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

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

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

Expression of AR, ERα, and ERβ in breast cancer and RCC cell lines, and the expression of ERα and ERβ in kidney, RCC, and breast cancer tissue samples.

<p>(A) (B) In breast cancer cell lines, MCF-7 showed AR, ERα, and ERβ expression, while HBL100 showed no ERα expression and only low ERβ expression. In RCC cell lines, no AR expression was observed. Except A498, the RCC cell lines showed higher ERβ expression than the breast cancer cell lines. Expression of ERβ was the highest in 786-O. (C) IHC results for the observation of ERα and ERβ expression in kidney, RCC, and breast cancer tissue samples. (C-a) Cytoplasm and nuclei of the kidney tissue showed high expression of ERβ. (C-b) In RCC tissue, ERβ expression was mainly found in the cytoplasm. (C-c) Low expression of ERβ was observed in the cytoplasm of breast cancer tissue. (C-d) (C-e) No ERα expression was found in the kidney or RCC tissue. (C-f) ERα expressed in the nuclei of breast cancer tissue. Densitometry analyses for protein quantification were done using Image J 1.46x software (<a href="http://rsb.info.nih.gov/ij/download.html" target="_blank">http://rsb.info.nih.gov/ij/download.html</a>). The experiments were repeated at least three times. MCF7 expression was the baseline expression for the quantification comparison of ER. The results were analyzed with t-test.</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

WNT10A Plays an Oncogenic Role in Renal Cell Carcinoma by Activating WNT/β-catenin Pathway

<div><p>Renal cell carcinoma (RCC) is a malignancy with poor prognosis. WNT/β-catenin signaling dysregulation, especially β-catenin overactivation and WNT antagonist silencing, is associated with RCC carcinogenesis and progression. However, the role of WNT ligands in RCC has not yet been determined. We screened 19 WNT ligands from normal kidney and RCC cell lines and tissues and found that WNT10A was significantly increased in RCC cell lines and tissues as compared to that in normal controls. The clinical significance of increase in WNT10A was evaluated by performing an immunohistochemical association study in a 19-year follow-up cohort comprising 284 RCC and 267 benign renal disease (BRD) patients. The results of this study showed that WNT10A was dramatically upregulated in RCC tissues as compared to that in BRD tissues. This result suggests that WNT10A, nuclear β-catenin, and nuclear cyclin D1 act as independent risk factors for RCC carcinogenesis and progression, with accumulative risk effects. Molecular validation of cell line models with gain- or loss-of-function designs showed that forced WNT10A expression induced RCC cell proliferation and aggressiveness, including higher chemoresistance, cell migration, invasiveness, and cell transformation, due to the activation of β-catenin-dependent signaling. Conversely, WNT10A siRNA knockdown decreased cell proliferation and aggressiveness of RCC cells. In conclusion, we showed that WNT10A acts as an autocrine oncogene both in RCC carcinogenesis and progression by activating WNT/β-catenin signaling.</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

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