Estrogen-dependent Endometrial Cancer Research Articles

MiR-196a-5p facilitates progression of estrogen-dependent endometrial cancer by regulating FOXO1.

Estrogen-dependent endometrial cancer mainly occurs in younger pre-menopausal and post-menopausal women and threatens their health. Recently, microRNAs (miRNAs) have been considered as novel targets in endometrial cancer treatment. Therefore, we aimed to explore the effect of miRNA (miR)-196a-5p in estrogen-dependent endometrial cancer. 17β-estradiol (E2; 2.5, 5, 10 and 20 nM) was used to treat RL95-2, HEC-1B and ECC-1 cells followed by cell viability assessment using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The level of miR-196a-5p was measured by reverse transcription-quantitative PCR (RT-qPCR). We then transfected miR-196a-5p mimic/inhibitor and Forkhead box protein O1 (FOXO1) small interfering RNA (siRNA) into E2-treated cells. Apoptotic cells were measured by flow cytometry. Wound healing and Transwell assays were implemented to assess migration and invasion. Bioinformatics and luciferase reporter assays were applied to confirm the interaction between miR-196a-5p and FOXO1. Immunoblotting determined the levels of FOXO1, Bcl-2, Bax, Caspase 3. E2 promoted cell viability and miR-196a-5p expression in RL95-2 and ECC-1 cells. miR-196a-5p mimic enhanced cell viability, migration and invasion but suppressed apoptosis and FOXO1, whilst miR-196a-5p inhibitor blocked these processes. In addition, miR-196a-5p upregulated Bcl-2, but down regulated Bax and Caspase 3 expression, an effect that was reversed by miR-196a-5p inhibitor. We determined that miR-196a-5p targeted FOXO1, and that si-FOXO1 blocked the effects of miR-196a-5p inhibitor on viability, apoptosis, migration and invasion of E2-treated RL95-2 and ECC-1 cells. Our findings suggested potential diagnostic and therapeutic applications for miR-196a-5p and its FOXO1 target in patients suffering from estrogen-dependent endometrial cancer.

Magnetic resonance spectroscopy associations with clinicopathologic features of estrogen-dependent endometrial cancer

BackgroundWe studied the magnetic resonance spectroscopy (MRS) associations with clinicopathologic features of estrogen-dependent endometrial cancer (type I EC).MethodsTotally 45 patients with type I EC who underwent preoperative multi-voxel MRS at 3.0 T were enrolled. The mean ratio of the Cho peak integral to the unsuppressed water peak integral (Cho/water) of the tumor was calculated. The Cho/water and apparent diffusion coefficient (ADC) of type I EC with and without local invasion, as well as with different levels of Ki-67 staining index (SI) (≤ 40% and > 40%), were compared. Correlation test was used to examine the relationship of Cho/water, as well as mean ADC, with Ki-67 SI, tumor stage, and tumor grade.ResultsThe mean Cho/water of EC with Ki-67 SI ≤ 40% (2.28 ± 0.93) × 10−3 was lower than that with Ki-67 SI > 40% (4.08 ± 1.00) × 10−3 (P < 0.001). The mean Cho/water of EC with deep and superficial myometrial invasion was (3.41 ± 1.26) × 10−3 and (2.43 ± 1.11) × 10−3, respectively (P = 0.011). There was no significant difference in Cho/water between type I EC with and without cervical invasioin ([2.68 ± 1.00] × 10−3 and [2.77 ± 1.28] × 10−3, P = 0.866). The mean Cho/water of type I EC with and without lymph node metastasis was (4.02 ± 1.90) × 10−3 and (2.60 ± 1.06) × 10−3, respectively (P = 0.014). The Cho/water was positively correlated with the Ki-67 SI (r = 0.701, P < 0.001). There were no significant differences in ADC among groups (all P > 0.05).ConclusionMRS is helpful for preoperative assessment of clinicopathological features of type I EC.

17β-Estradiol promotes endometrial cancer proliferation and invasion through IL-6 pathway.

Accumulating evidence revealed that the leading risk factor of endometrial cancer is exposure to endogenous and exogenous estrogens, while the exact mechanism underlying estrogen contribution to endometrial cancer progression has not been elucidated clearly. Interleukin (IL)-6 has been verified to be critical for tumor progression in several human cancers. In this study, we provided evidence that 17β-estradiol (E2) could significantly promote endometrial cancer cells viability, migration and invasion through activation of IL-6 pathway, which involved in its downstream pathway and target genes (p-Stat3, Bcl-2, Mcl-1, cyclin D1 and MMP2). Meanwhile, utilization of IL-6-neutralizing antibody could partially attenuate the increased cancer growth and invasion abilities in Ishikawa and RL95-2 endometrial cancer cell lines and an orthotopic endometrial cancer model. We established a causative link between estrogen and IL-6 signaling activation in the development of endometrial cancer. The molecular mechanism defined in this study provided the evidence that E2 promotes endometrial carcinoma progression via activating the IL-6 pathway, indicating that interruption of IL-6 might be an essential therapeutic strategy in estrogen-dependent endometrial cancer.

Metformin inhibits estrogen-dependent endometrial cancer cell growth by activating the AMPK-FOXO1 signal pathway.

Metformin is an oral biguanide commonly used for treating type II diabetes and has recently been reported to possess antiproliferative properties that can be exploited for the prevention and treatment of a variety of cancers. The mechanisms underlying this effect have not been fully elucidated. Our study shows a marked loss of AMP‐activated protein kinase (AMPK) phosphorylation and nuclear human Forkhead box O1 (FOXO1) protein in estrogen‐dependent endometrial cancer (EC) tumors compared to normal control endometrium. Metformin treatment suppressed EC cell growth in a time‐dependent manner in vitro; this effect was cancelled by cotreatment with an AMPK inhibitor, compound C. Metformin decreased FOXO1 phosphorylation and increased FOXO1 nuclear localization in Ishikawa and HEC‐1B cells, with non‐significant increase in FOXO1 mRNA expression. Moreover, compound C blocked the metformin‐induced changes of FOXO1 and its phosphorylation protein, suggesting that metformin upregulated FOXO1 activity by AMPK activation. Similar results were obtained after treatment with insulin. In addition, transfection with siRNA for FOXO1 cancelled metformin‐inhibited cell growth, indicating that FOXO1 mediated metformin to inhibit EC cell proliferation. A xenograft mouse model further revealed that metformin suppressed HEC‐1B tumor growth, accompanied by downregulated ki‐67 and upregulated AMPK phosphorylation and nuclear FOXO1 protein. Taken together, these data provide a novel mechanism of antineoplastic effect for metformin through the regulation of FOXO1, and suggest that the AMPK–FOXO1 pathway may be a therapeutic target to the development of new antineoplastic drugs.

Perspectives on targeting the phosphatidylinositol 3-kinase pathway for personalized medicine in endometrial and ovarian cancers

Abstract Endometrial and ovarian cancers show similar genetic and pathological backgrounds. In particular, high frequencies of activating mutations in the phosphatidylinositol 3-kinase (PI3K) pathway, including mutations in PIK3CA and PTEN , are found in both estrogen-dependent endometrial cancer (type I endometrioid carcinomas) and ovarian clear cell and endometrioid carcinomas. In this review, we focus on the PI3K pathway as a potential molecular target for personalized therapies in endometrial and ovarian cancers. We found that targeting the PI3K/mammalian target of rapamycin (mTOR) pathway produced anti-tumor effects in endometrial cancer upon suppression of the PI3K pathway. The presence of KRAS mutations may be a marker for resistance to the inhibition of the PI3K/mTOR pathway. However, the combination of a PI3K/mTOR pathway inhibitor and a MAPK pathway inhibitor, such as a MEK inhibitor, has been shown to suppress cell proliferation synergistically in certain endometrial cancers. In addition, PI3K/mTOR pathway inhibition sensitized endometrial cancer cells to ionizing radiation and produced anti-tumor effects in ovarian clear cell carcinomas in both in vitro and in vivo studies. Moreover, inhibition reduced the phosphorylation levels of MDM2 (a negative regulator of TP53), stabilized TP53, and induced TP53-mediated apoptosis. The activation of TP53 was associated with increased phosphorylation of TP53 on Ser-46, and its downstream target genes, such as TP53AIP1 . These findings demonstrate that targeting of the PI3K pathway in both endometrial and ovarian clear cell carcinomas warrants further investigation, including in clinical trials.

Steroid Sulfatase Inhibitor Reduces Proliferation of Ishikawa Endometrial Cancer Cells in Co-Culture Systems

Objectives: Estrogens significantly contribute toward the growth and development of endometrial cancers. Two principal pathways have been implicated in the final steps of estrogen synthesis: the steroid sulfatase (STS) and aromatase pathways. In this study, we aimed to evaluate the possible effects of tumor-stromal interactions on local estrogen biosynthesis in endometrial cancer. We also assessed the biological effects of inhibitors of steroid sulfatase and aromatase in the co-culture system compared with usual monocultures. Methods/Materials: We isolated stromal cells from endometrial cancer patients to examine local biosynthesis of estrogens and tumor-stromal interactions. Next we examined the effects of steroid sulfatase inhibitor and aromatase inhibitor in monoculture of endometrial cancer cell line (Ishikawa) and in a co-culture system involving an Ishikawa cells and stromal cells. Results: Estrogen receptor and steroid sulfatase mRNA levels in cancer cells were significantly higher in the co-cultures compared with the monocultures of endometrial cancer cells. Estradiol and androstenediol concentrations were also significantly higher in the co-cultured cells. Proliferation of the cancer cells was significantly increased through the steroid sulfatase pathway, which metabolizes androgens, estrone sulfate, and estradiol sulfate as its substrates. However, its proliferation was significantly decreased by the treatment of steroid sulfatase or aromatase inhibitors. The significant growth inhibition by the steroid sulfatase and aromatase inhibitors were also observed in the co-culture system. Conclusions: We evaluated the effects of STS inhibitor and aromatase inhibitors on the proliferation of estrogen-dependent endometrial cancer cells. Considering that intratumoral estrogen metabolism plays an important role, our co-culture systems provide an environment similar to that of the tumor in living patients in terms of metabolism and synthesis of intratumoral estrogens. The results of this study may aid in achieving improved clinical responses from patients treated with STS inhibitors.

Critical Tumor Suppressor Function Mediated by Epithelial Mig-6 in Endometrial Cancer

Endometrial cancer is preceded by endometrial hyperplasia, unopposed estrogen exposure, and genetic alterations, but the precise causes of endometrial cancer remain uncertain. Mig-6, mainly known as a negative regulator of the EGF receptor, is an important mediator of progesterone signaling in the uterus, where it mediates tumor suppression by modulating endometrial stromal-epithelial communications. In this study, we investigated the function of Mig-6 in the uterine epithelium using a tissue-specific gene knockout strategy, in which floxed Mig-6 (Mig-6(f/f)) mice were crossed to Wnt7a-Cre mice (Wnt7a(cre+)Mig-6(f/f)). Wnt7a(cre+)Mig-6(f/f) mice developed endometrial hyperplasia and estrogen-dependent endometrial cancer, exhibiting increased proliferation in epithelial cells as well as apoptosis in subepithelial stromal cells. We documented increased expression of NOTCH1 and BIRC3 in epithelial cells of Wnt7a(cre+)Mig-6(f/f) mice and decreased expression of the progesterone receptor (PR) in stromal cells. Progesterone therapy controls endometrial growth and prevents endometrial cancer, but the effectiveness of progesterone as a treatment for women with endometrial cancer is less clear. We noted that the hyperplasic phenotype of Wnt7a(cre+)Mig-6(f/f) mice was prevented by progesterone treatment, whereas this treatment had no effect in PR(cre/+)Mig-6(f/f) mice where Mig-6 was deleted in both the epithelial and stromal compartments of the uterus. In contrast, activation of progesterone signaling in the stroma regulated proliferation and apoptosis in the epithelium via suppression of ERα signaling. In summary, our results establish that epithelial Mig-6 functions as a critical tumor suppressor that mediates the ability of progesterone to prevent the development of endometrial cancer.

MK2206, an orally active allosteric AKT inhibitor, reverses progestin resistance in endometrial cancer

Objective: Progestin resistance is a major obstacle to fertility sparing therapy in endometrial cancer. Recurrent endometrial cancer is often resistant to hormonal or chemotherapy. The mechanism behind the lack of response to progestin in some patients is not well understood, however, downregulation of the progesterone receptor (PR) is thought to play a role. The PTEN tumor suppressor gene is mutated in the majority of cases of estrogen dependent endometrial cancer and this mutation results in the hyperactivation of the PI3K/AKT pathway. The AKT pathway, which is involved in promoting cell survival, is known to play a major role in a wide spectrum of solid tumors. We evaluated the effect of MK2206, a novel allosteric inhibitor of AKT currently being used in Phase I trials of solid tumors, on progestin responsiveness in different models of endometrial cancer.

MicroRNA regulation of the expression of the estrogen receptor in endometrial cancer

microRNAs (miRNAs) are non-coding RNA transcripts thought to be instrumental in controlling eukaryotic cell function. This study aimed to identify miRNAs associated with the differential expression of estrogen receptor α (ERα) and progesterone receptor (PR) in uterine endometrial cancer. First, the pathology of Ishikawa and KLE cell lines was identified by transplant and biopsy. Expression levels of ERα and PR and their response to estrogen and progesterone were examined, and total RNA was isolated to identify differentially expressed miRNAs by microarray analysis. miRNAs targeting ESR1 and PGR were predicted by miRANDA and TargetScan software, and their expression levels in cell lines and in patient samples were validated by real-time RT-PCR. Ishikawa and KLE represent estrogen-dependent and estrogen-independent endometrial cancers, respectively. As identified by miRNA microarray, 126 miRNAs were differentially expressed between the two cell lines; hsa-miR-100 and hsa-miR-99a were predicted to target ESR1, and hsa-miR-378 and hsa-miR-768-3p to target PGR. The differential expression of these miRNAs between Ishikawa and KLE was consistent in vivo and in vitro. Hsa-miR-100 was significantly down-regulated in the estrogen-dependent endometrial cancer samples as compared to the estrogen-independent samples and thus has the potential to target ESR1.

Function of Estrogen-Related Receptor α in Human Endometrial Cancer

The estrogen-related receptor alpha (ERRalpha) is an orphan member of the nuclear receptor superfamily that is closely related to estrogen receptor alpha (ERalpha). ERRalpha binds an estrogen response element (ERE), directly competes with ERalpha for binding ERE, and represses ERE-dependent transcription in MCF-7 cells, ER-positive breast cancer cells. We investigated whether ERRalpha modulate some ER-dependent activities in endometrial cancer. We investigated protein and mRNA expression of ERRalpha in endometrial cancer using immunohistochemistry and RT-PCR, respectively. After transient transfection using the ERRalpha expression vector (pCI-ERRalpha) or ERRalphaSi, which suppressed the expression of endogenous ERRalpha, Ishikawa cells were assayed for ERE-dependent luciferase activity. Cells stably overexpressing ERRalpha were generated and compared with estrogen-dependent and -independent cell growth. ERRalpha was detected in human endometrial cancer tissues by immunohistochemistry. An RT-PCR study showed that mRNA of ERRalpha was expressed in four endometrial cancer cell lines (Ishikawa, Hec1a, KLE, and SNGII) and 11 human endometrial tissues. Overexpression of ERRalpha repressed estrogen-induced ERE-dependent transcriptional activity in Ishikawa cells. After transfection with ERRalphaSi1, the expression of endogenous ERRalpha decreased to 0.5-fold, and estrogen-induced ERE luciferase activity increased to 1.5-fold. The cells stably overexpressing ERRalpha grew up more slowly than control cells in the presence of 10 nm estradiol. ERRalpha is expressed in human endometrial cancer tissues and cell lines and suppresses ERE-dependent transcriptional activity in the presence of estrogen. ERRalpha modulates estrogen-induced activity in estrogen-dependent endometrial cancer.

Expression analysis of estrogen-metabolizing enzymes in human endometrial cancer

Estrogen-dependent endometrial cancer is related to unopposed and prolonged estrogen stimulation. We examined the expression of estrogen-metabolizing enzymes in correlation with the ERα and ERβ estrogen receptors in human endometrial Ishikawa adenocarcinoma cells and in endometrial cancer specimens and adjacent normal endometrium from the same patients. Real-time PCR analysis revealed that both estrogen receptors and selected estrogen-metabolizing enzymes were expressed in the Ishikawa cells and in endometrial tissue. We detected higher expression of ERα than ERβ, higher expression of sulfatase than sulfotransferase and low expression of aromatase in the Ishikawa cells and the tissue, as well as higher levels of type 2 17β-hydroxysteroid dehydrogenase (17β-HSD) in normal and diseased tissue than in the Ishikawa cells. When we compared the expression in endometrial cancer samples and in the adjacent normal endometrium, ERα and ERβ, sulfatase and sulfotransferase were seen to be downregulated in the majority of the cancerous tissue specimens.