Endometrial Cancer-Associated FGF18 Expression Is Reduced by Bazedoxifene in Human Endometrial Stromal Cells In Vitro and in Murine Endometrium

Clare A Flannery; Andrew G Fleming; Gina H Choe; Hanyia Naqvi; Margaret Zhang; Anu Sharma; Hugh S Taylor

doi:10.1210/en.2016-1233

. 2016 Jun 6;157(10):3699–3708. doi: 10.1210/en.2016-1233

Endometrial Cancer-Associated FGF18 Expression Is Reduced by Bazedoxifene in Human Endometrial Stromal Cells In Vitro and in Murine Endometrium

Clare A Flannery ¹, Andrew G Fleming ¹, Gina H Choe ¹, Hanyia Naqvi ¹, Margaret Zhang ¹, Anu Sharma ¹, Hugh S Taylor ^1,^✉

PMCID: PMC5045514 PMID: 27267714

Abstract

Endometrial cancer develops during exposure to estrogen unopposed by progesterone. Traditional formulations for menopausal hormone therapy include a progestin in women with a uterus. However, progestin exposure increases breast cancer risk in postmenopausal women. Alternatives to progestin include bazedoxifene (BZA), a selective estrogen receptor modulator, which prevents estrogen induced endometrial hyperplasia in clinical trials. Molecular mechanisms responsible for BZA's antiproliferative effect are not fully elucidated. We profiled endometrial adenocarcinoma, hyperplasia, and normal proliferative endometrium for differential expression in genes known to be regulated by estrogens or progesterone. Fibroblast growth factor (FGF)18, a paracrine growth factor promoting epithelial proliferation, was significantly increased in adenocarcinoma. Progesterone represses FGF18 by inducing heart and neural crest derivatives expressed transcript 2 (HAND2) in stromal cells. Notably, we confirmed lower HAND2 mRNA in adenocarcinoma, along with higher FGF tyrosine kinase receptor 2 and E74-like factor 5, collectively promoting FGF18 activity. We hypothesized BZA reduces epithelial proliferation by inhibiting FGF18 synthesis in stromal cells. To determine whether BZA regulates FGF18, we treated primary stromal cells with BZA or vehicle. In vitro, BZA reduced FGF18, but did not affect, HAND2. CD1 female mice received either BZA, conjugated estrogen (CE), or combined BZA/CE for 8 weeks. CE-treated mice had nearly 3-fold higher FGF18 expression. In contrast, BZA-treated mice, alone or with CE, had similar FGF18 as controls. Unexpectedly, BZA, alone or with CE, reduced HAND2 more than 80%, differing from progesterone regulation. Reduction of FGF18 is a potential mechanism by which BZA reduces endometrial proliferation and hyperplasia induced by estrogens. However, BZA works independently of HAND2, revealing a novel mechanism for progestin-free hormone therapy in postmenopausal women.

The incidence of endometrial cancer peaked in the 1980s in the United States, largely due to the use of unopposed estrogen therapy in postmenopausal women. The use of combined progestin and estrogens in hormone therapy prevented endometrial hyperplasia, but the progestin component was later associated with an increased risk of breast cancer (see references 7 –9 below). Given the beneficial effect of estrogens on bone preservation and menopausal symptoms, the combination of conjugated estrogens (CEs) and a selective estrogen receptor modulator is a refined way of protecting against estrogen-induced endometrial hyperplasia using a progestin-free therapy. Alternative hormone strategies are especially important now due to the increasing prevalence of obesity and diabetes, which are independent risk factors for the development of breast and endometrial cancer.

Bazedoxifene (BZA) is a tissue-selective estrogen receptor modulator, with agonist activity on the bone and antagonist activity in the breast and endometrium (1 –4). BZA in combination with CEs is approved for the treatment of vasomotor symptoms and prevention of osteoporosis in postmenopausal women (5, 6). BZA has no effect on mammographic breast density in clinical trials (7). In rodent models, BZA does not promote growth of mammary glands and inhibits breast cancer growth (8, 9). Consistently, BZA alone or in combination with estrogen, inhibits endometrial proliferation in clinical and preclinical studies (10 –13). However, the molecular mechanisms responsible for BZA's endometrial effect are not fully elucidated.

In normal endometrial physiology, estrogen stimulates glandular proliferation through several mechanisms. A paracrine mechanism of estrogen action in the uterus was first demonstrated through a cell-selective estrogen receptor α knockout rodent model. Using various combinations of transplanted uteri tissue from ERα knockout and wild-type mice, Cooke et al (14) showed estrogen action through ERα in stromal cells stimulates epithelial cell proliferation. Similarly, progesterone works through a paracrine mechanism to inhibit epithelial cell proliferation. Progesterone induces the nuclear transcription factor, heart and neural crest derivatives expressed transcript 2 (HAND2) in endometrial stromal cells, leading to a decrease in stromal cell production of fibroblast growth factor (FGF)18 (15). By decreasing FGF18 levels, progesterone removes the stimulus for epithelial proliferation (15). FGFs are well characterized, inducing cell proliferation upon binding to FGF tyrosine kinase receptors (FGFRs) and initiating a phosphorylation cascade (16, 17). Increased activity of FGF is implicated in several cancers (17, 18). Endometrial adenocarcinoma develops in part due to a lack of inhibition of FGF18 synthesis, as a result of lower HAND2 expression due to hypermethylation of HAND2 (19). Further, hypermethylation of HAND2 in endometrial hyperplasia lesions correlates with resistance to progestin therapy (19), which is a mainstay medical therapy of endometrial hyperplasia.

To pursue the molecular mechanism by which BZA inhibits endometrial proliferation, we profiled gene expression of endometrial endometrioid adenocarcinoma, complex hyperplasia, and normal proliferative tissue to identify differences in estrogen and progesterone regulated genes. We identified differential expression of several genes in the FGF pathway and confirmed higher FGF18 and lower HAND2 in adenocarcinoma tissue vs normal tissue. We hypothesized that BZA reduces endometrial gland proliferation by inhibiting FGF18 synthesis from stromal cells. Because HAND2 prevents FGF18 expression, we examined BZA regulation of HAND2.

Materials and Methods

Endometrial adenocarcinoma, hyperplasia, and normal proliferative endometrium collection

Endometrial tissue was collected into RNAlater stabilization reagent (QIAGEN) at time of procedure, or for pathological samples, at the time of histological frozen section, within 10 minutes of hysterectomy. Diagnoses were confirmed after light microscopic examination of hematoxylin and eosin-stained slides by a gynecologic pathologist. The collected tissue specimen (n = 17) was immediately adjacent to the tissue viewed by a pathologist determining the presence of endometrial hyperplasia or adenocarcinoma. Normal endometrial biopsies were collected from healthy reproductive age women (n = 6), who were not receiving any hormonal therapy, and cycling in the proliferative phase as per last menstrual period and endometrial dating. RNAlater samples were stored at −80^οC, and later processed for whole-tissue gene analysis. For cell isolation and primary culture, normal endometrial tissue was received from 4 women of reproductive age who were not receiving any hormonal therapy, and processed immediately. The collection of normal and pathological endometrium was approved by the Yale Human Investigations Committee. Pathological endometrium and associated clinical data was collected through the Yale Gynecologic Oncology Tissue Bio-Repository.

The clinical parameters for these patients (n = 23), including the histological classification and surgical staging of each tumor are summarized in Table 1. Pathological tissue was classified histologically as per the World Health Organization (20). All hyperplasia tissue included in this study was complex, with or without atypia. All cancer tissue was type 1 endometrioid adenocarcinoma, with or without squamous differentiation. For adenocarcinoma specimens, the International Federation of Gynecology and Obstetrics surgical staging system (21) is reported as stage IA (invasion to <50% of myometrium), n = 6; stage IB (invasion to ≥50% of myometrium), n = 4; or stage IIIC2 (including para-aortic lymph node involvement), n = 3.

Table 1.

Clinical Parameters of the Normal Proliferative, Hyperplasia, and Adenocarcinoma Tissues Included in the Microarray Analysis and qRT-PCR Confirmation

Case Number	Age (y)	Final Histology	Stage	Included in Microarray
1	45	Normal, proliferative		Yes
2	41	Normal, proliferative		Yes
3	43	Normal, proliferative		Yes
4	32	Normal, proliferative		Yes
5	38	Normal, proliferative		Yes
6	39	Normal, proliferative		Yes
7	61	Complex hyperplasia with atypia		Yes
8	55	Complex hyperplasia with atypia		Yes
10	59	Focal complex hyperplasia without atypia		No
11	53	Focal complex hyperplasia without atypia		No
12	54	Focal complex hyperplasia with atypia		No
13	55	Endometrioid adenocarcinoma, endometrioid type, F2N2	IA	Yes
14	65	Endometrioid adenocarcinoma, endometrioid type with squamous differentiation	IA	Yes
15	74	Endometrioid adenocarcinoma, endometrioid type, F3N3	IIIC2	Yes
16	50	Endometrioid adenocarcinoma, endometrioid type, F2N2	IB	Yes
17	58	Endometrioid adenocarcinoma, endometrioid type, F3N2	IIIC2	Yes
18	72	Endometrioid adenocarcinoma, endometrioid type, F2	IB	Yes
19	80	Endometrioid adenocarcinoma, endometrioid type with squamous differentiation, F2N2	IB	No
20	53	Endometrioid adenocarcinoma, endometrioid type, F1N1	IA	No
21	57	Endometrioid adenocarcinoma, endometrioid type with squamous differentiation, F2N2	IA	No
22	32	Endometrioid adenocarcinoma, endometrioid type, F1N2	IA	No
23	63	Endometrioid adenocarcinoma, endometrioid type, F2N2	IB	No

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Primary cell culture and in vitro assay

To evaluate the effect of BZA on normal endometrium, endometrial stromal cells were isolated from normal endometrium (n = 4) as previously described (22) and grown to confluence in 5mM glucose DMEM, 10% calf serum, 1% amphotericin B, and 1% penicillin/streptomycin. Stromal cells were serum starved for 4 hours in phenol-free DMEM, then treated with BZA 1nM, 10nM, or 100nM or estrogen receptor antagonist ICI 182,780 ICI 1μM, or vehicle (1% ethanol) for 4 hours. RNA was extracted using the RNeasy Mini kit (QIAGEN).

Animal care and treatment

In order to investigate whether BZA regulates FGF18 through a HAND2-independent manner, we pursued an in vivo model with treatments of BZA alone or in combination with CE to mimic human exposure. Ethical guidelines for the use of animals were followed, as established by Institutional Animal Care and Use Committee, Yale University, and the United States Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. CD1 female mice from Charles River Laboratories were kept under regulated light and darkness cycles of 12 hours. At 16 weeks of age, the mice were treated daily with either BZA, a combination of BZA and CE, CE alone, or vehicle for 8 weeks. BZA was administered by ip injection to include a dose curve of 1, 2, 3, or 5 mg/kg per day in dimethyl sulfoxide (10%) + sesame oil (90%), with 4 mice in each group. For combination treatment groups (n = 4 in each group), BZA was injected ip at the same doses of 1, 2, 3, or 5 mg/kg per day in combination with CE 3 mg/kg per day via oral gavage. Ten mice received only CE 3 mg/kg per day, and 10 mice received ip injections of dimethyl sulfoxide (10%) + sesame oil (90%). After the completion of treatments, mice were euthanized and the uteri collected and snap frozen in TRIzol reagent (Invitrogen) for RNA extraction.

RNA extraction

Tissues were homogenized in TRIzol and precipitated with isopropanol. Total RNA was purified via RNeasy spin columns (QIAGEN) and treated with ribonuclease-free deoxyribonuclease for 10 minutes at room temperature. For in vitro studies, RNA was extracted from cultured cells using RNeasy Mini kit (QIAGEN). RNA concentration and purity analysis was determined via Nanodrop 2000 (Thermoscientific), and samples for microarray underwent further quality control of a RNA Integrity Analysis. All RNA samples included in the microarray had RNA Integrity Number values of more than 8.

Microarray gene analysis

We performed a microarray using Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray, through the Yale Keck Laboratory. RNA from each human endometrium was run on a separate chip, including normal proliferative (n = 6), complex endometrial hyperplasia (n = 2), and adenocarcinoma (n = 6). Of note, 4 tissues were initially included in the complex hyperplasia group in the microarray, but 2 tissues were found to be outliers on principal components analysis. As part of the assessment of these outliers, the histopathology of all tissues included in the microarray were reviewed with a gynecologic pathologist. One outlier was found to be very heterogenic endometrium, with small foci of hyperplasia and adenocarcinoma among normal tissue, and excluded from further analysis. A second outlier reported as hyperplasia on frozen section, was realized as adenocarcinoma at final reporting and reanalyzed as part of the adenocarcinoma group.

Gene expression analysis was performed by the Biostatistics Resource Keck Laboratory at Yale University, with Partek Genomics Suite (6.4; Partek, Inc). Affymetrix CEL files were imported by using the Robust Multichip Average method, which involves 4 steps: background correction of the perfect match values, quintile normalization across all of the chips in the experiment, Log2 transformation, and median polish summarization. Within each condition, outliers were identified using principle component analysis and excluded from all further analysis. Differentially expressed genes were selected with threshold of fold change more than 2 or less than −2, and false discovery rate (FDR) of 0.05 in ANOVA test. To identify high-yield targets for mechanistic studies, genes with significant differential expression were sorted by greatest fold change and P < 1 × 10⁻⁵. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to validate the microarray.

Gene Ontology biological processes, biological functions, pathway, and networks associated with Identified differentially expressed genes were identified using MetaCore GeneGO server (https://portal.genego.com/). P values were calculated based on hypergeometric distribution and reflects the probability for a pathway to arise by chance. Process, functions, pathway, and networks with a Benjamini-Hochberg multiple testing correction P ≤ .05 were considered significant.

Reverse transcription and real-time quantitative PCR analysis

For verification and validation of the microarray analysis, qRT-PCR was performed on RNA samples included in the microarray and additional RNA samples for the analysis of complex hyperplasia (n = 5) and type 1 endometrioid adenocarcinoma tissues (n = 11). qRT-PCR was also performed on RNA isolated from in vitro and in vivo studies. Primer sequences were designed via Primer Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast; NCBI) or from the literature (18) and synthesized at the W.M. Keck Foundation Oligo Synthesis Resource (Yale University, New Haven, CT). Optimal primer concentrations were determined by efficiency testing using either human or mouse reference gene RNA (Supplemental Table 1). qRT-PCR was performed using 12.5 ng of cDNA, reverse transcribed in triplicate using assay-specific primer concentration of 500nM, 250nM, or 125nM, SYBR Green containing deoxynucleotide solution mix, fluorescein, and reverse transcriptase (Bio-Rad), and amplified in a Bio-Rad CFX96 detection system (Bio-Rad) under the following cycling conditions: 95°C for 1 minute, 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 25 seconds, 95°C for 1 minute, and 55°C for 65 seconds. Gene expression levels were normalized to β-actin.

Statistical analysis

Microarray gene expression was analyzed by the Biostatistics Resource Keck Laboratory at Yale University, using GeneChip Operating Software (Affymetrix) to identify differences in expression levels in endometrial adenocarcinoma, hyperplasia, and normal proliferative tissue. Gene expression was normalized using quantile normalization method, and then corrected for multiple testing error using FDR < 0.05, with significance defined as fold change more than 2 or less than −2. Gene expression obtained by qRT-PCR was normalized to β-actin and graphically represented in a scatter plot, with each point representing the mean of triplicate values for an individual tissue. The mean and standard error of the mean are also shown in error bars. qRT-PCR data for complex hyperplasia was underpowered for adequate statistical comparison with normal tissue, but remains represented graphically due to its clinical significance. Statistical significance for tissue and in vivo experiments was determined by the Mann-Whitney test for nonparametric data using GraphPad Prism (GraphPad Software, Inc). Gene expression data from in vitro studies was log transformed and analyzed via Student's paired, 2-tailed t test using GraphPad Prism.

Results

Differential global gene expression in endometrial adenocarcinoma, hyperplasia, and normal proliferative tissue

In order to determine a mechanism of action for BZA's antiproliferative effect in the endometrium, we sought to identify genes which are differentially expressed between cancer and normal endometrial tissue, and are known to be regulated by either estrogens or progesterone. Hence, we performed a microarray analysis of endometrial endometrioid adenocarcinoma, complex hyperplasia, and normal proliferative endometrial tissue. The principal component analysis shows a tight and separate clustering of adenocarcinoma and normal proliferative tissue groups (Figure 1A). Hyperplasia samples were relatively dispersed, consistent with known heterogeneity in histology (23). A total of 1053 genes were differentially regulated between adenocarcinoma and normal proliferative tissue, with 440 genes of more than or equal to 2-fold increased expression and 613 genes of less than or equal to −2-fold decreased expression (FDR < 0.05). In the comparison between complex hyperplasia and normal proliferative tissues, 1898 genes were differentially regulated, with 572 genes of more than or equal to 2-fold increased expression and 1326 genes of less than or equal to −2-fold decreased expression (FDR < 0.05). A total of 137 genes were common between the differentially expressed genes identified in the comparisons between adenocarcinoma and normal, and between hyperplasia and normal, as represented in a Venn diagram (Figure 1B). Response to estradiol stimulus was the third most significantly altered process per enrichment analysis using the GO processes (Supplemental Table 2). Top processes of differentially regulated genes also included cell adhesion, extracellular matrix organization, cyclic nucleotide biosynthetic process, negative regulation of cell proliferation, and angiogenesis. Pathway analysis, not unexpectedly, also revealed alterations in genes involved in cell adhesion, extracellular matrix organization, epithelial-to-mesenchymal transition, and cell cycle regulation (Supplemental Table 3).

Figure 1. — A, Principal components analysis (PCA) mapping of endometrial adenocarcinoma (red spheres, n = 6), complex hyperplasia (green spheres, n = 2), and normal proliferative endometrium (blue spheres, n = 6) included in the microarray analysis. B, Venn diagram showing the overlap of differentially expressed genes identified in the comparison between adenocarcinoma and normal endometrium (red) and between hyperplasia and normal endometrium (green).

Given the large number of differentially regulated genes between adenocarcinoma and normal tissue, we ranked the up- and down-regulated genes with the greatest fold change and rigorous P < 1 × 10⁻⁵. We report the top 15 up-regulated (Table 2) and top 15 down-regulated (Table 3) differentially expressed genes. Most these genes are involved in signaling and regulation of cell cycle, seen in many cancers and associated with proliferation. FGF18 was selected as the most likely candidate for affecting cancer outcome due to the known relationship of FGFs in cancer pathogenesis (17), and the recent report of hypermethylated HAND2 as pathogenic in endometrial cancer (19). In addition, FGF18 is regulated by progesterone (15, 24, 25). RT-PCR confirmed that FGF18 expression was increased by 10.8-fold (P = .0003) in adenocarcinoma vs normal proliferative tissue (Figure 2A). FGF18 binds to several FGF receptors, including FGFR2 (17). Mean FGFR2 was increased in adenocarcinoma tissue by 4.9-fold (P < .05) (Figure 2B).

Table 2.

Top 15 Up-Regulated Genes in Endometrial Adenocarcinoma Relative to Normal Proliferative Endometrium

Gene Symbol	Gene Title	Fold Change	P Value
TFF3	Trefoil factor 3 (intestinal)	35.77	2.44E-06
CXCL2	Chemokine (C-X-C motif) ligand 2	29.39	3.98E-05
TFAP2A	Transcription factor AP-2α (activating enhancer-binding protein 2α)	29.25	2.82E-09
MMP12	Matrix metallopeptidase 12 (macrophage elastase)	18.55	2.76E-05
GABBR1/UBD	γ-aminobutynic acid (GABA) B receptor, 1/ubiquitin D adrenomedullin	13.84	1.77E-05
ADM	Adrenomedullin	13.60	3.76E-05
ELF5	E74-like factor 5 (ets domain transcription factor)	12.74	3.70E-05
CCNA1	Cyclin A1	11.72	3.76E-05
TMC5	Transmembrane channel-like 5	10.81	4.19E-05
ANKRD22	Ankyrin repeat domain 22	9.33	1.36E-05
FGF18	Fibroblast growth factor 18	8.28	4.97E-05
RASSF6	Ras association (RalGDS/AF-6) domain family member 6	8.01	1.55E-05
NTRK2	Neurotrophic tyrosine kinase, receptor, type 2	7.59	7.08E-07
DSC2	Desmocollin 2	7.52	7.28E-06
GLYATL2	Glycine-N-acyltransferase-like 2	7.18	8.58E-05

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From microarray results sorted by the greatest fold change and P < 1 × 10⁻⁵.

Table 3.

Top 15 Down-Regulated Genes in Endometrial Adenocarcinoma Relative to Normal Proliferative Endometrium

Gene Symbol	Gene Title	Fold Change	P Value
OLFM1	Olfactomedin 1	−26.47	1.22E-08
RBP7	Retinol-binding protein 7, cellular	−20.33	2.47E-08
RORB	RAR-related orphan receptor B	−16.60	5.20E-06
CACNA1D	Calcium channel, voltage-dependent, L type, α-1D subunit	−13.77	3.98E-06
TWIST2	Twist homolog 2 (Drosophila)	−12.50	1.44E-06
ARHGAP20	ρGTPase activating protein 20	−11.90	4.32E-06
ALDH1A2	Aldehyde dehydrogenase 1 family, member A2	−11.28	4.16E-06
PNMA2	Paraneoplastic Ma antigen 2	−11.16	4.56E-08
LSAMP	Limbic system-associated membrane protein	−10.99	5.56E-07
TCEAL7	Transcription elongation factor A (SII)-like 7	−10.67	1.02E-06
CACNA1G	Calcium channel, voltage-dependent, T type, α-1G subunit	−10.46	3.16E-07
MSANTD3-TMEFF1	Myb/SANT-like DNA-binding domain containing 3-transmembrane protein with EGF-like and 2 follistatin-like domains 1	−10.14	2.15E-07
WNT4	Wingless-type MMTV integration site family, member 4	−10.01	2.67E-08
IGF1	IGF-1 (somatomedin C)	−10.00	4.50E-06
UST	Uronyl-2-sulfotransferase	−9.49	4.36E-06

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From microarray results sorted by the largest negative fold change and P < 1 × 10⁻⁵.

Figure 2. — Scatter plot of tissue mRNA expression levels shown with mean ± SEM error bars, as normalized to β-actin in normal proliferative endometrium (•, n = 6), complex hyperplasia (■, n = 2 or 4), and endometrial adenocarcinoma (▴, n = 6 or 11). A, FGF18. B, FGFR2. C, HAND2. D, ELF5 was undetectable in 4 normal, 1 hyperplasia, and 1 adenocarcinoma tissues. *, P < .04 vs normal; **, P = .002 vs normal.

We also examined regulators and down-stream effectors of FGF18. HAND2 is a progesterone regulated transcription factor that reduces FGF18 expression. Mean HAND2 expression was reduced by 93% (P = .0001) in adenocarcinoma vs normal proliferative tissue (Figure 2C). Similar to FGF18, E74-like factor 5 (ELF5) was present in the highest 15 differentially expressed genes in the microarray (Table 2). ELF5 is a transcription factor regulated by members of the FGF family through FGFR2 (26, 27) as well as progesterone (28). ELF5 plays a role in stem cell self-renewal in trophoblasts (27, 29) and epithelial cell differentiation in breast and lung (26, 30) but has not yet been described in the endometrium. By RT-PCR, we found ELF5 expression was detectable in 10 of 11 adenocarcinoma samples, but only 2 of 6 normal proliferative samples. For the samples expressing ELF5, the mean expression in adenocarcinoma samples was greater than 450-fold higher than normal samples (P = .02) (Figure 2D).

IGF-1 was one of the top 15 down-regulated genes in adenocarcinoma tissue (Table 3), and was confirmed with RT-PCR to be reduced by 90% compared with normal tissue (P = .002) (Supplemental Figure 1). In normal endometrium, IGF-1 is regulated by estrogens and is a downstream mediator of estrogen proliferative action (31). IGF-1 is a ligand of the IGF-1 tyrosine kinase receptor, which has been implicated in endometrial cancer (32).

Gene expression for complex hyperplasia tissues are shown along with adenocarcinoma and normal endometrium (Figure 2), although the hyperplasia data are underpowered for statistical comparisons.

BZA suppresses FGF18 expression in human endometrial stromal cells

Based on evidence that FGF18 has a pathogenic role in endometrial cancer, we investigated whether BZA affects FGF18 expression in primary human endometrial stromal cells. BZA treatment resulted in a significant 13%–20% reduction of FGF18 expression (P < .05), occurring at all doses relative to vehicle (Figure 3A). Because progesterone reduces FGF18 expression through an up-regulation of HAND2, we also quantified HAND2 expression in vitro. Surprisingly, HAND2 expression was not significantly altered by either high or low BZA doses relative to vehicle (P = NS), demonstrating that the suppression of FGF18 was independent of HAND2 (Figure 3B). Fulvestrant, an estrogen receptor antagonist, was used as a control, and did not significantly alter either FGF18 or HAND2 expression relative to vehicle (P = NS).

Figure 3. — In vitro analysis of BZA activity in human endometrial stromal cells. Primary stromal cells (n = 4) were cultured and treated for 4 hours with a dose curve of 1nM, 10nM, or 100nM BZA, 1μM fulvestrant (ICI), or vehicle (EtOH). FGF18 (A) and HAND2 (B) mRNA levels shown as mean (±SEM) normalized to β-actin mRNA levels; *, P < .05 vs vehicle; **, P ≤ .008 vs vehicle.

BZA suppresses FGF18 expression in the murine uterus in vivo

Because our in vitro model suggested that BZA regulates the paracrine factor FGF18 in stromal cells, we then evaluated BZA activity in an in vivo model to enable stromal-epithelial interactions. CD1 female mice were treated with vehicle, CE, 4 doses of BZA, or a combination of BZA and CE to determine whether BZA reduced FGF18 expression in vivo. The doses of BZA included 3 mg/kg, a dose similar to the human therapeutic dose in addition to lower and higher doses (33). After 8 weeks of treatment, CE-treated mice had a 2.8-fold higher mean FGF18 expression than control mice (P = .002) (Figure 4A). In contrast, BZA-treated mice had similar FGF18 expression compared with control mice. In mice receiving combined CE and BZA, FGF18 expression was also similar to control mice (P = NS). Combination BZA and CE treatment resulted in FGF18 levels significantly lower than seen after CE exposure (P = .003), indicating that BZA suppressed CE-mediated induction of FGF18. This suppression occurred at all doses of BZA. BZA treatment consistently resulted in FGF18 levels that were no different from controls.

CE-treated mice had lower but not statistically different HAND2 expression relative to control mice (P = NS) (Figure 4B). BZA treatment resulted in significant 91%–94% reduction in HAND2 expression (P < .01), which was maximal at the lowest dose used. In mice receiving combined CE and BZA, HAND2 expression was also markedly reduced by 87%–93% (P < .04) compared with control mice at all but the highest 5-mg dose of BZA. BZA suppression of CE-mediated induction of FGF18 occurred in the absence of HAND2, indicating a novel HAND2-independent regulation of FGF18.

Because ELF5 is a transcription factor regulated by FGF as well as progesterone, we also quantified ELF5 in mouse uteri to determine whether reductions in FGF18 was associated with lower ELF5 levels. In contrast to human endometrium, ELF5 was present in all mouse uteri. Although CE-treated mice had higher FGF18 levels than vehicle, they did not have higher ELF5 levels relative to vehicle (P = NS). Vehicle-treated mice had a wide range of ELF5 expression, whereas CE and BZA treatment resulted in a narrower range of ELF5 expression. We found ELF5 was reduced in 4 of 8 groups treated with BZA (P < .05) or combined CE and BZA (P < .05), relative to CE-treated mice (Figure 4C). This effect was not dose dependent.

The proliferation marker proliferating cell nuclear antigen was lower in mice treated with BZA relative to mice treated with CE (P < .01) or vehicle (P < .05) (Supplemental Figure 2). Mice receiving combined CE and BZA had similar mean proliferating cell nuclear antigen levels as BZA-treated mice.

Discussion

We show that FGF18 and FGF receptor 2 expression levels are elevated in endometrial adenocarcinoma, whereas the negative regulator of FGF18 synthesis, HAND2, is reduced relative to normal proliferative tissue. In addition, the expression of ELF5, a nuclear transcription factor down stream of FGF, was higher in adenocarcinoma tissue. These findings implicate FGF18 activity in endometrial adenocarcinoma. BZA inhibited FGF18 expression in vitro and in vivo, thus preventing endometrial proliferation, similar to the effect of a progestin. BZA's direct effect on FGF18 was not dose dependent at the concentrations used, indicating that therapeutic BZA doses are equally effective in blocking estrogen induced effects as higher doses of BZA.

In contrast to progesterone, BZA did not exert an effect on FGF18 through HAND2. BZA had no direct effect on HAND2 expression in vitro, although BZA treatment in vivo resulted in a significant reduction of HAND2. Because HAND2 represses FGF18 synthesis, the decrease in HAND2 in response to BZA does not explain the simultaneous decrease in FGF18. Our results are consistent with in vitro studies by Cho et al, who found that estradiol did not alter HAND2 expression nor inhibit progesterone's regulation of HAND2 (24). Collectively, these findings indicate 2 independent pathways for negative regulation of FGF18 in the endometrium. Although progesterone stimulates HAND2 expression in stromal cells to reduce FGF18 levels, BZA acts through a HAND2-independent pathway to decrease FGF18 expression.

HAND2 is frequently hypermethylated in endometrial adenocarcinoma (19), conferring resistance to progesterone induction of HAND2 expression. Indeed, levels of hypermethylation in endometrial adenocarcinoma correlate with resistance to progestin therapy (19). BZA may provide an alternative to progestin therapy for endometrial hyperplasia.

We are the first to show that estrogens regulate FGF18 in the endometrium. Li et al (15) found that ovariectomized mice had undetectable FGFR phosphorylation in the uterus, indicating a relationship between estrogen and FGF signaling. Estrogen action through ER has been found to regulate other FGFs in prostate stromal cells (34). We found BZA blocked estrogen induced FGF18 expression, consistent with BZA's action as a selective antagonist of the estrogen receptor in endometrium. We have previously shown that BZA down regulates estrogen receptor expression (3) and other investigators showed that BZA treatment leads to degradation of the estrogen receptor (35). Together, these mechanisms likely account for BZA's antiestrogen effect. However, our in vitro studies showed BZA treatment reduces FGF18, whereas ICI, an estrogen receptor antagonist also known to cause degradation of the estrogen receptor (36, 37), did not significantly alter FGF18 levels. Although estrogens increase FGF18, it is likely that an ER interaction specific to BZA leads to decreased FGF18, and not simply an estrogen receptor antagonist effect.

Our study of adenocarcinoma, hyperplasia, and normal endometrium supports the FGF pathway as pathogenic in the development of adenocarcinoma. Our findings on reduced HAND2 in hyperplasia and adenocarcinoma tissues are similar to Jones et al (19). Further, we showed significant increases in FGF18, FGFR2, and ELF5 expression, strongly suggesting activation of this pathway. FGFR2 mutations are reported in 10% of endometrial cancers, and result in either altered ligand specificity or enhanced kinase activity (17, 38 –40). FGFR2 is recently implicated in maintaining and inducing proliferation of tumor initiating cells in breast cancer (41), and is overexpressed in triple-negative breast tumors (42). Downstream of FGFR2 is ELF5, which we found was undetectable in most normal proliferative endometrial samples while being markedly elevated in most adenocarcinomas. In breast cancer cells, ELF5 promotes hormone independence, by limiting the ability of progesterone to inhibit cell proliferation (28) and promoting an estrogen-independent phenotype which is unresponsive to antiestrogen therapy (43). ELF5 expression in endometrial adenocarcinoma is likely functioning through a similar mechanism. ELF5 expression was reduced in several groups of BZA-treated mice. The impact of estrogen on ELF5 expression remains unclear, and appears unassociated with CE-mediated induction of FGF18 levels. To our knowledge, the expression or potential ominous role of ELF5 in the endometrium has not been previously described.

In conclusion, we show that FGF18 is regulated by estrogens in vivo. BZA directly inhibits FGF18 expression in stromal cells and blocks estrogen induced FGF18 expression, by a mechanism independent of HAND2. Our studies identify a mechanism by which BZA prevents endometrial proliferation, providing reassurance about the long-term safety of combination BZA-CE therapy in postmenopausal women. In addition, BZA provides an alternative hormone strategy in women with obesity and type 2 diabetes, who are at increased risk for breast and endometrial cancer.

Acknowledgments

We thank assistance in tissue collection from Pinar Kodaman, MD, PhD of Yale Reproductive Endocrinology, Michelle Montagna of the Yale Gynecologic Oncology Tissue Bio-Repository, and Sharif Sakr, MD; Pei Hui, MD, PhD and Natalia Buza, MD from the Department of Pathology for reviewing each endometrial hyperplasia and adenocarcinoma specimen at time of frozen section; and Xiting Yan, PhD and Xiaoqing Yu, PhD of the Biostatistics Resource Keck Laboratory at Yale University for the statistical analysis of the microarray.

This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development Award K08HD071010 (to C.A.F.) and by the Eunice Kennedy Shriver National Institute of Child Health and Human Development Awards U54HD52668 and R01HD076422 and a grant from Pfizer (to H.S.T.).

Disclosure Summary: C.A.F., A.G.F., G.H.C., H.N., M.Z., and A.S. have nothing to disclose. H.S.T. reports receiving commercial research support from Pfizer and serving as a scientific consultant for Pfizer.

Footnotes

Abbreviations:

BZA: bazedoxifene
CE: conjugated estrogen
ELF5: E74-like factor 5
ER: estrogen receptor
FDR: false discovery rate
FGF: fibroblast growth factor
FGFR: FGF tyrosine kinase receptor
HAND2: heart and neural crest derivatives expressed transcript 2
ICI: estrogen receptor antagonist ICI 182,780
NS: nonsignificant
qRT-PCR: real-time quantitative polymerase chain reaction.

References

1. Pickar JH, Mirkin S. Tissue-selective agents: selective estrogen receptor modulators and the tissue-selective estrogen complex. Menopause Int. 2010;16(3):121–128. [DOI] [PubMed] [Google Scholar]
2. Kulak Júnior J, Kulak CA, Taylor HS. SERMs in the prevention and treatment of postmenopausal osteoporosis: an update. Arq Bras Endocrinol Metabol. 2010;54(2):200–205. [DOI] [PMC free article] [PubMed] [Google Scholar]
3. Naqvi H, Sakr S, Presti T, Krikun G, Komm BS, Taylor HS. Treatment with bazedoxifene and conjugated estrogens results in regression of endometriosis in a murine model. Biol Reprod. 2014;90(6):121. [DOI] [PMC free article] [PubMed] [Google Scholar]
4. Kulak J, Jr, Fischer C, Komm B, Taylor HS. Treatment with bazedoxifene, a selective estrogen receptor modulator, causes regression of endometriosis in a mouse model. Endocrinology. 2011;152(8):3226–3232. [DOI] [PMC free article] [PubMed] [Google Scholar]
5. Taylor HS, Ohleth K. Using bazedoxifene plus conjugated estrogens for treating postmenopausal women: a comprehensive review. Menopause. 201;19(4):479–485. [DOI] [PubMed] [Google Scholar]
6. Komm BS, Mirkin S, Jenkins SN. Development of conjugated estrogens/bazedoxifene, the first tissue selective estrogen complex (TSEC) for management of menopausal hot flashes and postmenopausal bone loss. Steroids. 2014;90:71–81. [DOI] [PubMed] [Google Scholar]
7. Harvey JA, Holm MK, Ranganath R, Guse PA, Trott EA, Helzner E. The effects of bazedoxifene on mammographic breast density in postmenopausal women with osteoporosis. Menopause. 2009;16(6):1193–1196. [DOI] [PubMed] [Google Scholar]
8. Song Y, Santen RJ, Wang JP, Yue W. Effects of the conjugated equine estrogen/bazedoxifene tissue-selective estrogen complex (TSEC) on mammary gland and breast cancer in mice. Endocrinology. 2012;153(12):5706–5715. [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Wardell SE, Nelson ER, Chao CA, McDonnell DP. Bazedoxifene exhibits antiestrogenic activity in animal models of tamoxifen-resistant breast cancer: implications for treatment of advanced disease. Clin Cancer Res. 2013;19(9):2420–2431. [DOI] [PMC free article] [PubMed] [Google Scholar]
10. Palacios S, de Villiers TJ, Nardone Fde C, et al. Assessment of the safety of long-term bazedoxifene treatment on the reproductive tract in postmenopausal women with osteoporosis: results of a 7-year, randomized, placebo-controlled, phase 3 study. Maturitas. 2013;76(1):81–87. [DOI] [PubMed] [Google Scholar]
11. Kulak J, Jr, Ferriani RA, Komm BS, Taylor HS. Tissue selective estrogen complexes (TSECs) differentially modulate markers of proliferation and differentiation in endometrial cells. Reprod Sci. 2013;20(2):129–137. [DOI] [PMC free article] [PubMed] [Google Scholar]
12. Ethun KF, Wood CE, Cline JM, Register TC, Appt SE, Clarkson TB. Endometrial profile of bazedoxifene acetate alone and in combination with conjugated equine estrogens in a primate model. Menopause. 2013;20(7):777–784. [DOI] [PMC free article] [PubMed] [Google Scholar]
13. Mirkin S, Komm BS, Pan K, Chines AA. Effects of bazedoxifene/conjugated estrogens on endometrial safety and bone in postmenopausal women. Climacteric. 2013;16(3):338–346. [DOI] [PubMed] [Google Scholar]
14. Cooke PS, Buchanan DL, Young P, et al. Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium. Proc Natl Acad Sci USA. 1997;94(12):6535–6540. [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Li Q, Kannan A, DeMayo FJ, et al. The antiproliferative action of progesterone in uterine epithelium is mediated by Hand2. Science. 2011;331(6019):912–916. [DOI] [PMC free article] [PubMed] [Google Scholar]
16. Itoh N, Ornitz DM. Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease. J Biochem. 2011;149(2):121–130. [DOI] [PMC free article] [PubMed] [Google Scholar]
17. Wesche J, Haglund K, Haugsten EM. Fibroblast growth factors and their receptors in cancer. Biochem J. 2011;437:199–213. [DOI] [PubMed] [Google Scholar]
18. Wei W, Mok SC, Oliva E, Kim SH, Mohapatra G, Birrer MJ. FGF18 as a prognostic and therapeutic biomarker in ovarian cancer. J Clin Invest. 2013;123(10):4435–4448. [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Jones A, Teschendorff AE, Li Q, et al. Role of DNA methylation and epigenetic silencing of HAND2 in endometrial cancer development. PLoS Med. 2013;10(11):e1001551. [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Silverberg SG. Uterine corpus. In: Scully RE, Bonfiglio TA, Kurman RJ, Silverberg SG, Wilkinson EJ, eds. Histological Typing of Female Genital Tract Tumours. 2nd ed New York, NY: Springer-Verlag; 1994;13. [Google Scholar]
21. Pecorelli S. Revised FIGO staging for carcinoma of the vulva, cervix, and endometrium. Int J Gynaecol Obstet. 2009;105(2):103–104. [DOI] [PubMed] [Google Scholar]
22. Yang H, Zhou Y, Edelshain B, Schatz F, Lockwood CJ, Taylor HS. FKBP4 is regulated by HOXA10 during decidualization and in endometriosis. Reproduction. 2012;143(4):531–538. [DOI] [PubMed] [Google Scholar]
23. Mills AM, Longacre TA. Endometrial hyperplasia. Semin Diagn Pathol. 2010;27(4):199–214. [DOI] [PubMed] [Google Scholar]
24. Cho H, Okada H, Tsuzuki T, Nishigaki A, Yasuda K, Kanzaki H. Progestin-induced heart and neural crest derivatives expressed transcript 2 is associated with fibulin-1 expression in human endometrial stromal cells. Fertil Steril. 2013;99(1):248–255. [DOI] [PubMed] [Google Scholar]
25. Pawar S, Hantak AM, Bagchi IC, Bagchi MK. Minireview: steroid-regulated paracrine mechanisms controlling implantation. Mol Endocrinol. 2014;28(9):1408–1422. [DOI] [PMC free article] [PubMed] [Google Scholar]
26. Metzger DE, Xu Y, Shannon JM. Elf5 is an epithelium-specific, fibroblast growth factor-sensitive transcription factor in the embryonic lung. Dev Dyn. 2007;236(5):1175–1192. [DOI] [PubMed] [Google Scholar]
27. Ng RK, Dean W, Dawson C, et al. Epigenetic restriction of embryonic cell lineage fate by methylation of Elf5. Nat Cell Biol. 2008;10(11):1280–1290. [DOI] [PMC free article] [PubMed] [Google Scholar]
28. Hilton HN, Kalyuga M, Cowley MJ, et al. The antiproliferative effects of progestins in T47D breast cancer cells are tempered by progestin induction of the ETS transcription factor Elf5. Mol Endocrinol. 2010;24(7):1380–1392. [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Hemberger M, Udayashankar R, Tesar P, Moore H, Burton GJ. ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta. Hum Mol Genet. 2010;19(12):2456–2467. [DOI] [PubMed] [Google Scholar]
30. Chakrabarti R, Wei Y, Romano RA, DeCoste C, Kang Y, Sinha S. Elf5 regulates mammary gland stem/progenitor cell fate by influencing notch signaling. Stem Cells. 2012;30(7):1496–1508. [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Kashima H, Shiozawa T, Miyamoto T, et al. Autocrine stimulation of IGF1 in estrogen-induced growth of endometrial carcinoma cells: involvement of the mitogen-activated protein kinase pathway followed by up-regulation of cyclin D1 and cyclin E. Endocr Relat Cancer. 2009;16(1):113–122. [DOI] [PubMed] [Google Scholar]
32. Bruchim I, Sarfstein R, Werner H. The IGF hormonal network in endometrial cancer: functions, regulation, and targeting approaches. Front Endocrinol (Lausanne). 2014;5:76. [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Crabtree JS, Peano BJ, Zhang X, Komm BS, Winneker RC, Harris HA. Activity of three selective estrogen receptor modulators on hormone-dependent responses in the mouse uterus and mammary gland. Mol Cell Endocrinol. 2008;287:40–46. [DOI] [PubMed] [Google Scholar]
34. Smith P, Rhodes NP, Ke Y, Foster CS. Upregulation of estrogen and androgen receptors modulate expression of FGF-2 and FGF-7 in human, cultured, prostatic stromal cells exposed to high concentrations of estradiol. Prostate Cancer Prostatic Dis. 2002;5:105–110. [DOI] [PubMed] [Google Scholar]
35. Lewis-Wambi JS, Kim H, Curpan R, Grigg R, Sarker MA, Jordan VC. The selective estrogen receptor modulator bazedoxifene inhibits hormone-independent breast cancer cell growth and down-regulates estrogen receptor α and cyclin D1. Mol Pharmacol. 2011;80(4):610–620. [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Wijayaratne AL, Nagel SC, Paige LA, et al. Comparative analyses of mechanistic differences among antiestrogens. Endocrinology. 1999;140(12):5828–5840. [DOI] [PubMed] [Google Scholar]
37. Wittmann BM, Sherk A, McDonnell DP. Definition of functionally important mechanistic differences among selective estrogen receptor down-regulators. Cancer Res. 2007;67(19):9549–9560. [DOI] [PubMed] [Google Scholar]
38. Dutt A, Salvesen HB, Chen TH, et al. Drug-sensitive FGFR2 mutations in endometrial carcinoma. Proc Natl Acad Sci USA. 2008;105(25):8713–8717. [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Byron SA, Gartside M, Powell MA, et al. FGFR2 point mutations in 466 endometrioid endometrial tumors: relationship with MSI, KRAS, PIK3CA, CTNNB1 mutations and clinicopathological features. PLoS One. 2012;7(2):e30801. [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Liu T, Willmore-Payne C, Wallander ML, Layfield LJ. Utilization of unlabeled probes for the detection of fibroblast growth factor receptor 2 exons 7 and 12 mutations in endometrial carcinoma. Appl Immunohistochem Mol Morphol. 2011;19(4):341–346. [DOI] [PubMed] [Google Scholar]
41. Kim S, Dubrovska A, Salamone RJ, et al. FGFR2 promotes breast tumorigenicity through maintenance of breast tumor-initiating cells. PLoS One. 2013;8(1):e51671. [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Schneider BP, Winer EP, Foulkes WD, et al. Triple-negative breast cancer: risk factors to potential targets. Clin Cancer Res. 2008;14(24):8010–8018. [DOI] [PubMed] [Google Scholar]
43. Kalyuga M, Gallego-Ortega D, Lee HJ, et al. ELF5 suppresses estrogen sensitivity and underpins the acquisition of antiestrogen resistance in luminal breast cancer. PLoS Biol. 2012;10(12):e1001461. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B1] 1. Pickar JH, Mirkin S. Tissue-selective agents: selective estrogen receptor modulators and the tissue-selective estrogen complex. Menopause Int. 2010;16(3):121–128. [DOI] [PubMed] [Google Scholar]

[B2] 2. Kulak Júnior J, Kulak CA, Taylor HS. SERMs in the prevention and treatment of postmenopausal osteoporosis: an update. Arq Bras Endocrinol Metabol. 2010;54(2):200–205. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3. Naqvi H, Sakr S, Presti T, Krikun G, Komm BS, Taylor HS. Treatment with bazedoxifene and conjugated estrogens results in regression of endometriosis in a murine model. Biol Reprod. 2014;90(6):121. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4. Kulak J, Jr, Fischer C, Komm B, Taylor HS. Treatment with bazedoxifene, a selective estrogen receptor modulator, causes regression of endometriosis in a mouse model. Endocrinology. 2011;152(8):3226–3232. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5. Taylor HS, Ohleth K. Using bazedoxifene plus conjugated estrogens for treating postmenopausal women: a comprehensive review. Menopause. 201;19(4):479–485. [DOI] [PubMed] [Google Scholar]

[B6] 6. Komm BS, Mirkin S, Jenkins SN. Development of conjugated estrogens/bazedoxifene, the first tissue selective estrogen complex (TSEC) for management of menopausal hot flashes and postmenopausal bone loss. Steroids. 2014;90:71–81. [DOI] [PubMed] [Google Scholar]

[B7] 7. Harvey JA, Holm MK, Ranganath R, Guse PA, Trott EA, Helzner E. The effects of bazedoxifene on mammographic breast density in postmenopausal women with osteoporosis. Menopause. 2009;16(6):1193–1196. [DOI] [PubMed] [Google Scholar]

[B8] 8. Song Y, Santen RJ, Wang JP, Yue W. Effects of the conjugated equine estrogen/bazedoxifene tissue-selective estrogen complex (TSEC) on mammary gland and breast cancer in mice. Endocrinology. 2012;153(12):5706–5715. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9. Wardell SE, Nelson ER, Chao CA, McDonnell DP. Bazedoxifene exhibits antiestrogenic activity in animal models of tamoxifen-resistant breast cancer: implications for treatment of advanced disease. Clin Cancer Res. 2013;19(9):2420–2431. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10. Palacios S, de Villiers TJ, Nardone Fde C, et al. Assessment of the safety of long-term bazedoxifene treatment on the reproductive tract in postmenopausal women with osteoporosis: results of a 7-year, randomized, placebo-controlled, phase 3 study. Maturitas. 2013;76(1):81–87. [DOI] [PubMed] [Google Scholar]

[B11] 11. Kulak J, Jr, Ferriani RA, Komm BS, Taylor HS. Tissue selective estrogen complexes (TSECs) differentially modulate markers of proliferation and differentiation in endometrial cells. Reprod Sci. 2013;20(2):129–137. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12. Ethun KF, Wood CE, Cline JM, Register TC, Appt SE, Clarkson TB. Endometrial profile of bazedoxifene acetate alone and in combination with conjugated equine estrogens in a primate model. Menopause. 2013;20(7):777–784. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13. Mirkin S, Komm BS, Pan K, Chines AA. Effects of bazedoxifene/conjugated estrogens on endometrial safety and bone in postmenopausal women. Climacteric. 2013;16(3):338–346. [DOI] [PubMed] [Google Scholar]

[B14] 14. Cooke PS, Buchanan DL, Young P, et al. Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium. Proc Natl Acad Sci USA. 1997;94(12):6535–6540. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15. Li Q, Kannan A, DeMayo FJ, et al. The antiproliferative action of progesterone in uterine epithelium is mediated by Hand2. Science. 2011;331(6019):912–916. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16. Itoh N, Ornitz DM. Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease. J Biochem. 2011;149(2):121–130. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17. Wesche J, Haglund K, Haugsten EM. Fibroblast growth factors and their receptors in cancer. Biochem J. 2011;437:199–213. [DOI] [PubMed] [Google Scholar]

[B18] 18. Wei W, Mok SC, Oliva E, Kim SH, Mohapatra G, Birrer MJ. FGF18 as a prognostic and therapeutic biomarker in ovarian cancer. J Clin Invest. 2013;123(10):4435–4448. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19. Jones A, Teschendorff AE, Li Q, et al. Role of DNA methylation and epigenetic silencing of HAND2 in endometrial cancer development. PLoS Med. 2013;10(11):e1001551. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20. Silverberg SG. Uterine corpus. In: Scully RE, Bonfiglio TA, Kurman RJ, Silverberg SG, Wilkinson EJ, eds. Histological Typing of Female Genital Tract Tumours. 2nd ed New York, NY: Springer-Verlag; 1994;13. [Google Scholar]

[B21] 21. Pecorelli S. Revised FIGO staging for carcinoma of the vulva, cervix, and endometrium. Int J Gynaecol Obstet. 2009;105(2):103–104. [DOI] [PubMed] [Google Scholar]

[B22] 22. Yang H, Zhou Y, Edelshain B, Schatz F, Lockwood CJ, Taylor HS. FKBP4 is regulated by HOXA10 during decidualization and in endometriosis. Reproduction. 2012;143(4):531–538. [DOI] [PubMed] [Google Scholar]

[B23] 23. Mills AM, Longacre TA. Endometrial hyperplasia. Semin Diagn Pathol. 2010;27(4):199–214. [DOI] [PubMed] [Google Scholar]

[B24] 24. Cho H, Okada H, Tsuzuki T, Nishigaki A, Yasuda K, Kanzaki H. Progestin-induced heart and neural crest derivatives expressed transcript 2 is associated with fibulin-1 expression in human endometrial stromal cells. Fertil Steril. 2013;99(1):248–255. [DOI] [PubMed] [Google Scholar]

[B25] 25. Pawar S, Hantak AM, Bagchi IC, Bagchi MK. Minireview: steroid-regulated paracrine mechanisms controlling implantation. Mol Endocrinol. 2014;28(9):1408–1422. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26. Metzger DE, Xu Y, Shannon JM. Elf5 is an epithelium-specific, fibroblast growth factor-sensitive transcription factor in the embryonic lung. Dev Dyn. 2007;236(5):1175–1192. [DOI] [PubMed] [Google Scholar]

[B27] 27. Ng RK, Dean W, Dawson C, et al. Epigenetic restriction of embryonic cell lineage fate by methylation of Elf5. Nat Cell Biol. 2008;10(11):1280–1290. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28. Hilton HN, Kalyuga M, Cowley MJ, et al. The antiproliferative effects of progestins in T47D breast cancer cells are tempered by progestin induction of the ETS transcription factor Elf5. Mol Endocrinol. 2010;24(7):1380–1392. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29. Hemberger M, Udayashankar R, Tesar P, Moore H, Burton GJ. ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta. Hum Mol Genet. 2010;19(12):2456–2467. [DOI] [PubMed] [Google Scholar]

[B30] 30. Chakrabarti R, Wei Y, Romano RA, DeCoste C, Kang Y, Sinha S. Elf5 regulates mammary gland stem/progenitor cell fate by influencing notch signaling. Stem Cells. 2012;30(7):1496–1508. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31. Kashima H, Shiozawa T, Miyamoto T, et al. Autocrine stimulation of IGF1 in estrogen-induced growth of endometrial carcinoma cells: involvement of the mitogen-activated protein kinase pathway followed by up-regulation of cyclin D1 and cyclin E. Endocr Relat Cancer. 2009;16(1):113–122. [DOI] [PubMed] [Google Scholar]

[B32] 32. Bruchim I, Sarfstein R, Werner H. The IGF hormonal network in endometrial cancer: functions, regulation, and targeting approaches. Front Endocrinol (Lausanne). 2014;5:76. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33. Crabtree JS, Peano BJ, Zhang X, Komm BS, Winneker RC, Harris HA. Activity of three selective estrogen receptor modulators on hormone-dependent responses in the mouse uterus and mammary gland. Mol Cell Endocrinol. 2008;287:40–46. [DOI] [PubMed] [Google Scholar]

[B34] 34. Smith P, Rhodes NP, Ke Y, Foster CS. Upregulation of estrogen and androgen receptors modulate expression of FGF-2 and FGF-7 in human, cultured, prostatic stromal cells exposed to high concentrations of estradiol. Prostate Cancer Prostatic Dis. 2002;5:105–110. [DOI] [PubMed] [Google Scholar]

[B35] 35. Lewis-Wambi JS, Kim H, Curpan R, Grigg R, Sarker MA, Jordan VC. The selective estrogen receptor modulator bazedoxifene inhibits hormone-independent breast cancer cell growth and down-regulates estrogen receptor α and cyclin D1. Mol Pharmacol. 2011;80(4):610–620. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36. Wijayaratne AL, Nagel SC, Paige LA, et al. Comparative analyses of mechanistic differences among antiestrogens. Endocrinology. 1999;140(12):5828–5840. [DOI] [PubMed] [Google Scholar]

[B37] 37. Wittmann BM, Sherk A, McDonnell DP. Definition of functionally important mechanistic differences among selective estrogen receptor down-regulators. Cancer Res. 2007;67(19):9549–9560. [DOI] [PubMed] [Google Scholar]

[B38] 38. Dutt A, Salvesen HB, Chen TH, et al. Drug-sensitive FGFR2 mutations in endometrial carcinoma. Proc Natl Acad Sci USA. 2008;105(25):8713–8717. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B39] 39. Byron SA, Gartside M, Powell MA, et al. FGFR2 point mutations in 466 endometrioid endometrial tumors: relationship with MSI, KRAS, PIK3CA, CTNNB1 mutations and clinicopathological features. PLoS One. 2012;7(2):e30801. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B40] 40. Liu T, Willmore-Payne C, Wallander ML, Layfield LJ. Utilization of unlabeled probes for the detection of fibroblast growth factor receptor 2 exons 7 and 12 mutations in endometrial carcinoma. Appl Immunohistochem Mol Morphol. 2011;19(4):341–346. [DOI] [PubMed] [Google Scholar]

[B41] 41. Kim S, Dubrovska A, Salamone RJ, et al. FGFR2 promotes breast tumorigenicity through maintenance of breast tumor-initiating cells. PLoS One. 2013;8(1):e51671. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B42] 42. Schneider BP, Winer EP, Foulkes WD, et al. Triple-negative breast cancer: risk factors to potential targets. Clin Cancer Res. 2008;14(24):8010–8018. [DOI] [PubMed] [Google Scholar]

[B43] 43. Kalyuga M, Gallego-Ortega D, Lee HJ, et al. ELF5 suppresses estrogen sensitivity and underpins the acquisition of antiestrogen resistance in luminal breast cancer. PLoS Biol. 2012;10(12):e1001461. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Endometrial Cancer-Associated FGF18 Expression Is Reduced by Bazedoxifene in Human Endometrial Stromal Cells In Vitro and in Murine Endometrium

Clare A Flannery

Andrew G Fleming

Gina H Choe

Hanyia Naqvi

Margaret Zhang

Anu Sharma

Hugh S Taylor

Abstract

Materials and Methods