Developmental Exposure to Diethylstilbestrol Alters Uterine Gene Expression That May Be Associated With Uterine Neoplasia Later in Life
2007 Study Abstract
Previously, we described a mouse model where the well-known reproductive carcinogen with estrogenic activity, diethylstilbestrol (DES), caused uterine adenocarcinoma following neonatal treatment. Tumor incidence was dose-dependent reaching >90% by 18 mo following neonatal treatment with 1000 μg/kg/d of DES. These tumors followed the initiation/promotion model of hormonal carcinogenesis with developmental exposure as initiator, and exposure to ovarian hormones at puberty as the promoter. To identify molecular pathways involved in DES-initiation events, uterine gene expression profiles were examined in prepubertal mice exposed to DES (1, 10, or 1000 μg/kg/d) on days 1–5 and compared to controls. Of more than 20 000 transcripts, approximately 3% were differentially expressed in at least one DES treatment group compared to controls; some transcripts demonstrated dose–responsiveness. Assessment of gene ontology annotation revealed alterations in genes associated with cell growth, differentiation, and adhesion. When expression profiles were compared to published studies of uteri from 5-d-old DES-treated mice, or adult mice treated with 17β estradiol, similarities were seen suggesting persistent differential expression of estrogen responsive genes following developmental DES exposure. Moreover, several altered genes were identified in human uterine adenocarcinomas. Four altered genes [lactotransferrin (Ltf), transforming growth factor beta inducible (Tgfb1), cyclin D1 (Ccnd1), and secreted frizzled-related protein 4 (Sfrp4)], selected for real-time RT-PCR analysis, correlated well with the directionality of the microarray data. These data suggested altered gene expression profiles observed 2 wk after treatment ceased, were established at the time of developmental exposure and maybe related to the initiation events resulting in carcinogenesis.
The results presented in this study showed significant alterations in uterine gene expression following neonatal DES treatment at 1, 10, and 1000 μg/kg/d that occur at least 2 wk after treatment ceased. We have previously reported that neonatal DES exposure at these same doses caused uterine adenocarcinoma at 12 mo of age in 19%, 29%, and 63% of the mice treated with the respective doses; the tumor incidence reached >90 % at 18 mo at the highest DES dose. These tumors were dependent on ovarian hormones at puberty because mice ovariectomized before puberty did not develop uterine tumors. Our current data support the idea that early exposure to DES changes the transcriptional profile of the uterus so that it responds abnormally to ovarian hormones at puberty which may then lead to the development of uterine cancer later in life. The changes in gene expression that we identified in the uterus were consistent with developmental DES exposure being an initiating event in an initiation/promotion model of hormonal carcinogenesis because the alterations were detected before puberty. Gene categories which showed significantly enriched differential expression in ontological biological processes include cell cycle, adhesion, growth and development, as well as cell differentiation. Because multiple gene categories are represented, this suggests complex mechanisms are involved in uterine hormonal carcinogenesis.
We compared our data from 19-d-old prepubertal mice exposed neonatally to DES to previously published data obtained from 5-d-old DES-treated mice. Although we examined changes that occurred in response to three increasing doses of DES, we only compared data from our dose of DES-1000 (2 μg/kg/d) which was the same as that published by Huang et al. There were numerous changes in common between the two studies with 15 genes showing increased expression and 10 genes showing decreased expression. Our findings suggest that these genes may be particularly important because they represent a subset of genes that are permanently altered, not just transiently altered in response to DES immediately following DES treatment. Some of these genes include estrogen-regulated proteins such as Ltf, C3, and Ccnd1 as well as Sprr2f which have been shown to be involved in epithelial cell differentiation. Further, we identified 11 genes that were increased in one study but decreased in the other. These genes probably represent genes that are transiently altered by DES treatment or are differentially regulated by other factors active in uterine differentiation processes. In addition to these genes, Huang et al. described decreased expression of Msx-2, a homoeodomain transcription factor and a member of the Wnt signaling pathway although it was not consistently decreased across all of their samples. In a more recent study, they also reported repression of Msx-2 by DES and a role for the gene in female reproductive tract differentiation . In our study, the expression of the Msx-2 gene was increased in all three DES dose groups. This difference between studies can probably be explained by the time after DES treatment. While it has been shown that Msx-2 is involved in uterine differentiation, the contribution to the etiology of uterine cancer remains to be determined. Previously, we have also shown that other genes and their proteins such as c-fos and p21 were increased during the time of DES treatment but then returned to control levels by puberty supporting the idea that some genes like Ltf, C3, Ccnd1, and Sprr2f are permanently imprinted, but others like Msx-2 are not. This points out that the timing of gene expression is very important and can be altered by exposure to DES resulting in abnormal uterine differentiation whether genes are permanently imprinted or not.
We further compared our data from CD-1 mice to a previous report with C57 Bl/6 mice that examined the direct response of the mature uterus to an estrogenic stimulus . A remarkable similarity was found between the 24-h estradiol treatment and our DES-1 treatment group which was collected 2 wk after neonatal treatment at 19 d of age. Of all the genes that were considered statistically significant, over half were commonly increased or decreased in our lowest DES-treated group (DES-1) and the adult estradiol group. These two groups are probably the most similar because estrogen responsiveness of the uterus can be demonstrated in the DES-1 treatment group whereas, the DES-10 and DES-1000 treated mice often exhibit limited ability to respond to estrogen, as well as, exhibit altered uterine cellular differentiation. The overall similarities between the two studies suggest that a substantial estrogenic component remains present long after the last DES treatment on day 5. From the gene ontology analysis, many of these genes were involved in cell-cycle regulation.
For example, expression of Ccnd1, a cell-cycle regulatory gene important in the G1/S phase of the cell cycle, was decreased in the neonatally DES-treated uterus at 19 d as well as at 5 d directly following treatment and in the mature uterus 4 h following estrogen treatment. Further analysis of Ccnd1 expression with real-time RT-PCR showed the decreased expression of this gene in individual DES-treated animals. The alteration of cell-cycle genes, like Ccnd1, may contribute to abnormal cell division and hormone responses at puberty thus resulting in neoplasia later in life.
Expression of Ltf, an estrogen regulated gene, was increased as expected in the mature estradiol stimulated uterus and the 5-d-old DES-treated uterus as determined by microarray analysis; it was also increased in all three of our DES dose groups. Real-time RT-PCR supported these findings although there were variable responses in individual mice. Several studies have shown that the mRNA for this gene, as well as, the protein were over-expressed during the time of neonatal DES treatment . Further, mice treated developmentally with DES showed high levels of lactotransferrin (also referred to as lactoferrin) protein later in life even in the absence of estrogen stimulation . Our current study is consistent with these findings. In addition, we have previously shown that the promoter region of the Ltf gene has an altered methylation pattern following DES exposure suggesting that DES permanently imprints this gene. Interestingly, our current data show a dose responsive increase in Ltf expression which correlates with a dose responsive increase in uterine cancers seen later in life. Whether lactoferrin plays a direct role in the initiation and/or promotion of uterine cancer is an ongoing investigation but it is interesting that it has been reported to have growth promoter effects in uterine cells.
In addition to Ltf, the expression of another estrogen regulated gene, C3 was increased 1.5-fold in the DES-1000 group. While, our analysis did not identify all estrogen-responsive genes as reported in the mature uterus, this may be due to the age of the tissues, differences in mouse strains, time differential of 2 wk following DES treatment, or the differences in the threshold of our analysis. Estrogen-regulated genes that did not show a significant difference in our study but were reported to be responsive by others include epidermal growth factor (Egf) , EGF receptors (Egfr) , progester-one receptor (Pr) , and heat shock protein 27 (Hsp27) . Further, another group of estrogen-regulated genes not found to be significantly changed in our study include c-fos, c-jun, and c-myc but this is most probably because of the transient expression of these genes because they are expressed minutes to hours after estrogen exposure and not likely to be present 2 wk after treatment. Our findings do not lessen the role of these genes in the development of neoplasia but it does suggest that the expression of these genes is not permanently altered in the initiation phase of uterine hormonal carcino-genesis, although the response to hormone stimulation later in life may be altered.
A large category of differentially expressed genes following neonatal DES treatment included genes involved in cell adhesion. Many of the genes and gene products in this category are involved in cell–cell communication which can be disrupted in the progression to cancer. We identified several collagens and integrins found in this category. These genes showed the highest expression level in the highest dose of DES treatment supporting disruption of tissue architecture, which we have previously described . For example, we have previously shown that high doses of DES exhibit alterations in uterine morphology such as squamous metaplasia which indicates abnormal cellular differentiation . Another gene involved in cell adhesion Tgfbi is decreased at the highest dose of DES. Further study of this gene with real-time RT-PCR confirmed the microarray data suggesting that this gene may play an important role in the initiation event.
Cell growth and development as well as cellular differentiation pathways were also altered following neonatal DES treatment. Genes in these categories include those involved in Wnt signaling and Homeo-box (Hox) genes. These have been previously reported to be altered following developmental exposure to DES. These genes are involved in reproductive tract tissue patterning and cellular differentiation. Alterations in Hox gene expression are molecular mechanisms by which DES can affect reproductive tract differentiation. Another interesting gene is Sfrp4 which has been shown to function as a modulator of Wnt signaling through direct interaction with Wnt genes; Sfrp4 is also increased following estrogen treatment. Our data with real-time RT-PCR analysis clearly demonstrates the increased expression of this gene, especially following the highest dose of DES. Alterations in the expression of this gene in the uterus may lead to disruption of the Wnt signaling pathways resulting in tissue dysmorphogenesis such as malformation early in life and/or uterine neoplasia later in life.
A striking overlap in differentially expressed genes was revealed when comparing the murine neonatal DES response with altered genes found in human uterine endometrial cancer. Of the genes found in common, Sfrp4and Igf-1 were particularly interesting because they were also identified in the estrogen-regulated set of genes. The altered expression of these genes in the prepubertal mouse prior to uterine cancer formation and in human uterine cancer implicates their role in hormonal carcinogenesis.
Another interesting observation was the variability in gene expression of individual mice following neonatal DES exposure as seen in the real-time RT-PCR data. As we have previously reported, lactoferrin expression in the uterus of control 19-d-old mice was low and did not vary among animals. However, in this study, the expression of several genes including lactoferrin was quite variable at 19 d of age following neonatal exposure to DES. This was not probably caused by endogenous ovarian hormones of puberty because the controls had very low levels of lactoferrin at this age and none of the mice showed morphological signs of puberty. The varying levels of lactoferrin observed following neonatal DES treatment were probably because of early altered imprinting, therefore some animals would be expected to be more affected than others.
In summary, developmental exposure to DES resulted in altered gene expression pathways which included an estrogen-regulated component. Because uterine tumors do not occur in animals that are ovariectomized before puberty, this implies that the induction of tumors arises from the combination of neonatal exposure to DES and subsequent pubertal exposure to endogenous estrogens. The current data suggest that DES acts during development by altering estrogen-regulated pathways and subsequent tissue response to estrogen signaling later in life, possibly resulting in cancer. It appears that neonatal DES treatment alters uterine cell proliferation and differentiation by controlling genes normally regulated by estrogen. Although the majority of the morphological and pathological effects of DES in the uterus have been shown to be mediated through estrogen receptor (ER)α (Esr1) mechanisms, little is known about the downstream targets of ERα in this process especially those involved in the development of uterine adenocarcinoma. This study identifies some early molecular events that DES alters which may be associated with an abnormal hormonal response resulting in neoplasia later in life.
The mechanisms involved in abnormal cellular responses of the uterus following neonatal DES treatment are not well understood but the information from the current study may prove useful in elucidating the pathways involved in the initiation events of uterine hormonal carcinogenesis. Further, data from this study will help develop a molecular fingerprint that can be used to predict the long-term effects of DES exposure including neoplasia which is transmitted to subsequent generations.
Sources and more information
- Full study (free access) : Developmental Exposure to Diethylstilbestrol Alters Uterine Gene Expression That May Be Associated With Uterine Neoplasia Later in Life, Molecular Carcinology, NCBI PubMed PMC2254327, 2007 September.
- Global differential gene expression cluster analysis. Panel A: Hierarchical agglomerative clustering (cosine correlation similarity measure) was performed on genes exhibiting a log (ratio) P < 0.001 and fold change of 1.5 between DES-treated uteri featured image credit NCBI PMC2254327/figure/F1.