Fig. 5

YAP-TEAD transcriptionally promoted expression of FAK upstream regulatory factor, thrombospondin 1 (THBS1). (a) Analysis of TEAD ChIP-sequence data of MCF7 cells from the ENCODE database (GSM1010860) via the ChIP-seek tool. SL14575 and SL16341 were two bio-replications of the ChIP-sequence data. (b) Peaks in promoter-TSS category from (a) were exacted and annotated. A total of 192 genes whose promoter was potentially combined with TEAD4 were identified. (c) Gene expression profiling was performed in MCF7 cells overexpressing the YAP-S127A mutant compared with empty vector. Expression values of the 192 genes from (b) were exacted and presented in a heat map. (d) The 192 genes were divided into four categories according to the expression fold change (FC) in MCF7-YAP-S127A vs. MCF7-EV cells. Upregulated: genes that were upregulated by the YAP-S127A mutant with a FC ≥ 2; Unchanged: genes with an expression fold change between the two groups of less than two; Downregulated: genes were downregulated by the YAP-S127A mutant with a FC ≥ 2; Undetected: genes that were not detected by the expression profiling. (e) Gene ontology analysis (biological processes) was performed for the 30 upregulated genes from (d). “Cell adhesion” was the first enrichment category and contained 6 genes. (f) Using the STRING program to analyse potential interactions between FAK (also known as PTK) and the 6 upregulated genes that were included in the “cell adhesion” category (THBS1, HABP2, L1CAM, BCAM, CYR61 and CTGF). THBS1 appeared to be highly correlated to FAK (confidence score: 0.849). (g) Dual luciferase reporter assays showed that THBS1 promoter activity could be significantly enhanced by YAP, both in HEK293T and MCF7 cells. *p < 0.05 by Student’s t-test. The experiments were performed in triplicate. (h) Dual luciferase reporter assays showed that compared to the YAP-S94A mutant, YAP-S127A could significantly increase THBS1 promoter activity in HEK293T and MCF7 cells. *p < 0.05 and **p < 0.01 by ANOVA. The experiments were performed in triplicate. (i) Through overexpressing the YAP-S127A mutant, the combination of YAP protein and THBS1 promoter was significantly increased in MCF7 cells. Chromatin and proteins were cross-linked, and mouse monoclonal anti-YAP antibodies were used for pulldown. The promoter of THBS1 was amplified and verified via agarose gel electrophoresis. Mouse IgG was used as a negative control. (j) Quantitative real-time PCR showed mRNA levels of YAP target genes (CTGF, CYR61) and THBS1 in MCF7-EV, MCF7-YAP-S127A and MCF7-S94A cells. The YAP-S127A mutant could significantly induce THBS1 and YAP target gene expression. GAPDH was used as an internal control. *p < 0.05 by ANOVA. The experiments were performed in triplicate. (k) Knockdown of endogenous YAP significantly downregulated THBS1, CTGF and CYR61 expression in MDA-MB-231 cells. GAPDH was used as an internal control. *p < 0.05 by ANOVA. The experiments were performed in triplicate. (l) Western blot showed that compared with the YAP-S94A mutant, overexpression of the YAP-S127A mutant significantly induced THBS1 expression. (m) Knockdown of endogenous YAP expression significantly decreased THBS1 protein levels in MDA-MB-231 cells. (n) Verteporfin could inhibit THBS1 expression in MDA-MB-231 cells. Cells were exposed to verteporfin (10 μM) for 24 h before the Western blot assay was performed. (o) THBS1 expression was positively associated with YAP in clinical breast cancer specimens (R = 0.382, p < 0.01). Gene correlation analysis was based on the TCGA breast invasive carcinoma dataset and was analysed via the R2: Genomics Analysis and Visualization Platform. The degrees of freedom (df) was 1095