Proposed model to explain an increase in breast cancer risk in daughters, and possibly granddaughters and great granddaughters, of mothers who took diethylstilbestrol during pregnancy
Gene expression can be altered as a consequence of mutations or epigenetic changes. In contrast to gene mutations within the DNA, epigenetic changes involve post-transcriptional modifications; that is, methylation of gene promoter regions, histone modifications, deposition of certain histone variants along specific gene sequences and microRNA (miRNA) expression. Although both changes are heritable, an important distinction between the two is that mutations are not reversible, but epigenetic modifications generally are.
Probably the most common mechanism of epigenetic gene silencing is methylation, and it might also be the most important. DNA methyltransferases (DNMTs) catalyze the methylation of genomic DNA by adding a methyl group (CH3) onto the 5-carbon of the cytosine ring within CpG dinucleotides. Histone modifications are complex, as they involve not just histone methylation but also acetylation, deacetylation and other post-translational changes. These modifications occur in the amino-terminal tails of histones and affect the ‘openness’ of the chromatin, which determines whether a gene is expressed or silenced (for example, acetylation allows transcription, while deacetylation represses transcription). Trimethylation of histone H3 at lysine K27 is catalyzed by the Polycomb group (PcG) protein enhancer of Zeste-2 (EZH2) and results in gene silencing. PcG/H3K27me3 interact with DNMTs, and together they establish and maintain silencing of PcG target genes. Over 2,000 different PcG target genes have been identified and they include some tumor suppressor genes. Many of the PcG target genes regulate cell fate, including apoptosis, proliferation and stem cell differentiation. As discussed in more detail below, methylation of PcG target genes is linked to increased breast cancer risk.
DNMTs may be key players in regulating histones and the entire epigenomic machinery, since DNA methylation events often precede histone modifications. Upregulation of DNMTs increases the expression of EZH2 and other polycombs; this may happen by DNMTs inducing methylation of non-coding miRNAs that target the polycombs.
We and others have observed that the expression of DNMTs is persistently altered in estrogen-regulated tissues following estrogenic exposures during early life. In utero exposure to DES is reported to increase the expression of DNMT1 in the epididymis and uterus . We found that DNMT1 expression is increased in the mammary glands of adult rat offspring of dams exposed to ethinyl estradiol during pregnancy. These changes provide a key regulatory layer to influence gene expression in the mammary gland and perhaps breast tumors of individuals exposed to DES or other estrogenic compounds in utero.
In utero DES exposure alters methylation patterns of several genes in estrogen’s target tissues, including Hox genes, c-fox, and Nsbp1, but it has not been studied whether changes in methylation patterns occur in the mammary gland. We have explored changes in methylation in the mammary glands of adult rats exposed in utero to the synthetic estrogen ethinyl estradiol using global sequencing approaches. Among the genes that exhibited increased promoter methylation were several PcG target genes, suggesting that a maternal exposure to synthetic estrogens during pregnancy causes long-lasting changes in the methylation of genes that regulate cell fate, including stem cell differentiation.
As an increase in EZH2 expression in the mammary glands of mice exposed to DES in utero has been reported, histone modifications also seem to be influenced by maternal exposure to synthetic estrogens during pregnancy. Jefferson and colleagues recently investigated whether upregulation of lactoferrin and sine oculis homeobox 1 (Six1) in the uterus of adult mice exposed to DES neonatally is caused by histone modifications. Their data indicate that neonatal DES exposure induces changes during the early postnatal period in the expression of multiple chromatin-modifying proteins but these changes do not last to adulthood. However, alterations in epigenetic marks at the Six1 locus in the uterus were persistent. Similarly, changes in the methylation of Nsbp1 and expression of DNMTs in the uterus of DES-exposed offspring are different in the early postnatal period compared to adulthood. This suggests that some epigenetic alterations are further influenced by factors operating during postnatal development, such as a surge of estrogens and progesterone from the ovaries at puberty onset.
Maternal exposures during pregnancy have been found to induce persistent changes in miRNA expression in the offspring. miRNAs are short non-coding single-stranded RNAs composed of approximately 21 to 22 nucleotides that regulate gene expression by sequence-specific base-pairing with the 3’ untranslated region of target mRNAs. miRNA binding induces post-transcriptional repression of target genes, either by inducing inhibition of protein translation or by inducing mRNA degradation. Expression of many miRNAs is suppressed by estrogens. Although the effects of maternal DES exposure during pregnancy on miRNA expression in the offspring have not been investigated, it is known that many other manipulations, such as maternal low protein diet, alter miRNA patterns among the offspring. We recently found that in utero exposure to ethinyl estradiol lowers the expression of many of the same miRNAs in the adult mammary gland as are downregulated by E2 in MCF-7 human breast cancer cells. Since miRNAs can be silenced by methylation or as a result of increased PcG expression, and they target DNMTs, histone deacetylases and polycomb genes, the observed increase in DNMT expression, histone marks and EZH2 in the in utero DES-exposed offspring may be a result of epigenetic silencing of miRNAs that target them.
2014 Study Conclusions
In summary, women exposed to DES in utero are destined to be at an increased risk of developing breast cancer, and this risk may extend to their daughters and granddaughters as well. It is of critical importance to determine if the increased risk is driven by epigenetic alterations in genes that increase susceptibility to breast cancer and if these alterations are reversible.
Sources and more information
- Full text (free access) : Maternal exposure to diethylstilbestrol during pregnancy and increased breast cancer risk in daughters, Breast Cancer Research, NCBI PubMed, PMC4053091, 2014 Apr 30.
- Proposed model to explain an increase in breast cancer risk in daughters, and possibly granddaughters and great granddaughters, of mothers who took diethylstilbestrol during pregnancy featured image credit PMC4053091/figure/F1.