DES silencing of KLF9 may contribute to the etiology of endometrial cancers initiated by aberrant ESR1 signaling
2010 Study Abstract
Inappropriate early exposure of the hormone-responsive uterus to estrogenic compounds is associated with increased risk for adult reproductive diseases including endometrial cancers. While the dysregulation of estrogen receptor-α (ESR1) signaling is well-acknowledged to mediate early events in tumor initiation, mechanisms contributing to sustained ESR1 activity later in life and leading to induction of oncogenic pathways remain poorly understood. We previously showed that the transcription factor Krüppel-like Factor 9 (KLF9) represses ESR1 expression and activity in Ishikawa endometrial glandular epithelial cells. We hypothesized that KLF9 functions as a tumor suppressor and loss of its expression enhances ESR1 signaling. Here, we evaluated the contribution of KLF9 to early perturbations in uterine ESR1 signaling pathways elicited by the administration of synthetic estrogen diethylstilbestrol (DES) to wild-type (WT) and Klf9 null (KO) mice on postnatal days (PND) 1–5. Uterine tissues collected at PND84 were subjected to histological, immunological, and molecular analyses. Compared to WT, KO mice demonstrated larger endometrial glands and lower endometrial gland numbers; DES exposure exacerbated these differences. KLF9 loss of expression resulted in increased glandular ESR1 immunoreactivity with DES, without effects on serum estradiol levels. QPCR analyses indicated altered expression of uterine genes commonly dysregulated in endometrial cancers (Akt1, Mmp9, Slpi, Tgfβ1) and those involved in growth regulation (Fos, Myc, Tert, Syk), with loss of Klf9, alone or in concert with DES. Our data support a molecular network between KLF9 and ESR1 in the uterus and suggest that silencing of KLF9 may contribute to endometrial dysfunction initiated by aberrant estrogen action.
- RNA isolation and quantitative RT-PCR (QPCR)
- DES-associated growth effects with Klf9 mutation
- DES-associated alterations in uterine parameters with Klf9 mutation
- Developmental expression of uterine KLF9
- KLF9 regulation of ESR1 expression with DES-treatment
- Altered uterine gene expression in WT and Klf9 KO mice with DES treatment
- Early effects of KLF9 mutations on gene expression
In this study, we established a central role for KLF9 in the response of uterine endometrial cells to early perturbations in ESR1 signaling. Using the well-characterized DES-induced model for dysregulation of ESR1 function leading to pathology, we showed that loss of Klf9 expression alone altered uterine structural parameters (LE height, gland numbers, gland size) comparable to those in DES-treated WT mice and increased endometrial gland sensitivity to DES. We demonstrated that Klf9 null mutation enhanced glandular ESR1 expression with DES, and this was positively associated with increased expression of Slpi, a gene encoding an endometrial glandular secreted protein whose over-expression is implicated in the pathogenesis of many cancers, including that of the endometrium, due to its pro-proliferative actions. Finally, we demonstrated that unlike ESR1, whose uterine expression was found to be an early target of DES, occurring within days of DES administration and which remained dysregulated later in life, KLF9 expression was undetectable in the developing uterus (PND5, PND11) and was unaffected by DES later in life. Our results suggest that a primary site of KLF9 action is the endometrial glandular compartment where Klf9 ablation promoted long-term DES effects on ESR1 and Slpi expression and on glandular morphology (gland size and number). Given that endometrial cancer originates from endometrial glands and consistent with our previous report of increased ESR1 expression with estradiol-17β treatment upon siKLF9 knockdown in Ishikawa (glandular epithelial) cells, we propose that KLF9 constitutes an important regulator of ESR1 signaling in endometrial glands and that loss of this regulation may predispose to increased risk for endometrial cancers.
Our findings that early (postnatal) DES exposure altered adult uterine phenotype (PND84) are consistent with previously reported long-term consequences of DES-induced early developmental perturbations for this tissue in mice (Couse et al. 2001). The precise mechanism by which early disruption of ESR1 signaling leads to abnormalities in uterine function and phenotype as adults, remains poorly understood. Epigenetic changes involving modifications to chromatin structure due to aberrant histone methylation, histone acetylation, and DNA methylation, are increasingly considered to underlie the onset and progression of cancers (Ellis et al. 2009). Consistent with this, neonatal DES exposure has been recently reported to alter genomic DNA methylation due to changes in expression of uterine DNA methyltransferases. Our data showing the lack of co-expression of KLF9 with ESR1 in uteri of mice in the first two weeks of life suggest that KLF9 mediates glandular ESR1 expression in adult uteri subsequent to and/or independent of early events arising from epigenetic modifications of ESR1. KLF9 may alter nuclear ESR1 expression through effects on ESR1 stability, nuclear localization, and/or promoter activity in the adult uteri, the latter consistent with our previous findings that reduced KLF9 expression prevented the recruitment of ESR1 to its own promoter to elicit ESR1 negative autoregulation. While our data does not address the stage in uterine development wherein loss of Klf9 expression on ESR1 signaling is manifested, we suggest that this likely occurs as early as PND28 based on the comparable patterns of uterine Slpi expression in PND28 and PND84 DES-exposed Klf9 null mice, when compared to corresponding-aged WT mice, and given that Slpi is an ESR1-induced gene. Further studies will address ESR1 expression in Klf9 null uteri of younger (prepubertal) mice. Interestingly, while our previous study of the human endometrial cancer cell line HEC-1A, which lack detectable ESR1 expression, demonstrated induction of SLPI expression with KLF9 over-expression , results presented here indicate negative regulation of Slpi expression by KLF9 in ESR1-expressing cells with perturbed ESR1 signaling. Taken together, our data suggest that while the contribution of epigenetic mechanisms to dysregulated ESR1 expression with DES exposure cannot be excluded, KLF9 involvement in the regulation of ESR1 and Slp1 expression is most likely a consequence of its action at the level of the ESR1 promoter.
To determine if changes in estrogen-responsive parameters were attributable to altered ovarian function, we measured both serum E2 and ovarian weights of adult WT and Klf9 null mice with and without early DES exposure. The comparable E2 levels in both genotypes as well as the lack of significant differences in ovarian wet weights as a function of genotype and DES administration support our previous reports of the lack of major KLF9 effects on the ovary. Thus, ESR1/KLF9-dependent effects noted in glandular epithelial cells is largely ovarian-independent and likely a consequence of direct uterine targeting by KLF9.
We used a combination of candidate gene and focused gene array approaches to identify genes whose expression was differentially altered in adult (PND84) WT and Klf9 mutant uteri, based on their relationships to KLF9, ESR1, and/or cancer risk. Of the uterine genes confirmed to be regulated with loss of Klf9 expression, alone or as a function of DES exposure, two major categories are proposed: one category is comprised of genes that are regulated by KLF9, independent of ESR1 (DES) signaling (Klf13, Myc, Tert, Syk, Akt1), while the second category includes genes that are influenced by KLF9/ESR1 cross-regulation (Fos, Slp1). The increased expression of pro-proliferative/cell survival, ESR1-regulated genes Akt1, and Tert with loss of Klf9 expression is consistent with the potential tumor suppressor function of KLF9, as suggested by the reported loss of KLF9 expression in endometrial tumors. Moreover, the coincident elevated expression of Myc and Tert mRNAs is consistent with the observation by Wu and colleagues (1999) that MYC upregulates Tert promoter activity. Fos and Slpi are estrogen-regulated genes, whose gene products exhibit pro-proliferative activities, hence, their increased expression with Klf9 mutation coincident with increased ESR1 expression (with DES exposure) suggests a pathway linking the loss of KLF9-mediated ESR1 negative regulation to enhanced proliferation and provide further support to potential KLF9 tumor suppressor activity. The increased expression of Klf13 with Klf9 null mutation implicates a regulatory loop between these highly-related family members, as suggested from our previous studies. Pten mutations are among the earliest identifiable aberrations in endometrial carcinoma ; hence, our findings of a robust decrease in Pten transcript levels in DES-exposed WT uteri relative to control WT are consistent with increased incidence of endometrial carcinoma in peri-natal DES-exposed mice. While we demonstrated only a trend for decreased uterine Pten expression in Klf9 null mutants without and with DES exposure relative to WT mice, Pten transcript levels for Klf9 null mutants without DES exposure were numerically comparable to those of DES-exposed WT mice, suggesting that dysregulation of Pten expression by early DES exposure is partially mimicked by Klf9 null mutation. In this regard, recent data showing Slpi upregulation in the uteri of Pten heterozygous mice which develop atypical hyperplasias and carcinomas in situ and that conditional deletion of endometrial Pten resulted in the rapid development of endometrial carcinoma in mice are consistent with our predicted linkages between expression of Klf9, Slpi and Pten and uterine tumorigenesis. Studies evaluating uterine tumor development in Klf9 null mice with and without postnatal DES exposure will be critical to addressing the link between KLF9 and uterine endocarcinoma and are ongoing in our laboratory.
In summary, our data suggest a novel role for the transcription factor KLF9 in modulating uterine responses to ESR1-dependent DES actions, the latter leading to a dysfunctional uterus. Our in vivo model superimposing Klf9 null mutation with early (postnatal) DES exposure has revealed a molecular network between KLF9 and ESR1 operative in the uterus involving key estrogen-regulated uterine morphological and molecular indices that may constitute early predictors of uterine tumor development. While direct evidence for KLF9 as a tumor suppressor awaits further studies, our results raise the intriguing possibility that the silencing of KLF9 either by genetic and/or epigenetic mechanisms, as demonstrated for many tumor suppressors and oncogenes, may contribute to increased susceptibility to developing endometrial cancers initiated by aberrant estrogen action.