMD Chemicals Inc, Cat#OP92). Secondary antibodies were 1:1000 anti-mouse Alexa488 conjugated antibody (Molecular Probe, Cat#A-11008) and 1:1000 anti-Rabbit Alexa594 conjugated antibody (Molecular Probe, Cat#A-11005). Staining patterns were visualized via fluorescence microscopy (Carl Zeiss, Thornwood, NY). A total of at least 50 cells were counted per field, and a total of 10 fields were assessed. For BRCA1 localization, cells were assessed as having predominantly nuclear staining (N), predominantly cytoplasmic staining (C), or mixed nuclear/cytoplasmic staining (N/C).
Whole cell lysates were prepared using radioimmunoprecipitation lysis buffer (150mM NaCl, 50mM Tris, pH 8.0, 5mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 1.0% Nonidet P-40) with protease and phophatase inhibitor cocktails (Sigma) and subjected to SDS-PAGE analysis. BRCA1 antibody (Ab-1) was used at 1:1000 dilution. Caspase antibodies [cleaved caspase-3 (Asp175) (Cell Signaling Technology, Cat#9661), cleaved caspase-9 (Asp330) (Cell Signaling Technology, Cat#9501), caspase-8 (H-134) (Santa Cruz, Cat#SC-7890)] were used at 1:1000 dilution. Hsp70 (1:1000, Santa Cruz, Cat#SC-66048) or actin (1:1000, Santa Cruz, Cat#SC-47778) levels were also analyzed by western blot as a loading control.
The colony-forming ability following treatment was evaluated in MCF7 and HCC1937 cells as previously described (12). Briefly, cells subjected to the indicated treatment and doses were maintained in culture in plates for 10–15 days. Colonies in plates were fixed with 70% EtOH and stained with 1% methylene blue. Colonies consisting of >50 cells were scored using a microscope. Survival fraction was calculated as: (number of colonies / number of cells plated) / (number of colonies for corresponding control / number of control cells plated).
HCC1937 breast cancer cells were transfected with pcDNA3 vector control, wild type BRCA1, or various mutant BRCA1 constructs using FuGene6 per the manufacturer’s recommendations (Roche). Twenty four hours post-transfection, cells were subjected to either mock or 10 Gy IR. Forty hours post-IR, cells were harvested, and apoptotic cells were stained using the Annexin V-FITC Apoptosis Detection kit (BD PharMingen, San Diego, CA). The percentage of apoptotic cells was determined by flow cytometry.
Analysis of cleaved caspase 3, 8, or 9 was also performed in transfected cells as above subjected to either mock or 6 Gy IR. Forty-eight hours following IR, whole cell lysates were prepared and subjected to Western blot analysis for cleaved caspase 3, 8, or 9.
Data were analyzed via one way analysis of variance (ANOVA) followed by a Bonferroni or Dunnett post test using GraphPad Prism version 4.02 for Windows (GraphPad Software, San Diego, CA).
Previously, we and others have shown that in response to DNA damage, BRCA1 shuttles between the nucleus and cytoplasm through the importin and CRM1 pathways, respectively (5, 6). Additionally, it has been demonstrated that the protein BARD1 prevents BRCA1 export to the cytosol through its binding to the N-terminal region of BRCA1. This, in turn, masks the BRCA1 NES and blocks BRCA1 interaction with CRM1/exportin (6).
Given the central role of BRCA1 in promoting the repair of DSBs, we sought to determine whether BRCA1 nuclear/cytosolic shuttling regulates its function in DSB repair. We first analyzed Rad51 foci, an in vivo functional marker of homologous recombination (HR) repair activity, in relation to BRCA1 localization in MCF7 human breast cancer cells. The subcellular localization of BRCA1 was assessed by immunohistochemical staining. Similar to previous studies by us and others (5, 6), cells were categorized into three groups based on BRCA1 staining pattern: strictly nuclear (N), strictly cytoplasmic (C), and mixed nuclear and cytoplasmic staining (N/C). Rad51 nuclear foci were subsequently determined in cells of each category (Figure 1A). As shown in Figure 1B, IR-induced Rad51 foci were found predominantly in MCF7 cells with BRCA1 present in the nucleus (90% of cells with nuclear BRCA1, 70% of cells with mixed nuclear/cytoplasmic BRCA1). In contrast, the percentage of cells with IR-induced Rad51 foci were drastically diminished in cells with BRCA1 not present in nucleus (10% of cells with cytoplasmic BRCA1). These results suggest that repair of DSBs is strongly associated with nuclear BRCA1.
To determine whether DSB repair is dependent on BRCA1 in the nucleus, we next examined whether depletion of nuclear BRCA1 could attenuate DSB repair. We thus exogenously expressed the small peptide “tr-BRCA1,” a truncated form (1–301 aa) of BRCA1 that contains the NES and BARD1 binding site (13), in MCF7 cells. Translocation of BRCA1 by tr-BRCA1 was subsequently determined by immunohistochemical staining. As shown in Figure 1C, expression of tr-BRCA1 alone in MCF7 cells effectively decreased nuclear BRCA1 by 2-fold compared to vector control (40% versus 20%). As a reference control, vector-transfected MCF7 cells were irradiated and exhibited a similar 2-fold nuclear export of BRCA1 in response to 5 Gy IR as previously reported (5). IR, however, did not further enhance tr-BRCA1’s effects on BRCA1 localization (18% versus 15% nuclear staining). This suggests that tr-BRCA1 is as efficient as IR in shifting BRCA1 to the cytosol.
Having verified that tr-BRCA1 can efficiently drive BRCA1 to the cytosol, we next confirmed its effects on DNA repair. We reasoned that compromised DNA repair by the expression of tr-BRCA1 would result in increased levels of persistent DSBs following IR. In these experiments, we examined IR-induced γ-H2AX foci, a commonly used in situ marker of DNA DSBs, with or without tr-BRCA1. As shown in Figure 1D, expression of tr-BRCA1 significantly decreases DSB repair efficiency as evidenced by persistent γ-H2AX foci compared to control cells (1 hr post-IR: 32% versus 25%, p<0.001; 8 hr post-IR: 25% versus 12%, p<0.001). These results suggest that BRCA1-mediated DSB repair depends on nuclear BRCA1. Additionally, in support for a role of BRCA1 localization/shuttling in its repair function, we have previously shown that tr-BRCA1 significantly decreased HR efficiency in MCF7 human breast cancer cells (14).
Given that integral cell survival processes such as DNA repair are dependent on nuclear BRCA1 (Figure 1), we hypothesized that depletion of nuclear BRCA1 could potentially serve as a molecular strategy to render cells more susceptible to genotoxic agents. Support of this hypothesis has been demonstrated by sensitization of tumor cells to IR or cisplatin when DSB repair is deficient (15). Because tr-BRCA1 can modify BRCA1 localization and DSB repair function (Figure 1C, 1D, and (14)), we next examined whether expression of tr-BRCA1 could sensitize cells to DNA damaging agents by decreasing the nuclear functions of BRCA1 in DSB repair. The survival fractions as measured by colony formation assays were used to determine cytotoxicity. As shown in Figure 2, increasing cytosolic translocation of BRCA1 by tr-BRCA1 expression in MCF7 breast cancer cells significantly enhanced the cytotoxic response to IR (Figure 2A; p<0.001) or cisplatin (Figure 2B; p<0.001) compared to vector control cells. These effects were also observed in HCT116 human colon cancer cells (data not shown). To determine whether the cytotoxic effects of tr-BRCA1 are specific to its effects on BRCA1, radiosensitization was assessed in the BRCA1-deficient human breast cancer cell line HCC1937, which is hemizygous for BRCA15382insC mutation. This mutation renders the BRCA1 protein unstable and predominantly cytosolic (16). As shown in Figure 2C, tr-BRCA1 did not radiosensitize HCC1937 cells at 4 Gy IR, the dose at which tr-BRCA1 already significantly enhanced radiosensitivity in MCF7 breast cancer cells (Figure 2A). This lack of radiosensitization by tr-BRCA1 in BRCA1-deficient cells suggests a dependence of these effects on BRCA1.
If compromised DSB repair is the predominant underlying mechanism for tr-BRCA1-mediated sensitization of cells to DNA damage, then mutations that abolish BRCA1’s repair function would similarly render cell susceptible to DNA damage. We and others have shown that the DNA damage response kinase Chk2 regulates BRCA1’s repair function by phosphorylating BRCA1 on serine 988. Mutation of this Chk2 phosphorylation site renders cells deficient in DSB repair (17, 18). Thus, to determine whether cytotoxicity mediated by tr-BRCA1 is conferred by decreasing BRCA1’s repair function, we utilized the previously established isogenic BRCA1-deficient HCC1937 cells that stably express either wild type BRCA1 (HCCwt-BRCA1) or BRCA1 mutated at the Chk2 phosphorylation site (HCCBRCA1-S988A) (18). As previously reported and as shown in Figure 3A (black data points), restoration of wild type BRCA1 in HCC1937 cells resulted in a 5-fold decrease in radiosensitivity at 6 Gy IR compared to HCC1937 cells with control pcDNA3 vector (12). This IR resistance has been hypothesized to be due to the enhanced efficiency of cells to repair IR-induced DNA DSBs. Similar effects were seen in response to the DNA damaging agent cisplatin (75µM) (Figure 3B, black data points). Surprisingly, HCCBRCA1-S988A exhibited IR and cisplatin resistance similar to HCCwt-BRCA1 cells despite being deficient in DSB repair (Figures 3A and 3B, red data points). Additionally, this differential response to DNA damage-induced cytotoxicity among the various BRCA1 constructs is not due to differences in protein expression (Figure 3C) or cell cycle effects (data not shown). These results did not support our hypothesis that compromised DSB repair is the predominant underlying mechanism for enhanced sensitivity of cells to DNA damaging agents. Instead, our observations imply a potential dissociation of BRCA1’s function in DSB repair and cellular cytotoxicity.
It has been shown that BRCA1 is required for DNA damage-induced apoptosis via the caspase 3 pathway (19). Furthermore, cytoplasmic BRCA1 accumulation alone induces apoptosis (7). We have also previously shown that DNA damage-induced BRCA1 nuclear export is dependent on p53 (5). Thus, an alternative hypothesis is that in addition to inhibition of repair function, BRCA1 nuclear export/cytosolic translocation is also required to regulate cell death processes following DNA damage. Thus, we next examined whether the observed resistance to DNA damage in HCCwt-BRCA1 and HCCBRCA1-S988A cells was due to dysregulated BRCA1 nuclear/cytosolic shuttling secondary to the endogenous p53 deficiency of HCC cells, given that p53 controls DNA damage-induced BRCA1 nuclear export. To test this notion, we assessed nuclear shuttling of BRCA1 in HCC1937 cells. Interestingly, in contrast to IR-induced BRCA1 nuclear export seen in p53-proficient MCF7 cells ((5) and Figure 1C), the p53-deficient HCCwt-BRCA1 cells exhibited predominantly nuclear BRCA1 with a diminished magnitude of IR-induced BRCA1 nuclear export (Figure 3D). Similarly, mutation of BRCA1 at the Chk2 phosphorylation site did not significantly alter BRCA1 localization compared to wild type BRCA1 (Figure 3D). These data suggest that, in addition to repair of damaged DNA, the resistance to DNA damage-induced cytotoxicity in HCCwt-BRCA1 and HCCBRCA1-S988A cells may be associated with diminished shuttling of BRCA1 from the nucleus to the cytosol.
To further test this alternative hypothesis that BRCA1 nuclear export following DNA damage confers the enhanced cytotoxicity, we utilized HCC1937 cells that express BRCA1 C64G (HCCBRCA1C64G), which carries a mutation within the N-terminal RING domain, or BRCA1 P1749R (HCCBRCA1P1749R), which carries a mutation within the C-terminal BRCT domain. These mutations have been previously shown to not only affect BRCA1 localization (20) but also render cells deficient in repairing IR-induced DNA DSBs (12). In contrast to the HR-deficient Chk2 kinase mutant above, HCCBRCA1P1749R cells expressed predominantly cytoplasmic BRCA1 with or without IR, while HCCBRCA1C64G cells exhibited pronounced BRCA1 nuclear export in response to IR (Figure 4A).
Interestingly, HCCBRCA1C64G cells, which are proficient at IR-induced BRCA1 nuclear export, and HCCBRCA1P1749R cells, which exhibit predominantly cytoplasmic BRCA1, possessed an increased sensitivity to IR and cisplatin similar to HCCVector cells when compared to HCCwt-BRCA1 and HCCBRCA1-S988A cells (Figures 4B and 4C). These effects are not due to differences in BRCA1 expression levels (Figure 4D). Thus, our data support the notion that, in addition to impaired DNA repair, increased cytosolic BRCA1 is a critical determinant of enhanced cytotoxicity from DNA damage.
We next reasoned that if cytosolic accumulation of BRCA1 is the critical determinant in DNA damage-induced cytotoxicity, this effect should not be limited to the type of DNA lesions which specifically require BRCA1-dependent HR-mediated repair. To further test the hypothesis that increased cytosolic BRCA1 is a crucial determinant of enhanced cytotoxicity from DNA damage, we analyzed sensitivity of cells to UV-induced DNA damage, which is predominantly repaired via the nucleotide excision repair (NER) pathway. MCF7 cells with or without the ectopic expression of tr-BRCA1, which we have shown to induce BRCA1 cytosolic localization independent of DNA damage (Figure 1C), were subjected to UV exposure. The survival fractions as measured by colony formation assays were used to determine cytotoxicity. As shown in Figure 5, increasing cytosolic translocation of BRCA1 by tr-BRCA1 expression in MCF7 breast cancer cells significantly enhanced the cytotoxic response to UV (Figure 5; p<0.001) compared to vector control cells. These results again support the hypothesis that cytosolic BRCA1 confers enhanced cytotoxicity to DNA damage.
One potential mechanism by which BRCA1 subcellular localization controls DNA damage-induced cytotoxicity may be the previously reported role of cytoplasmic BRCA1 in the induction of apoptosis (7). To test this possibility, we analyzed the effect of the various BRCA1 mutants on IR-induced apoptosis as a function of their subcellular location. As shown in Figure 6A, HCC1937 cells that express BRCA1 mutants located predominantly in the cytosol exhibited the greatest IR-induced apoptosis as measured by the positivity of AnnexingV staining. Further support for involvement of the apoptotic pathways is shown in Figure 6B. In particular, cells which express cytosolic localized BRCA1 mutants exhibit a 2 fold increase in cleavage of caspase 3 (Figure 6C). These results are in agreement with the cytotoxicity shown in Figure 4.
There are 2 major apoptotic processes, consisting of the intrinsic and extrinsic pathways (21). The extrinsic pathway is activated via pro-apoptotic ligand-mediated stimulation of cellular death receptors. This, in turn, results in cleavage of caspase 8 and induction of apoptotic response. In contrast, the intrinsic pathway is triggered by stress signals from within the cell which ultimately results in cleavage of the initiator caspase 9 and subsequent programmed cell death.
To further investigate the mechanism by which cytosolic BRCA1 mediates IR-induced apoptosis, cleavage of caspase 8 and 9 following IR was examined. As shown in Figure 7A and 7B, an increased IR-induced cleavage of caspase 9 is observed when there is a predominance of cytosolic BRCA1. Interestingly, no IR-induced cleavage of caspase 8 is observed (data not shown). This is consistent with a role of cytosolic BRCA1 in the intrinsic apoptotic pathway. These results support the notion that cytosolic localization/shuttling of BRCA1 may regulate apoptotic processes through the intrinsic pathway following DNA damage. Further support of this idea is evident in the vector control HCC1937 cells, which harbor the BRCA15382insC mutation that renders the BRCA1 protein predominantly cytosolic.
Taken together, these data suggest that DSB repair deficiency alone may not be sufficient for radiosensitization, and that nuclear export-induced loss of DSB repair in combination with cytoplasmic accumulation of BRCA1 appears to be required to fully launch the DNA damage-induced cytotoxic response.
In this report, we investigated the regulatory role of BRCA1 localization on its DNA repair function as well as on the cytotoxic response to DNA damage. Specifically, we showed that DNA repair was dependent on nuclear BRCA1. Targeted translocation of BRCA1 to the cytoplasm suppressed BRCA1’s function in DSB repair and conferred enhanced cytotoxicity to DNA damaging agents, including IR and cisplatin. Evidence for decreased HR capacity and enhanced sensitivity to DNA damaging agents has been previously reported (15). Surprisingly, our genetic study using various BRCA1 mutants deficient in either repair or nuclear/cytosolic shuttling revealed a dissociation of DNA damage-induced cytotoxicity from BRCA1’s DNA repair function but a dependence on BRCA1 shuttling. These results imply that BRCA1 localization not only controls repair but also regulates additional cell death processes in response to DNA damage. Understanding this facet of the molecular pathways regulated by BRCA1, in particular BRCA1-mediated regulation of cell death, could shed light on one potential mechanism by which BRCA1 serves as a tumor suppressor.
Using various BRCA1 mutants, we found that one of the main determinants of cellular sensitivity to genotoxic therapy may be DNA damage-induced BRCA1 nuclear export and/or cytosolic accumulation. The BRCA1-deficient HCC1937 cells exhibit exquisite sensitivity to DNA damaging agents. Interestingly, these cells do express a mutant 5382insC BRCA1 that is exclusively cytosolic, which supports the notion that cytoplasmic BRCA1 confers the enhanced cytotoxic response to DNA damage. Stable expression of wild type BRCA1 in these cells (HCCwt-BRCA1) confers increased therapeutic resistance that is thought to be due to enhanced repair of therapy-induced DNA damage. However, a similar resistance is seen in isogenic cells expressing a BRCA1 mutation at the Chk2-phosphorylation site (HCCBRCA1-S988A). Contrary to HCCwt-BRCA1 cells, HCCBRCA1-S988A cells are HR deficient, which argues against the idea that DSB repair alone is sufficient and can fully account for the resistance to DNA damage observed in these cells.
A potential explanation may be that the cell cycle checkpoint function of wild type BRCA1 is responsible for rescuing the sensitivity of HCC1937 cells. However, the BRCA1 S988A mutation is deficient in S-phase checkpoint (17), making this explanation less likely. We have also examined another BRCA1 mutant, S1423A/S1524A, which renders BRCA1 resistant to ATM-mediated phosphorylation. Cells stably expressing this mutant have a deficient G2 checkpoint but are proficient in HR (22). BRCA1 S1423A/S1524A localizes predominantly in the nucleus, does not respond to DNA damage-induced nuclear export, and restores the resistance of HCC1937 cells to IR or cisplatin (data not shown, Wang et al. manuscript submitted). This is also in accordance with previous reports that demonstrate that checkpoint dysfunction does not abrogate cellular sensitivity to IR (22).
Sensitivity to DNA damaging agents, however, is restored upon disruption of nuclear BRCA1 localization, as evidenced by BRCA1 N- (C64G) and C-terminal (P1749R) mutations. These cells not only are deficient in HR-mediated repair of DSBs but also exhibit a robust DNA-damage-induced BRCA1 nuclear export (C64G) or a predominantly cytosolic distribution of BRCA1 (P1749R) (20). Interestingly, the cytosolic translocation/localization of these BRCA1 mutants is not dependent on p53. The C64G mutation in BRCA1 resides in the ring domain of BRCA1, which interacts with BARD1 (6, 7). This interaction prevents BRCA1 nuclear export by masking the BRCA1 NES. It is possible that the C64G mutation disrupts BRCA1-BARD1 interaction following DNA damage to allow for p53-independent BRCA1 shuttling.
In contrast, the P1749R mutation in BRCA1 resides in the BRCT domain of BRCA1, which interacts with other DNA repair proteins such as BACH1 to localize BRCA1 to sites of DNA damage (23, 24). The BRCT domain also interacts with p53 (25, 26), and p53 dysfunction has been shown to induce BRCA1 nuclear accumulation and disrupt BRCA1 nuclear export following DNA damage (Jiang et al., manuscript submitted). This mutation (P1749R) may render BRCA1 unable to interact with its partner proteins and thus results in a predominantly cytosolic localization, as seen in this and other studies(20).
Nevertheless, these data thus suggest that the unrepaired DSBs alone resulting from loss of BRCA1’s repair function may not be sufficient to fully affect the cytotoxicity and sensitivity of these cells to DNA damaging agents but rather may rely on the subsequent nuclear export/cytoplasmic accumulation of BRCA1. This is not discordant with previous observations that repair of DSBs is critical for cellular sensitivity to DNA damage. Instead, our novel finding implies that cytosolic translocation of BRCA1 following DNA damage may be the process that links failed repair of DNA damage to the induction and execution of cell death processes.
Specifically, disruption of DNA damage-induced cytosolic translocation of BRCA1 inhibited the apoptotic pathway by 2 fold as measured by Annexin V positivity and cleavage of caspase 3 and 9. The magnitude of regulation of apoptosis does not reach the levels of cytoxicity as measured by colony formation assays. As multiple pathways other than apoptosis can affect the colony forming ability of cells, such as inhibition of cell proliferation, cell cycle arrest, mitotic catastrophe, and autophagy, it is likely that cytosolic translocation of BRCA1 may regulate multiple cytotoxic pathways. These studies are currently ongoing.
Further support of a role of cytosolic BRCA1 in conferring cytoxicity following DNA damage was found in cells subjected to UV damage (Figure 5). Cells expressing tr-BRCA1, which drives BRCA1 to the cytosol, possess an enhanced sensitivity to UV. As UV-damage is mediated by the NER pathway, this result emphasizes the importance of cytosolic BRCA1 in the cytotoxic response. However, a transcriptional role of nuclear BRCA1 in the regulation of the repair of UV-mediated damage (27) cannot be ruled out.
Based on our findings, we speculate that nuclear depletion and cytosolic accumulation of BRCA1 may:
1) completely abolish all (including unknown) aspects of BRCA1’s nuclear functions. In addition to DNA repair and checkpoints, for example, sequestration of BRCA1 away from the nucleus could block BRCA1’s interaction with other nuclear protein partners that initiate or regulate cell death processes and thus prevent activation of other DNA damage response pathways;
2) result in a loss of nuclear function in combination with a gain of cytosolic function in mediating cell death, such as BRCA1 localization to the mitochondria (28); or
3) change the ratio of nuclear:cytoplasmic BRCA1, resulting in a switch from repair and survival activity in the nucleus to cell death processes in the cytosol.
Interestingly, recent reports suggest an importance of cytosolic BRCA1 in facilitating cell death pathways (19, 28). Similarly, BRCA1 nuclear export has been shown to stimulate apoptosis, while nuclear sequestration of BRCA1 inhibits apoptosis (7). These findings further substantiate our observation that the enhanced cytotoxic response to DNA damaging agents is dependent on BRCA1 nuclear shuttling/cytosolic accumulation of BRCA1. Cytoplasmic BRCA1 has also been shown to interact with the centrosome. This additional function contributes to BRCA1’s tumor suppressor activities and maintenance of genomic stability (reviewed in (29)). It is intriguing to hypothesize that BRCA1 executes its tumor suppressor function not only by its critical role in repair in the nucleus but also signals to the apoptotic machinery in the cytoplasm and thereby eliminates cells when DNA damage cannot be successfully repaired. Ongoing investigations to test these notions may provide further insight regarding the role of BRCA1 nuclear-cytoplasmic shuttling in the control of its function and determination of cell fate (survival vs. death).
BRCA1 is essential in maintaining genomic stability and controlling the cellular response to genotoxic stress. Precise regulation of these BRCA1 functions is of obvious importance from an oncologic and cell survival perspective. One emerging target is BRCA1 localization and shuttling, as sequestration of BRCA1 away from the nucleus may switch BRCA1 function from repair in the nucleus to activation of cell death signals in the cytoplasm. These data point to the potential use of BRCA1 shuttling as a novel avenue by which manipulation of BRCA1 localization can control cellular function and sensitivity to therapy. Furthermore, BRCA1 shuttling itself may be a functional biomarker to predict tumor response to therapy.
This work was supported by grants R01 CA118158-02 from the National Institutes of Health (to F.X.) and RR0813 from the Radiological Society of North America (to E.S.Y.). We thank Melissa Stauffer, PhD, of Scientific Editing Solutions, for editorial assistance.