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

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

The combined effects of estrogen and ΔNp63 knockdown on integrin β4 expression and AKT activation.

<p>(A) After MCF-7 and MDA-MB-231 cells were treated with 10nM estrogen or 0.1% ethanol for 0, 1, 2, 4, and 8 h, integrin β4 protein was detected by western blotting. After estrogen treatment, integrin β4 peaked at 2 h in MCF-7 cells but revealed similar levels among 5 time points in MDA-MB-231 cells. (B) After siΔNp63 or scrambled siRNA were transfected into MCF-7 and MDA-MB-231 cells for 48 h, siΔNp63 specifically inhibited the protein expression of ΔNp63 but not TAp63. (C) After siΔNp63 or scrambled siRNA were transfected into MCF-7 and MDA-MB-231 cells for 48 h, cells were co-treated with 10nM estrogen or 0.1% ethanol for 2 h. In MCF-7 cells, estrogen induced integrin β4 expression and AKT activation, whereas siΔNp63 reduced integrin β4 expression and AKT activation. In MDA-MB-231 cells, estrogen conferred no inductive effect on integrin β4 protein level, but siΔNp63 also reduced integrin β4 expression and AKT activation. 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 (+/+). (D) ERα transcriptional activation of the <i>ΔNp63</i> promoter was evaluated with a luciferase reporter assay using the full-length <i>ΔNp63</i> promoter. In MCF-7 cells, estrogen treatment induced higher luciferase activity than 0.1% ethanol treatment, but no difference was observed in the empty vector-transfected cells, regardless of treatment with 10nM estrogen or 0.1% ethanol. (E)A ChIP assay was performed to evaluate whether ERα directly binds to the most conserved ERE within the <i>ΔNp63</i> promoter. In estrogen-treated MCF-7 cells, the 234-bp PCR product of an ERE-containing fragment was more observable than that of the 0.1% ethanol-treated control group, and no PCR product was observed in the non-immunized rabbit IgG control. (F) ΔNp63 transcriptional activation for the <i>ITGB4</i> promoter was evaluated with a luciferase reporter assay using the full-length <i>ITGB4</i> promoter. In MCF-7 cells, estrogen treatment induced higher luciferase activity than 0.1% ethanol treatment, whereas siΔNp63 reduced luciferase activity under both 10nM estrogen and 0.1% ethanol treatment conditions. There was no difference observed in the empty vector-transfected cells, regardless treatment with 10nM estrogen or 0.1% ethanol. 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