DNA damage response markers are differentially expressed in BRCA-mutated breast cancers

Etiologically, BC is classified into sporadic and familial forms, with the latter forming up to 5–10 % of invasive BC. BRCA-related familial BC is caused by germline mutations either in the BRCA1 or BRCA2 genes and is associated with an increased risk of developing breast and other cancers [18]. However, in sporadic BC, mutational inactivation of BRCA1/2 is a rare occurrence, as inactivation requires both gene alleles to be mutant or totally deleted. However, non-mutational functional suppression of BRCA1/2 could result from various mechanisms, such as hypermethylation of the BRCA1 promoter or silencing of BRCA2 by other proteins [19, 20]. Moreover, BRCA genes function in a highly coordinated manner in concert with a complex array of genes to carry out high-fidelity repair of DNA damages. However, the expression of these DNA damage repair genes in clinical BC samples and their associations with clinico-pathological parameters have so far yielded controversial findings.

We have used two well-characterised cohorts of invasive BC with updated comprehensive biomarkers, including DNA damage response proteins, and outcome data to assess the pathobiological and clinical features of tumours harbouring BRCA1 and BRCA2 mutations compared to sporadic BC.

Regarding their clinico-pathological criteria, BRCA1/2 tumours when compared with sporadic tumours were significantly poorly differentiated/higher grade with less tubule formation and more nuclear pleomorphism. BRCA1 tumours had a higher mitotic frequency than sporadic and BRCA2 tumours. The vast majority of BRCA1 tumours showed higher proliferative index as assessed by Ki67LI compared to sporadic cancers. However, BRCA2 and BRCA1 tumours were neither different from each other regarding their Ki67LI, nor the BRCA2 tumours were significantly different from the sporadic tumours. These findings go in line with previous reports [21, 22]. Moreover, medullary-like tumours were significantly more frequent in BRCA1-mutated tumours. Although the latter has long been recognised [23], it underscores the potential significance of histologic observations, medullary histologic criteria reported by pathologists in patients’ clinical care [24]. Nevertheless, BRCA2 tumours were not significantly different from sporadic cases in terms of histological type. However, lobular tumours were more common in BRCA2 tumours than in BRCA1 tumours.

The majority of BRCA1-mutated tumours were ER negative compared to sporadic and BRCA2 tumours with similar expression pattern observed for PgR. Moreover, TN tumours were significantly more represented in the BRCA1-mutated tumours compared with both sporadic and BRCA2 tumours. Furthermore, BRCA1 tumours displayed significantly higher proportions of basal BC than sporadic and BRCA2 tumours as evidenced by significant session of the basal markers CK5/6, CK14, and EGFR. Accordingly, TN-basal tumours were significantly associated with BRCA1-mutated tumours compared with sporadic or BRCA2 tumours. These findings are in agreement with our previous report [16] and those of gene microarray studies [25]. Additionally, BRCA1-mutated BCs were not significantly different from TN-non-basal or TN-basal sporadic tumours in any of the known clinico-pathological parameters. These findings describing the clinico-pathological associations of BRCA1/2 tumours are in agreement with those previously described [26]. BRCA2 tumours appear to show a phenotype between sporadic and BRCA1‐associated BC. In other words, the IHC profile of BRCA1-mutated BC is distinctively different from sporadic BC more than BRCA2-mutated tumours.

Regarding the biomarkers’ profile of DNA damage repair markers in the studied series, there was, as expected, a significantly reduced expression of BRCA1 and BRCA2 proteins in both BRCA1- and BRCA2-mutated BC compared with sporadic cases. Moreover, BRCA1 protein was significantly more expressed in BRCA1-mutated tumours compared to BRCA2-mutated tumours. However, BRCA2 protein did not show significant difference in expression between BRCA1- versus BRCA2-mutated tumours.

PARP1 is a known key facilitator of DNA repair and is implicated in pathways of carcinogenesis. It is a 113 kDa nuclear enzyme which is cleaved into two fragments (89 and 24 kDa) during apoptotic cell death [27]. In this study, only non-cleaved PARP1 showed significantly different expression between the studied series, while the cleaved PARP1, whose levels are known to be increased in apoptosis, did not show any significantly different expression. The vast majority of BRCA1 (93.5 %) and BRCA2 (88.5 %) tumours were significantly positive for PARP1 (non-cleaved) expression compared to only half of the sporadic cases. With BRCA1 or BRCA2 loss of function due to mutations, cells become deficient in DNA DSB repair. This in turn activates PARPs whose catalytic activity is immediately stimulated by DNA breaks. It is not, therefore, surprising for the BRCA1/2 cases in our series overexpressing PARP1 (non-cleaved), which appears to be the reactive response of cells when DNA damage repair by BRCA gene is defective, and has been recently reported in BRCA1 mutant breast cancer cell lines and BRCA1-mutated BC cases [28]. Our results are in line with previous studies [27, 29, 30]. The relatively high percentages of sporadic BC positive for PARP1 (non-cleaved) prompted some authorities to suggest the potential therapeutic benefits of PARP1 inhibitors not only in familial BRCA-mutated BC but in sporadic cancers as well [31]. On the other hand, PARP1 cleaved isoform which has been regarded as a useful hallmark of activated cellular apoptotic machinery [9] did not show any significantly different expression between the studied series, with the vast majority of cases in all series showing positive expression. In our recent report, cleaved PARP1 was found to be highly significantly associated with other DNA repair proteins including RAD51, CHK1, CHK2, and others [32]. This overexpression of cleaved PARP1 in our series could point out to its roles in DNA repair, in addition to the recently reported functions of transcriptional regulation of other molecular regulators [33].

BARD1 gene encodes a protein that forms heterodimers with BRCA1 N-terminal region. This stable BARD1/BRCA1 complex is crucial for BRCA1 tumour suppression and coordinates a diverse range of cellular pathways such as DNA repair, transcriptional regulation to maintain genomic stability, and others [34]. BARD1 in this study was significantly down-regulated in the BRCA-mutated series compared to sporadic BC series. Nearly all cases of the latter showed positive expression, while three-quarters and up to half of the BRCA1-mutated cases and BRCA2-mutated cases were BARD1 positive, respectively. The concomitant reduction of BARD1 expression in BRCA1 mutant cases underscores that the participation of BARD1 may be required for proper functioning of BRCA1-mediated tumour suppressor activity aiming ultimately to maintain chromosome integrity through HR mechanism [10].

RAD51 is a key component of DNA damage repair by HR mechanism associated with the activation of DSB DNA repair. It binds to single- and double-stranded DNA giving rise to a RAD51 nucleoprotein filament, which is essential for strand-pairing reactions during DNA recombination [11, 35]. Both BRCA1 and BRCA2 co-localise with RAD51 at sites of DNA damage and activate HR repair of DSB mediated by RAD51 [36]. In this study, significantly lower RAD51 expression was observed in BRCA1-mutated tumours relative to BRCA2 and sporadic BC. Thus, BRCA1 dysfunction caused by inactivating mutations appears to deregulate nuclear RAD51 levels. Similar findings were reported in BRCA1 mutant ovarian cancer cell lines which displayed lowest levels of BRCA1 and RAD51 [37]. Moreover, lower RAD51 nuclear expression was observed in prostatic carcinomas associated with BRCA1/2 mutations than sporadic cancers [38]. Reduced nuclear expression of RAD51 has been reported concomitantly with reduced nuclear expression of BRCA1 and BRCA2 protein expression in early invasive BC [39]. However, in our study, BRCA2 mutated tumours were not significantly different from the sporadic BC. Although BRCA2 is needed for RAD51 nuclear localisation [40], other BRCA2-independent mechanisms involving other proteins, for instance RAD51C, have also been proposed [6].

The tumour suppressor P53 showed closely comparable low expression in BRCA1/2 and sporadic tumours with no significant differences between the studied series. In other words, up to three-quarters to around four-fifths of the studied series have negative P53 expression. These results of low P53 expression in BRCA mutant BC are in agreement with the findings reported by Zakhartseva and co-authors in invasive BC [41]. However, they are contradicting other studies describing the collaborative synergistic functionality of P53 mutations for the tumourigenic influence of BRCA2 loss [42] and BRCA1 loss [43]. P53 inactivation through protein-truncating mutations, rather than hotspot mutations, has been reported to be one of the mechanisms which accompany BRCA loss, which could not be always detected by IHC expression of P53 protein. This could, at least in part, explain the low levels of P53 expression, and hence inactivation, in our BRCA mutant series. Accordingly, this has prompted some authorities to report on the inherent weakness of immunohistochemical detection of TP53 inactivation that could lead to misdiagnosis in significant proportions of BRCA1 mutant tumours [43].

Ku70/Ku80 is a heterodimer known to play crucial roles in regulating diverse cellular processes including NHEJ, transcription regulation, and DNA replication [12]. In this study, Ku70/Ku80 was similarly highly expressed in the studied series, where all BRCA2-mutated and 95.6 % of BRCA1-mutated BC cases showed positive expression. These results are in agreement with our recent report using IHC on invasive BC cases and reverse phase protein array on cell lysates from BRCA1-deficient BC cell lines [44]. These figures might indicate over-activation of non-homologous end joining (NHEJ) known to be mediated by Ku70/Ku80 as a back-up alternative mechanism for DNA DSB repair in cases when BRCA1/2 are mutated with altered HR DNA repair pathway.

Currently, it is widely accepted that the biologic processes carried out by BRCA1 are disrupted by numerous mechanisms in sporadic cancers especially the TN and basal-like BC. Therefore, we have restricted the analysis to compare TN sporadic BC with BRCA1/2-mutated tumours. Expectedly, there were no statistically significant differences between the TN and basal-like phenotypes regarding any of the clinico-pathological parameters. Interestingly, both had large proportions of large tumour size and the vast majority were high-grade and moderate/poor NPI. Of the DNA repair markers studied, PARP1 (non-cleaved) showed significantly more positivity in BRCA1-mutated tumours. Although more than 90 % of the latter showed PARP1 overexpression, up to 53 % of the TN sporadic tumours were also PARP1 (non-cleaved) positive. Moreover, both phenotypes showed more BRCA1/2-negative, RAD51-negative, and Ku70/Ku80-positive expression. The same pattern of association were maintained when the analyses were further restricted between TN BRCA1-mutated cases and TN sporadic BC. In other terms, in both phenotypes, the HR protein RAD51 showed reduced expression, while the NHEJ protein Ku70/Ku80 was similarly overexpressed. Furthermore, BARD1 and the tumour suppressor P53 showed significantly lower expression in BRCA1/2 mutant and TN BRCA1/2 mutant tumours compared with TN sporadic BC cases. Collectively, these findings refer to the properties that define ‘BRCAness’ of sporadic BC. These are the traits that some sporadic cancers could share with those occurring in either BRCA1 or BRCA2 mutation carriers [45, 46]. These shared properties between the TN sporadic and familial cancers might have important implications in clinical management of these cancers. This highlights the potential therapeutic benefit of PARP1 inhibitors, based on the hypothesis of synthetic lethality, in TN sporadic as well as BRCA1-mutated BC [47].

In conclusion, the results of this study demonstrate, at a translational level, the complexity of DNA repair mechanisms in BRCA-mutated tumours and the presence of a degree of disruption of these pathways especially in TN BC. Moreover, our results support the hypothesis that DSBs are repaired by one or more alternative pathways and they are not independent of each other as evidenced by the reciprocal relationship between markers of HR and NHEJ of DNA DSB. Furthermore, DSB DNA repair is assisted by PARP1 expression in BRCA-mutated tumours, whereas the loss of DSB repair via RAD51 is predominant in BRCA1 rather than BRCA2 BC.