Expressions And Regulation Analysis Among PPARγ, ERα And ERβ In Endometrial Carcinoma

ObjectiveEndometrial carcinoma (EC) is one of the most common gynecologic malignant tumors worldwide and is increasing in frequency. In some developed countries, it has become the first gynecologic malignancy. Approximately70-80%of sporadic endometrial carcinomas are distinguished as type Ⅰ carcinomas. Type Ⅰ EC is considered to be an estrogen dependent cancer and is associated with endometrial hyperplasia, hyperestrogenism, and expression of estrogen receptor (ER). However, hormone therapy on the basis of ER antagonism is not effective for all patients of ER-positive EC. Thus, we speculate that there may be other signaling pathways play important roles on EC tumorigenesis and tumor progression.Peroxisome proliferator-activated receptor gamma (PPARy) is a member of the superfamily of nuclear receptors. PPARy is important in lipid and glucose metabolism, adipose differentiation, inflammatory responses, macrophage differentiation, and energy homeostasis. In addition, the known risk factors for EC include obesity, type II diabetes mellitus and hypertension, thus aberrant PPARy expression may be the important molecular mechanism for EC carcinogenesis. Endometrial carcinoma is representative of hormone dependent gynecologic cancers. However, attempts to treat female hormone dependent cancers with anti-hormonal treatments have not been effective, except in early stage cancers. The estrogen receptors (ERs) are ligand-dependent transcription factors and belong to a superfamily of steroid nuclear receptors. Estrogen receptors (ERα and ERβ), which mediate estrogen actions, regulate cell growth and differentiation of a variety of normal tissues and hormone-responsive tumors through interaction with cellular factors. The complex biological effects mediated by ERa and ERβ involve communication between many proteins and signaling pathways. Recently, an increasing physiologic significance has been attributed to the crosstalk among nuclear receptors, which was observed at several levels of the signal transduction cascades. PPARy and ERs belong to a family of nuclear hormone receptors that were shown to affect transcriptional activity of each other. Despite these efforts, the exact interactions of PPARy, ERa and ERβ in endometrial carcinoma remain obscure. To elucidate the possible mechanism of this process, we therefore evaluated PPARy, ERa and ERv expressions in endometrial carcinomas and analyzed the correlations among them, to explore the possible mechanism related to EC, furthermore, to find out a new target for treatment of endometrial carcinoma.MethodsWe collected45endometrial adenocarcinomas (including14well differentiated adenocarcinomas,17moderately differentiated adenocarcinomas,14poorly differentiated adenocarcinomas) and13normal endometrium controls from surgical pathology specimens. Immunohistochemical method and western blot method were used to detect the expression of PPARy, ERa, ERβ in normal endometrial, well differentiated endometrial carcinoma, moderately differentiated endometrial carcinoma, and poorly differentiated endometrial carcinoma. The correlations among the expressions of these three different proteins were analyzed using Pearson (homogeneity of variance) correlation analysis method. Using ECC-1and KLE cells as model systems, we did transient transfection using PPARy expression vector (PPARy expression vector group) and PPARy small interference RNA (siRNA) to up or down regulate PPARy expression, and to detect its effect on the expression of ERa and ERβ. Then qRT-PCR and western blot were used to show mRNA and protein level changes after cell transfection. As a biological counterpart, we further examined the effects on cell migration, invasion and proliferation through in vitro transwell invasion assay and CCK-8assay. Then, we did transient transfection using ERa and ERβ expression vectors to up-regulate ERa and ERβ expression. Their modulatory role in the PI3K/AKT/mTOR pathway was evaluated by determining phosphorylation levels of PI3K p85a and key proteins. The results are expressed as the mean±standard deviation (SD) of at least three independent experiments. Two group comparisons were performed with Student’s t-test and multiple group comparisons were performed using one-way ANOVA. Small sample group comparisons were performed using nonparametric Wilcoxon Mann-Whitney test. Differences in P values of<0.05were considered statistically significant. All calculations were performed using SPSS17.0software.Results1. Expression of PPARy was significantly lower in endometrial carcinoma than in normal endometrium (P<0.05), and intimately associated with clinicopathologic variables (P<0.05). Expression of ERa was gradually reduced in moderately differentiated and poorly differentiated endometrial carcinoma (P<0.05). However, no significant differences were detected between normal endometrium and well differentiated endometrial carcinoma (P>0.05). Expression of ERβ was only decreased in poorly differentiated endometrial carcinoma (P<0.05) and no significant associations were detected between ERβ and clinicopathologic variables (P>0.05).2. Pearson correlation analysis showed that PPARy expression was positive significantly correlated with ERa expression (P<0.05). There were no correlations between the expression of ERa and ERβ, ERγ and PPARy (P>0.05).3. Up-regulating PPARy expression associated with a decreased ERa expression, and down-regulating PPARy expression associated with an increased ERa expression. In vitro migration and invasion assay indicated that the migratory and invasive cell numbers of PPARy expression group were significantly decreased while the migratory and invasive cell numbers of were PPARy siRNA group increased significantly compared with control groups (P<0.05). CCK-8assay showed that cell viability in PPARy expression vector group was lower than control groups, and in PPARy siRNA group, cell viability was higher than control groups, the differences were statistically significant (P<0.05). However, regulating PPARy expression had no obvious effects on ERp expression. 4. Up-regulating ERa expression in EC cells enhanced PI3K activity. Western blot showed that the protein levels of p-p85a, p-AKT and p-mTOR were increased significantly compared with control groups (P<0.05). Of note, no similar effects were noticeable between ERβ and PI3K activity (P>0.05).Conclusions1. Expressions of PPARy and ERa are significantly associated with clinicopathologic stage of EC. PPARy expression is negatively associated with ERa expression and PPARy may play essential roles in endometrial tumorigenesis and tumor progression through regulating ERa expression.2. Up-regulating PPARy expression can inhibit EC cell migration, invasion and proliferation abilities, and down-regulating PPARy expression can promote EC cell migration, invasion and proliferation abilities.3. Up-regulating ERa expression in EC cells can activate PI3K/AKT/mTOR signaling pathway and may be a possible mechanism for ERa regulates the capability of cell invasion and proliferation in EC.4. Regulating PPARy and ERa expression may set new development of novel therapeutic method in EC treatment.