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
). 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
). The BRCT domain also interacts with p53 (25
), 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 (). 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
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
). 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.