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

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

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

Effects of estrogen on ΔNp63 and TAp63 expression in MCF-7 and MDA-MB-231 cells.

<p>MCF-7 and MDA-MB-231 cells were cultured in 10% FBS/DMEM with 10nM estrogen or 0.1% ethanol for 0, 1, 2, 4, and 8 h, and protein and mRNA expression was detected by (A) western blotting and real-time RT-PCR for (B) ΔNp63 and (C) TAp63. In MCF-7, ΔNp63 expression peaked at 2 h after estrogen treatment at both the protein and mRNA levels, and estrogen treatment did not significantly affect TAp63 protein and mRNA expression levels. In MDA-MB-231, estrogen treatment did not affect ΔNp63 and TAp63 expression in both protein and mRNA levels. All data are the mean ± SD of triplicate experiments.</p

Pseudopodium patterns of co-treatment of estrogen and ΔNp63 knockdown.

<p>After siΔNp63 or scrambled siRNA was transfected into MCF-7 cells for 48 h, the cells were co-treated with 10nM estrogen or 0.1% ethanol. A wound-healing assay was conducted to compare the pseudopodium patterns at 24 h. Only the cells treated with 10nM estrogen showed protruding pseudopodia, and siΔNp63 reduced the pseudopodium patterns, regardless of the presence or absence of estrogen.</p

The combined effects of estrogen and ΔNp63 knockdown on cell motility.

<p>(A, B) After siΔNp63 or scrambled siRNA transfected into MCF-7 and MDA-MB-231 cells for 48 h, cells were co-treated with 10nM estrogen or 0.1% ethanol and then subjected to a wound-healing assay. Wound widths were recorded at 0, 24, and 48 h. Compared with the scrambled siRNA and 0.1% ethanol co-treated controls, the wounds of MCF-7cells co-treated with scrambled siRNA and 10nM estrogen were completely healed, but siΔNp63 reduced MCF-7 cell healing, regardless of the presence or absence of estrogen. In MDA-MB-231, wounds were completely healed at 48 h irrespective of treatment with 10nM estrogen or 0.1% ethanol, and siΔNp63 also reduced MDA-MB-231 cell healing regardless of the presence or absence estrogen. Statistical analysis was performed in four groups, 0.1% ethanol with scrambled siRNA (-/-), 0.1% ethanol with siΔNp63 (-/+), 10nM estrogen with scrambled siRNA (+/-), and 10nM estrogen with siΔNp63 (+/+). (C, D) After the transfected cells were co-treated with 10nM estrogen or 0.1% ethanol for 24 h, similar results were observed in a transwell assay. In MCF-7 cells, estrogen treatment induced the highest cell migratory effect, but siΔNp63 reduced cell migratory ability, regardless of the presence or absence of estrogen. And in MDA-MB-231 cells, estrogen treatment conferred no inductive effect on cell migration, and siΔNp63 also reduced cell migratory ability, regardless of the presence or absence of estrogen. All data are the mean ± SD of triplicate experiments.</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

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