Progesterone inhibits epithelial-to-mesenchymal transition in endometrial cancer

Background

Every year approximately 74,000 women die of endometrial cancer, mainly due to recurrent or metastatic disease. The presence of tumor infiltrating lymphocytes (TILs) as well as progesterone receptor (PR) positivity has been correlated with improved prognosis. This study describes two mechanisms by which progesterone inhibits metastatic spread of endometrial cancer: by stimulating T-cell infiltration and by inhibiting epithelial-to-mesenchymal cell transition (EMT).

Methodology and Principal Findings

Paraffin sections from patients with (n = 9) or without (n = 9) progressive endometrial cancer (recurrent or metastatic disease) were assessed for the presence of CD4+ (helper), CD8+ (cytotoxic) and Foxp3+ (regulatory) T-lymphocytes and PR expression. Progressive disease was observed to be associated with significant loss of TILs and loss of PR expression. Frozen tumor samples, used for genome-wide expression analysis, showed significant regulation of pathways involved in immunesurveillance, EMT and metastasis. For a number of genes, such as CXCL14, DKK1, DKK4, PEG10 and WIF1, quantitive RT-PCR was performed to verify up- or downregulation in progressive disease. To corroborate the role of progesterone in regulating invasion, Ishikawa(IK) endometrial cancer cell lines stably transfected with PRA (IKPRA), PRB(IKPRB) and PRA+PRB (IKPRAB) were cultured in presence/absence of progesterone (MPA) and used for genome-wide expression analysis, Boyden- and wound healing migration assays, and IHC for known EMT markers. IKPRB and IKPRAB cell lines showed MPA induced inhibition of migration and loss of the mesenchymal marker vimentin at the invasive front of the wound healing assay. Furthermore, pathway analysis of significantly MPA regulated genes showed significant down regulation of important pathways involved in EMT, immunesuppression and metastasis: such as IL6-, TGF-β and Wnt/β-catenin signaling.

Conclusion

Intact progesterone signaling in non-progressive endometrial cancer seems to be an important factor stimulating immunosurveilance and inhibiting transition from an epithelial to a more mesenchymal, more invasive phenotype.     

Nuclear progesterone receptor A and B isoforms in mouse fallopian tube and uterus: implications for expression, regulation, and cellular function

Progesterone and its interaction with nuclear progesterone receptors (PR) PR-A and PR-B play a critical role in the regulation of female reproductive function in all mammals. However, our knowledge of the regulation and possible cellular function of PR protein isoforms in the fallopian tube and uterus in vivo is still very limited. In the present study, we revealed that equine chorionic gonadotropin (eCG) treatment resulted in a time-dependent increase in expression of both isoforms, reaching a maximal level at 48 h in the fallopian tube. Regulation of PR-A protein expression paralleled that of PR-B protein expression. However, in the uterus PR-B protein levels increased and peaked earlier than PR-A protein levels after eCG treatment. With prolonged exposure to eCG, PR-B protein levels decreased, whereas PR-A protein levels continued to increase. Furthermore, subsequent treatment with human (h)CG decreased the levels of PR protein isoforms in both tissues in parallel with increased endogenous serum progesterone levels. To further elucidate whether progesterone regulates PR protein isoforms, we demonstrated that a time-dependent treatment with progesterone (P(4)) decreased the expression of PR protein isoforms in both tissues, whereas decreases in p27, cyclin D(2), and proliferating cell nuclear antigen protein levels were observed only in the uterus. To define the potential PR-mediated effects on apoptosis, we demonstrated that the PR antagonist treatment increased the levels of PR protein isoforms, induced mitochondrial-associated apoptosis, and decreased in epidermal growth factor (EGF) and EGF receptor protein expression in both tissues. Interestingly, immunohistochemistry indicated that the induction of apoptosis by PR antagonists was predominant in the epithelium, whereas increase in PR protein expression was observed in stromal cells of both tissues. Taken together, these observations suggest that 1) the tissue-specific and hormonal regulation of PR isoform expression in mouse fallopian tube and uterus, where they are potentially involved in regulation of mitochondrial-mediated apoptosis depending on the cellular compartment; and 2) a possible interaction between functional PR protein and growth factor signaling may have a coordinated role for regulating apoptotic process in both tissues in vivo.     

Transforming growth factor-beta1 attenuates expression of both the progesterone receptor and Dickkopf in differentiated human endometrial stromal cells

TGFbeta1 is thought to be intimately involved in cyclic tissue remodeling and inflammatory events associated with menstruation. Menstruation is initiated by progesterone withdrawal; however, the underlying mechanisms are not well understood. In the present study, we have tested the hypothesis that locally produced TGFbeta1 may influence expression of progesterone receptor (PR) or the Wnt antagonist Dickkopf-1 (DKK) with consequential impact on regulation of menstruation. Endometrial stromal cells (ESC) were isolated from endometrial biopsy samples collected from patients undergoing gynecological procedures for benign indications. Treatment of differentiated ESC with TGFbeta1 (10 ng/ml) significantly inhibited the expression of mRNAs encoding PR and DKK. TGFbeta1 also attenuated the protein expression of PR and secretion of DKK proteins in culture supernatants. Neutralization of endogenous TGFbeta1 signaling abolished the TGFbeta1-induced effects, significantly increased expression of PR, and increased DKK protein release levels to that of differentiated ESCs, confirming the specificity of the TGFbeta1 effect. Additionally, in vitro decidualization of ESCs significantly augmented DKK protein release. Moreover, although TGFbeta1 was capable of signaling via the Sma- and mothers against decapentaplegic (MAD)-related protein (SMAD) pathway, the inhibitory effect on DKK was SMAD independent. Conversely, the inhibitory effect of TGFbeta1 on PR was dependent on SMAD signal transduction. In conclusion, these results suggest that local TGFbeta1 signaling can potentiate progesterone withdrawal by suppressing expression of PR and may coordinate tissue remodeling associated with menstruation by inducing Wnt-signaling via inhibition of DKK, which we found to be up-regulated as a consequence of decidualization of ESCs.     

Regulation of progesterone receptors and decidualization in uterine stroma of the estrogen receptor-alpha knockout mouse

Regulation of progesterone receptor (PR) in uterine stroma (endometrial stroma plus myometrium) by estrogen was investigated in estrogen receptor-alpha (ERalpha) knockout (alphaERKO) mice. 17 beta-Estradiol (E(2)) increased PR levels in uterine stroma of ovariectomized alphaERKO mice, and ICI 182 780 (ICI) inhibited this E(2)-induced PR expression. Estrogen receptor-beta(ER beta) was detected in both uterine epithelium and stroma of wild-type and alphaERKO mice by immunohistochemistry. In organ cultures of alphaERKO uterus, both E(2) and diethylstilbestrol induced stromal PR, and ICI inhibited this induction. These findings suggest that estrogen induces stromal PR via ERbeta in alphaERKO uterus. However, this process is not mediated exclusively by ERbeta+, because in ERbeta knockout mice, which express ERalpha, PR was up-regulated by E(2) in uterine stroma. In both wild-type and alphaERKO mice, progesterone and mechanical traumatization were essential and sufficient to induce decidual cells, even though E(2) and ERalpha were also required for increase in uterine weight. Progesterone receptor was strongly expressed in decidual cells in alphaERKO mice, and ICI did not inhibit decidualization or PR expression. This study suggests that up-regulation of PR in endometrial stroma is mediated through at least three mechanisms: 1) classical estrogen signaling through ERalpha, 2) estrogen signaling through ERbeta, and 3) as a result of mechanical stimulation plus progesterone, which induces stromal cells to differentiate into decidual cells. Each of these pathways can function independently of the others.     

Ontogenic variations in the content and distribution of progesterone receptor isoforms in the reproductive tract and brain of chicks

Progesterone participates in the regulation of several functions in chicks such as ovulation, gonadal differentiation, and sexual and nesting behaviors. Many progesterone actions are mediated by specific intracellular receptors (PR) which are ligand-induced transactivators. Two PR isoforms that are functionally distinct in their ability to activate genes and regulate distinct physiological processes have been described in chicks: a full length form PR-B and the N-terminally truncated one PR-A which lacks the amino-terminal 128 amino acids of PR-B. PR isoforms have been detected in several tissues of both the adult and the embryo chick such as brain, ovary and oviduct. PR isoforms expression ratio varies among progesterone target tissues and under different hormonal and environmental conditions such as those presented during avian sexual maturity and the seasons of the year. These data let us to conclude that progesterone actions in brain, ovary, and oviduct highly depend on PR isoforms expression pattern and regulation.     

雌、孕激素受体及C-erbB2在子宫内膜癌中的表达和意义

目的探讨雌、孕激素受体（Estrogen Receptor ER、Progesterone Receptor PR）及C-erbB2在子宫内膜癌中的表达与临床病理参数的关系。方法采用免疫组织化学技术检测56例子宫内膜癌患者子宫内膜中ER、PR及C-erbB2的表达,并对56例患者的临床病理资料进行回顾性分析。结果子宫内膜癌患者内膜ER、PR及C-erbB2阳性率分别为41.07%、57.14%、53.57%。子宫内膜癌中ER表达与肿瘤分化程度、年龄、有无淋巴结转移呈负相关;PR表达与肿瘤组织学类型、分化程度、年龄、浸润肌层深度及有无淋巴结转移均呈负相关;而C-erbB2的表达与内膜癌分化程度、有无淋巴结转移呈正相关。结论激素依赖型子宫内膜癌ER、PR及C-erbB2表达水平均反映了子宫内膜癌的生物学行为,三者的测定对估计预后和指导术后辅助治疗具有重要意义。     

Effects of metformin on the expression of GLUT4 in endometrium of obese women with polycystic ovary syndrome

The objective was to explore the effects of metformin on the expression of endometrial glucose transporter 4 (GLUT4) and analyze the related factors of GLUT4 in patients with polycystic ovary syndrome (PCOS). This study included 20 obese patients with PCOS (PCOS group) and 20 obese patients who had infertility caused by oviducal or pelvic factors but had no PCOS (control group). Follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol-2 (E(2)), testosterone (T), fasting serum glucose (FSG), fasting insulin serum (FINS), homeostasis model assessment-insulin resistance (HOMA-IR), and endometrial GLUT4 expression were determined in the two groups. In PCOS group, patients were given 500 mg of metformin three times per day for 3 mo, and then the parameters above were determined again and compared with that before metformin treatment. The parameters above also were compared between PCOS and control groups. The correlation of GLUT4 with its related factors was analyzed. The levels of T, FINS, and HOMA-IR were higher in PCOS group than in the control group (P < 0.01). The levels of protein and mRNA of endometrial GLUT4 were lower in the PCOS group than in the control group (P < 0.001). The expression of protein and mRNA of endometrial GLUT4 increased after metformin treatment (P < 0.001). HOMA-IR was negatively correlated with GLUT4 expression (P = 0.027). In patients with PCOS, the levels of protein and mRNA of endometrial GLUT4 were lower compared with that in non-PCOS women, and HOMA-IR was strongly associated with endometrial GLUT4 expression. Metformin may up-regulate endometrial GLUT4 expression to improve endometrial IR.     

Expression and hormonal regulation of calcyclin-binding protein (CacyBP) in the mouse uterus during early pregnancy

Calcyclin-binding protein (Siah-1-Interacting Protein, CacyBP/SIP), is a calcium signaling protein involved in the degradation of beta-catenin, however, little is known about its role in reproductive biology. The present study was to character its temporospatial expression pattern and regulation in mouse uterus and to investigate whether it plays a role in the regulation of normal endometrial events. While prominently expressed in both luminal and glandular epithelia, CacyBP underwent dynamic changes during early pregnancy. CacyBP expression was observed weakly from days 1-4. An intense accumulation in luminal and glandular epithelia as well as decidua surrounding the embryo at later stages (days 5-7) was observed. Most notably, CacyBP accumulation in trophoblast was pronounced at day 7. Using ovariectomized and pseudopregnant mice, we found that progesterone (P(4)) and 17beta-estradiol (E(2)) led to increased expression of CacyBP gene and this could be abolished by Ru486 and tamoxifen, respectively. Antisense oligonucleotides (ODNs) against CacyBP significantly inhibited cultured endometrial stromal cells' (ESCs) apoptosis induced by UV irradiation. Injection of antisense ODNs into mouse uterine horn severely impaired the number of implanted blastocysts. Taken together, our results suggested that CacyBP expression was positively regulated by P(4) and E(2). CacyBP may be involved in the regulation of endometrial cell apoptosis during early pregnancy and play an important role in mouse endometrial events such as pregrancy establishment.