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. 2015 Jul 10;6(19):17685-97.
doi: 10.18632/oncotarget.3779.

The interplay between TEAD4 and KLF5 promotes breast cancer partially through inhibiting the transcription of p27Kip1

Chunyan Wang  1   2   3 , Zhi Nie  3 , Zhongmei Zhou  1 , Hailin Zhang  1 , Rong Liu  1 , Jing Wu  1   2   4 , Junying Qin  1   2 , Yun Ma  3 , Liang Chen  3 , Shumo Li  3 , Wenlin Chen  5 , Fubing Li  1   2 , Peiguo Shi  1   2 , Yingying Wu  3 , Jian Shen  3 , Ceshi Chen  1
Affiliations

The interplay between TEAD4 and KLF5 promotes breast cancer partially through inhibiting the transcription of p27Kip1

Chunyan Wang et al. Oncotarget. .

Abstract

Growing evidence suggests that YAP/TAZ are mediators of the Hippo pathway and promote breast cancer. However, the roles of YAP/TAZ transcription factor partners TEADs in breast cancer remain unclear. Here we found that TEAD4 was expressed in breast cancer cell lines, especially in triple negative breast cancers (TNBC) cell lines. TEAD4 binds to KLF5. Knockdown of either TEAD4 or KLF5 in HCC1937 and HCC1806 cells induced the expression of CDK inhibitor p27. Depletion of either TEAD4 or KLF5 activated the p27 gene promoter and increased the p27 mRNA levels. Depletion of p27 partially prevents growth inhibition caused by TEAD4 and KLF5 knockdown. TEAD4 overexpression stimulated proliferation in vitro and tumor growth in mice, while stable knockdown of TEAD4 inhibited proliferation in vitro and tumor growth in mice. Thus TEAD4 and KLF5, in collaboration, promoted TNBC cell proliferation and tumor growth in part by inhibiting p27 gene transcription. TEAD4 is a potential target and biomarker for the development of novel therapeutics for breast cancer.

Keywords: Hippo pathway; KLF5; TEAD4; TNBC; p27.

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Figures

Figure 1
Figure 1. TEAD4 interacts with KLF5 and suppresses the p27 gene expression in TNBC cell lines
A. Protein expression levels of TEAD1–4 in breast epithelial cell lines by WB. β-actin serves as the loading control. B. TEAD4 specifically interacts with KLF5 in HEK293FT cells. All TEADs were tagged with Flag and immunoprecipitated with the anti-Flag antibody. Exogenous KLF5 was only immunoprecipitated by Flag-TEAD4. TEAD1-L and -S are two different TEAD1 isoforms. C. TEAD4 and KLF5 suppress the protein levels of p27 in both HCC1937 and HCC1806. TEAD4 and KLF5 were silenced by two different siRNAs in both cell lines. The p27 and β-actin protein levels were quantified by the IMAGE J software. The normalized p27 protein levels are shown below the p27 panel.
Figure 2
Figure 2. TEAD4 overexpression promotes TNBC cell proliferation and tumor growth
A. Stable overexpression of TEAD4 decreased the p27 protein expression level in HCC1937. B. TEAD4 overexpression significantly promoted HCC1937 cell growth, as determined by the SRB assay. **p < 0.01, t-test. C. TEAD4 overexpression significantly (p < 0.01) increased DNA synthesis in HCC1937 cells, as measured with a Click-iT EdU Alexa Fluor Imaging Kit. D. TEAD4 overexpression significantly increased G1/S cell cycle transition in HCC1937 cells, as measured by PI staining and flow cytometry. a&c, the cells were arrested at the G1 phase by serum starvation. b&d, the arrested cells were stimulated with 10% serum for 4 hours. E. TEAD4 promotes cell proliferation in part through KLF5 in HCC1937 cells. KLF5 was transiently knocked down in HCC1937-pBabe and HCC1937–pBabe-TEAD4 cells. Western blot results are shown on the left side and cell viability data are shown on the right side. **, p < 0.01, n.s., not significant, t-test. F. Tumor masses harvested from HCC1937-pBabe and –pBabe-TEAD4 after tumors had grown for 6 weeks. TEAD4 overexpression significantly increases xenograft weight (right panel). G. TEAD4 overexpression significantly promotes tumor growth in female NOD-SCID mice.
Figure 3
Figure 3. TEAD4 knockdown inhibited HCC1806 cell proliferation and tumor growth
A. Stable knockdown of TEAD4 increased the p27 protein level. B. TEAD4 knockdown significantly inhibited cell growth, as determined by the SRB assay. *p < 0.05, **p < 0.01, t-test. C. TEAD4 knockdown inhibited DNA synthesis by using the Click-iT EdU Alexa Fluor Imaging Kit. D. Quantitative results of panel C, **p < 0.01, t-test. E. Tumor masses harvested from HCC1806-Lucsh and –TEAD4sh#3 after tumors had grown for 21 days. F. TEAD4 knockdown significantly decreases xenograft tumor weight. G. TEAD4 knockdown significantly suppressed tumor growth in female nude mice.
Figure 4
Figure 4. TEAD4 and KLF5 promote cell proliferation partially by suppressing the p27 gene transcription together in TNBC cell lines
A. Knockdown of TEAD4 by two different siRNAs upregulated p27 mRNA levels in both HCC1937 and HCC1806, as measured by qRT-PCR. B. Knockdown of KLF5 by two different siRNAs upregulated p27 mRNA levels in both HCC1937 and HCC1806, as measured by qRT-PCR. C. Knockdown of TEAD4 and KLF5 activated the p27 gene promoter in HCC1806 cells. TEAD4 and KLF5 knockdown were evaluated by WB. Luciferase activities were measured by the dual-luciferase assay kit. D. Both TEAD4 and KLF5 bind to the p27 gene promoter as determined by ChIP assays in HCC1806. E. Knockdown of p27 rescued the TEAD4 and KLF5 knockdown-induced growth arrest in HCC1806, as determined by the SRB assay. **p < 0.01, t-test. F. Knockdown of KLF5 and TEAD4 together additively increased the p27 protein levels in HCC1806.
Figure 5
Figure 5. TEAD4 and KLF5 promote cell migration in HCC1806 cells
A. Knockdown of TEAD4 by two different shRNAs decreased wound healing at 22 hours. B. Knockdown of KLF5 by two different shRNAs dramatically decreased wound healing at 21 hours. (note: The cell density is higher than that in panel A) C. Knockdown of TEAD4 by two different shRNAs decreased transwell cell migration (24 h). Quantative data is shown on the right side. **p < 0.01, t-test.
Figure 6
Figure 6. TEAD4 expression in human breast tumors
A. 121 sample slides were stained with anti-ERα, PR, HER-2 and TEAD4 antibodies. Examples of IHC staining in breast carcinomas are shown. TEADs protein expression is negatively associated with the patient age. B. Kaplan-Meier plotter was used to analyze the breast cancer RNA seq data from the TCGA databse. High expression levels of TEAD4 mRNA are significantly associated with patient distant metastasis free survival and relapse free survival.
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