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Metformin (MET) is increasingly implicated in reducing the incidence of multiple cancer types in patients with diabetes. However, similar effects of MET in non-diabetic women with endometrial cancer (EC) remain unknown. In a pilot study, obese non-diabetic women diagnosed with type 1, grade 1/2 EC, and consenting to participate were randomly assigned to receive MET or no MET (control (CON)) during the pre-surgical window between diagnosis and hysterectomy. Endometrial tumors obtained at surgery (MET, n = 4; CON, n = 4) were analyzed for proliferation (Ki67), apoptosis (TUNEL), and nuclear expression of ERα, PGR, PTEN, and KLF9 proteins in tumor glandular epithelial (GE) and stromal (ST) cells. The percentages of immunopositive cells for PGR and for KLF9 in GE and for PTEN in ST were higher while those for ERα in GE but not ST were lower, in tumors of MET vs. CON patients. The numbers of Ki67- and TUNEL-positive cells in tumor GE and ST did not differ between groups. In human Ishikawa endometrial cancer cells, MET treatment (60 μM) decreased cell numbers and elicited distinct temporal changes in ESR1, KLF9, PGR, PGR-B, KLF4, DKK1, and other tumor biomarker mRNA levels. In the context of reduced KLF9 expression (by siRNA targeting), MET rapidly amplified PGR, PGR-B, and KLF4 transcript levels. Our findings suggest that MET acts directly in EC cells to modify steroid receptor expression and signaling network and may constitute a preventative strategy against EC in high-risk non-diabetic women.

Despite the well-accepted notion that early maternal influences persist beyond fetal life and may underlie many adult diseases, the risks imposed by the maternal environment on breast cancer development and underlying biological mechanisms remain poorly understood. In this study, we investigated whether early exposure to blueberry (BB) via maternal diet alters oncogene Wnt1-induced mammary tumorigenesis in offspring. Wnt1-transgenic female mice were exposed to maternal Casein (CAS, control) or blueberry-supplemented (CAS + 3%BB) diets throughout pregnancy and lactation. Offspring were weaned to CAS and mammary tumor development was followed until age 8 months. Tumor incidence and latency were similar for both groups; however, tumor weight at killing and tumor volume within 2 weeks of initial detection were lower (by 50 and 60%, respectively) in offspring of BB- versus control-fed dams. Dietary BB exposure beginning at weaning did not alter mammary tumor parameters. Tumors from maternal BB-exposed offspring showed higher tumor suppressor (Pten and Cdh1) and lower proproliferative (Ccnd1), anti-apoptotic (Bcl2) and proangiogenic (Figf, Flt1 and Ephb4) transcript levels, and displayed attenuated microvessel density. Expression of Pten and Cdh1 genes was also higher in mammary tissues of maternal BB-exposed offspring. Mammary tissues and tumors of maternal BB-exposed offspring showed increased chromatin-modifying enzyme Dnmt1 and Ezh2 transcript levels. Body weight, serum insulin and serum leptin/adiponectin ratio were lower for maternal BB-exposed than control tumor-bearing offspring. Tumor weights and serum insulin were positively correlated. Results suggest that dietary influences on the maternal environment contribute to key developmental programs in the mammary gland to modify breast cancer outcome in adult progeny.

Breast cancer is the leading cause of cancer deaths in women. Diet and lifestyle are major contributing factors to increased breast cancer risk. While mechanisms underlying dietary protection of mammary tumor formation are increasingly elucidated, there remains a dearth of knowledge on the nature and precise actions of specific bioactive components present in foods with purported health effects. The 43-amino acid peptide lunasin (LUN) is found in soybeans, is bioavailable similar to the isoflavone genistein (GEN), and thus may mediate the beneficial effects of soy food consumption. Here, we evaluated whether LUN displays common and distinct actions from those of GEN in non-malignant (mouse HC11) and malignant (human MCF-7) mammary epithelial cells. In MCF-7 cells, LUN up-regulated tumor suppressor phosphatase and tensin homolog deleted in chromosome ten (PTEN) promoter activity, increased PTEN transcript and protein levels and enhanced nuclear PTEN localization, similar to that shown for GEN in mammary epithelial cells. LUN-induced cellular apoptosis, akin to GEN, was mediated by PTEN, but unlike that for GEN, was p53-independent. LUN promoted E-cadherin and β-catenin non-nuclear localization similar to GEN, but unlike GEN, did not influence the proliferative effects of oncogene Wnt1 on HC11 cells. Further, LUN did not recapitulate GEN inhibitory effects on expansion of the cancer stem-like/progenitor population in MCF-7 cells. Results suggest the concerted actions of GEN and LUN on cellular apoptosis for potential mammary tumor preventive effects and highlight whole food consumption rather than intake of specific dietary supplements with limited biological effects for greater health benefits.

Endometriosis is characterized by progesterone resistance and associated with infertility. Krüppel-like factor 9 (KLF9) is a progesterone receptor (PGR)-interacting protein, and mice null for Klf9 are subfertile. Whether loss of KLF9 expression contributes to progesterone resistance of eutopic endometrium of women with endometriosis is unknown.The aims were to investigate 1) KLF9 expression in eutopic endometrium of women with and without endometriosis, 2) effects of attenuated KLF9 expression on WNT-signaling component expression and on WNT inhibitor Dickkopf-1 promoter activity in human endometrial stromal cells (HESC), and 3) PGR and KLF9 coregulation of the stromal transcriptome network.Transcript levels of KLF9, PGR, and WNT signaling components were measured in eutopic endometrium of women with and without endometriosis. Transcript and protein levels of WNT signaling components in HESC transfected with KLF9 and/or PGR small interfering RNA were analyzed by quantitative RT-PCR and Western blot. KLF9 and PGR coregulation of Dickkopf-1 promoter activity was evaluated using human Dickkopf-1-luciferase promoter/reporter constructs and by chromatin immunoprecipitation. KLF9 and PGR signaling networks were analyzed by gene expression array profiling.Eutopic endometrium from women with endometriosis had reduced expression of KLF9 mRNA together with those of PGR-B, WNT4, WNT2, and DKK1. KLF9 and PGR were recruited to the DKK1 promoter and modified each other's transactivity. In HESC, KLF9 and PGR coregulated components of the WNT, cytokine, and IGF gene networks that are implicated in endometriosis and infertility.Loss of KLF9 coregulation of endometrial stromal PGR-responsive gene networks may underlie progesterone resistance in endometriosis.

Endometrial cancer is the most commonly diagnosed female genital tract malignancy. Kruppel-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 methyl- transferase 1 (DNMT1) and histone deacetylase 3 (HDAC3). Expression of estrogen receptor alpha (ESR1) and the tumor- suppressor phosphatase and tensin homolog deleted in chromo- some 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 progres- sion of established neoplasms.

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 had shown previously 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 that 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 (PNDs) 1–5. Uterine tissues collected at PND84 were subjected to histological, immunological, and molecular analyses. Compared with WT mice, KO mice demonstrated larger endometrial glands and lower endometrial gland numbers; DES exposure exacerbated these differences. Loss of KLF9 expression resulted in increased glandular ESR1 immunoreactivity with DES, without effects on serum estradiol levels. Quantitative RT-PCR analyses indicated altered expression of uterine genes commonly dysregulated in endometrial cancers (Akt1, Mmp9, Slpi, and Tgfβ1) and of those involved in growth regulation (Fos, Myc, Tert, and 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 dysfunctions initiated by aberrant estrogen action.

Estrogen, acting through its cognate receptor estrogen receptor-α (ESR1), is a critical regulator of uterine endometrial epithelial proliferation. Although the dynamic communication between endometrial stromal (ST) and epithelial cells is considered to be an important component in this process, key molecular players in particular compartments remain poorly defined. Here, we used mice null for Krüppel-like factor 9 (KLF9) to evaluate the contribution of this nuclear protein in ST–epithelial interactions underlying proliferative effects of estrogen. We found that in ovariectomized mice administered estradiol-17β (E2) for 24 h,Klf9null mutation resulted in lack of E2-induced proliferative response in all endometrial compartments. We demonstrated a negative association betweenKlf9expression and nuclear levels of ESR1 transcriptional corepressor prohibitin (PHB) 2 in uterine ST and epithelial cells of E2-treated wild-type (WT) andKlf9null mice. In early pregnancy uteri of WT mice, the temporal pattern ofKlf9transcript levels was inversely associated with that ofPhb2. Deletion ofKlf9up-regulated uterinePhb2expression and increased PHB2 nuclear localization in endometrial ST and epithelial cells, with no effects on the expression of the relatedPhb1. In the human endometrial ST cell line treated with E2for 24 h,Klf9siRNA targeting augmented PHB2 transcript and increased nuclear PHB2 protein levels, albeit this effect was not to the extent seenin vivowithKlf9null mutants. Our findings suggest a novel mechanism for control of estrogen-induced luminal epithelial proliferation involving ST KLF9 regulation of paracrine factor(s) to repress epithelial expression of corepressor PHB2.

Female reproductive tract pathologies arise largely from dysregulation of estrogen and progesterone receptor signaling, leading to aberrant cell proliferation, survival, and differentiation. The signaling pathways orchestrated by these nuclear receptors are complex, require the participation of many nuclear proteins serving as key binding partners or targets, and involve a range of paracrine and autocrine regulatory circuits. The members of the Krüppel-like factor (KLF) family of transcription factors are ubiquitously expressed in reproductive tissues and have been increasingly implicated as critical co-regulators and integrators of steroid hormone actions. Herein, we explore the involvement of KLF family members in uterine pathology, describe their currently known molecular mechanisms, and discuss their potential as targets for therapeutic intervention.

The small intestine participates in lipid digestion, metabolism and transport. Cytosolic malic enzyme 1 (ME1) is an enzyme that generates NADPH used in fatty acid and cholesterol biosynthesis. Previous work has correlated liver and adipose ME1 expression with susceptibility to obesity and diabetes; however, the contributions of intestine-expressed ME1 to these conditions are unknown. We generated transgenic (Tg) mice expressing rat ME1 in the gastrointestinal epithelium under the control of the murine villin1 promoter/enhancer. Levels of intestinal ME1 protein (endogenous plus transgene) were greater in Tg than wildtype (WT) littermates. Effects of elevated intestinal ME1 on body weight, circulating insulin, select adipocytokines, blood glucose, and metabolism-related genes were examined. Male Tg mice fed a high-fat (HF) diet gained significantly more body weight than WT male littermates and had heavier livers. ME1-Tg mice had deeper intestinal and colon crypts, a greater intestinal 5-bromodeoxyuridine labeling index, and increased expression of intestinal lipogenic (Fasn, Srebf1) and cholesterol biosynthetic (Hmgcsr, Hmgcs1), genes. The livers from HF diet-fed Tg mice also exhibited an induction of cholesterol and lipogenic pathway genes and altered measures (Irs1, Irs2, Prkce) of insulin sensitivity. Results indicate that gastrointestinal ME1 via its influence on intestinal epithelial proliferation, and lipogenic and cholesterologenic genes may concomitantly impact signaling in liver to modify this tissue's metabolic state. Our work highlights a new mouse model to address the role of intestine-expressed ME1 in whole body metabolism, hepatomegaly, and crypt cell proliferation. Intestinal ME1 may thus constitute a therapeutic target to reduce obesity-associated pathologies.

Late-term pregnancy may lead to maternal and neonatal morbidity and mortality. Mice null for the progesterone receptor co-regulator Krüppel-like Factor 9 (KLF9) exhibit delayed parturition and increased incidence of neonatal deaths.Our aim is to evaluate the contribution of myometrial KLF9 to human parturition.Myometrial biopsies were obtained from women with term (37 to ≤41 wk) and late-term (41 wk) pregnancies during cesarean delivery and assessed for gene and protein expression. Human myometrial cells transfected with nontargeting or KLF9 small interfering RNAs (siRNA) were treated with the progesterone antagonist RU486 and analyzed for pro-inflammatory chemokine/cytokine gene expression.The study took place in a University-affiliated tertiary care hospital and University research laboratory.Term patients (n = 8) were in spontaneous active labor whereas late-term patients (n = 5) were either in or were induced to active labor, prior to elective cesarean delivery.Steroid hormone receptor, contractility, and inflammation-associated gene expression in myometrial biopsies and in siKLF9-transfected, RU486-treated human myometrial cells was associated with KLF9 expression levels.Myometrium from women with late-term pregnancy showed lower KLF9, total PGR, and PGR-A/PGR-B isoform expression. Transcript levels of select chemokines/cytokines were up- (CSF3, IL1, IL12A, TGFB2) and down- (CCL3, CCL5, CXCL1, CXCL5, IL15) regulated in late-term relative to term myometrium. Knock-down of KLF9 expression in RU486-treated human myometrial cells modified the expression of PGR and labor-associated cytokines, relative to control siRNA-treated cells.Myometrial KLF9 may contribute to the onset of human parturition through its regulation of PGR expression and inflammatory signaling networks.

The ovarian hormones estrogen and progesterone promote uterine receptivity and successful pregnancy through their cognate receptors functioning in concert with context-dependent nuclear coregulators. Previously, we showed that the transcription factor Kruppel-like factor (KLF) 9 is a progesterone receptor (PGR) coactivator in the uterus and that mice null for Klf9 exhibit subfertility and reduced progesterone sensitivity. The highly related family member KLF13 displays increased expression in uteri of pregnant and nonpregnant 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. In the present study, we demonstrate that Klf13 deficiency in mice did not compromise female fertility and pregnancy outcome. Klf13 null females had litter sizes, numbers of implanting embryos, uterine morphology, and ovarian steroid hormone production comparable to those of wild-type (WT) counterparts. Further, pregnant WT and Klf13 null females at Day Postcoitum (DPC) 3.5 had similar uterine Pgr, estrogen receptor, and Wnt-signaling component transcript levels. Nuclear levels of KLF9 were higher in Klf13 null than in WT uteri at DPC 3.5, 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 knockdown by small interfering RNA targeting reduced decidualization-associated PRL expression, whereas KLF9 and KLF13 knockdowns alone reduced transcript levels of WNT4 and BMP2, respectively. Results suggest that KLF9 and KLF13 functionally compensate in peri-implantation uterus for pregnancy success.

Background Obesity and associated hormonal disturbances are risk factors for colon cancer. Cytosolic Malic Enzyme (ME1) generates NADPH used for lipogenesis in gastrointestinal (GI), liver and adipose tissues. We have reported that inclusion of soy protein isolate (SPI) in the diet lowered body fat content and colon tumor incidence of rats fed AIN-93G diet, while others have demonstrated SPI inhibition of rat hepatic ME1 expression. The present study examined the individual and combined effects of dietary SPI and absence of ME1 on: 1) serum concentrations of hormones implicated in colon cancer development, 2) expression of lipogenic and proliferation-associated genes in the mouse colon and small intestine, and 3) liver and adipose expression of lipogenic and adipocytokine genes that may contribute to colon cancer predisposition. Methods Weanling wild type (WT) and ME1 null (MOD-1) male mice were fed high-fat (HF), iso-caloric diets containing either casein (CAS) or SPI as sole protein source for 5 wks. Somatic growth, serum hormone and glucose levels, liver and adipose tissue weights, GI tissue parameters, and gene expression were evaluated. Results The MOD-1 genotype and SPI-HF diet resulted in decreases in: body and retroperitoneal fat weights, serum insulin, serum leptin, leptin/adiponectin ratio, adipocyte size, colon mTOR and cyclin D1 mRNA abundance, and jejunum FASN mRNA abundance, when compared to WT mice fed CAS-HF. Regardless of diet, MOD-1 mice had reductions in liver weight, liver steatosis, and colon crypt depth, and increases in adipose tissue expression of IRS1 and IRS2, compared to WT mice. SPI-HF diet reduced ME1 gene expression only in retroperitoneal fat. Conclusions Data suggest that the pharmacological targeting of ME1 or the inclusion of soy protein in the diet may provide avenues to reduce obesity and its associated pro-tumorigenic endocrine environment and improve insulin sensitivity, potentially disrupting the obesity-colon cancer connection.

The inability of the uterine epithelium to enter a state of receptivity for the embryo to implant is a significant underlying cause of early pregnancy loss. We previously showed that mice null for the progesterone receptor (PGR)-interacting protein Krüppel-like factor (KLF) 9 are subfertile and exhibit reduced uterine progesterone sensitivity. KLF9 expression is high in predecidual stroma, undetectable in decidua, and enhanced in uteri of mice with conditional ablation of bone morphogenetic protein 2 (BMP2). Given the individual importance of KLF9 and BMP2 for implantation success, we hypothesized that the establishment of uterine receptivity involves KLF9 and BMP2 functional cross-regulation. To address this, we used early pregnant wild-type and Klf9 null mice and KLF9 small interfering RNA-transfected human endometrial stromal cells (HESCs) induced to differentiate under standard conditions. Loss of KLF9 in mice and HESCs enhanced BMP2 expression, whereas recombinant BMP2 treatment of HESCs attenuated KLF9 mRNA levels. IGFBP1 and KLF9-related KLF13 expression were positively associated with BMP2 and inversely associated with KLF9. Prolonged, but not short-term, knockdown of KLF9 in HESCs reduced IGFBP1 expression. Mouse uterine Igfbp1 expression was similarly reduced with Klf9 ablation. PGR-A and PGR-B expression were positively associated with KLF9 in predecidual HESCs but not decidualizing HESCs. KLF13 knockdown attenuated BMP2 and PGR-B and abrogated BMP2-mediated inhibition of KLF9 expression. Results support cross-regulation among BMP2, KLF9, and KLF13 to maintain progesterone sensitivity in stromal cells undergoing differentiation and suggest that loss of this regulatory network compromises establishment of uterine receptivity and implantation success.

Krüppel-like factors (KLFs), of which there are currently 17 known protein members, belong to the specificity protein (Sp) family of transcription factors and are characterized by the presence of Cys2/His2 zinc finger motifs in their carboxy-terminal domains that confer preferential binding to GC/GT-rich sequences in gene promoter and enhancer regions. While previously regarded to simply function as silencers of Sp1 transactivity, many KLFs are now shown to be relevant to human cancers by their newly identified abilities to mediate crosstalk with signaling pathways involved in the control of cell proliferation, apoptosis, migration, and differentiation. Several KLFs act as tumor suppressors and/or oncogenes under distinct cellular contexts, underscoring their prognostic potential for cancer survival and outcome. Recent studies suggest that a number of KLFs can influence steroid hormone signaling through transcriptional networks involving steroid hormone receptors and members of the nuclear receptor family of transcription factors. Since inappropriate sensitivity or resistance to steroid hormone actions underlies endocrine-related malignancies, we consider the intriguing possibility that dysregulation of expression and/or activity of KLF members is linked to the pathogenesis of endometrial and breast cancers. In this review, we focus on recently described mechanisms of actions of several KLFs (KLF4, KLF5, KLF6, and KLF9) in cancers of the mammary gland and uterus. We suggest that understanding the mode of actions of KLFs and their functional networks may lead to the development of novel therapeutics to improve current prospects for cancer prevention and cure.

Diet is highly linked to breast cancer risk, yet little is known about its influence on mammary epithelial populations with distinct regenerative and hence, tumorigenic potential. To investigate this, we evaluated the relative frequency of lineage-negative CD29(hi)CD24(+), CD29(lo)CD24(+) and CD29(hi)Thy1(+)CD24(+) epithelial subpopulations in pre-neoplastic mammary tissue of adult virgin MMTV-Wnt1-transgenic mice fed either control (Casein) or soy-based diets. We found that mammary epithelial cells exposed to soy diet exhibited a lower percentage of CD29(hi)CD24(+)Lin(-) population, decreased ability to form mammospheres in culture, lower mammary outgrowth potential when transplanted into cleared fat pads, and reduced appearance of tumor-initiating CD29(hi)Thy1(+)CD24(+) cells, than in those of control diet-fed mice. Diet had no comparable influence on the percentage of the CD29(lo)CD24(+)Lin(-) population. Global gene expression profiling of the CD29(hi)CD24(+)subpopulation revealed markedly altered expression of genes important to inflammation, cytokine and chemokine signaling, and proliferation. Soy-fed relative to casein-fed mice showed lower mammary tumor incidence, shorter tumor latency, and reduced systemic levels of estradiol 17-β, progesterone and interleukin-6. Our results provide evidence for the functional impact of diet on specific epithelial subpopulations that may relate to breast cancer risk and suggest that diet-regulated cues can be further explored for breast cancer risk assessment and prevention.