Estrogen-induced hypomethylation and overexpression of YAP1 facilitate breast cancer cell growth and survival

Abstract

Increased expression of Yes-associated protein-1 (YAP1) was shown to correlate with reduced survival in breast cancer (BC) patients. However, the exact mechanism of YAP1 regulation in BC cells remains ambiguous. Genomic sequence search showed that the promoter region of the YAP1 gene contains CpG Islands, hence the likelihood of epigenetic regulation by DNA methylation. To address this possibility, the effect of estrogen (17β estradiol; E2) on YAP1 gene expression and YAP1 promoter methylation status was evaluated in BC cells. The functional consequences of E2 treatment in control and YAP1-silenced BC cells were also investigated. Our data showed that E2 modulates YAP1 expression by hypomethylation of its promoter region via downregulation of DNA methyltransferase 3B (DNMT3B); an effect that seems to facilitate tumor progression in BC cells. Although the effect of E2 on YAP1 expression was estrogen receptor (ER) dependent, E2 treatment also upregulated YAP1 expression in MDA-MB231 and SKBR3 cells, which are known ER-negative BC cell lines but expresses ERα. Functionally, E2 treatment resulted in increased cell proliferation, decreased apoptosis, cell cycle arrest, and autophagic flux in MCF7 cells. The knockdown of the YAP1 gene reversed these carcinogenic effects of E2 and inhibited E2-induced autophagy. Lastly, we showed that YAP1 is highly expressed and hypomethylated in human BC tissues and that increased YAP1 expression correlates negatively with DNMT3B expression but strongly associated with ER expression. Our data provide the basis for considering screening of YAP1 expression and its promoter methylation status in the diagnosis and prognosis of BC.

Keywords: Breast cancer; Carcinogenesis; DNA methylation; Epigenetics; Estrogen; Yes-associated protein-1 gene.

Graphical abstract

Figure 1

Expression of YAP1 in breast cancer cell lines and human breast cancer tissue samples. (A) YAP1 mRNA expression was measured in MCF7 cells treated with escalating concentration (5–20 nM) of estrogen (E2) and increase duration from 2 to 24 h. (B) YAP1 protein expression was measured by western blotting at 24 h post-E2 (5–20 nM) treatment. (C) YAP1 protein expression was measured in ER-negative breast cancer cell lines (MDA-MB231 and SKBR3). (D) Status of estrogen receptor alpha (ERα) was detected in the three breast cancer cell lines used. (E) Expression of YAP1 mRNA in human breast cancer tissues compared with YAP1 expressed in the MCF7 cell line. Out of at least 2 independent experiments, only one representative western blot figure is shown. In each bar graph, the data represent mean ± SEM. #P = 0.0017, 2-way ANOVA for multiple comparisons; *P < 0.01, 2-tailed t test compared with control cells. C = control; E2 = Estrogen; V = vehicle.

Figure 2

YAP1 immunohistochemical expression. (A) Normal breast tissue showing YAP1 expression in the nuclei of the myoepithelial cells only with faint low-intensity cytoplasmic expression in the luminal epithelial cells. (B) Ductal carcinoma in situ (DCIS) cells showing low-intensity cytoplasmic expression and no evidence of nuclear expression. (C and D) Luminal tumors showing positive YAP1 nuclear expression in tumor cells with a high-intensity cytoplasmic expression. (E) Luminal tumors showing low-intensity cytoplasmic expression in malignant cells with less than 20% of the nuclei demonstrating positive YAP1 expression. (F) Triple-negative breast cancer tumor cells showing total negativity for YAP1 nuclear expression and faint or no cytoplasmic expression. Black scale bar represents 200 µm.

Figure 3

Effect of estrogen (E2) and YAP1 knockdown on breast cancer cell growth.(A) Phase-contrast microscopic images of MCF7 cell growth and proliferation upon treatment with E2 with or without siYAP1 at increasing concentration. (B) Flow cytometry using Annexin V-FITC/PI staining showing apoptosis in MCF7 cells treated with E2 with or without siYAP1. (C) Flow cytometric analysis of cell-cycle regulation in MCF7 cells treated with E2 with or without siYAP1. For each experiment, one representative figure is shown out of at least 3 independent experiments. Black scale bar represents 200 µm.

Figure 4

Effect of estrogen, PPT, siYAP1, and siERα/β on autophagic flux in breast cancer cells.(A) Western blotting showing expression of LC3A/B conversion and YAP1 in E2 treated MCF7 cells with or without siYAP1. (B) Expression of LC3A/B conversion, YAP1, ERα/β, upon treatment with ER agonist (PPT; 1 µM), and E2 (20 nM) with or without siERα/β. (C and D) Autophagosome signals showing autophagic flux in MCF7 cells treated with AZA (1 µM), E2 (5–20 nM), and E2 (20 nM) with or without siYAP1 and siERα/β. Rapamycin (Rapa; 1 µM) was used as positive control showing the induction of autophagy. Out of at least 2 independent experiments, only one representative western blot figure is shown. In the bar graph, the data represents mean ± SEM. *P-value is < 0.01 compared with control cells. White scale bar represents 200 µm. DR represents densitometry ratio for protein expression relative to actin.

Figure 5

Effect of estrogen (E2) on DNA methylation and expression of YAP1.(A) Expression of YAP1 mRNA upon treatment with E2 (20 nM) and/or AZA 1.0 µM. (B) DNA methylation levels of YAP1 promoter region in MCF7 cells treated with E2 (5–20 nM), AZA 1.0 µM and both E2 and AZA in combination. (C) Protein expression of DNA methyltransferases (DNMT1, 3A, and 3B) in MCF7 cells treated with E2 (5–20 nM) for 24 h, treated with vehicle or left untreated. (D) Flowcytometric analysis of DNMT3B in MCF7 cells treated with E2 (5–20 nM), AZA 1.0 µM, and both E2 and AZA in combination. (E) Protein expression of YAP1 and DNMT3B in siDNMT3B, siERα and siERβ transfected MCF7 cells treated with E2 (20 nM) for 24 h. (F) YAP1 mRNA expression and G. YAP1 promoter methylation levels in siDNMT3B, siERα and siERβ transfected MCF7 cells treated with E2 (20 nM) for 24 h. For all experiments, one representative figure out of 2 independent experiments is shown. In the bar graph, the data represents mean ± SEM. #P = 0.0122, ANOVA; significant after multiple comparisons; *P < 0.01, 2-tailed t test compared with control cells. C = control; E2 = Estrogen; V = vehicle.

Figure 6

In silico analysis of DNMT3B and YAP1 expression and YAP1 promoter DNA methylation levels.(A and B) Gene expression omnibus (GEO) was used to obtain and perform analysis of microarray dataset containing MCF7 cells treated with estrogen (E2) for 12, 24, and 48 hours. YAP1 and DNMT3B mRNA expression was measured in E2 treated versus control cells (n = 3 for each treatment). (C) TCGA datasets were filtered to obtain DNMT3B and YAP1 mRNA expression and YAP1 promoter methylation levels in primary human breast cancer tissues. A total of 783 cancer samples were included in the final analysis and are presented as a heat map. Red and blue colors were used to represent high or low mRNA expression, respectively. Blue, black, and yellow colors were used to represent hypo-, un- or hypermethylation, respectively. (D) A dot scatter plot was generated to present the correlation/association of YAP1 and DNMT3B mRNA expression in human breast cancer tissue samples. (Color version of figure is available online.)