EZH2 is an independent marker of recurrence and metastasis in women with breast cancer (Kleer et al., 2003
). EZH2 overexpression occurs mainly in the basal-type tumors, characterized by ER, PR, and Her-2/neu negative status as well as low levels of BRCA1 protein (Bachmann et al., 2006
; Collett et al., 2006
). A major novel finding presented in our study that EZH2 down-regulation in aggressive ER negative breast cancer cells greatly decreases their proliferative capacity and rate of progression through the cell cycle. Specifically, we found that EZH2 knockdown prolonged the doubling time of ER negative breast cancer cell lines, and caused an arrest at the G2/M transition of the cell cycle, with corresponding changes in mitotic Cdc25C, Cdc2 and Cdc2-Tyr15 phosphorylation. Consequently, EZH2 knockdown led to a decreased number of breast cancer cells undergoing mitosis. In support of these data, ectopic expression of EZH2 in the non-tumorigenic MCF10A cells caused a striking increase in the mitotic fraction. Work from our and other laboratories support these findings. Braken et al demonstrated that ectopic overexpression of EZH2 promotes cell proliferation in human diploid fibroblasts, and that EZH2 siRNA inhibits BrdU incorporation suggesting a role for EZH2 for progression through the cell cycle (Bracken et al., 2003
). Varambally et al reported that EZH2 siRNA induces a G2/M arrest in prostate cell lines (Varambally et al., 2002
). Using tissue samples, it has been shown that EZH2 overexpression is significantly associated with increased proliferative activity determined by detection of the Ki-67 antigen in ductal carcinoma in situ
and invasive carcinomas of the breast (Bachmann et al., 2006
; Ding et al., 2006
There have been intensive studies concerning the oncogenic mechanism underlying EZH2 overexpression. Both repression and activation of genes that regulate fundamental processes have been implicated, and new mechanisms that do not involve gene transcription have been proposed (Shi et al., 2007
; Su et al., 2005
; Tonini et al., 2004
). However, direct demonstration of EZH2’s function in in vivo
breast tumorigenesis is currently lacking. In the present study, we show that EZH2 modulates breast cell proliferation and tumor growth in vivo
. Targeted down-regulation of EZH2 in aggressive ER negative breast cancer cells resulted in a significant reduction of mammary tumor size as well as better survival. Although the histological features of the EZH2 inhibited and control tumors were similar, we found that EZH2 knockdown decreased the mitotic activity of breast cancer cells. Our xenograft mouse models thus suggest that EZH2 inhibiting compounds may have potential utility in treating ER negative breast cancer patients, and provide the foundation for the design of pre-clinical models.
In addition to its role in hereditary breast cancer, BRCA1 expression is reduced in up to 40 % of sporadic breast carcinomas (Turner et al., 2004
; Turner et al., 2007
; Wilson et al., 1999
; Yoshikawa et al., 2000
). Although BRCA1 promoter methylation is responsible for BRCA1 reduction in 10–15 % of sporadic breast carcinomas, it does not explain BRCA1 deficiency in the remainder of the tumors and it has been suggested that a group of ER basal-like breast carcinomas are characterized by BRCA1 deficiency (Turner et al., 2004
; Turner et al., 2007
). Discovery of novel strategies to restore BRCA1 expression and function is highly desirable to develop therapies for women carrying BRCA1-deficient tumors.
It has been proposed that the function BRCA1 in cell cycle regulation complements its role in DNA damage response, by allowing time for DNA repair to occur more effectively (Mullan et al., 2006
; Venkitaraman, 2002
). We demonstrate that EZH2 knockdown in ER negative breast cancer cell lines causes up-regulation of BRCA1 protein levels with a concomitant increase in pBRCA1 s1423; the total amount of the latter being crucial for G2/M arrest (Cortez et al., 1999
; Xu et al., 2001
). Consistently, ectopic overexpression of EZH2 in benign breast cells decreased nuclear BRCA1 and pBRCA1 s1423 protein levels. The in vivo
relevance of these findings is underscored by our xenograft mouse models showing that EZH2 knockdown increased BRCA1 and pBRCA1 s1423 proteins in the nuclei of breast cancer cells in situ
. Of note, our data demonstrate that the observed effects of EZH2 down-regulation on breast cancer cell proliferation and G2/M transition require BRCA1, as BRCA1 inhibition was sufficient to completely rescue the decrease in cell proliferation and the delay in G2 caused by EZH2 down-regulation.
Ectopic overexpression of BRCA1 in breast cancer cells has been shown to cause G2/M arrest by regulating the levels and activity of Cdc2/Cyclin B1 complex and the dual phosphatase Cdc25C, both of which are essential for G2/M checkpoint control (MacLachlan et al., 2000
; Yan et al., 2005
). At the G2/M transition, hyperphosphorylation of Cdc25C on several sites within its regulatory N-terminal domain coincides with mitotic activation of Cdc25C (Bonnet et al., 2008
; Bulavin et al., 2003
; Roshak et al., 2000
; Strausfeld et al., 1994
). Once activated, Cdc25C is able to dephosphorylate Ccd2-Tyr15, which increases the activity of the Cdc2/Cyclin B1 complex and results in entry into mitosis (Dunphy, 1994
). Providing strong evidence in support of EZH2 participation in BRCA1-mediated G2/M checkpoint control, EZH2 knockdown decreased nuclear mitotic Cdc25C, increased Cdc2 and Cdc2-Tyr15 phosphorylation, and decreased total and phosphorylated Cyclin B1. Importantly, BRCA1 knockdown reverted the effects of EZH2 down-regulation on the levels of both mitotic Cdc25C, and total and phosphorylated Cyclin B1.
Our experiments show that EZH2 knockdown is sufficient to increase BRCA1 levels in breast cancer cells and trigger similar effects in G2/M as those reported after ectopic expression of BRCA1 using an adenovirus vector in the absence of ionizing irradiation (MacLachlan et al., 2000
; Yan et al., 2005
). We propose that EZH2 knockdown in breast cancer cells reduces their growth by enhancing the cell cycle regulatory effects of BRCA1, slowing the transition from G2 to M phases, and allowing more time for DNA repair to occur. This hypothesis is supported by our experiments showing that breast cancer cells with EZH2 knockdown have increased levels of pBRCA1 s1423 protein, which is crucial for G2/M arrest and is activated during the DNA damage response (Xu et al., 2001
). Even though the precise mechanism by which EZH2 modulates BRCA1 protein levels will require further studies, unpublished preliminary data from our laboratory suggest that it does not involve BRCA1 gene transcription since EZH2 knockdown had no effect on BRCA1 messenger RNA. This finding is not surprising in light of studies showing that EZH2 regulates the activity of Cdc42 without directly affecting its transcription (Su et al., 2005
). Furthermore, we have no evidence by co-immunoprecipitation of a direct interaction between EZH2 and BRCA1 proteins (data not shown). A functional connection of EZH2 and BRCA1 proteins is currently under investigation.
The relevance of the association between EZH2 and BRCA1 proteins to human breast cancer is highlighted by the finding that 76 % of ER negative invasive carcinomas overexpress EZH2 and are negative for pBRCA1 s1423 protein. Previous studies have demonstrated that ER negative breast carcinomas have significantly lower BRCA1 protein than ER positive tumors (Turner et al., 2007
). However, the expression of pBRCA1 s1423 has not been reported. Our study shows for the first time that pBRCA1 s1423 levels are significantly associated with the ER status of invasive breast carcinomas as invasive carcinomas with negative ER exhibit significantly higher levels of pBRCA1 s1423 protein.
In conclusion, our results demonstrate a previously undescribed function of EZH2 during ER negative breast cancer progression by showing that EZH2 knockdown decreases tumor proliferation and growth in vivo and in vitro, and influences the transition from G2 phase to mitosis. We provide the first link between EZH2 and BRCA1 proteins and show that EZH2 knockdown depends on BRCA1 up-regulation to decrease breast cancer proliferation and progression through G2 phase (). Our data raise a novel hypothesis that restoration of BRCA1 function by EZH2 knockdown may effectively decrease tumor progression enabled by BRCA1 deficiency, and provide a new lead toward future developments of specific strategies to restore BRCA1 levels and function, and for possible prevention of ER negative tumors.