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

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

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

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

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

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

Estrogen Enhances the Cell Viability and Motility of Breast Cancer Cells through the ERα-ΔNp63-Integrin β4 Signaling Pathway

<div><p>Estrogen induces ERα-positive breast cancer aggressiveness via the promotion of cell proliferation and survival, the epithelial-mesenchymal transition, and stem-like properties. Integrin β4 signaling has been implicated in estrogen/ERα-induced tumorigenicity and anti-apoptosis; however, this signaling cascade poorly understood. ΔNp63, an N-terminally truncated isoform of the p63 transcription factor, functions as a transcription factor of integrinβ4 and therefore regulates cellular adhesion and survival. Therefore, the aim of the present study was to investigate the estrogen-induced interaction between ERα, ΔNp63 and integrin β4 in breast cancer cells. In ERα-positive MCF-7 cells, estrogen activated ERα transcription, which induced ΔNp63 expression. And ΔNp63 subsequently induced integrin β4 expression, which resulted in AKT phosphorylation and enhanced cell viability and motility. Conversely, there was no inductive effect of estrogen on ΔNp63-integrinβ4-AKT signaling or on cell viability and motility in ERα-negative MDA-MB-231 cells. ΔNp63 knockdown abolishes these estrogen-induced effects and reduces cell viability and motility in MCF-7 cells. Nevertheless, ΔNp63 knockdown also inhibited cell migration in MDA-MB-231 cells through reducing integrin β4 expression and AKT phosphorylation. In conclusion, estrogen enhances ERα-positive breast cancer cell viability and motility through activating the ERα-ΔNp63-integrin β4 signaling pathway to induce AKT phosphorylated activation. Those findings should be useful to elucidate the crosstalk between estrogen/ER signaling and ΔNp63 signaling and provide novel insights into the effects of estrogen on breast cancer progression.</p></div