Previous reports demonstrated that RAP80 plays an important role in DDR signaling. RAP80 is part of a BRCA1 protein complex and facilitates the translocation of the complex to DSBs (13
). This translocation is at least in part mediated through the interaction of the UIMs of RAP80 with K63- and K6-linked ubiquitinated γH2AX and other histones. Recently, Hu, et al. (30
) reported that knockdown of RAP80 by siRNAs affected the retention of BRCA1 at IRIF rather then the initial recruitment of this complex. Our results showing diminished retention of BRCA1 complexes at IRIF at later times after DNA damage in MEFs derived from RAP80−/−
embryos is in agreement with these observations. It was suggested that RAP80 complexes contribute to a process that down-modulates BRCA1 homologous recombination function and thereby suppressing exaggerated BRCA1-driven HR (30
). This was corroborated by observations showing that the down-regulation of RAP80 by siRNA or expression of a functionally deficient RAP80 mutant in U2OS cells result in a hyper-recombination phenotype accompanied by increased chromosomal abnormalities, the majority of which consist of sister chromatid breaks and sister chromatid exchange (21
). Our analysis of chromosome spreads of iMEFs from WT and RAP80−/−
mice indicated that the number of sister chromatid exchanges was significantly increased in IR exposed RAP80−/−
iMEFs (Fig. S3
) and further supports a role for RAP80 in the down-modulation of homologous recombination functions of BRCA1.
We further demonstrated that MEFs derived from RAP80−/−
embryos undergo premature senescence. Moreover, RAP80−/−
MEFs as well as RAP80−/−
thymocytes, exhibited an increased sensitivity to several DNA damaging agents indicative of a less efficient DDR. Interesting the sensitivity to DNA damaging agents was p53 dependent. In addition, cultures of non-irradiated RAP80−/−
MEFs contained a higher number of cells with γH2AX foci compared to WT cells and an increase in CtIP and BACH1 foci has been reported in U2OS cells in which RAP80 is down-regulated (21
). These observations would be consistent with more extensive end processing of spontaneous single and double strand breaks in RAP80-deficient cells and with the concept that loss of RAP80 function leads to deficient DSB repair and increased chromosome instability.
A major role of DDR proteins in mammalian cells is to maintain genomic integrity. Impairment of the DDR process results in genome instability and increased cancer risk. In this study, we examined whether the loss of RAP80 expression affected cancer risk in several different murine cancer models. Our data showed that by 20 months the percentage of spontaneously developing lymphomas in RAP80−/−
mice was significantly greater than in WT mice (52% versus 3.6%, respectively). IR exposure accelerated lymphoma formation in both WT and RAP80−/−
mice; however, RAP80−/−
mice remained more susceptible. Similar findings were recently reported by Wu et al.
). These observations indicate that loss of RAP80 enhances the susceptibility to lymphoma development consistent with the hypothesis that RAP80 functions as tumor suppressor.
It is well established that p53 acts as a tumor suppressor and plays an important role in determining heritable cancer risk (31
). Inactivation of p53 acts cooperatively with genetic alterations in other tumor suppressor or oncogenes to increase cancer risk (43
). In this study, we showed that RAP80−/−
double knockout mice exhibited a greater decrease in tumor-free survival compared to p53−/−
single knockout mice indicating that the loss of RAP80 acts synergistically with the loss of p53 in promoting spontaneous lymphoma development. No statistically significant difference was observed in tumor-free survival between irradiated p53−/−
mice, which is likely due to the very small latency in tumor development. Heterozygous p53 knockout mice also exhibit a predisposition for developing lymphoma, sarcoma, and carcinoma and exposure to IR considerably reduces the latency to tumor formation and survival (37
). We showed that p53+/−
mice were significantly more susceptible to the development of IR-induced thymic lymphomas than p53+/−
mice (78.5% versus 42.8%, respectively). These observations suggest that inactivating RAP80 and p53 mutations cooperate in promoting tumorigenesis. The latter is consistent with the concept that RAP80 functions as a tumor suppressor. In agreement with previous observations (18
), we found that the ability of several p53 activators to induce cell death and the expression of p53 target genes was significantly enhanced in RAP80−/−
MEFs. As proposed for its modulation of BRCA1 (30
), RAP80 might suppress the ability of p53 to transactivate apoptotic target genes in the presence of DNA stress and damage signals thereby increasing the time to repair.
In humans, germline mutations in BRCA1 and BRCA2 account for 5–7% of familial breast cancers (10
). Because RAP80 is part of a BRCA1 complex, we were particularly interested in determining whether RAP80−/−
mice were more susceptible to DMBA-induced mammary cancer development. We found that RAP80−/−
mice were significantly more susceptible to DMBA-induced mammary cancer development than WT mice. In addition to mammary cancer, DMBA treatment also enhanced the formation of lymphomas and ovarian cancer. Our results show that total tumor-free survival was significantly reduced in RAP80−/−
mice. These observations are consistent with our conclusion that loss of RAP80 enhances cancer risk. Several recent studies investigating an association between RAP80 mutations and breast cancer risk in humans reported that truncating mutations of the RAP80 gene are not associated with familial breast cancer, while RAP80 haplotype or rare missense mutations may be weakly or moderately associated with elevated risk of breast cancer (45
). A separate study found an association between a RAP80 haplotype and elevated ovarian cancer risk (5
). Screening of Finnish BRCA1/BRCA2 mutation-negative breast cancer families revealed the presence of several RAP80 variants (21
); however, whether these are associated with increased cancer risk remains to be established. Overall these studies appear to indicate that the mutations identified within RAP80 to-date do not play a major role in increasing the risk of breast cancer. In summary, our study indicates that the loss of RAP80 enhances genome instability and increases the susceptibility to cancer development in several mouse models of human cancer. The impact of RAP80 on DNA repair and maintenance of genomic stability are consistent with the concept that RAP80 can function as a tumor suppressor.