DNA demethylation by 5-aza-2-deoxycytidine treatment abrogates 17 beta-estradiol-induced cell growth and restores expression of DNA repair genes in human breast cancer cells

Abstract

Prolonged exposure to elevated levels of estrogen is a risk factor for breast cancer. Though increased cell growth and loss of DNA repair capacity is one of the proposed mechanisms for estrogen-induced cancers, the mechanism through which estrogen induces cell growth and decreases DNA repair capacity is not clear. DNA hypermethylation is known to inactivate DNA repair genes and apoptotic response in cancer cells. Therefore, the objective of this study was to determine the role of DNA hypermethylation in estrogen-induced cell growth and regulation of DNA repair genes expression in breast cancer cells. To achieve this objective, the estrogen-responsive MCF-7 cells either pretreated with 5-aza-2-deoxycytidine (5-aza-dC) or untreated (as control) were exposed to 17 beta-estradiol (E2), and its effect on cell growth and expression of DNA repair genes were measured. The result revealed that 5-aza-dC abrogates the E2-induced growth in MCF-7 cells. An increased expression of OGG1, MSH4, and MLH1 by 5-aza-dC treatment alone, suggest the DNA hypermethylation as a potential cause for decreased expression of these genes in MCF-7 cells. The decreased expression of ERCC1, XPC, OGG1, and MLH1 by E2 alone and its restoration by co-treatment with 5-aza-dC further suggest that E2 reduces the expression of these DNA repair genes potentially through promoter hypermethylation. Reactivation of mismatch repair (MMR) gene MLH1 and abrogation of E2-induced cell growth by 5-aza-dC treatment suggest that estrogen causes increased growth in breast cancer cells potentially through the inhibition of MMR-mediated apoptotic response. In summary, this study suggests that estrogen increases cell growth and decreases the DNA repair capacity in breast cancer cells, at least in part, through epigenetic mechanism.

Introduction

Breast cancer is the second leading cause of cancer related death in women and is a major public health issue. The physiological concentration of estrogen is required for the normal growth and maintenance of various reproductive and non-reproductive organs in the body [1]. A large number of evidence from epidemiological, clinical, and experimental studies suggests that besides it normal function, the prolonged exposure to elevated levels of estrogen is associated with the development of breast cancer [2], [3]. The endogenous estrogen 17 beta-estradiol (E2) acts as proliferation and survival factors in breast epithelial cells that ultimately promotes the breast cancer development [4], [5]. Though the involvement of estrogen in the etiology of breast cancer is well established, the mechanism of estrogen-induced breast carcinogenesis is still unclear. Loss of DNA repair capacity resulting in accumulation of mutations and genomic instability is one of the characteristic features of cancer cells. Aberrant expression of DNA repair genes has been reported from human breast cancers [6], [7], [8] as well as in estrogen-induced cancers [9]. Either complete loss or reduced expression of mismatch repair (MMR) gene MSH2 in human sporadic breast cancer samples has also been reported [10]. Mutations in DNA repair gene DNA polymerase-β and its decreased expression in synthetic estrogen diethylstilbestrol (DES)-induced renal tumors has been reported [9]. Mutations in microsatellite repeat sequences [11], [12], and Alu repeat sequences [13] has also been reported from DES-induced renal tumors in Syrian hamsters. Recently, we have reported mutations in a new gene ETRG1 in DES-induced renal tumors in Syrian hamsters [14]. These evidences suggest that the loss of DNA repair capacity resulting in accumulation of mutations and genomic instability as one of the mechanisms for the development of estrogen-induced cancers. However, the mechanism for reduced expression of DNA repair genes in human breast cancers and estrogen-induced cancers is unclear. It is well known that neoplastic cells simultaneously harbor widespread global hypomethylation and regional hypermethylation that contribute to tumor progression [15]. Accumulating evidences suggest that aberrant promoter methylation causes loss of gene expression that can provide selective growth advantage to neoplastic cells [16]. For example, promoter hypermethylation causing loss of expression of VHL in renal cell carcinoma [17], and BRCA1 in sporadic breast and ovarian tumors [18] has been reported. Promoter hypermethylation linked to reduced expression of DNA repair genes, MLH1 in colon [19], [20] and sporadic breast cancers [10], and MGMT [21], [22] in colon cancers has also been reported. The promoter hypermethylation-associated loss of DNA repair capacity can predispose cells to mutational events and may provide a selective advantage to neoplastic cells [16].

In addition to the post-replication DNA repair function, the MMR system plays important role in cellular response to DNA damage, cell cycle arrest, and apoptosis [23]. The loss of MMR capacity has been shown to cause increased cell survival by decreased apoptotic response and increased resistance to chemotherapeutic drugs [24].

In vivo study with ACI rat model have shown that exposure to elevated estrogen levels results in hyperproliferative changes in parallel with epigenetic changes, such as, upregulation of DNA methyltransferases and hyperacetylation of histone residues [25]. However, the effects of estrogen-induced epigenetic changes on cell growth and genomic instability are not clear. Therefore, the objective of this study was to determine the potential role of DNA methylation in mediating the effect of estrogen on cell growth and expression of DNA repair genes in estrogen-responsive MCF-7 human breast cancer cells.