Oncogenic Ras regulates BRIP1 expression to induce dissociation of BRCA1 from chromatin, inhibit DNA repair, and promote senescence

doi:10.1016/j.devcel.2011.10.010

Dev Cell. Author manuscript; available in PMC 2012 December 13.

Published in final edited form as:

Dev Cell. 2011 December 13; 21(6): 1077–1091.

Published online 2011 December 1. doi: 10.1016/j.devcel.2011.10.010

PMCID: PMC3241855

NIHMSID: NIHMS332739

Oncogenic Ras regulates BRIP1 expression to induce dissociation of BRCA1 from chromatin, inhibit DNA repair, and promote senescence

Zhigang Tu,¹ Katherine M. Aird,¹ Benjamin G. Bitler,¹ Jasmine P. Nicodemus,¹ Neil Beeharry,² Bing Xia,³ Tim J. Yen,² and Rugang Zhang^1,²^*

¹Women’s Cancer Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111

²Epigenetics and Progenitor Cells Keystone Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111

³The Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, NJ 08903

^* Corresponding Author: Rugang Zhang, Ph.D., W446, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, Tel: 215-728-7108, Fax: 215-728-3616, Email: rugang.zhang/at/fccc.edu

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Summary

Here, we report a cell-intrinsic mechanism by which oncogenic RAS promotes senescence while predisposing cells to senescence bypass by allowing for secondary hits. We show that oncogenic RAS inactivates the BRCA1 DNA repair complex by dissociating BRCA1 from chromatin. This event precedes senescence-associated cell cycle exit and coincides with the accumulation of DNA damage. Downregulation of BRIP1, a physiological partner of BRCA1 in the DNA repair pathway, triggers BRCA1 chromatin dissociation. Conversely, ectopic BRIP1 rescues BRCA1 chromatin dissociation and suppresses RAS-induced senescence and the DNA damage response. Significantly, cells undergoing senescence do not exhibit a BRCA1-dependent DNA repair response when exposed to DNA damage. Overall, our study provides a molecular basis by which oncogenic RAS promotes senescence. Since DNA damage has the potential to produce additional "hits" that promote senescence bypass, our findings may also suggest one way a small minority of cells might bypass senescence and contribute to cancer development.

Introduction

Activation of oncogenes (such as RAS) in primary mammalian cells typically triggers a cascade of molecular and cellular events, which ultimately culminates in a state of irreversible cell growth arrest (Campisi, 2005). This process is termed oncogene-induced senescence and is an important tumor suppression mechanism in vivo (Campisi, 2005). Paradoxically, the definition of an oncogene is a gene that actively promotes tumorigenesis. The mechanism underlying this paradox remains poorly understood.

Senescent cells display several hallmark morphological and molecular characteristics. These cells are positive for senescence-associated β-galactosidase (SA-β-gal) activity (Dimri et al., 1995). In addition, chromatin in the nuclei of senescent human cells often re-organizes to form specialized domains of facultative heterochromatin called senescence-associated heterochromatin foci (SAHF) (Braig et al., 2005; Narita et al., 2006; Narita et al., 2003; Zhang et al., 2007a; Zhang et al., 2005). SAHF contain markers of heterochromatin, including di- and tri-methylated lysine 9 histone H3 (H3K9Me2/H3K9Me3), histone H2A variant mH2A and HMGA (Narita et al., 2006; Narita et al., 2003; Zhang et al., 2005). SAHF formation contributes to senescence-induced cell cycle exit by directly sequestering and silencing proliferation-promoting genes (Narita et al., 2003; Zhang et al., 2007a).

Oncogene-induced senescence is often characterized by the accumulation of DNA damage; in particular, DNA double-strand breaks (DSBs) (Bartkova et al., 2006; Di Micco et al., 2006). For example, oncogenic RAS mutants induce DNA damage by triggering aberrant DNA replication (Di Micco et al., 2006). However, it remains to be determined whether impaired DNA repair contributes to the accumulation of DNA damage observed during oncogene-induced senescence.

BRCA1 plays an important role in DNA DSB repair (Scully and Livingston, 2000). Germline mutations in the BRCA1 gene predispose women to breast and ovarian cancer (Scully and Livingston, 2000), and inactivation of BRCA1 contributes to cancer development by causing genomic instability (Turner et al., 2004). BRCA1 interacts with various DNA damage repair proteins through its two C-terminus BRCA1 C-terminal (BRCT) repeats. The BRCT repeats of BRCA1 recognize cognate partners by binding to their phosphoserine residues (Manke et al., 2003; Yu et al., 2003), and their binding partners include BRCA1-interacting protein 1 (BRIP1), CtIP and RAP80/Abraxas (Wang et al., 2007; Yu et al., 2003; Yu et al., 1998). In addition, BRCA1 interacts with partner and localizer of BRCA2 (PALB2), which is necessary for localization of BRCA2 to DNA DSBs (Xia et al., 2006). Functional BRCA1 is required for localizing/sustaining PALB2 at sites of DNA DSBs and error-free homologous recombination repair (Livingston, 2009; Sy et al., 2009; Zhang et al., 2009). A role for BRCA1 in senescence is implied by findings from the BRCA1 exon 11 knockout mouse whose cells exhibit signs of senescence (Cao et al., 2003). These observations suggest that senescence and tumorigenesis pathways may converge on BRCA1-associated DNA damage responses.

Here, we report a cell-intrinsic mechanism by which oncogenic RAS promotes senescence but at the same time predisposes cells to secondary hits, which ultimately leads to senescence bypass.

Results

BRCA1 becomes dissociated from chromatin during oncogenic RAS-induced senescence

Senescent cells are characterized by the accumulation of DNA DSB (Bartkova et al., 2006; Di Micco et al., 2006; Halazonetis et al., 2008), and one of the critical players in DSB repair is BRCA1 (Scully and Livingston, 2000). To test the hypothesis that changes in BRCA1 function occur during oncogene-induced senescence, we first examined changes in the sub-cellular distribution of BRCA1 during RAS-induced senescence of IMR90 primary human fibroblasts (Figure S1A). BRCA1 immunofluorescence (IF) staining was performed in proliferating (control) and senescent IMR90 primary human fibroblasts induced by RAS. Notably, BRCA1 was excluded from SAHF in senescent cells (Figure 1A). In addition, similar results were obtained using multiple anti-BRCA1 antibodies (one rabbit polyclonal and two individual mouse monoclonal antibodies) (data not shown).

Figure 1

Oncogene-induced dissociation of BRCA1 from chromatin occurs prior to the oncogene-induced cell cycle exit and coincides with the accumulation of DNA damage during senescence

We next fractionated protein from proliferating (control) and senescent IMR90 cells into soluble and chromatin fractions (Mendez and Stillman, 2000; Narita et al., 2006) and tested each for the presence of BRCA1. Compared with control cells, BRCA1 levels were dramatically decreased in the chromatin fractions of senescent IMR90 cells (Figure 1B and S1B). We next sought to exclude the possibility that BRCA1 chromatin dissociation was caused by supra-physiological levels of RAS. To do so, we titrated RAS expression in IMR90 cells to levels lower than those in the classic bladder cancer cell line T24 that harbors an oncogenic RAS mutation (H-RAS^G12V, the same RAS mutant used in the current study) (Hurlin et al., 1989) (Figure S1C). Importantly, we observed a decrease in BRCA1 levels in the chromatin fractions of RAS-infected IMR90 cells with lower RAS expression than the T24 cells (Figure S1C). Interestingly, BRCA1 levels in the chromatin fractions of T24 cells were also notably lower when compared to IMR90 cells, albeit similar levels of total BRCA1 levels were observed (Figure S1C). Together, our data show that the physiological levels of oncogenic RAS observed in cancerous cells are sufficient to dissociate BRCA1 from chromatin in primary cells.

Next, we asked whether BRCA1 chromatin dissociation is unique to IMR90 cells induced to senesce by RAS. To answer this question, primary WI38 and BJ human fibroblasts, which both senesce after oncogenic RAS is expressed (Ye et al., 2007; Zhang et al., 2005) (data not shown), were infected with control or RAS-encoding retrovirus. Compared with controls, BRCA1 levels were dramatically decreased in the chromatin fractions of RAS-infected WI38 and BJ cells (Figure S1D). Taken together, these data demonstrate that BRCA1 becomes dissociated from chromatin during RAS-induced senescence.

BRCA1 chromatin dissociation precedes the cell cycle exit during RAS-induced senescence

We next sought to determine whether BRCA1 chromatin dissociation occurs early or late during RAS-induced senescence. Towards this goal, we conducted a detailed time-course analysis of chromatin-associated BRCA1, senescence-associated cell cycle exit (determined by BrdU incorporation or expression of cyclin A or serine 10 phosphorylated histone H3 (pH3S10)) and other markers of senescence (such as formation of SAHF, mH2A foci and H3K9Me2 foci) in control and RAS-infected IMR90 cells (Figure 1C-H). Strikingly, as early as day 1 (Figure S1A), BRCA1 was largely dissociated from chromatin (Figure 1F and S1K), which is well before the senescence-associated cell cycle exit and accumulation of markers of senescence (Figure 1C-H).

BRCA1 forms discrete nuclear foci during the S/G2 phases of the cell cycle in normal cycling cells (Durant and Nickoloff, 2005; Scully et al., 1997a; Xu et al., 2001). Therefore, we sought to determine whether RAS expression impairs BRCA1 foci formation in cycling cells. Towards this goal, at day 2, control and RAS-infected IMR90 cells were labeled with BrdU to identify S phase cells. We next pre-extracted soluble proteins from control and RAS-infected cells and stained these cells with antibodies against BRCA1 and BrdU. Indeed, the intensity of BRCA1 foci in BrdU-positive cells was significantly weaker in RAS-infected IMR90 cells compared with controls (p=0.009) (Figure 1I-J). This is not simply a consequence of DNA damage because this did not occur in IR-treated cells (Figure S1E-F). Further, control and RAS-infected IMR90 cells were stained with antibodies against BRCA1 and cyclin A, a marker of the S/G2 phases of the cell cycle (Erlandsson et al., 2000; Sartori et al., 2007). Consistently, BRCA1 foci were either negative or notably weaker in cyclin A-positive RAS-infected cells compared with controls (Figure S1G-H). Finally, FACS analysis revealed that RAS-infected cells accumulated at the S and G2/M phases of the cell cycle compared with controls at this stage (day 2) (Figure 1K and S1I-J). From these results, we conclude that oncogene-induced dissociation of BRCA1 from chromatin precedes the oncogene-induced cell cycle exit during senescence.

BRCA1 chromatin dissociation coincides with the accumulation of DNA damage

We next asked whether BRCA1 chromatin dissociation plays a role in the DNA damage accumulation observed during RAS-induced senescence. Strikingly, accumulation of markers of DNA damage, including formation of γH2AX and 53BP1 foci, accumulation of γH2AX in chromatin fractions, and upregulation of p53 displayed the identical kinetics as dissociation of BRCA1 from chromatin in RAS-infected IMR90 cells (Figure 1F, 1L-M and S1K-O). For example, as early as day 1, γH2AX had already accumulated in chromatin fractions (Figure 1F and S1K), γH2AX and 53BP1 foci were increased and the p53 expression levels were upregulated in RAS-infected IMR90 cells compared with controls (Figure 1L-M, S1L-O). We conclude that RAS expression results in concomitant BRCA1 chromatin dissociation and DNA damage accumulation.

To determine whether BRCA1 chromatin dissociation is a RAS specific effect, IMR90 cells were infected with control, RAS, BRAF or myristylated AKT1 (myr-AKT1) encoding retrovirus. Ectopic expression of RAS, BRAF and myr-AKT1 were confirmed by western blot (data not shown). Notably, expression of RAS, BRAF and myr-AKT1 all induced expression of markers of senescence in IMR90 cells (Figure S2A) (Krizhanovsky et al., 2008; Michaloglou et al., 2005; Xue et al., 2007; Zhang et al., 2005). Next, we examined BRCA1 protein levels in total cell lysates and chromatin fractions of control, RAS, BRAF and myr-AKT1 infected IMR90 cells by western blot. Strikingly, compared with controls, BRCA1 levels decreased dramatically in the chromatin fractions of RAS and BRAF infected IMR90 cells, but not in myr-AKT1 infected IMR90 cells (Figure 2A). These results suggest that BRCA1 chromatin dissociation is dependent upon specific oncogenic pathways and is not simply a consequence of senescence.

Figure 2

Oncogene-induced BRCA1 chromatin dissociation correlates with DNA damage accumulation and SAHF formation

We next sought to determine whether BRCA1 chromatin dissociation coincides with DNA damage accumulation or is associated with SAHF formation. Towards this goal, control, RAS, BRAF and myr-AKT1 -infected IMR90 cells were stained with DAPI to visualize SAHF and with an antibody to mH2A, which is a component of SAHF (Zhang et al., 2005). Both RAS and BRAF, but not myr-AKT1, induced formation of SAHF and mH2A foci (Figure 2B). Additionally, compared with controls, both RAS and BRAF expression induced formation of γH2AX and 53BP1 foci and increased levels of chromatin-associated γH2AX and 53BP1 (Figure 2C-E). In contrast, myr-AKT1, which did not induce BRCA1 chromatin dissociation, failed to induce formation of γH2AX and 53BP1 foci or increase the levels of chromatin-associated γH2AX and 53BP1 (Figure 2C-E). Consistently, it has been recently reported that cell senescence induced by myr-AKT1 is not associated with SAHF formation or the DNA damage response (Kennedy et al., 2011). Similarly, PTEN knockdown induced expression of markers of senescence but had no effects on BRCA1 chromatin association and also failed to trigger formation of γH2AX foci or SAHF (Figure S2B-G). Likewise, it has been previously shown that senescence induced by loss of PTEN is not associated with the accumulation of DNA damage (Alimonti et al., 2010). The lack of both DNA damage and BRCA1 chromatin dissociation in the PTEN knockdown cells is consistent with the idea that BRCA1 chromatin dissociation contributes to DNA damage accumulation.

We next directly measured the extent of DNA damage in control, RAS, BRAF and myr-AKT1 infected IMR90 cells using the comet assay. Compared with controls, there was a significant increase in DNA damage in RAS and BRAF infected IMR90 cells (p<0.05), whereas the difference between control and myr-AKT1 infected IMR90 cells was not significant (p>0.05) (Figure 2F-G). The combined data suggest that dissociation of BRCA1 from chromatin coincides with DNA damage accumulation, correlates with SAHF formation, and is independent of PI3K/AKT signaling.

BRCA1 knockdown induces senescence

We next sought to test whether BRCA1 knockdown is sufficient to drive SAHF formation and senescence in IMR90 cells. Towards this goal, three individual short hairpin RNA to the human BRCA1 (shBRCA1) gene with different degrees of BRCA1 knockdown efficacy were utilized. BRCA1 knockdown efficiency was confirmed by IF staining and western blot (Figure 3A-B). Two individual shBRCA1 (#2 and #3), which efficiently knocked down BRCA1, induced SAHF formation and expression of SA-β-gal activity (Figure 3C-F). Notably, an shBRCA1 (#1) that knocked down BRCA1 with ~40% efficacy at the total protein level had no effect on SAHF formation or expression of SA-βgal activity (Figure 3A-F). Interestingly, similar to the hyper-proliferation observed in RAS-infected IMR90 cells prior to cell cycle exit (e.g. Figure 1G-H and S5) (Di Micco et al., 2006), knockdown of BRCA1 in IMR90 cells also triggers a minor but statistically significant hyper-proliferation prior to the cell cycle exit as demonstrated by increased BrdU incorporation (Figure S3A). Consistent with a previous report (Krum et al., 2010), we observed an increase in expression of γH2AX as well as formation of γH2AX foci following BRCA1 knockdown (Figure S3B-C). We conclude that knockdown of BRCA1 is sufficient to drive senescence.

Figure 3

BRCA1 knockdown induces senescence

Downregulation of BRIP1 expression triggers BRCA1 chromatin dissociation

Next, we investigated the molecular mechanism underlying BRCA1 chromatin dissociation. Notably, a fragment of the C-terminus of BRCA1 (AA 1314-1863) containing two BRCT repeats was sufficient to be dissociated from chromatin in IMR90 cells infected with RAS (Figure S4A), suggesting that factors that interact with the BRCT repeats of BRCA1 may trigger BRCA1 chromatin dissociation.

BRCT repeats of BRCA1 bind to BRIP1, CtIP and RAP80/Abraxas (Wang et al., 2007; Yu et al., 2003; Yu et al., 1998). Compared with controls, BRIP1 levels in both total cell lysates and the chromatin fractions of RAS-infected cells were dramatically decreased (Figure 4A-B and Figure S4B). In contrast, expression levels of CtIP and RAP80 did not overtly change in RAS-infected cells at the same time (Figure 4A). Notably, RAS-induced downregulation of BRIP1 occurred prior to the cell cycle exit, as reflected by the kinetics of cyclin A and pH3S10 expression (Figure 4B). Together, these results suggest that downregulation of BRIP1 may trigger BRCA1 chromatin dissociation in RAS-infected cells.

Figure 4

BRIP1 repression triggers BRCA1 chromatin dissociation

BRIP1 was first identified as a BRCA1 physiological binding partner (Cantor et al., 2001). We next sought to determine whether BRIP1 is downregulated at the mRNA level in RAS-infected IMR90 cells. Expression of BRIP1 and BRCA1 mRNA in control and RAS-infected IMR90 cells was examined by qRT-PCR. Compared with controls, BRIP1 mRNA levels were significantly decreased as early as day 1 in RAS-infected cells (Figure 4C). However, the BRCA1 mRNA expression level did not statistically change at the same time (Figure 4C). This is consistent with the idea that RAS-induced BRIP1 repression triggers BRCA1 chromatin dissociation prior to RAS-induced cell cycle exit.

We next sought to determine the mechanism underlying BRIP1 downregulation in RAS-infected cells. BRIP1 mRNA expression is downregulated in RAS-infected cells (Figure 4C), but the stability of BRIP1 mRNA is not decreased by RAS expression (data not shown), suggesting that BRIP1 may be regulated at the transcriptional level. Consistently, the activity of a 400 bp (-300 bp - +100 bp) fragment of the proximal human BRIP1 gene promoter was significantly suppressed in RAS-infected cells (Figure S4C). Notably, deletion of a critical B-Myb binding site from the BRIP1 promoter blocked the RAS-mediated suppression of promoter activity, suggesting that B-Myb plays a critical role in suppressing BRIP1 expression in response to RAS (Figure S4C). Interestingly, it has previously been demonstrated that B-Myb suppresses oncogenic RAS induced senescence (Masselink et al., 2001). Consistently, we observed downregulation of B-Myb in RAS-infected cells prior to the RAS-induced cells cycle exit (i.e., as early as 12 hours) compared with controls (Figure S4D). Significantly, using an anti-B-Myb antibody, chromatin immunoprecipitation studies demonstrated that the binding of B-Myb to the promoter of the human BRIP1 gene was significantly reduced in RAS-infected cells compared with controls (Figure 4D). Additionally, it has recently been demonstrated that B-Myb expression is suppressed by upregulation of microRNA 29 (mir29) during senescence (Lafferty-Whyte et al., 2009; Martinez et al., 2011). Consistently, expression of mir29 was upregulated in RAS-infected cells as early as 6 hours compared with controls (Figure S4E). We conclude that RAS-mediated inhibition of B-Myb contributes to the downregulation of BRIP1 during RAS-induced senescence.

Next, we asked whether knockdown of BRIP1 drives BRCA1 chromatin dissociation and senescence. Three individual shRNAs to the human BRIP1 gene (shBRIP1) were developed, and the efficacy of BRIP1 knockdown was confirmed by western blot (Figure 4E). BRCA1 levels in the chromatin fractions of shBRIP1 expressing cells were greatly reduced when compared to controls (Figure 4E). In addition, we observed an increase in expression of γH2AX in shBRIP1 expressing cells (Figure S4F). Notably, BRIP1 knockdown induced expression of markers of senescence, including SA-β-gal activity and SAHF formation (Figure 4F-G). Consistent with this, BRIP1 knockdown increased levels of p16, p21, p53 and hypophosphorylated pRB, demonstrating activation of both the p53 and the pRB pathways (Figure S4G). To limit off-target effects, we sought to determine whether BRIP1 restoration in shBRIP1-expressing cells could prevent senescence induced by BRIP1 knockdown. Indeed, ectopic expression of an shBRIP1-resistant wild type BRIP1 significantly reduced the expression of markers of senescence induced by shBRIP1 (Figure 4H-J), indicating the specificity of the senescence phenotype induced by BRIP1 knockdown. We conclude that BRIP1 knockdown is sufficient to dissociate BRCA1 from chromatin and induce senescence.

The interaction between BRIP1 and the BRCT repeats of BRCA1 depends on phosphorylation of BRIP1 at serine 990 (S990) (Yu et al., 2003). Thus, we determined whether rescuing of senescence by BRIP1 depends on the phosphorylation of BRIP1 at S990. For this purpose, we made a serine to alanine mutant (S990A) that mimics the non-phosphorylated state of BRIP1 (Yu et al., 2003). Compared with wild type BRIP1, BRIP1 S990A failed to rescue the senescence induced by BRIP1 knockdown (Figure 4H-J). This result suggests that the interaction between BRCA1 and BRIP1 plays a critical role in regulating senescence.

Ectopic BRIP1 rescues BRCA1 chromatin dissociation and suppresses RAS-induced senescence

We next asked whether ectopic BRIP1 might rescue BRCA1 chromatin dissociation and suppress oncogene-induced senescence. Towards this goal, IMR90 cells were co-transduced with a retrovirus encoding RAS to induce senescence and a retrovirus encoding a Myc-tagged wild type BRIP1 or control. Compared with controls, BRIP1 expression notably rescued the levels of BRCA1 in the chromatin fractions of RAS-infected cells (Figure 5A, Lane 2 vs. Lane 3). This was not due to a lower RAS expression level because RAS was expressed at a higher level in ectopic BRIP1 expressing cells compared to controls (Figure 5A, Lane 2 vs. Lane 3). In addition, the total BRCA1 protein level was not increased by ectopic BRIP1 (Figure 5A, Lane 2 vs. Lane 3), implying that the increased level of chromatin-associated BRCA1 was not due to increased levels of total BRCA1.

Figure 5

Ectopic BRIP1 rescues BRCA1 chromatin dissociation and suppresses oncogene-induced senescence and DNA damage response

Notably, ectopic BRIP1 restored BRCA1 foci formation in cyclin A-positive RAS-infected cells (Figure 5B-C). Interestingly, ectopic BRIP1 did not affect RAS-induced hyper-proliferation (Figure S5), a trigger of the DNA damage response (Di Micco et al., 2006), suggesting that ectopic BRIP1 may instead affect DNA repair. In addition, expression of ectopic BRIP1 in RAS-infected cells suppressed expression of markers of senescence including SA-β-gal activity, SAHF formation and senescence-induced cell growth arrest and inhibited DNA damage response revealed by decreased γH2AX foci formation when compared to controls (Figure 5D-H). These data further support the conclusion that BRIP1 plays a major role in BRCA1 chromatin association during RAS-induced senescence.

We next sought to determine whether suppression of BRCA1 chromatin dissociation by BRIP1 depends on the phosphorylation of BRIP1 at S990. Compared with wild type BRIP1, the BRIP1 S990A mutant failed to rescue BRCA1 chromatin dissociation in RAS-infected cells (Figure 5A, Lane 3 vs. Lane 4) and was also impaired in restoring BRCA1 foci in cyclin A-positive RAS-infected cells (Figure 5B-C). These results imply that the interaction between BRIP1 and BRCA1 is necessary for suppression of BRCA1 chromatin dissociation by BRIP1. Notably, the BRIP1 S990A mutant failed to suppress senescence and its associated DNA damage accumulation in RAS-infected IMR90 cells (Figure 5D-G). We conclude that suppression of senescence by ectopic BRIP1 is dependent on its interaction with BRCA1.

Oncogenic RAS impairs the BRCA1-mediated DNA repair response prior to RAS- induced cell cycle exit during senescence

We next asked whether oncogene-induced BRCA1 chromatin dissociation inactivates the BRCA1-mediated DNA repair response. Upon DNA damage BRCA1 foci largely disappear in normal cycling cells, although new damage-induced foci form hours after DNA damage during the S/G2 phase of the cell cycle (Chen et al., 1998; Durant and Nickoloff, 2005; Scully et al., 1997a; Xu et al., 2001). To determine the effects of oncogenic RAS expression on the BRCA1-mediated DNA repair response, at day 2, control and RAS-infected IMR90 cells were treated with 2 Gy of ionizing radiation (IR) to induce DNA DSBs. Notably, IR did not prevent BRCA1 chromatin dissociation in RAS-infected cells (Figure 6A). This was not due to a lack of DNA damage induction by IR in RAS-infected cells as nearly 100% of both control and RAS-infected cells were positive for γH2AX foci (Figure 6B). We next examined formation of damage-induced BRCA1 foci in the S/G2 phases of cycling cells by co-staining cells with antibodies against BRCA1 and cyclin A five hours after IR treatment (Peng et al., 2006; Zhang et al., 2009). As expected, BRCA1 foci were significantly induced upon IR treatment in controls (Figure 6C-D). However, formation of damage-induced BRCA1 foci was severely impaired in RAS-infected IMR90 cells when exposed to IR (Figure 6C-D).

Figure 6

BRCA1-mediated DNA repair response is impaired prior to the oncogene-induced cell cycle exit

Functional BRCA1 is required for relocating/sustaining BRCA2 and its partner protein PALB2 at damage-induced foci (Sy et al., 2009; Zhang et al., 2009), which are critical for BRCA1-mediated DNA DSB repair (Huen et al., 2010). Consistently, formation of damage-induced BRCA2 and PALB2 foci was also significantly impaired in RAS-infected cells upon IR treatment compared to controls (Figure 6E-G). Based on these results, we conclude that oncogenic RAS impairs the BRCA1-mediated DNA repair response prior to the RAS-induced cell cycle exit during senescence.

DNA damage promotes senescence bypass in RAS-infected cells

The impaired ability of BRCA1 to repair DNA damage promotes genomic instability, facilitates acquisition of oncogenic alterations and ultimately drives tumorigenesis (Turner et al., 2004). Thus, we anticipated that impaired BRCA1-mediated DNA repair might lead to DNA damage accumulation and allow for accumulation of secondary hits that might promote senescence bypass. Consistently, we reproducibly observed rare foci of senescence-bypassed cells in RAS-infected IMR90 cells (Figure S6A). RAS remains overexpressed in those cells (Figure S6B), suggesting that senescence bypass is not due to loss of ectopic RAS expression. To directly test our hypothesis, control and RAS-infected IMR90 cells were treated with 2 Gy IR at day 2, and the extent of DNA damage was measured by the comet assay. At this time point, BRCA1 was largely dissociated from chromatin in RAS-infected proliferative cells (Figure 1F). There was increased DNA damage in IR-treated RAS-infected cells, which was significantly greater than either IR-treated control cells or RAS-infected cells without IR treatment (p<0.05) (Figure 7A-B). Notably, ectopic expression of wild type BRIP1, but not the BRIP1 S990A mutant, suppressed the DNA damage accumulation in IR-treated RAS-infected cells (Figure 7C-D).

Figure 7

DNA damage promotes senescence bypass

We next sought to determine whether this decreased ability to repair DNA might lead to senescence bypass. We tested this by focus formation and cell growth assays. Indeed, IR treatment consistently induced senescence bypass as evidenced by both focus formation and apparent cell growth in IR-treated cells compared to controls (Figure 7E-F). Notably, senescence bypassed cells formed colonies under anchorage-independent growth condition in soft-agar (Figure 7G-H). As a negative control, IR did not promote the proliferation of control cells (Figure 7E and S6D-E). To eliminate the possibility that senescence bypass observed in IR-treated cells was due to loss of ectopic RAS, these cells were isolated and analyzed by western blot for exogenous RAS expression. Compared with controls, RAS remained greatly overexpressed in the senescence-bypassed cells (Figure 7I). In addition, pRB was hyperphosphorylated and p53 expression was reduced in the senescence-bypassed cells (Figure 7J), suggesting that inactivation of the key senescence-promoting pRB and p53 pathways contributes to the senescence bypass induced by IR treatment. Notably, IR treatment has no effect on senescence-associated cell growth arrest once RAS-infected cells have exited from cell cycle (e.g., at day 7) (Figure S6C-E). This result suggests that the senescence bypass observed in IR-treated RAS-infected cells is not due to the pre-existence of senescence-resistant cells. Ectopic BRIP1 suppresses senescence (Figure 5D-H), which prevented us from determining whether rescuing BRCA1 chromatin dissociation by ectopic BRIP1 inhibits IR treatment-induced senescence bypass in RAS-infected cells.

If senescence bypass in IR-treated cells is achieved through accumulation of DNA damage and acquisition of secondary hits due to BRCA1 chromatin dissociation induced by BRIP1 repression, we anticipated that BRIP1 might remain downregulated and BRCA1 might remain dissociated from chromatin in the senescence-bypassed cells. Indeed, compared with controls, BRIP1 remained downregulated and BRCA1 remained dissociated from chromatin in the senescence-bypassed cells (Figure 7K). These results further support the idea that BRCA1 chromatin dissociation is not merely a consequence of senescence-associated cell cycle exit because the senescence-bypassed cells are highly proliferative. In addition, markers of DNA damage (such as γH2AX and 53BP1 foci) were expressed at higher levels in the senescence-bypassed cells compared to controls (Figure 7L-N). This is consistent with the idea that BRCA1 chromatin dissociation contributes to DNA damage accumulation in those cells. Together, these results suggest that the loss of BRCA1-mediated DNA repair may also allow for subsequent hits that ultimately enable a small fraction of Ras-induced cells to bypass senescence.

Discussion

Our study reveals that oncogenic RAS induces dissociation of BRCA1 from chromatin prior to the cell cycle exit during RAS-induced senescence. This dissociation of BRCA1 from chromatin coincides with DNA damage accumulation. Downregulation of BRIP1, a BRCA1 binding partner, contributes to BRCA1 chromatin dissociation. Conversely, ectopic BRIP1 rescues BRCA1 chromatin dissociation and suppresses RAS-induced senescence. Significantly, the BRCA1-mediated DNA repair response is impaired prior to the RAS-induced cell cycle exit, which renders cells susceptible to the accumulation of secondary hits, which may in some instances ultimately allow senescence bypass.

The role of BRCA1 chromatin dissociation during oncogene-induced senescence

DNA damage persists in senescent cells (Rodier et al., 2009), suggesting that defects in DNA repair may contribute to the accumulation of DNA damage observed during senescence. Our data reveal that BRCA1 chromatin dissociation coincides with DNA damage accumulation, which occurs as early as the hyper-proliferation phase in RAS-infected cells (Figure 1F-H and 1L-M,). A previous report by Di Micco et al. showed that the accumulation of DNA damage closely follows the hyper-proliferation phase in RAS-infected cells during senescence (Di Micco et al., 2006). The basis for this minor discrepancy between these two reports remains to be determined. It could be due to quantitative approaches used in the current study, which make it easier to reveal subtle differences at early stages (Figure 1L-M). We showed that BRCA1 chromatin dissociation correlates with the DNA damage response in both RAS and BRAF-infected cells (Figure 2). Further, BRCA1 knockdown in primary human cells triggers the DNA damage response and induces senescence (Figure 3 and S3B-C). Finally, suppression of BRCA1 chromatin dissociation by ectopic BRIP1 suppresses the DNA damage induced by oncogenic RAS (Figure 5B-E). Together, these results support the notion that BRCA1 chromatin dissociation contributes to the accumulation of DNA damage during oncogene-induced senescence.

Herein, we demonstrated that RAS-induced dissociation of BRCA1 from chromatin precedes SAHF formation (Figure 1C-F), and BRCA1 knockdown drives SAHF formation (Figure 3). Further, BRCA1 chromatin dissociation correlates with SAHF formation in IMR90 cells expressing RAS and BRAF oncogenes (Figure 2A-B). Consistent with this idea that BRCA1 antagonizes heterochromatin formation and/or maintenance, it has been previously demonstrated that targeting BRCA1 to an amplified lac operator-containing chromosome region in the mammalian genome results in large-scale chromatin unfolding (Ye et al., 2001). We were unable to ectopically express wild type BRCA1 in primary human cells (data not shown), which prevented us from determining whether ectopically expressed BRCA1 might suppress SAHF formation. However, ectopic BRIP1 was able to rescue BRCA1 chromatin dissociation and suppress SAHF formation (Figure 5D-E). Together, these findings support the idea that BRCA1 chromatin dissociation promotes senescence by contributing to SAHF formation.

Suppression of the DNA damage response by ectopic BRIP1 inhibits SAHF formation (Figure 5B-E). Conversely, knockdown of BRCA1, which induces DNA damage, drives SAHF formation (Figure 3 and S3). In addition, AKT or shPTEN, neither of which dissociate BRCA1 from chromatin, also fail to induce a DNA damage response or SAHF formation (Figure 2 and S2). Together, these data suggest that DNA damage response triggered by BRCA1 chromatin dissociation is required for SAHF formation. Indeed, there is evidence to suggest that formation of SAHF limits the degree of DNA damage response during oncogene-induced senescence (Di Micco et al., 2011). However, the DNA damage response is not sufficient for SAHF formation, which also requires activation of p16/pRB and HIRA/PML pathways (Narita et al., 2003; Ye et al., 2007; Zhang et al., 2007a; Zhang et al., 2005). Overall, these results support the notion that DNA damage is necessary but not sufficient for SAHF formation.

The role of BRIP1 repression during oncogene-induced senescence

Stable BRIP1 knockdown reduces damage-induced BRCA1 foci formation and BRIP1-deficient cells demonstrate defects in the number and intensity of BRCA1 foci (Peng et al., 2006). This suggests that BRIP1 plays an important role in BRCA1-mediated DNA repair. BRIP1 has been shown to be significantly downregulated in response to RAS expression in primary human fibroblasts based on gene expression microarray analysis, while CtIP and RAP80 are not among the list of genes whose expression is significantly changed in the same analysis (Mason et al., 2004). Interestingly, BRIP1, but not CtIP or RAP80/Abraxas, regulates DNA replication and is required for timely S phase progression (Huen et al., 2010; Kumaraswamy and Shiekhattar, 2007), implying the BRCA1/BRIP1 complex may regulate senescence by altering DNA replication. In support of this idea, RAS-induced senescence is characterized as a DNA damage response triggered by aberrant DNA replication (Di Micco et al., 2006). Further, B-Myb, a key regulator of BRIP1 expression (Figure 4D and S4C-D), is critical for proper DNA replication and regulates the DNA damage response (Ahlbory et al., 2005; Lorvellec et al., 2010). Notably, BRIP1 is expressed at higher levels in advanced breast carcinomas, and its overexpression correlates with a higher cell proliferation index (Eelen et al., 2008), supporting the notion that high levels of BRIP1 may contribute to cell proliferation by suppressing senescence (Figure 5). Taken together, these data imply a key role for BRIP1 in accumulation of DNA damage during oncogene-induced senescence.

The role of BRCA1 chromatin dissociation in senescence bypass

Oncogene-induced BRCA1 chromatin dissociation precedes the cell cycle exit during senescence (Figure 1). Significantly, oncogenic RAS impairs BRCA1-mediated DNA repair response prior to the cell cycle exit during senescence (Figure 6 and and7).7). This allows for the creation of a large time window for cells to accumulate secondary oncogenic hits prior to the senescence-associated cell cycle exit, which ultimately leads to senescence bypass in a minority of cells while the vast majority of cells eventually exit from the cell cycle and become senescent. Consistent with this model, RAS-expressing cells accumulate significantly greater DNA damage after IR treatment, and IR treatment promotes senescence bypass in RAS-infected cells (Figure 7). The mechanism we uncovered here may help explain the paradox of why activation of oncogenes (such as RAS) promotes senescence but at the same time predisposes cells to transformation.

Experimental Procedures

Chromatin isolation and chromatin immunoprecipitation

Chromatin was prepared according to published methods (Mendez and Stillman, 2000; Narita et al., 2006). Soluble proteins in supernatant 1 (SN1, cytoplasmic) and 2 (SN2, nuclear) and chromatin-bound proteins in the chromatin fraction were detected by western blot.

Chromatin immunoprecipitation in control and RAS-infected IMR90 cells was performed at day 6 as previously described (Zhang et al., 2007b) using a monoclonal anti-B-Myb antibody (Santa Cruz) or an isotype matched IgG control. Immunoprecipitated DNA was analyzed using SYBR green quantitative-PCR (SA Biosciences) against the human BRIP1 gene promoter region containing the B-Myb binding site using the following primers: Forward 5′-ATAAAGCGGAGCCCTGGAAGAGAA-3′ and Reverse 5′-ATTCGTCTCGGGTTGTGTG-GTTGA-3′.

Comet assay

The comet assay was performed with the CometAssay (Trevigen) kit following the manufacturer’s instructions. DNA damage was measured as the artificial Olive Moment using Cometscore software downloaded from http://www.tritekcorp.com. To determine significance, the t-test was performed using Graph Prism software (http://www.graphpad.com).

Anchorage-independent growth in soft-agar and focus formation assay

Anchorage-independent growth in soft-agar was performed as previously described (Li et al., 2010). For focus formation, four days after initial infection, control or oncogenic H-RAS^G12V encoding retrovirus infected BJ-hTERT cells were treated with 2 Gy IR. Cells were cultured for six days to eliminate the apoptotic cells induced by IR. Then, control and H-RAS^G12V expressing cells with or without IR treatment were seeded into 6-well plates at a density of 3000 cells/well in triplicate. Two weeks later, the plates were stained with 0.05% crystal violet in PBS (Kuilman et al., 2008).

Retrovirus and lentivirus infections

Retrovirus production and transduction were performed as described previously (Ye et al., 2007a; Zhang et al., 2005) using Phoenix cells to package the infection viruses (Dr. Gary Nolan, Stanford University). Lentivirus was packaged using the Virapower Kit from Invitrogen following the manufacturer’s instructions and as described previously (Li et al., 2010; Ye et al., 2007a). Cells infected with viruses encoding a drug resistant gene to puromycin or neomycin were selected in 1μg/ml and 500μg/ml, respectively, of the corresponding agent.

Immunofluorescence, BrdU labeling, FACS and SA-β-gal staining

Immunofluorescence staining and BrdU labeling for cultured cells was performed as described previously using antibodies described above (Zhang et al., 2007a; Zhang et al., 2007b; Zhang et al., 2005). Soluble protein pre-extraction with detergent was carried out as described previously (Taddei et al., 2001). Briefly, cells were incubated with PBS supplemented with 0.5% Triton X-100 for 5 min at room temperature followed by fixation in 4% paraformaldehyde (Sigma) for 10 min. Fixed cells were incubated with a rabbit anti-BRCA1 antibody for 1 h at room temperature and visualized by incubating the cells with goat anti-rabbit Cy3 (Jackson ImmunoResearch, 1:5000) secondary antibody followed by detection of BrdU using a FITC labeled anti-BrdU antibody (BD Biosciences). FACS was performed as previously described (Ye et al., 2007a), and FlowJo software was used to analyze cell cycle distribution. SA-β-gal staining was performed as previously described (Dimri et al., 1995).

For additional information about cell culture methods, RT-PCR and luciferase assays, as well as antibodies and plasmids used in this study, please see the supplemental Experimental Procedures

Supplementary Material

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Acknowledgments

We thank Dr. Kathy Wilson and Igor Makhlin for reagents, other lab members for critical discussion, and Dr. Xinying Zhuang for technical assistance. We thank Drs. Hua-Ying Fan, Erica Golemis and Maureen Murphy for critical reading of the manuscript. Author contributions: T.Z. performed most of the experiments, designed the experiments and drafted the manuscript. K.M.A contributed to Figures 4H, S2C-D, S2F-G and S4C. B.G.B contributed to Figure 4D. J.P.N contributed to initial observations that led to Figure 1A. N.B., B.X. and T.J.X. provided critical materials and/or reagents. R.Z. conceived the study, designed experiments and wrote the manuscript. R.Z. is an Ovarian Cancer Research Fund (OCRF) Liz Tilberis Scholar. This work was supported in part by a NCI FCCC-UPenn ovarian cancer SPORE (P50 CA083638) pilot project and SPORE career development award (to R.Z.), a DOD ovarian cancer academy award (OC093420 to R.Z.), and an OCRF program project (to R.Z.). B.G.B is supported by a NCI postdoctoral training grant (CA-009035-35). We would like to acknowledge Anna Pecherskaya and Margret Einarson for help with quantitative image analysis and Emmanuelle Nicolas for help with microRNA qRT-PCR analysis.

Footnotes

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