Aberrant expression of DNA damage response proteins is associated with breast cancer subtype and clinical features

Abstract

Landmark studies of the status of DNA damage checkpoints and associated repair functions in preneoplastic and neoplastic cells has focused attention on importance of these pathways in cancer development, and inhibitors of repair pathways are in clinical trials for treatment of triple negative breast cancer. Cancer heterogeneity suggests that specific cancer subtypes will have distinct mechanisms of DNA damage survival, dependent on biological context. In this study, status of DNA damage response (DDR)-associated proteins was examined in breast cancer subtypes in association with clinical features; 479 breast cancers were examined for expression of DDR proteins γH2AX, BRCA1, pChk2, and p53, DNA damage-sensitive tumor suppressors Fhit and Wwox, and Wwox-interacting proteins Ap2α, Ap2γ, ErbB4, and correlations among proteins, tumor subtypes, and clinical features were assessed. In a multivariable model, triple negative cancers showed significantly reduced Fhit and Wwox, increased p53 and Ap2γ protein expression, and were significantly more likely than other subtype tumors to exhibit aberrant expression of two or more DDR-associated proteins. Disease-free survival was associated with subtype, Fhit and membrane ErbB4 expression level and aberrant expression of multiple DDR-associated proteins. These results suggest that definition of specific DNA repair and checkpoint defects in subgroups of triple negative cancer might identify new treatment targets. Expression of Wwox and its interactor, ErbB4, was highly significantly reduced in metastatic tissues vs. matched primary tissues, suggesting that Wwox signal pathway loss contributes to lymph node metastasis, perhaps by allowing survival of tumor cells that have detached from basement membranes, as proposed for the role of Wwox in ovarian cancer spread.

Fig. 1

Graphical representation of expression of DDR proteins in TN cancers. In studies of the breast cancers on the SFBCAS TMA, DDR proteins were expressed more frequently in TN and basal-like tumors. The numbers to the lower left of each graph represent the cut-off percentages for scoring a tumor positive or negative; i.e., for pChk2, tumors were scored positive if >25% of nuclei were positive for expression and were scored negative if <25% of nuclei were expressing pChk2. For γH2AX the cut-off was at 50% and for BRCA1 at 90%. Because percentages were calculated using denominators that included cases for which scores were not available, the percentages will not necessarily sum to 100% across the expression groups for individual markers

Fig. 2

Expression of specific proteins in breast cancers by IHC analysis. Photographs (×400) were taken of immunohistochemical detection of each of the specific proteins assessed on the TMA and representative photographs are shown for each protein

Fig. 3

Boxplot of DDR scores, for number of aberrantly expressed DDR-associated proteins in TN vs. non-TN breast cancers. The DDR-associated proteins assessed were: altered or activated expression of nuclear BRCA1, γH2AX, pChk2, Fhit, p53; i.e., proteins involved in recognition of DNA breaks (γH2AX), in checkpoint activation in response to breaks (pChk2, p53), or in DNA repair (Fhit, BRCA1)

Fig. 4

Differentially expressed proteins in primary and metastatic breast cancers. Photographs (×400) of differentially expressed markers in matched primary and metastatic lymph node cancer tissue from a single patient

Fig. 5

DFS of patients with TN breast tumors. DFS of patients with TN breast tumors cohort was significantly worse than luminal subtypes or the other subtypes grouped together (not shown); also DFS for basal-like tumors was worse than other subtypes grouped together (not shown). Note that this TMA represents cases treated before the introduction of trastuzamab for ErbB2+++ cases

Fig. 6

DFS of breast cancers with low or no Fhit expression vs. cancers with moderate or high Fhit expression (P = 0.008)

Fig. 7

DFS of patients based on breast cancer expression levels of DDR proteins. The DDR-associated proteins assessed and used in this DFS analysis were altered or activated expression of nuclear BRCA1, γH2AX, pChk2, Fhit, p53; i.e., proteins involved in recognition of DNA breaks (γH2AX), in checkpoint activation in response to breaks (pChk2, p53), or DNA repair (Fhit, BRCA1). It is likely that subtype is the driver of these differences because the DDR proteins tend to be most frequently altered in TN cancers, though the analyses are limited by the relatively large percentage of censored observations and the small numbers of cases in some subgroups, as illustrated in Table S3