In this study, we provide evidence for a hitherto unknown mechanism of action for EZH2 in the development of breast cancer. We report that EZH2 overexpression results in specific downregulation of five RAD51 paralogs RAD51L1, RAD51L2, RAD51L3, XRCC2, and XRCC3, which are crucial for the normal function of the HR pathway of DNA DSB repair in mammalian cells [36,37,40
]. Coupled with this effect on gene expression, EZH2 overexpression attenuates RAD51 foci formation at sites of DNA damage, impairing the ability of human breast epithelial cells to repair DNA by HR. Our results show that EZH2 overexpression leads to a lower survival after DNA-damaging insults. We suggest that suppression of the RAD51 paralogs by EZH2 is a likely mechanism of EZH2-driven malignant transformation of breast epithelial cells.
EZH2 is a member of the PcG of early-onset gene repressors involved in maintaining heritable gene expression profiles, thus regulating cell type identity [22–25
]. There is compelling evidence that EZH2 overexpression leads to cancer. EZH2 is involved in the pathogenesis of myeloid leukemia, Hodgkin's disease, B-cell lymphoma, and multiple myeloma [9,10,12
]. Our group and other investigators have reported that EZH2 promotes the development and progression of breast cancer, prostate cancer, and other solid tumors [11,13–15
]. By performing an in silico
analysis of published cDNA datasets using ONCOMINE [54
], we found that, in addition to breast cancer, EZH2 mRNA is overexpressed in lung, liver, prostate, bladder, and adrenal carcinomas when compared to their normal epithelial counterparts (data not shown). These data suggest that misexpression of EZH2 may be a fundamental event in the transition from normal state to cancer in several organs. At present, the mechanism of action of EZH2 in cancer is unknown.
There are several lines of evidence supporting a role for the RAD51
gene family in cancer development, especially in light of the functional links between RAD51 and the BRCA
]. In breast cancer, it has been shown that BRCA2
mutation carriers are at greater risk for breast cancer when they also carry the RAD51 variant G135CD [55–57
]. XRCC2 codon 188 variant may carry an increased risk for breast cancer [58
]. The variant Glu233Gly of RAD51L3 may increase the risk of breast cancer in families without BRCA
gene mutations [59
]. The emerging association between the RAD51
gene family and cancer is not surprising given the critical role of RAD51 and its paralogs in HR repair, which is essential for the maintenance of genomic stability and tumor avoidance. Cell lines deficient in XRCC2 and XRCC3 have a high frequency of chromosomal instability, with especially high frequencies of chromosome exchange and aneuploidy [32,46,47,60
]. XRCC2- and XRCC3-deficient hamster cells have been shown to have increased missegregation of chromosomes resulting in aneuploidy, likely due to a centrosome defect [46,47
]. It has been suggested that loss of RAD51 paralog genes leads, in particular, to a chromosomal segregation defect that results in aneuploidy [32,46,47
]. Studies have shown that aneuploidy causes imbalances in groups of proteins involved in chromosome segregation, synthesis, and repair, possibly leading to malignant transformation of the cell [27–30
In the present study, we found that EZH2 overexpression in human mammary epithelial cells resulted in a drastic downregulation of the transcripts of the five RAD51 paralogs involved in HR repair (RAD51L1
, and XRCC3
). Notably, the expression of the sixth paralog, DMC1
, and of RAD51 (data not shown) was not affected by EZH2. In light of these data, it is likely that EZH2 may play a role in the transcriptional regulation of the RAD51 paralogs and thus modulate the function of HR DNA repair. In Drosophila
, PcG exert their function through a DNA motif called Polycomb response element (PRE) [61,62
]. Although a mammalian PRE has not been found yet, a recent study found that the EZH2 complex is able to associate with the hDAB2IP promoter in prostate cells and is able to recruit HDAC1 to the promoter region [19
]. Whether the EZH2 complex also associates with the promoters of the RAD51 paralogs is intriguing and warrants investigation.
Consistent with the observed decrease in RAD51L1, RAD51L2, RAD51L3, XRCC2, and XRCC3, EZH2-overexpressing mammary epithelial cells had an attenuated formation of RAD51 repair foci after treatment with etoposide. EZH2 overexpression resulted in a marked decrease in the number of cells with repair foci when compared to controls. The attenuated RAD51 foci formation most likely led to unrepaired chromosomal breaks. Although most cells would succumb to the damage, a subset of cells may survive and acquire additional genetic alterations, which may lead to neoplastic transformation.
We next tested the hypothesis that attenuated DNA repair foci formation may lead to defective HR. Indeed, EZH2-overexpressing cells exhibited decreased survival ability following etoposide and ionizing radiation, both of which cause DNA DSBs that necessitate HR repair [42,63–67
]. EZH2 overexpression not only affected HR repair in the spontaneously immortalized human mammary epithelial cell line MCF10A, but had a similar effect on two breast cancer cell lines MCF7 and SUM102. Furthermore, EZH2-overexpressing MCF10A cells exhibited an increase in the number of chromosomes when compared to controls. These results are not surprising given the important role of HR not only in DNA repair, but also in the resolution of crossover during cell divisions [32
]. Taken together, our data suggest that EZH2 overexpression occurs early in neoplastic transformation and may be important for the development of prevention strategies for breast cancer based on blockade of EZH2. Our data open the way to future investigations on the detection of EZH2 overexpression in histologically normal breast epithelial cells as a harbinger of carcinoma. This could result in the development of a new clinically applicable tissue-based test to determine the individual risk of breast cancer before histologic atypia is evident.
Our data are in agreement with previous studies demonstrating that aneuploidy is particularly seen in association with defects in HR, but not in association with defects in other DNA repair mechanisms such as NHEJ repair [32,46,47
]. These data suggest further investigations to elucidate whether EZH2 overexpression induces random or specific chromosomal alterations. Our data strengthen the emerging link between decreased expression of the RAD51 paralogs, aneuploidization, and cancer development.
In summary, we have identified and delineated a new functional role for EZH2 in the HR mechanism of DNA repair, which may cause aneuploidy in breast epithelial cells. We provide a mechanistic basis for our initial observations implicating EZH2 in promoting breast cancer development and also uncover a role for EZH2 in the regulation of a central pathway in DNA repair and in maintaining the stability of the genome.