Expression of EGF signaling pathway downstream proteins in RCC cells after ERβ changed.

<p>(A) Expression of EGF signaling pathway downstream proteins in 786-O after siERβ transfection. (B) Expression of EGF signaling pathway downstream proteins in A498 after ERβ overexpression.</p

Effect of ERβ downregulation on the cell growth rate.

<p>(A) Western blot results after transfection of siERβ (siERβ1, siERβ2, siERβ3, or siERβ123) or scramble control (nonsense siRNA sequence that would not affect ERβ expression). Cells transfected with siERβ showed ERβ protein downregulation. (B) mRNA expression reduced after 786-O transfection with different concentrations of siERβ. (C) Cell growth of cells transfected with scramble control or siERβ after estrogen stimulation (10 nM) was observed by MTT assay.</p

Kaplan–Meier survival curve: the correlation between ERβ expression and RCC prognosis, assessed by univariate survival analysis.

<p>(A) Correlation analysis of ERβ expression and the overall survival of RCC (B) Correlation analysis of ERβ expression and DFS of RCC.</p

Expression of apoptotic signaling pathway downstream proteins in 786-O after ERβ downregulation and in A498 after ERβ overexpression.

<p>Expression of apoptotic signaling pathway downstream proteins in 786-O after ERβ downregulation and in A498 after ERβ overexpression.</p

Correlation between the risk factors of RCC and the expression of ERβ.

*<p>Categorized as low (≤mean) and high (>mean) was separated at ERβ >35% positivity (high) and ERβ ≤35% positivity (low).</p>†<p>Based on the logistic regression model. Statistical significance (p<0.05) is shown in boldface. n.a.: not analyzed.</p><p>Abbrev: RCC: renal cell carcinoma, BRD: benign renal disease, OR: odds ratio, CI: confidence interval; ER: estrogen receptor.</p

Estrogen Inhibits Renal Cell Carcinoma Cell Progression through Estrogen Receptor-β Activation

<div><p>Renal cell carcinoma (RCC) originates in the lining of the proximal convoluted tubule and accounts for approximately 3% of adult malignancies. The RCC incidence rate increases annually and is twofold higher in males than in females. Female hormones such as estrogen may play important roles during RCC carcinogenesis and result in significantly different incidence rates between males and females. In this study, we found that estrogen receptor β (ERβ) was more highly expressed in RCC cell lines (A498, RCC-1, 786-O, ACHN, and Caki-1) than in breast cancer cell lines (MCF-7 and HBL-100); however, no androgen receptor (AR) or estrogen receptor α (ERα) could be detected by western blot. In addition, proliferation of RCC cell lines was significantly decreased after estrogen (17-β-estradiol, E2) treatment. Since ERβ had been documented to be a potential tumor suppressor gene, we hypothesized that estrogen activates ERβ tumor suppressive function, which leads to different RCC incidence rates between males and females. We found that estrogen treatment inhibited cell proliferation, migration, invasion, and increased apoptosis of 786-O (high endogenous ERβ), and ERβ siRNA-induced silencing attenuated the estrogen-induced effects. Otherwise, ectopic ERβ expression in A498 (low endogenous ERβ) increased estrogen sensitivity and thus inhibited cell proliferation, migration, invasion, and increased apoptosis. Analysis of the molecular mechanisms revealed that estrogen-activated ERβ not only remarkably reduced growth hormone downstream signaling activation of the AKT, ERK, and JAK signaling pathways but also increased apoptotic cascade activation. In conclusion, this study found that estrogen-activated ERβ acts as a tumor suppressor. It may explain the different RCC incidence rates between males and females. Furthermore, it implies that ERβ may be a useful prognostic marker for RCC progression and a novel developmental direction for RCC treatment improvement.</p> </div

Firgure 6. Change in invasion ability after ERβ downregulation or overexpression.

<p>(A) After transfection with siERβ or pcDNA3.1-ERβ, the cells passing through the Transwell to the lower membrane were observed. (B) (C) Quantification of cell numbers that passed through the Transwell.</p

Effect of estrogen on cell growth in the RCC cell line.

<p>In the CC cell lines 786-O, RCC-1, Caki-1, and ACHN, cell growth slowed down in the cells treated with estrogen (10 nM) compared with the control (ethanol). Only A498 was not affected by estrogen treatment. The experiments were repeated at least three times.</p

Univariate and multivariate analyses of prognostic factors and RCC survival.

*<p>Categorized as low (≤mean) and high (>mean) was separated at ERβ >22% positivity (high) and ERβ ≤22% positivity (low).</p>†<p>Analyzed with the Cox hazard regression model. Statistical significance (p<0.05) is shown in boldface. n.a.: not analyzed.</p><p>Abbrev: RCC: renal cell carcinoma, OR: odds ratio, CI: confidence interval, OS: overall survival, DFS: disease-free survival, ER: estrogen receptor.</p

Change in migration ability after ERβ downregulation or overexpression.

<p>(A) After transfection with siERβ or pcDNA3.1-ERβ, the cells passing through the Transwell to the lower membrane were observed. (B) (C) Quantification of cells numbers that passed through the Transwell.</p