PARP inhibitors have gained recent attention due to their highly selective killing of BRCA1/2-mutated and DNA double-strand break (DSB) repair–deficient tumors. Unfortunately, the majority of sporadic breast cancers carry wild-type BRCA1/2 and are proficient in DSB repair. We and others have shown that BRCA1 is a nuclear/cytoplasm shuttling protein that is transiently exported from the nucleus to the cytosol upon various stimuli. Thus, we hypothesized that depletion of nuclear BRCA1 would compromise DSB repair and subsequently render sporadic tumors susceptible to PARP inhibition. Indeed, in human sporadic breast cancer cells with functional BRCA1 and proficient DSB repair, a transient nuclear depletion of BRCA1 and subsequent homologous recombination repair deficit was induced with either truncated BRCA1 or irradiation. This rendered these human sporadic breast cancer cells susceptible to PARP inhibition. These observations were confirmed genetically using mislocated BRCA1 mutants as well as in vivo in mice bearing breast tumor xenografts. These data support the potential strategy of targeting BRCA1 location to convert BRCA1-proficient sporadic tumors to be susceptible to the synthetic lethal combination with PARP inhibitors. Cancer Res; 72(21); 5547–55. ©2012 AACR.
PARP inhibitors induce synthetic lethality by targeting homologous recombination (HR)-mediated DNA repair–deficient tumors (1–3). However, this approach is only applicable to about 10% of cancers. Thus, much effort has been undertaken to expand the use of PARP inhibitors beyond the realm of BRCA-associated tumors by combining with agents that alter the DNA damage/repair pathways. Indeed, PARP inhibitors have been reported to enhance cytotoxicity in sporadic tumors when combined with other DNA-damaging agents, such as with platinum-based chemotherapy in breast cancer (4). Unfortunately, these combinations have so far failed to be efficacious in clinical trials (5, 6). Thus, novel therapeutic approaches are warranted.
We and others have previously shown that the tumor suppressor BRCA1, a nuclear–cytoplasmic shuttling protein, is critical for DNA damage repair and induction of apoptosis. The function of BRCA1 is regulated by a variety of mechanisms including transcriptional control, phosphorylation, and protein–protein interactions (7). Its subcellular localization is controlled by a nuclear localization signal (NLS)-mediated nuclear import via the importin receptor pathway and a nuclear export signal (NES)-facilitated nuclear export through a CRM1-dependent pathway (7). Importantly, cytoplasmic relocalization of BRCA1 protein is one mechanism whereby BRCA1 function is regulated in response to DNA damage.
We have previously shown that sequestering BRCA1 from the nucleus to the cytoplasm compromises its nuclear function in DNA repair and results in enhanced cytotoxic response to ionizing radiation (IR) and other DNA-damaging agents (7–9). Because PARP inhibitors have been reported to induce synthetic lethality in DNA repair–deficient cells, we hypothesized that inducing BRCA1 cytoplasmic translocation in sporadic breast cancer, which generates a double-strand break (DSB) repair deficiency, will confer enhanced cytotoxicity to the PARP inhibitor ABT-888. Consistent with our hypothesis, ectopic expression of truncated BRCA1 (tr-BRCA1) sequestered endogenous BRCA1 to the cytosol, suppressed HR-mediated DSB repair, and subsequently rendered human sporadic breast cancer cells to become susceptible to the PARP inhibitor ABT-888. These observations were confirmed genetically using mislocated mutants of BRCA1. Furthermore, as a potential therapeutic strategy, we used IR to generate a transient depletion of nuclear BRCA1 and an HR repair deficiency. This subsequently conferred breast tumor cytotoxicity to ABT-888. Importantly, these findings were validated in mice bearing breast tumor xenografts. These data support the potential strategy of using radiotherapy to make BRCA1-proficient tumor cells susceptible to systemic DNA-damaging agents such as PARP inhibitors. Furthermore, this novel strategy may also be feasible in other tumor types.
Materials and Methods
MCF7 (HTB-22, American Type Culture Collection), MCF7DRGFP, MCF7 cells stably expressing BRCA1 short hairpin RNA (shRNA; obtained courtesy of Dr. Simon Powell, Memorial Sloan Kettering, New York, NY) were used in this study. Please refer to Supplementary Materials and Methods for details. The genetic background, including expression and function of key proteins as well as the growth characteristics and their response to genotoxic agents, was tested most recently in December 2011 using western blot analysis, immunohistochemical, and colony formation assays. The PARP inhibitor ABT-888 (Enzo Life Sciences) was used in our study.
Tr-BRCA1 has been described previously (10, 11). Wild-type (WT) BRCA1 YFP and NES-mutant BRCA1 YFP were kindly provided by Dr. Beric Hendeson (Westmead Millennium Institute, New South Wales, Australia) and has been previously described (12). NLS-mutant BRCA1 YFP was created using a site-directed mutagenesis kit (Invitrogen) and had AA503-A506 mutation.
BRCA1 location, Rad51, and DNA DSBs were assayed as described previously (8–10, 13, 14).
Clonogenic survival assay
Cell survival was evaluated by the colony formation assay as previously described (10).
Tumor growth delay
Tumor growth delay and immunohistochemical detection of γ-H2AX and BRCA1 were conducted as described previously (8, 15). Please refer to Supplementary Methods for details. All animal procedures were approved by the Vanderbilt University Institutional Animal Care and Use Committee (Nashville, TN; protocol M/08/154).
The data were analyzed via ANOVA followed by a Bonferroni posttest using GraphPad Prism version 4.02 (GraphPad Software). Data were presented as average ± SEM.
Targeting endogenous WT-BRCA1 to the cytosol using tr-BRCA1 confers susceptibility to ABT-888 in breast cancer cells
BRCA1 shuttles between the nucleus and cytoplasm through the importin and CRM1 pathways, respectively (7, 16). It has been shown that the protein BARD1 prevents BRCA1 export to the cytosol through its binding to the NH2-terminal region of BRCA1. This, in turn, masks the NES-BRCA1 and blocks BRCA1 interaction with CRM1/exportin (10, 16). Exogenous expression of the small peptide “tr-BRCA1,” a truncated form (1–301 amino acids) of BRCA1 that contains the BARD1-binding site and competes with endogenous full-length BRCA1 in binding with BARD1, has been previously shown to effectively induce BRCA1 cytoplasmic translocation (10). To target BRCA1 to the cytosol, we transiently expressed tr-BRCA1 in MCF7 cells, a well-characterized breast cancer cell line with WT-BRCA1 and WT-p53 (7, 9, 10). Changes in BRCA1 location following tr-BRCA1 was subsequently determined by immunohistochemical staining. Similar to our previous studies (7, 9, 10), cells were categorized into 3 groups on the basis of BRCA1 staining pattern: strictly nuclear (N), strictly cytoplasmic (C), and mixed nuclear and cytoplasmic staining (NC; refs. 9, 10). As shown in Fig. 1A, expression of tr-BRCA1 alone in MCF7 cells effectively decreased endogenous nuclear BRCA1 by 2-fold compared with vector control (49% vs. 24%) with a concomitant 3-fold increase in cytosolic fraction of BRCA1 (27% vs. 73%). This suggests that tr-BRCA1 is effective in shifting BRCA1 to the cytosol, away from its nuclear repair substrates.
Having verified that tr-BRCA1 could efficiently drive endogenous BRCA1 to the cytosol in MCF7 cells, we next confirmed its effects on HR-mediated DNA repair. MCF7 DRGFP cells, which carry a single copy of a chromosomally integrated HR repair substrate as described previously (7, 9) and in Fig. 1B, were transiently transfected with tr-BRCA1 and subsequently transfected with ISce-1 endonuclease, which generates a DSB within the nonfunctional GFP gene. Only HR-mediated DSB repair restores GFP function. Thus, GFP-positive cells, indicative of HR-mediated repair, were sorted for quantification of HR capacity. As shown in Fig. 1B, a 4-fold decrease in GFP-positive cells was observed in tr-BRCA1–transfected cells compared with vector alone. These HR-mediated repair effects were not due to differences in cell viability or transfection efficiency, which were included as controls in all HR repair assays. These results suggest that cytosolic translocation of endogenous BRCA1 by tr-BRCA1 indeed induces a DSB repair deficiency in breast cancer cells.
We next assessed cytotoxicity of MCF7 cells expressing tr-BRCA1 in combination with various doses (1–10 μmol/L) of ABT-888 (13, 14). In these experiments, MCF7 cells were transfected with either YFP-tr-BRCA1 or YFP and subsequently sorted. Twenty-four hours later, they were exposed to various doses of ABT-888. As shown in Fig. 1C, ABT-888 alone failed to have any cytotoxic effect in vector-transfected cells whereas those expressing tr-BRCA1 displayed a dose-dependent cytotoxicity to ABT-888.
We reasoned that compromised DNA repair by the expression of tr-BRCA1 would result in increased levels of persistent DSBs. Indeed, as shown in Fig. 1D, increased DSBs as evidenced by persistent γ-H2AX foci were observed in tr-BRCA1 expressing cells treated with ABT-888 compared with vehicle control cells (24 hours after ABT-888 treatment, 70% vs. 5%, P < 0.001). No significant levels of γ-H2AX foci were detected in vector control cells with or without exposure to ABT-888 due to their repair proficiency.
To substantiate that the main determinant of susceptibility to PARP inhibition is the induction of DNA repair deficiency from sequestering BRCA1 to the cytosol, we next used a fluorescent YFP-tagged NES-BRCA1 genetic mutant [mutation at aa 86–90 (refs. 12, 17) and localized exclusively in the nucleus]. Tr-BRCA1 failed to induce cytosolic translocation (Supplementary Fig. S1A) or DNA damage following ABT-888 (Supplementary Fig. S1B) in this mutant. These results again substantiate the role of targeting BRCA1 to the cytosol and the subsequent inhibition of HR-mediated DSB repair as a mechanism by which tumor cell susceptibility to PARP inhibition can be achieved.
Genetic exclusion of nuclear BRCA1 confers susceptibility to ABT-888
To further validate whether the main determinant of susceptibility to PARP inhibition is the induction of DNA repair deficiency from sequestering BRCA1 to the cytosol, we next used YFP-tagged WT-BRCA1 as well as YFP-tagged NES (as above) and NLS mutants. The NLS-BRCA1 mutant (mutation at aa 503–506) is localized in the cytosol (12, 17). These plasmids were transiently transfected into MCF7 BRCA1 shRNA cells in which the endogenous WT-BRCA1 has been depleted through stably expressing BRCA1 shRNA. BRCA1 location was assessed 48 hours following transfection by staining cells for YFP as a surrogate for BRCA1, as the constructs were YFP tagged. Cells were scored as above. As shown in Fig. 2A, compared with cells expressing WT-BRCA1, cells expressing the NLS-BRCA1 mutant had predominantly cytosolic BRCA1 (87%). Conversely, NES-BRCA1 mutant transfected cells had predominantly nuclear BRCA1 (82%). Furthermore, the shuttling response of these BRCA1 mutants following IR was also assessed (Supplementary Fig. S2A). Similar to our previous observations (7), IR effectively decreased nuclear WT-BRCA1 by 2-fold compared with mock-radiated control (47% vs. 19%) whereas concomitantly increasing cytosolic BRCA1 (24% vs. 56%). In contrast, the NES-BRCA1 and NLS-BRCA1 failed to show any significant redistribution following IR.
On the basis of these results, and given the central role of BRCA1 in promoting the repair of DSBs, we next assessed whether the location mutants of BRCA1 similarly altered HR-mediated DSB repair capacity of cells. We first analyzed Rad51 foci, a well-established functional marker of HR repair activity. It has been shown that accumulation of DNA single-strand breaks (SSB) will be converted to DSBs during DNA replication. Cellular attempts to repair these DSBs lead to induction of HR-mediated repair and Rad51 foci. We thus reasoned that treatment with ABT-888 should induce HR repair in cells expressing the nuclear-localized BRCA1 isoforms (NES-BRCA1, WT-BRCA1) but not in cells with cytoplasmic BRCA1 (NLS-BRCA1). Consistent with our hypothesis, no significant levels of Rad51 foci–positive cells were detected in the vector or NLS-BRCA1 mutant cells (Fig. 2B). In addition, WT-BRCA1–transfected cells displayed elevated levels of Rad51 foci following exposure to ABT-888, indicative of activated HR-mediated DSB repair (1% vs. 8%, P < 0.001). Similar results were obtained in NES-BRCA1 mutant expressing cells following exposure to ABT-888 (1% vs. 8%, P < 0.001). ABT-888–induced Rad51 foci were also potentiated with IR in the WT- and NES-BRCA1 but not the NLS-BRCA1–expressing cells (Supplementary Fig. S2B). Furthermore, it was observed that after IR, the enhanced RAD51 foci in response to PARP inhibition was more robust in the NES-BRCA1 compared with WT-BRCA1–expressing cells, which correlates closely with the amount of BRCA1 being retained in the nucleus following IR. These results substantiate the observation that nuclear BRCA1 is required to activate HR-mediated repair and that targeting BRCA1 location can disrupt HR-mediated DSB repair.
On the basis of these observations, we hypothesized that expressing the NLS-BRCA1 mutant (cytosolic BRCA1), but not NES-BRCA1 mutant (nuclear BRCA1), in the MCF7 BRCA1 shRNA cells will result in synthetic lethality with ABT-888. To test this hypothesis, we conducted colony-forming assays with the various mutants in combination with various doses (1–10 μmol/L) of ABT-888. IR was used as a positive control with WT-BRCA1–expressing MCF7 cells. As shown in Fig. 2C, ABT-888 failed to induce any cytotoxicity in WT-BRCA1–expressing cells. Significantly increased levels of ABT-888–induced dose-dependent cell death were observed in the NLS-BRCA1 mutant–expressing cells that express predominantly cytosolic BRCA1. No ABT-888–induced cytotoxicity was observed in the MCF7 NES-BRCA1 mutant cells, which have nuclear accumulation of BRCA1. These results validate genetically that targeting BRCA1 location to the cytosol can augment tumor susceptibility to PARP inhibition via an induced DSB repair deficit.
Because PARP-induced cytotoxicity is mainly attributed to persistent DSBs, we next assessed DNA DSBs in treated cells expressing the various BRCA1 location mutants. As shown in Fig. 2D, in the vector alone transfected cells, significantly increased persistent DNA damage was observed 24 hours following ABT-888 treatment as shown by increased percentage of cells with γ-H2AX foci. This is consistent with a DSB repair deficiency due to absence of functional BRCA1. Similarly, a significantly increased persistent DNA damage was observed in the NLS-BRCA1 mutant expressing cells, which are deficient in HR repair due to cytosolic BRCA1. On the contrary, WT-BRCA1 or NES-BRCA1 mutant expressing cells failed to display increased persistent DNA damage following treatment with ABT-888, likely due to proficient HR repair. However, following IR, ABT-888–induced γ-H2AX foci were potentiated in the NLS-BRCA1 and WT-BRCA1 but not NES-BRCA1–expressing cells (Supplementary Fig. S2C). These results indicate that ABT-888–induced cytotoxicity following IR is due to persistent DNA damage from a DNA repair deficiency caused by altered BRCA1 location.
Radiation-induced cytosolic translocation of BRCA1 induces susceptibility to ABT-888 in WT-BRCA1 breast cancer cells
Our results thus far support the notion that targeting BRCA1 to the cytoplasm induces a DNA repair defect and subsequent susceptibility to ABT-888. As a potential clinical application of this novel finding, we hypothesized that IR, which we have reported to induce BRCA1 nuclear export (10, 16), will induce synthetic lethality with PARP inhibition (7, 10). To test this hypothesis, we first analyzed BRCA1 location via immunohistochemistry at various time points following 4 Gy IR in MCF7 breast cancer cells. Indeed, IR induced BRCA1 cytosolic translocation in MCF7 cells in a time-dependent manner, with a 2-fold decrease in nuclear BRCA1 compared with mock-radiated control (40% vs. 20%) and a concomitant increase in cytosolic BRCA1 (25% vs. 50%). The maximal shift was observed at 24 hours following IR (Fig. 3A). Next, we verified whether IR-induced decrease in nuclear BRCA1 compromises HR-mediated DSB repair capacity in MCF7DRGFP cells. As shown in Fig. 3B, 4 Gy IR resulted in a 2-fold decrease in HR repair efficiency (P < 0.001). Importantly, these effects on HR repair were not due to indirect changes in cell-cycle distribution (Supplementary Fig. S3A–S3D). Interestingly, NES-BRCA1 mutant rescued IR-induced reduction in HR-mediated repair reaffirming that IR can induce a transient DNA repair deficit by depleting nuclear BRCA1.
Our results thus far suggest that IR induces BRCA1 nuclear export and subsequently decreases HR-mediated DSB repair. We next tested whether IR can induce synthetic lethality with ABT-888 in MCF7 breast cancer cells. As shown in Fig. 3C, after normalizing for IR-induced toxicity, a differential dose-dependent susceptibility to IR and ABT-888 was observed in MCF7 human breast cancer cells (3-fold reduction in cell viability with combination treatment). As expected, treatment with the inhibitor alone failed to show any cytotoxic effect due to proficient HR repair. Furthermore, the NES-BRCA1 mutant, which is not exported following IR, abrogated synthetic lethality of IR and PARP inhibition.
We next hypothesized that the mechanism of synthetic lethality involved attenuation of HR by IR. Rad51 foci levels were analyzed via immunohistochemical staining as a surrogate marker for HR-mediated DSB repair in NES-BRCA1 mutant cells. As anticipated, a robust induction in Rad51 foci was observed in NES-BRCA1 mutant cells, which showed significant nuclear BRCA1, whereas no significant induction was observed in the WT-BRCA1 cells (Fig. 3D).
We also examined whether cells exposed to IR exhibit either increased levels of or persistent ABT-888–induced DSBs by assaying levels of γ-H2AX foci (13, 14, 18). A significant increase in unrepaired DSBs was observed in cells treated with ABT-888 and IR, as shown by increased percentage of cells with γ-H2AX foci (Fig. 3E) but, importantly, persistent γ-H2AX foci were not observed in the NES-BRCA1 mutant. These results indicate that IR-induced BRCA1 nuclear export creates a DSB repair deficiency, which results in increased levels of persistent DSBs in treated cells.
Targeting BRCA1 location with radiation delays tumor growth in breast cancer tumor xenografts
We next validated our intriguing observations in vivo in mice bearing MCF7 breast cancer xenografts. Tumors were exposed to 3 Gy or mock IR on day 0. Beginning on day 1, tumor-bearing mice were then treated with or without ABT-888 (25 mg/kg daily) for 5 days, at which point treatment ceased and tumors were followed through day 21. Consistent with results observed in cell culture, tumor growth was modestly inhibited upon treatment with either IR alone or ABT-888 alone as compared with mock-treated mice. However, growth of tumors treated with IR followed by 5 days of PARP inhibition was profoundly reduced as compared with untreated tumors or those treated with either agent alone (Fig. 4A).
In addition, accumulation of mitotic figures was observed at 24 hours after IR (Fig. 4B). These are indicative of DNA damage and typically precede mitotic catastrophe and cell death (19). By 6 days, IR-treated tumors displayed no mitotic figures but abundant apoptotic bodies (Fig. 4C). Interestingly, mitotic figures did not accumulate in tumors treated with ABT-888. Furthermore, apoptotic bodies were more predominant in IR + ABT-888–treated tissues than what was seen in response to either agent alone. Increased collagen scarring was also apparent in IR-treated tumors but was increased in tumors treated with IR + ABT-888, consistent with drastically reduced tumor cellularity.
Similar to our in vitro results, increased cytosolic BRCA1 was observed in tumors obtained from mice irradiated with 3 Gy IR (Fig. 4D, P < 0.001, Supplementary Fig. S4A). Furthermore, 2-fold increase in the level of unrepaired residual DNA DSBs was observed in these tumors treated with combination of IR + ABT-888, as measured by the amount of cells with persistent γ-H2AX foci (Fig. 4E, P < 0.001, Supplementary Fig. S4B). Thus, these in vivo results validate the role of BRCA1 location as a mechanism by which tumors can be rendered susceptible to PARP inhibition.
BRCA1 is a nuclear shuttling protein that is essential in maintaining genomic stability and controlling the cellular response to genotoxic stress. Precise regulation of these BRCA1 functions is vital 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. When nuclear, BRCA1 controls high fidelity repair of damaged DNA. In contrast, BRCA1 has been shown to enhance p53-independent apoptosis when cytoplasmic (8, 16). Interestingly, we have also previously reported that cytosolic translocation of BRCA1 and subsequent accumulation in the cytosol controls its DNA repair functions and regulates cell death processes following DNA damage (10). These data point to other potential mechanisms in addition to an induced DNA repair deficit that may explain the enhanced cytotoxicity to PARP inhibition following IR-mediated BRCA1 nuclear export, including the potential role of cytosolic BRCA1 in augmenting cell death pathways as well as other cytosolic functions of BRCA1.
Recent studies support the existence of a BRCAness phenotype in sporadic breast cancers, such as the highly aggressive triple-negative subtypes, without a BRCA mutation. This has been thought to confer sensitivity of PARP inhibitors (20–23). Whether this BRCAness phenotype involves aberrant localization of BRCA1 is an interesting question and warrants further investigation.
BRCA1 shuttling can be regulated via protein–protein interaction (24, 25). The BRCA1-associated RING domain protein (BARD1) binds and masks the NES- BRCA1 located at the N-terminal RING domain, thereby preventing nuclear export of BRCA1 (16, 17). By altering the interaction between BRCA1 and BARD1 with tr-BRCA1, we rendered breast cancer cells susceptible to PARP inhibition by effectively shifting BRCA1 to the cytosol (8–11, 16, 17). Finding the minimal region of tr-BRCA1 that can induce BRCA1 nuclear export could lead to the discovery of novel compounds that could augment tumor cytotoxicity to PARP inhibition in tumors. We and others have also reported the requirement of wild-type p53 on IR-, but not tr-BRCA1–induced BRCA1 cytosolic sequestration (7, 8, 10). Thus, with respect to the sensitization of sporadic breast cancer cells to PARP inhibition by targeting BRCA1 location, further preclinical investigation and consideration in clinical studies are needed to determine the role of p53 in this strategy.
In this study, IR was used to generate a DNA repair deficit. This is at first glance counterintuitive, as IR is a potent inducer of immediate DNA repair response. It is important to note that the repair deficit generated by IR was measured at a time well after repair of IR-induced DSBs had occurred. Because BRCA1 export is a later DNA damage response event, our repair assays were not conducted until 24 hours following IR. These data also indicate that the timing and sequence of treatment are important to achieve IR-induced tumor sensitization to PARP inhibition.
Our report suggests that BRCA1 cytoplasmic translocation can be a potential surrogate marker to predict tumor response to PARP inhibitor–based therapy. This is substantiated by our freshly resected human breast cancer specimen ex vivo results (data not shown) that show IR-induced BRCA1 cytoplasmic translocation. Further investigation is necessary to validate this method as a potential predictor of PARP inhibitor sensitivity following IR.
Disclosure of Potential Conflicts of Interest
F. Xia has honoraria from Speakers Bureau of Abott laboratory. No potential conflicts of interest were disclosed by the other authors.
Conception and design: E.S. Yang, S. Nowsheen, M.A. Rahman, F. Xia
Development of methodology: E.S. Yang, S. Nowsheen, M.A. Rahman, R.S. Cook, F. Xia
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): E.S. Yang, S. Nowsheen, M.A. Rahman, R.S. Cook
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): E.S. Yang, S. Nowsheen, M.A. Rahman, F. Xia
Writing, review, and/or revision of the manuscript: E.S. Yang, S. Nowsheen, M.A. Rahman, R.S. Cook, F. Xia
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E.S. Yang, S. Nowsheen, M.A. Rahman, F. Xia
Study supervision: E.S. Yang, F. Xia
This work was supported by a concept award from the Department of Defense (W81XWH-08-1-0571), Susan G. Komen Breast Cancer Research Award (BCTR0201704 to F. Xia), a research resident seed grant from the American Society for Therapeutic Radiology and Oncology, the Vanderbilt Institute for Clinical and Translational Research and the CTSA voucher grant, and IMPACT Award from the Department of Radiation Oncology, University of Alabama-Birmingham Comprehensive Cancer Center and University of Alabama-Birmingham School of Medicine (E.S. Yang).
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