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Endometrial stromal and glandular epithelial ESR1 expression levels as a function of Klf9 genotype and DES exposure. (A) Representative images of ESR1 immunostaining in stromal and glandular epithelial cells of Oil-treated PND84 Klf9 WT and KO mice. Images are at 400X magnification; scale bar, 10μm. Control (NTC) represents tissue section from Klf9 mutant mice, in the absence of primary antibody. Arrows indicate nuclear staining. PND84 stromal (B) and glandular epithelial (C) ESR1 quantification (mean±SEM) for n=3–4 mice per genotype per treatment (Oil or DES). Means with different superscripts differed at P<0.05 by Two-Way ANOVA followed by Tukey Test.
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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-alpha (ESR1) signaling is well acknowledged to mediate early events in tumor initiation, mechanisms contributing to sustai...
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Context 1
... the effects of DES, which signals through ESR1, on estrogen-responsive uterine parameters (e.g., LE height, gland number and size) differed with Klf9 genotype at PND84 (Fig. 3), the expression of uterine ESR1 as a function of KLF9 expression status and DES treatment was assessed by immunohistochemistry (Fig. 5A). Immunoreactive ESR1 protein was detectable in all endometrial cellular compartments. Notably, glands showed the highest staining, with most cells in each gland showing strong nuclear immunoreactivity. The numbers of nuclear stromal ESR1 (quantified as % ESR1-immunopositive cells relative to the total cells counted) were similar in ...
Context 2
... glands showed the highest staining, with most cells in each gland showing strong nuclear immunoreactivity. The numbers of nuclear stromal ESR1 (quantified as % ESR1-immunopositive cells relative to the total cells counted) were similar in Oil-treated WT and KO mice, with a significant increase noted for WT but not KO with early DES exposure (Fig. 5B). In the glandular compartment, nuclear ESR1-immunopositive cells were significantly higher only in DES-treated KO mice; Oil and DES-treated WT and control KO mice showed comparable numbers of nuclear ESR1 immunopositive cells (Fig. ...
Context 3
... in Oil-treated WT and KO mice, with a significant increase noted for WT but not KO with early DES exposure (Fig. 5B). In the glandular compartment, nuclear ESR1-immunopositive cells were significantly higher only in DES-treated KO mice; Oil and DES-treated WT and control KO mice showed comparable numbers of nuclear ESR1 immunopositive cells (Fig. ...
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Citations
... KLF9 is an evolutionarily well-conserved transcriptional regulator implicated as a tumor/metastasis suppressor in liver [24,25], endometrium [36,37], colon and rectum [38], breast [39], glioblastoma [40], ovary [41], and osteosarcoma [42]. Nevertheless, the underlying mechanism of its tumor-suppressive action for any given tissue remains undefined. ...
... It is interesting that sexual dimorphism was evident for expression of some hepatic gene upon loss of the Klf9 gene. KLF9 has not been extensively examined with regard to sexual dimorphism of liver gene expression, although it is a well-known glucocorticoidresponsive factor and circadian clock participant, as well as being a mediator of estrogen action (in concert with estrogen receptors) [30,36,37,59,84]; all of which factor into the regulation of the large sex-specific expressed gene sets in murine liver [85]. Additionally, in this regard, KLF9 is a member of an estrogen receptor α-mediated network of female regulatory genes in mouse liver [85]. ...
Simple Summary
The nuclear protein Krüppel-like factor 9 (KLF9) suppresses development of cancers in multiple tissues including the liver. However, a mechanistic basis for how KLF9 decreases cancer initiation/development has not been elucidated. Here, we used a mouse model in which the Klf9 gene was ablated and placed wild-type and mutant animals of both sexes on a high-fat, obesogenic diet. We then examined how the presence or absence of KLF9 affected adiposity, as well as indices of liver oxidative stress and inflammation, and systemic oxidative stress. Results demonstrate that KLF9 is a suppressor of liver oxidative stress and inflammation which may underlie its tumor suppressive actions in liver and other tissues.
Abstract
Obesity, oxidative stress, and inflammation are risk factors for hepatocellular carcinoma (HCC). We examined, in mice, the effects of Krüppel-like factor 9 (KLF9) knockout on: adiposity, hepatic and systemic oxidative stress, and hepatic expression of pro-inflammatory and NOX/DUOX family genes, in a high-fat diet (HFD) context. Male and female Klf9+/+ (wild type, WT) and Klf9−/− (knockout, KO) mice were fed HFD (beginning at age 35 days) for 12 weeks, after which liver and adipose tissues were obtained, and serum adiponectin and leptin levels, liver fat content, and markers of oxidative stress evaluated. Klf9−/− mice of either sex did not exhibit significant alterations in weight gain, adipocyte size, adipokine levels, or liver fat content when compared to WT counterparts. However, Klf9−/− mice of both sexes had increased liver weight/size (hepatomegaly). This was accompanied by increased hepatic oxidative stress as indicated by decreased GSH/GSSG ratio and increased homocysteine, 3-nitrotyrosine, 3-chlorotyrosine, and 4HNE content. Decreased GSH to GSSG ratio and a trend toward increased homocysteine levels were observed in the corresponding Klf9−/− mouse serum. Gene expression analysis showed a heightened pro-inflammatory state in livers from Klf9−/− mice. KLF9 suppresses hepatic oxidative stress and inflammation, thus identifying potential mechanisms for KLF9 suppression of HCC and perhaps cancers of other tissues.
... Some of the differentially expressed tfs including KLF9, FOXO3, NFAT3, and MSX1 (Table 7) and were shown to be transcriptional regulators of their respective promoters. KLF9 represses Esr1 [28] and is upregulated during diapause. FOXO3 has been shown to associate with ESR1 and ESR2 proteins and inhibit ER-dependent transcriptional activation [29]. ...
Embryonic diapause is an enigmatic phenomenon that appears in diverse species. Although regulatory mechanisms have been established, there is much to be discovered. Herein, we have made the first comprehensive attempt to elucidate diapause regulatory mechanisms using a computational approach. We found transcription factors unique to promoters of genes in diapause species. From pathway analysis and STRING PPI networks, the signalling pathways regulated by these unique transcription factors were identified. The pathways were then consolidated into a model to combine various known mechanisms of diapause regulation. This work also highlighted certain transcription factors that may act as 'master transcription factors' to regulate the phenomenon. Promoter analysis further suggested evidence for independent evolution for some of regulatory elements involved in diapause.
... The unopposed activity of uterine ESR-1 speed up the development and progression of endometrial cancers in humans and animal models (Di Cristofano and Ellenson 2007). The intricate ESR-1's regulation which occurs at many levels is predictive of the fact that perturbations in any or all of these mechanisms will result in reproductive dysfunctions and diseases (Simmons et al. 2010). ...
The present study is the first attempt made to investigate the effects of diabetes on expression and promoter DNA methylation of TGF-b1, ESR-1, and CDH-1 genes and also the effects of folic acid (FA) and vitamin E (Vit E) supplementations on improving diabetes mellitus. STZ-induced diabetic rats were treated with Vit E (200mg/kg/day) and FA (25mg/kg/day) for 8weeks and expression and DNA methylation of TGF-b1, ESR-1, and CDH-1 genes in uterus were analysed. Data indicated that diabetes increases the expression of TGFb-1 and ESR-1 and decreases CDH-1 expression and TGFb-1 promoter methylation in the uterus of rats. Vit E and FA improved the negative effects of diabetes by decreasing the expression of TGFb-1 and ESR-1 and increasing that of CDH-1 in diabetic rats. In conclusion, these findings emphasise that Vit E and FA supplementations could improve negative effects caused by diabetes on uterus function and fertility in diabetic rats.
... The unopposed activity of uterine ESR-1 speed up the development and progression of endometrial cancers in humans and animal models (Di Cristofano and Ellenson 2007). The intricate ESR-1's regulation which occurs at many levels is predictive of the fact that perturbations in any or all of these mechanisms will result in reproductive dysfunctions and diseases (Simmons et al. 2010). ...
2020): Effect of folic acid and vitamin E on promoter DNA methylation and expression of TGF-β1, ESR-1 and CDH-1 in the uterus of STZ-induced diabetic rats, Archives of Physiology and Biochemistry To link to this article: https://doi. ABSTRACT The present study is the first attempt made to investigate the effects of diabetes on expression and promoter DNA methylation of TGF-b1, ESR-1, and CDH-1 genes and also the effects of folic acid (FA) and vitamin E (Vit E) supplementations on improving diabetes mellitus. STZ-induced diabetic rats were treated with Vit E (200 mg/kg/day) and FA (25 mg/kg/day) for 8 weeks and expression and DNA methy-lation of TGF-b1, ESR-1, and CDH-1 genes in uterus were analysed. Data indicated that diabetes increases the expression of TGFb-1 and ESR-1 and decreases CDH-1 expression and TGFb-1 promoter methylation in the uterus of rats. Vit E and FA improved the negative effects of diabetes by decreasing the expression of TGFb-1 and ESR-1 and increasing that of CDH-1 in diabetic rats. In conclusion, these findings emphasise that Vit E and FA supplementations could improve negative effects caused by diabetes on uterus function and fertility in diabetic rats. ARTICLE HISTORY
... Evidence for the involvement of KLF9 in cancer was provided by previous studies. For example, the expression of KLF9 was shown to be inversely correlated with endometrial tumor grade [28], and its silencing was further proposed to contribute to the etiology of endometrial cancer initiated by aberrant ESR1 signaling [29]. Additional evidence supporting a role for KLF9 as a tumor suppressor from another report indicated that KLF9 inhibits the growth of hepatocellular carcinoma cells by modulating p53 [30]. ...
Growing evidence suggests that circular RNAs (circRNAs) play pivotal roles in cancer progression. In this study, bioinformatic analysis identified a dysregulated circRNA termed circPTPRA in bladder cancer (BC). By using qRT-PCR analysis, we verified that circPTPRA is down-regulated in clinical BC specimens compared with the matched non-tumor samples, while correlation analyses showed that low circPTPRA expression is associated with poor prognosis, advanced tumor stage and larger tumor size. Based on these findings, we conducted functional assays and revealed that circPTPRA inhibits BC cell proliferation in vitro and tumor growth in vivo. In addition, RNA pull-down, miRNA capture, FISH, and luciferase reporter assays demonstrated that circPTPRA can directly sponge miR-636. Cell transfection experiments showed that miR-636 promotes the proliferation of BC cells by decreasing the expression of Krüppel Like Factor 9 (KLF9) upon binding to the 3’UTR of its mRNA.
Further analysis confirmed that circPTPRA competitively sponges miR-636 to upregulate the KLF9 expression, leading to decreased proliferation of BC cells. Our investigation indicates that circPTPRA acts as a tumor suppressor in BC, and suggests that this circRNA may be a novel prognostic biomarker and therapeutic target in BC.
... In the inner ear, estrogen acts to respond to noise-induced damage, and loss of ERβ causes inner ear defects and hearing loss [79][80][81]. Klf9 opposes estrogen hormonal action by repressing estrogen receptor (ERα) expression and activity [82][83][84]. ERα is also known to down-regulate miR-140 in breast cancer stem cells suggesting miR-140 is downstream of estrogen action [83]. The combination of these findings may indicate that the function of mir-140 and Klf9 is important for estrogen action in the inner ear. ...
Both nuclear receptor subfamily 2 group F member 1 (NR2F1) and microRNAs (miRNAs) have been shown to play critical roles in the developing and functional inner ear. Based on previous studies suggesting interplay between NR2F1 and miRNAs, we investigated the coregulation between NR2F1 and miRNAs to better understand the regulatory mechanisms of inner ear development and functional maturation.
Using a bioinformatic approach, we identified 11 potential miRNAs that might coregulate target genes with NR2F1 and analyzed their targets and potential roles in physiology and disease. We selected 6 miRNAs to analyze using quantitative real-time (qRT) -PCR and found that miR-140 is significantly down-regulated by 4.5-fold (P=0.004) in the inner ear of NR2F1 knockout (Nr2f1(-/-) ) mice compared to wild-type littermates but is unchanged in the brain. Based on this, we performed chromatin-immunoprecipitation followed by qRT-PCR and confirmed that NR2F1 directly binds and regulates both miR-140 and Klf9 in vivo. Furthermore, we performed luciferase reporter assay and showed that miR-140 mimic directly regulates KLF9-3'UTR, thereby establishing and validating an example coregulatory network involving NR2F1, miR-140, and Klf9.
We have described and experimentally validated a novel tissue-dependent coregulatory network for NR2F1, miR-140, and Klf9 in the inner ear and we propose the existence of many such coregulatory networks important for both inner ear development and function.
... Relative expression data were calculated using the DDCt method of Livak and Schmittgen [38], and normalized to beta-actin (Actb) and matrix metallopeptidase 9 (Mmp9) expression using GeNorm [39]. Actb b-actin [68] F: 5 0 -GACAGGATGCAGAAGGAGATTACT R: 5 0 -TGATCCACATCTGCTGGAAGGT Mmp9 matrix metallopeptidase 9 [69] F: 5 0 -CGAACTTCGACACTGACAAGAAGT R: 5 0 -GCACGCTGGAATGATCTAAGC B2m b-2-microglobulin ENSMUS-T00000102476 F: 5 0 -CCTGGTCTTTCTGGTGCTTG R: 5 0 -TATGTTCGGCTTCCCATTCT Dnmt1 DNA methyltransferase(cytosine-5) 1 NM_010066 F: 5 0 -CGGCTCAAAGACTTGGAAAG R: 5 0 -TAGCCAGGTAGCCTTCCTCA Fgf2 fibroblast growth factor 2 NM_008006 F: 5 0 -AGCGGCTCTACTGCAAGAAC R: 5 0 -GCCGTCCATCTTCCTTCATA Gapdh Glyceraldehyde-3-phosphate dehydrogenase [70] F: 5 0 -TGTGTCCGTCGTGGATCTGA R: 5 0 -GCATCGAAGGTGGAAGAGTGG Gusb glucuronidase beta ENSMUS-G00000025534 F: 5 0 -CCAGCCACTATCCCTACTCA R: 5 0 -GCCACAGACCACATCACAAC Pten phosphate and tensin homolog ENSMUS-G00000013663 F: 5 0 -TTGAAGACCATAACCCACCA R: 5 0 -TACACCAGTCCGTCCCTTTC Vegfa vascular endothelial growth factor A [71] F: 5 0 -GCACTGGACCCTGGCTTTACT R: 5 0 -ACTTGATCACTTCATGGGACTTCTG Primers used for RT-PCR are listed along with gene IDs and names. Accession numbers from which these primers were designed are provided; publications are cited when applicable. ...
Abstract Multiple sclerosis (MS) is a complex disease influenced by genetic and environmental contributing factors. Endocrine disrupting compounds (EDCs) such as bisphenol A (BPA) affect gene expression and hormone-regulated systems throughout the body. We investigated the effects of BPA on Theiler's-virus induced demyelination (TVID), a mouse model of MS. Perinatal BPA exposure, combined with viral infection, resulted in a decreased level of viral antibodies, accelerated the onset of TVID symptoms, increased inflammation in both the spinal cord and digestive tract, and amplified immune-related gene expression changes induced by viral infection. These results demonstrate the effect of BPA on the trajectory of TVID, and illustrate how multiple factors collectively influence autoimmune disease.
... Growing evidence shows that KLF9 (Krüppel-like factor 9) also is a novel gene in the development of uterine endometrial cancer. In a study, Simmons et al. [7] identified that KLF9 mutant mice develop uterine endometrial cancer faster than normal mice. It has been shown that KLF9, as a basic transcription element-binding protein 1 (BTE-B1), regulates the transactivation of progesterone-responsive promoters and has been found to be down regulated in endometrial carcinoma and colorectal cancer [7]. ...
... In a study, Simmons et al. [7] identified that KLF9 mutant mice develop uterine endometrial cancer faster than normal mice. It has been shown that KLF9, as a basic transcription element-binding protein 1 (BTE-B1), regulates the transactivation of progesterone-responsive promoters and has been found to be down regulated in endometrial carcinoma and colorectal cancer [7]. KLF9 protein is a transcriptional regulator of uterine endometrial cell proliferation, adhesion, and differentiation processes essential for pregnancy success and which are subverted during tumorigenesis [8,9]. ...
... The network of endometrial genes controlled by KLF9 protein is largely unknown. Simmon et al. showed that over-expression of KLF9 in the human endometrial cancer cell line HEC-1-A alters cell morphology, proliferative indices, and differentiation when compared to KLF9 under-expressing HEC-1-A cells [7]. Simmons et al. found that the loss of expression of transcription factor KLF9 in human endometrial cells leads to aberrant growth regulation and underlies the progression of endometrial carcinoma [9]. ...
The current study was designed to explore the changes of the mRNA levels of the YT521, Forkhead box protein O1 (FOXO1), and Krüppel-like factor 9 (KLF9) proteins in human normal and cancerous endometrial tissue.
The study was conducted in 30 premenopausal patients diagnosed with endometrial cancer and 20 premenopausal women with no clinically documented abnormalities of the endometrium undergoing hysterectomy. Gene expression levels were assayed using quantitative real-time PCR.
The endometrial tissue FOXO1 mRNA level (0.82 +/- 0.27) of patients with endometrial cancer was significantly lower (p < 0.001) than controls (4.51 +/- 2.68). In subjects with endometrial cancer the KLF9 mRNA level (1.12 +/- 0.38) was lower (p < 0.001) when compared to controls (3.11 +/- 1.52). A remarkable (not significant, p = 0.069) increase was found in the YT521 mRNA level of patients' endometrial tissue (11.19 +/- 3.99) in comparison with the control subjects (8.82 +/- 5.01). No significant difference was detected for the FOXO1, KLF9 and YT521 mRNA levels of the endometrial tissue of patients with cancer at different stages.
It is suggested that the alteration of the gene expression profiles of FOXO1, KLF9 and YT521, which occur in human endometrial cancers likely play a crucial role in initiation of cancer.
... The extent to which KLF9 plays a role in uterine function was previously demonstrated from our studies of female Klf9 null mice. We showed that Klf9 null mutation resulted in smaller litters due to reduced numbers of implanted embryos, a shift in the normal window of embryo implantation, partial uterine P 4 resistance, and enhanced uterine estrogen sensitivity [20, 22, 23]. Klf9 null mutation also dramatically influenced LE proliferative, apoptotic, and PGR expression status [22, 24], consistent with LE as a target of stromal KLF9 through paracrine signaling. ...
... Whole uteri from early pregnant (DPC 3.5) and virgin (PND 56) WT and Klf13 (À/À) mice were fixed in 10% neutral buffered formalin as previously described [20] . Sections (thickness, 5 mm) were mounted on poly-L-lysinecoated slides (Fisher Scientific) and processed for hematoxylin/eosin staining following standard protocols [23, 24]. LE height was quantified using the Axiovert 200M microscope equipped with a Axiocam HRc camera and Axiovision software (Carl Zeiss, Inc.). ...
... Specifically, both Klf9 [22] and Klf13 (present study) null females exhibit normal steroid hormone production, as evidenced by their ability to normally cycle and achieve pregnancy. Although our results are not supported by the recent report that KLF9 and KLF13 are transcriptional activators of the steroidogenic genes LDLR, StAR, and CYP11A in ovarian granulosa cells [44], suggesting that loss of their respective expression should lead to reduced steroidogenesis, it is possible that the KLF9- regulated gene KLF4 [23] may take over the roles of KLF9 and KLF13 in a compensatory manner, as demonstrated for members of this family [25, 45] (present study). Alternatively, KLF9 and KLF13 may compensate for each other's function in the context of the ovary. ...
The ovarian hormones estrogen and progesterone promote uterine receptivity and successful pregnancy through their cognate receptors functioning in concert with context-dependent nuclear co-regulators. Previously, we showed that transcription factor Krüppel-like factor 9 (KLF9) is a progesterone receptor (PGR) co-activator in the uterus and mice null for Klf9 exhibit subfertility and reduced progesterone sensitivity. The highly-related family member KLF13 displays increased expression in uteri of pregnant and non-pregnant Klf9 null mice and similarly regulates PGR-mediated transactivation in endometrial stromal cells. However, a uterine phenotype with loss of Klf13 has not been reported. Herein, we demonstrate that Klf13 deficiency in mice did not compromise female fertility and pregnancy outcome. Klf13 null females had comparable litter sizes, numbers of implanting embryos, uterine morphology, and ovarian steroid hormone production to wildtype (WT) counterparts. Further, pregnant WT and Klf13 null females at day post-coitum (dpc) 3.5 had similar uterine Pgr, estrogen receptor, and Wnt-signaling component transcript levels. Nuclear levels of KLF9 were higher in dpc 3.5 Klf13 null than in WT uteri, albeit whole tissue KLF9 protein and transcript levels did not differ between genotypes. The lack of a similar induction of nuclear KLF9 levels in uteri of virgin Klf13((-/-)) mice relative to WT uteri was associated with lower stromal PGR expression. In differentiating human endometrial stromal cells, coincident KLF9/KLF13 knockdowns by siRNA targeting reduced decidualization-associated PRL expression while KLF9 and KLF13 knockdowns alone, respectively reduced WNT4 and BMP2 transcript levels. Results suggest that KLF9 and KLF13 functionally compensate in peri-implantation uterus for pregnancy success.
... Our laboratory has identified the transcription factor Krüppel-like factor (KLF) 9, an Sp-family member [9], as a transcriptional coregulator of ESR1 and PGR-B signaling in uterine cells [10][11][12][13][14][15]. KLF9 is expressed in uterine endometrial stroma and glandular, but not luminal, epithelium in mice [16], a finding recently confirmed for human endometrial cells [17]. ...
... KLF9 is expressed in uterine endometrial stroma and glandular, but not luminal, epithelium in mice [16], a finding recently confirmed for human endometrial cells [17]. Using mice null for KLF9 [16], we demonstrated the numerous contributions of KLF9 to uterine morphology; embryo implantation, and fertility; sensitivity to E 2 and P 4 ; and timing of parturition [12,14,15,[18][19][20]. These findings have raised the possibility of yet unidentified roles for KLF9 in the etiology of gynecological diseases such as endometriosis and EC, which are characterized by unopposed E 2 signaling coincident with increased P 4 resistance. ...
... To determine whether attenuated KLF9 expression in EC is associated with loss of growth control, the expression of a subset of growth-regulatory genes was evaluated in NE and EC by QPCR. We previously reported the aberrant expression of these genes in Klf 9-null mice with deregulated ESR1 signaling due to early postnatal exposure to diethylstilbestrol (DES) [15]. Loss of KLF9 expression in EC was accompanied by decreased expression of FBJ murine osteosarcoma viral oncogene homolog (FOS; P ¼ 0.03), a modest down-regulation of myelocytomatosis viral oncogene homolog (MYC; P ¼ 0.08), and a dramatic increase in telomerase reverse transcriptase (TERT; by 20-fold; P ¼ 0.02) transcript levels ( Fig. 2A). ...
Endometrial cancer is the most commonly diagnosed female genital tract malignancy. Krüppel-like factor 9 (KLF9), a member of the evolutionarily conserved Sp family of transcription factors, is expressed in uterine stroma and glandular epithelium, where it affects cellular proliferation, differentiation, and apoptosis. Deregulated expression of a number of Sp proteins has been associated with multiple types of human tumors, but a role for KLF9 in endometrial cancer development and/or progression is unknown. Here, we evaluated KLF9 expression in endometrial tumors and adjacent uninvolved endometrium of women with endometrial carcinoma. KLF9 mRNA and protein levels were lower in endometrial tumors coincident with decreased expression of family member KLF4 and growth-regulators FBJ murine osteosarcoma viral oncogene homolog (FOS) and myelocytomatosis viral oncogene homolog (MYC) and with increased expression of telomerase reverse transcriptase (TERT) and the chromatin-modifying enzymes DNA methyltransferase 1 (DNMT1) and histone deacetylase 3 (HDAC3). Expression of estrogen receptor alpha (ESR1) and the tumor-suppressor phosphatase and tensin homolog deleted in chromosome 10 (PTEN) did not differ between tumor and normal tissue. The functional relevance of attenuated KLF9 expression in endometrial carcinogenesis was further evaluated in the human endometrial carcinoma cell line Ishikawa by siRNA targeting. KLF9 depletion resulted in loss of normal cellular response to the proliferative effects of estrogen concomitant with reductions in KLF4 and MYC and with enhancement of TERT and ESR1 gene expression. Silencing of KLF4 did not mimic the effects of silencing KLF9 in Ishikawa cells. We suggest that KLF9 loss-of-expression accompanying endometrial carcinogenesis may predispose endometrial epithelial cells to mechanisms of escape from estrogen-mediated growth regulation, leading to progression of established neoplasms.
