Do epigenetic alterations precede the increased risk of breast neoplasia after in utero exposure to DES ?

In Utero Exposure to Diethylstilbestrol (DES) or Bisphenol-A (BPA) Increases EZH2 Expression in the Mammary Gland: An Epigenetic Mechanism Linking Endocrine Disruptors to Breast Cancer

2010 Study Abstract

Diethylstilbestrol (DES) and bisphenol-A (BPA) are estrogen-like endocrine-disrupting chemicals that induce persistent epigenetic changes in the developing uterus. However, DES exposure in utero is also associated with an increased risk of breast cancer in adult women. Similarly, fetal exposure to BPA induces neoplastic changes in mammary tissue of mice.

We hypothesized that epigenetic alterations would precede the increased risk of breast neoplasia after in utero exposure to endocrine disruptors. Enhancer of Zeste Homolog 2 (EZH2) is a histone methyltransferase that has been linked to breast cancer risk and epigenetic regulation of tumorigenesis. We examined the effect of BPA and DES on EZH2 expression and function in MCF-7 cells and in mammary glands of mice exposed in utero. DES and BPA treatment approximated human exposure. EZH2 functional activity was assessed by measuring histone H3 trimethylation.

Treatment of MCF-7 cells with DES or BPA led to a 3- and 2-fold increase in EZH2 mRNA expression, respectively (p < 0.05) as well as increased EZH2 protein expression. Mice exposed to DES in utero showed a >2-fold increase in EZH2 expression in adult mammary tissue compared with controls (p < 0.05). EZH2 protein was elevated in mammary tissue of mice exposed to DES or BPA. Histone H3 trimethylation was increased in MCF-7 cells treated with BPA or DES. Similarly, mice exposed to BPA or DES in utero showed increased mammary histone H3 trimethylation.

Developmental programming of EZH2 is a novel mechanism by which in utero exposure to endocrine disruptors leads to epigenetic regulation of the mammary gland.

Discussion

DES and BPA are endocrine-disrupting chemicals with well-known developmental effects. Many of their effects are mediated through their estrogen-agonist activity. However, many of the effects of these endocrine disruptors persist well after exposure, with lasting changes in gene expression that cannot be explained simply as a result of their direct estrogenic effects. We have previously shown that DES or BPA exposure causes epigenetic changes in HOXA10 expression in the reproductive tract by altered DNA methylation. Epigenetic changes as a result of exposure to EDC may predispose to malignancies in adulthood. In women, exposure to DES in utero is associated with an increased incidence of breast cancer as an adult. Rodent studies have shown that BPA exposure in utero causes molecular changes in mammary tissue, altering estrogen sensitivity and predisposing to mammary ductal hyperplasia and an increase in carcinoma in situ of the breast. Here we examine the mechanisms by which DES and BPA exert these epigenetic effects in mammary tissue.

Recently, epigenetic changes involving DNA methylation and chromatin remodeling as a result of histone modifications (methylation, acetylation, etc.) have been implicated in the process of carcinogenesis. Interestingly, nearly half of the tumor suppressor genes known to cause certain cancer syndromes can be activated by DNA promoter hypermethylation in sporadic cancers. As described above, histone methylation by EZH2 is a known epigenetic modifier in breast cancers.

Because of the possible role of EZH2 in the development of breast cancer, we investigated the effects of exposure to two well described EDCs, DES and BPA, on expression of EZH2. DES was administered to pregnant women at high dose, while BPA is a ubiquitous environmental contaminant. Here, plasma BPA concentrations in treated mice were found to be similar to those commonly reported in pregnant women. Exposure to either DES or BPA in human MCF-7 cells increased EZH2 mRNA and protein expression. After in utero exposure to DES or BPA, mice demonstrated lasting increases in EZH2 expression in the adult. DES exposure in utero caused an increase in EZH2 mRNA and protein levels. Interestingly, after in utero exposure to BPA, EZH2 protein expression was increased, while there was no apparent change in EZH2 mRNA in mice. This suggests that BPA increases EZH2 protein by increasing translation or decreasing protein degradation. BPA may decrease microRNAs specific to EZH2 mRNA, thus increasing protein translation without any significant changes in mRNA expression. Alternatively, BPA might stabilize EZH2 protein by a mechanism-decreasing protein catabolism.

We also assessed the functional significance of increased EZH2 protein levels by examining the expression of histone H3 (tri methyl K27). We found increases in histone H3 (tri methyl K27) in MCF-7 cells treated with DES or BPA. Mice exposed to DES or BPA in utero also showed increased histone H3 (tri methyl K27) levels. Because the epigenetic effect of EZH2 is mediated largely due to its histone methyltransferase activity at lysine 27 of histone H3, our data confirm that both DES exposure and BPA exposure in utero cause epigenetic alterations in mammary tissue.

In breast cells, an interesting target of EZH2-mediated histone methyltransferase activity is p57 (CDKN1C), a-cyclin dependent kinase inhibitor that functions in the maintenance of the cell cycle, which has recently been shown to be repressed by EZH2-mediated methylation of histone H3 at lysine 27 in breast cancer cells. Repression of this cell cycle regulator may lead to the increase in cell proliferation seen in breast tumors with elevations in EZH2. Another important target gene, repressed by EZH2-mediated histone methylation, is E-cadherin. E-Cadherin is important in cell–cell adhesion, and its disruption has been associated with increased invasiveness and metastases of cancers. E-Cadherin is transcriptionally silenced by EZH2 by methylation of histone H3 at lysine 27. The increased invasiveness seen in breast cancers with elevated EZH2 levels could therefore involve repression of E-cadherin.

In conclusion, we have demonstrated a novel mechanism by which endocrine-disrupting chemicals regulate developmental programming in the breast. Exposure to DES or BPA in utero alters mammary tissue expression of EZH2, a histone methyltransferase with known associations to tumorigenesis. EZH2 function, measured by examination of histone H3 (tri methyl K27), also increases as a result of exposure to DES or BPA. Increased expression of EZH2 within the breast, even in morphologically normal appearing tissue, may prove to be a marker of increased breast cancer risk. The increase in EZH2 expression and function shown here in mice after in utero exposure to these chemicals is a potential mechanism for the increased risk of breast cancer as a result of exposure to these EDCs. This study also generates important safety concerns about exposures to environmental endocrine disruptors such as BPA and suggests a potential need to monitor women exposed to these chemicals for the development of breast lesions as adults.

Sources and more information
  • Full study (free access) In Utero Exposure to Diethylstilbestrol (DES) or Bisphenol-A (BPA) Increases EZH2 Expression in the Mammary Gland: An Epigenetic Mechanism Linking Endocrine Disruptors to Breast Cancer, Journal List, HHS Author Manuscripts, NCBI PubMed PMC3140020, 2010 Jun.
  • DES and BPA exposure in vitro induce EZH2 mRNA expression in human mammary cells featured image credit PMC3140020/figure/F2.
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