Steroid-modulated proliferation of human endometrial carcinoma cell lines: any role for insulin-like growth factor signaling?

OBJECTIVES: Estrogen-stimulated proliferation of the normal and malignant human endometrium is balanced by the differentiating properties of progesterone. This study evaluated the role of insulin-like growth factor (IGF) signaling in steroid-induced modulation of endometrial cancer cell proliferation. METHODS: We used the human endometrial, estrogen-responsive ECC-1 and progesterone-responsive PRAB-36 cell lines. Proliferation studies with IGFs in combination with either estrogen or progesterone were conducted. Furthermore, the mRNA and protein expression of insulin-like growth factor-binding proteins (IGFBPs) was evaluated. RESULTS: Using the ECC-1 cell line, we observed that estrogen-induced proliferation is modulated via the IGF-receptor signaling pathway, and that IGF-1-induced stimulation of proliferation does not influence estrogen receptor signaling. Furthermore, expression of the main modulators of IGF action, the IGFBPs, was found to be regulated by estrogen and progesterone in both cell lines. IGFBP-4 was up-regulated by estrogen in the ECC-1 cell line, and IGFBP-3 and IGFBP-6 were down-regulated by progesterone in the PRAB-36 cell line. CONCLUSION: Estrogen-induced stimulation of proliferation of ECC-1 endometrial cancer cells is partly achieved via IGF signaling. Furthermore, the IGFBPs are regulated by estrogens as well as progestagens and could potentially play a role in the modulation of endometrial cancer cell proliferation.     

Differential gene expression in progesterone-sensitive and progesterone-insensitive endometrial carcinoma cells

High doses of progesterone are used in the treatment of advanced and recurrent endometrial cancer. Unfortunately the response rate is relatively low: 10-30%. The mechanisms involved in the development of insensitivity to progesterone treatment of endometrial cancer tissue are largely unknown. As tumour development is thought to be associated with a cascade of genetic alterations, it can be expected that genetic changes are involved in the development of progesterone insensitivity in endometrial carcinomas. We therefore started an investigation to identify, isolate and characterise progesterone-regulated genes involved in progesterone-induced growth inhibition in endometrial carcinoma cells. Using differential display PCR eight progesterone-regulated cDNA clones were identified in endometrial carcinoma cell lines. Four of these progesterone-regulated cDNA clones were regulated in the for growth progesterone-sensitive cell line IK-3H12 and not regulated in the for growth-insensitive cell line ECC-1. This indicates that these four cDNA clones represent potentially important genes, which could be involved in inhibition of growth of endometrial carcinoma tissue by progesterone.     

Genomic and nongenomic effects of estrogen signaling in human endometrial cells: involvement of the growth factor receptor signaling downstream AKT pathway

For the endometrium, estradiol and tamoxifen induce proliferation, and consequently, tamoxifen treatment of breast cancer results in a 2-fold to 7-fold increased risk for endometrial cancer. Here, the role of activation of growth factor receptor signaling in mediating the effects of estrogen and tamoxifen is determined. Microarray analysis of ECC-1 cells treated with estradiol or tamoxifen indicate that rapid responses to treatment (1 hour) are very distinct from long-term responses (>24 hours). Furthermore, estradiol and tamoxifen are observed to induce AKT activation. Comparing long-term estrogen- and tamoxifen-regulated genes with genes regulated by insulin-like growth factor 1 and amphiregulin reveals that the late effects of estrogen and tamoxifen signaling may partly be mediated via activation of growth factor receptor signaling pathways. It is hypothesized that both early and late effects of estrogen and tamoxifen signaling in the endometrium are partly mediated via the activation of growth factor receptor signaling, putatively at the level of AKT activation.     

Distinct functional differences of human progesterone receptors A and B on gene expression and growth regulation in two endometrial carcinoma cell lines

To study the functional differences between the two progesterone receptor isoforms (hPRA and hPRB) in human endometrial cancer, two new endometrial carcinoma cell lines were created-one expressing hPRA and one expressing hPRB.A well-differentiated, hPR-negative Ishikawa cell line was stably transfected with either hPRA or hPRB cDNA. Transfected cells were selected, and two cell lines expressing approximately equal amounts of receptor were isolated-one expressing hPRA (PRA-14) and one expressing hPRB (PRB-59).Cell growth experiments revealed a growth-inhibitory response to progestins (MPA and R5020) in the PRB-59 cells but not in the PRA-14 cells. Differences in expression of genes targeted by the two isoforms were studied using a cDNA expression array technique. A different set of genes appeared to be progesterone regulated in the PRA-14 cells than in the PRB-59 cells. None of the genes were regulated by both hPRA and hPRB. Insulin-like growth factor binding protein 3 expression was studied in more detail as an example of a gene regulated in PRB-59 cells but not in PRA-14 cells.We established a new model to study functional differences between the two hPR isoforms in human endometrial carcinoma cells. This model revealed distinctive differences in target gene regulation between the two hPR isoforms. Moreover, antiproliferative actions of progesterone on human endometrial cancer cells could be observed only in the PRB-expressing cell line.     

Progesterone-regulated gene expression in the primate endometrium

Progesterone action is essential for maturation of the endometrium to a receptive state for implantation in humans and nonhuman primates. The orchestration of progesterone-regulated gene expression is also temporally controlled during the secretory phase based on the limited window for implantation. The genes and gene networks affected by progesterone are likely to involve both activation and repression. Our laboratory has used the rhesus monkey as a model to study the regulation of genes known or suspected to be involved in endometrial maturation. In addition, we have used subtractive hybridization and differential display techniques to identify novel or unsuspected genes that are regulated by progesterone during endometrial maturation. Our studies have led us to propose a working model of progesterone action during the primate secretory phase that includes waves of gene activation and repression that culminate in a receptive endometrium.     

Progesterone-mediated regulation of catechol-O-methyl transferase expression in endometrial cancer cells

The effects of estrogen and progesterone on the expression of estrogen-metabolizing enzymes such as catechol-O-methyl transferase (COMT) are not known. COMT converts genotoxic catecholestrogens to anticarcinogenic methoxyestrogens in the endometrium. The aim of this study is to investigate the effect of progesterone on COMT expression in well-differentiated endometrial cancer cells. The wild-type Ishikawa cell line as well as progesterone receptor A- or progesterone receptor B-transfected Ishikawa cells were used for in vitro studies. The regulation of COMT expression by progesterone was studied using Western blots, Hoechst dye DNA proliferation studies, and wild-type and/or site-directed mutagenesis of COMT promoter 1-luciferase reporter gene. Progesterone upregulated COMT protein expression in Ishikawa cells through progesterone receptor A isoform. COMT promoter activity was differentially regulated by the 3 half-site progesterone response elements in the COMT promoter. High doses of 2-ME2 inhibited Ishikawa cell proliferation. These data suggest that COMT expression is hormonally regulated in well-differentiated human endometrial cancer cells. COMT regulation and 2-ME2 production in the endometrium may affect endometrial carcinogenesis.     

Effect of levonorgestrel and mifepristone on endometrial receptivity markers in a three-dimensional human endometrial cell culture model

OBJECTIVE: To investigate the effect of levonorgestrel and mifepristone on the expression of endometrial receptivity markers in a three-dimensional endometrial construct. DESIGN: In vitro study. SETTING: University hospital and research laboratory. PATIENT(S): Twelve fertile donors. INTERVENTION(S): Timed endometrial biopsy. MAIN OUTCOME MEASURE(S): Examine the effect of levonorgestrel along with another well-studied fertility-regulating drug, mifepristone, on the expression of endometrial receptivity factors in a three-dimensional stromal and epithelial cell coculture model by immunohistochemistry. RESULT(S): Both epithelial and stromal cells of in vitro endometrial construct showed the presence of estrogen receptor-alpha, estrogen receptor-beta, progesterone receptors-(A+B), vascular endothelial growth factor, leukemia inhibitory factor, interleukin-1 beta, and cyclooxygenase-2, whereas the expression of progesterone receptor-B (AR), integrin alpha(V)beta(3,) and MUC1 were confined to epithelial cells. Mifepristone up-regulated expression of epithelial estrogen receptor-beta and progesterone receptor-B and down-regulated stromal vascular endothelial growth factor and surface molecules MUC1 and integrin alpha(V)beta(3) as observed in vivo. Levonorgestrel had no effect on the expression of endometrial receptivity markers studied. CONCLUSION(S): This in vitro model expresses progesterone-regulated endometrial receptivity factors seen in the physiologic condition. Treatment with levonorgestrel did not affect the expression of these endometrial receptivity markers in contrast to mifepristone. This in vitro model holds the potential to study endometrial receptivity, the embryo-endometrial interaction, and develop new agents for fertility control.     

Danazol binds to progesterone receptors and inhibits the growth of human endometrial cancer cells in vitro

Based on our recent findings that danazol, an isoxazol derivative of ethinyltestosterone, has a profound growth-inhibitory effect on an established human endometrial adenocarcinoma cell line, the effects of danazol on cancer cells from human endometrial adenocarcinomas obtained by hysterectomy were investigated in the present study. Of the 22 uterine adenocarcinomas, estrogen, progesterone, and androgen receptors were found in 12, 14, and 4 tumors, respectively. Competitive binding studies showed that danazol specifically binds to progesterone and androgen receptors but not to estrogen receptors. Of the five cancer cells from five patients succeeded in primary cell culture, a marked inhibition of cell growth was demonstrated by addition of danazol in two cancer cells having progesterone but not androgen receptors. However, danazol did not affect the growth of the remaining three cancer cells lacking progesterone receptors. These results strongly suggest that danazol has a significant growth-inhibitory effect on human endometrial adenocarcinoma cells, possibly through progesterone receptors in the cells.     

Molecular tools to reestablish progestin control of endometrial cancer cell proliferation

OBJECTIVE: Endometrial cancers often arise in a setting of estrogen stimulation unopposed by the differentiating effects of progesterone. Our laboratory and others have previously shown that progesterone receptor down-regulation or perturbation of progesterone receptor isoform A or B expression is associated with the development of poorly differentiated endometrial cancers that are not growth inhibited by progestins. The purpose of these studies was to reestablish high progesterone receptor isoform A and B gene expressions in such endometrial cancer cells and to examine the effects of progestin treatment on cell growth and metastatic potential after this transformation. STUDY DESIGN: To induce high levels of expression of the progesterone receptor isoforms in KLE and Hec50 endometrial cancer cells, adenoviral vectors encoding the genes for progesterone receptor isoforms A and B were created. The characteristic ability of cancer cells to grow independently of anchorage to the surrounding solid matrix was measured by counting colony formation on soft agar for 8 to 14 days. Cell proliferation in response to a time course of progestin treatment was tested with flow cytometry. RESULTS: After treatment with a control vector without a progesterone receptor--encoding insert, no effect of progestin treatment on cell proliferation was found; after treatment with vectors encoding progesterone receptor isoform A or B, however, progestin treatment resulted in significant inhibition of cell growth. The anchorage-independent cell growth on soft agar assay showed that by 8 to 14 days the number of cell colonies was reduced by 50% relative to control preparations in the presence of progesterone receptor isoform A plus progestin (P <.0001, both Hec50 and KLE cell lines) and by 90% in the presence of progesterone receptor isoform B plus progestin (P <.0001, both Hec50 and KLE cell lines). Progestin treatment also resulted in a time-dependent reduction in cell proliferation as measured by flow cytometry. Although transfection with both progesterone receptor isoforms A and B reduced cell proliferation according to our assays, progesterone receptor isoform B caused a much more dramatic decrease in cell growth (P =.001, Hec50 cells; P <.0001, KLE cells). CONCLUSION: In poorly differentiated endometrial cancer cells that are resistant to progestin therapy, adenovirus-induced expressions of progesterone receptors A and B reestablish progestin control of endometrial cancer cell proliferation.     

Modulation of endometrial steroid receptors and growth regulatory genes by tamoxifen

OBJECTIVE: We investigated tamoxifen's effects on the expression of growth regulatory genes in the endometrium to identify the mechanism by which tamoxifen induces proliferation. METHODS: Using immunohistochemical techniques, we analyzed 39 endometrial specimens for expression of Ki-67, lactoferrin, transforming growth factor-alpha, tumor necrosis factor receptor-II, adrenomedullin, estrogen receptors, and progesterone receptors. Twenty specimens were obtained from postmenopausal breast cancer patients treated with tamoxifen (20 mg/day) for at least 6 months to include two endometrial adenocarcinoma specimens. Five secretory phase, three proliferative phase, and seven atrophic endometrial specimens were used as controls. In addition, four endometrial adenocarcinoma specimens were reviewed from patients not treated with tamoxifen. Intensity of immunostaining was quantified using digitized imaging techniques. RESULTS: Overexpression of both estrogen receptors and progesterone receptors, and an elevated proliferative index were the most consistent effects observed in benign endometrial specimens from tamoxifen-treated patients compared with atrophic controls (P <. 003). This staining pattern was also evident in adenocarcinomas from patients who received tamoxifen. Benign endometrium from tamoxifen-treated patients also expressed transforming growth factor-alpha, tumor necrosis factor receptor-II, lactoferrin, and adrenomedullin at levels comparable with those found in proliferative endometrial specimens. CONCLUSION: These data provide further documentation that the uterotropic effects of tamoxifen may be due, at least in part, to the induction of estrogen receptors and progesterone receptors, as well as other genes associated with the proliferative phase. Furthermore, analysis of estrogen receptors, progesterone receptors, and Ki-67 may be useful in identifying postmenopausal individuals on tamoxifen, who are at increased risk for developing endometrial cancer.     

Global gene expression profiling of proliferative phase endometrium reveals distinct functional subdivisions

The human endometrium follows a predictable pattern of development during the proliferative phase. Endometrial thickness increases after day 3 and then plateaus at days 9 to 10 of the menstrual cycle despite continued high serum levels of estrogen. We hypothesized that proliferative phase endometrium undergoes more than simple estrogen responsive growth, rather it is characterized by complex time-dependent functional activities reflected in differential gene expression. Nine endometrial RNA samples from healthy participants were subjected to microarray analysis and 15 samples were used for quantitative real-time polymerase chain reaction. The samples were divided into early, mid, or late proliferative phase. The early proliferative phase showed higher expression of genes including transforming growth factor β2, chemokine (C-C motif) ligand 18 (CCL18), and metallothionein 2A. The mid-proliferative phase was characterized by higher expression of heat shock proteins and implantation-associated genes including Indian hedgehog, secreted frizzled protein 4, and progesterone receptor. In the late proliferative phase, we identified increased angiotensin II receptor, type 2 and large decrease in expression of genes related to natural killer (NK) cell function. We demonstrate a unique gene expression signature at distinct time points within the proliferative phase. The early proliferative phase is characterized by tissue remodeling, angiogenesis, and modulation of inflammation; the mid-proliferative phase is characterized not only by proliferation in response to estrogens but also marks the onset of expression of genes required for endometrial receptivity and a dampening of estrogen responsiveness. In the late proliferative phase, changes in immune function and NK cells predominate. The proliferative phase is not simply a uniform period of estrogen responsive endometrial growth that can be considered as a single experimental time point when evaluating endometrial development; rather the proliferative phase is complex with differing functions and patterns of gene expression.     

雌、孕激素对人子宫内膜Pbx2蛋白表达的影响

目的:探讨人子宫内膜腺上皮细胞和基质细胞中雌、孕激素对Pbx2蛋白表达的影响。方法:采用免疫组织化学链霉菌抗生物素蛋白-过氧化物酶(SP)连接法检测人增生期、分泌中期和蜕膜期子宫内膜中Pbx2的表达和分布;体外培养人子宫内膜基质细胞和高分化子宫内膜癌细胞Ishikawa,分别加入雌激素、孕激素、雌、孕激素联合刺激48 h,并以不加雌、孕激素的基质细胞核和Ishikawa细胞为对照组,采用免疫组织化学、免疫印迹法测定各种条件下Ishikawa细胞中Pbx2蛋白的表达。结果:①在人各期子宫内膜组织中,Pbx2表达于腺上皮细胞和基质细胞的细胞核中;Pbx2在分泌中期和蜕膜期基质细胞中的表达显著高于增生期,但在腺上皮细胞中增生期组织的表达高于分泌中期和蜕膜期,差异均具有统计学意义(P<0.05)。②Western blotting结果显示,在孕激素和雌、孕激素联合处理Ishikawa细胞组中,Pbx2的表达显著低于对照组(P<0.05),雌激素处理后Pbx2的表达与其他各组间的表达无显著性差异(P>0.05),在人子宫内膜基质细胞(ESC)中,Pbx2的表达在雌、孕激素处理各组间比较均无统计学差异(P>0.05)。结论:在人子宫内膜腺上皮Ishikawa细胞中孕激素对Pbx2的表达具有下调作用,在人子宫内膜基质细胞中,Pbx2的调控可能是非雌、孕激素依赖性的。     

Estrogen and progesterone receptors and cyclooxygenase-2 expression in endometrial cancer, endometrial hyperplasia, and normal endometrium

OBJECTIVES: To determine whether cyclooxygenase-2 (COX-2) expression is seen in endometrial cancer, endometrial hyperplasia, and normal endometria and whether it correlates with expression of estrogen and progesterone receptors. METHODS: The study was a retrospective, IRB-approved analysis of biopsy samples from 14 patients with endometrial adenocarcinoma, 19 with endometrial hyperplasias, and 10 with normal endometrium. Excluded were samples from women with a history of pelvic radiation, NSAID use, or treatment with hormones during previous year. Immunohistochemical analyses were performed on formalin-fixed, paraffin-embedded tissues. Expression of COX-2, estrogen and progesterone receptors were scored according to the proportion of positive-staining cells: 1(+), <10%; 2(+), 10-50%; and 3(+), >50%. A score > or =2(+) was considered positive. Fisher's exact test and analysis of variance were used to compare proportions and continuous variables, respectively. RESULTS: Overexpression of COX-2 was seen in 4 (29%) of the endometrial cancers, 6 (32%) of the endometrial hyperplasia, and 4 (20%) of the normal endometria. These differences were not statistically significant (P = 0.90). No COX-2 expression was found in stromal tissue. Of 14 endometrial cancers, 7 (50%) expressed any COX-2, with 4 (29%) having an expression score of > or =2(+). Of 19 endometrial hyperplasias, 11 (58%) expressed any COX-2; with 6 (32%) having a score of > or =2(+). All 10 normal endometria showed only 1(+) expression. No significant differences were detected in COX-2 expression by grade or stage of cancer. Although 100% and 95% of both hyperplasia and normal endometrium samples expressed in estrogen and progesterone receptors, respectively, only 71% and 79% of endometrial cancers expressed estrogen and progesterone receptors (P = 0.01). A nonparametric trend was performed to detect a relationship, between COX-2 and estrogen receptor or progesterone receptor expression; no significant trend was found. CONCLUSIONS: In this study, the immunohistochemical analysis showed a trend toward increased COX-2 expression in endometrial cancer and hyperplasia compared to normal endometria. A larger sample size is needed to confirm these results. The increased COX-2 expression in hyperplasia may signify an early step in carcinogenesis. These findings may represent an important treatment opportunity for synergism in the hormonal therapy of endometrial cancer.     

High throughput silencing identifies novel genes in endometrioid endometrial cancer

Objective

To validate the gene expression profile obtained from the previous microarray analysis and to further study the biological functions of these genes in endometrial cancer. From our previous study, we identified 621 differentially expressed genes in laser-captured microdissected endometrioid endometrial cancer as compared to normal endometrial cells. Among these genes, 146 were significantly up-regulated in endometrial cancer.