Hippo component YAP promotes focal adhesion and tumour aggressiveness via transcriptionally activating THBS1/FAK signalling in breast cancer

Abstract

Background: Focal adhesion plays an essential role in tumour invasiveness and metastasis. Hippo component YAP has been widely reported to be involved in many aspects of tumour biology. However, its role in focal adhesion regulation in breast cancer remains unexplored.

Methods: Tissue microarray was used to evaluate YAP expression in clinical breast cancer specimens by immunohistochemical staining. Cell migration and invasion abilities were measured by Transwell assay. A cell adhesion assay was used to measure the ability of cell adhesion to gelatin. The focal adhesion was visualized through immunofluorescence. Phosphorylated FAK and other proteins were detected by Western blot analysis. Gene expression profiling was used to screen differently expressed genes, and gene ontology enrichment was performed using DAVID software. The gene mRNA levels were measured by quantitative real-time PCR. The activity of the THBS1-promoter was evaluated by dual luciferase assay. Chromatin immunoprecipitation (ChIP) was used to verify whether YAP could bind to the THBS1-promoter region. The prediction of potential protein-interaction was performed with the String program. The ChIP sequence data of TEAD was obtained from the ENCODE database and analysed via the ChIP-seek tool. The gene expression dataset (GSE30480) of purified tumour cells from primary breast tumour tissues and metastatic lymph nodes was used in the gene set enrichment analysis. Prognostic analysis of the TCGA dataset was performed by the SurvExpress program. Gene expression correlation of the TCGA dataset was analysed via R2: Genomics Analysis and Visualization Platform.

Results: Our study provides evidence that YAP acts as a promoter of focal adhesion and tumour invasiveness via regulating FAK phosphorylation in breast cancer. Further experiments reveal that YAP could induce FAK phosphorylation through a TEAD-dependent manner. Using gene expression profiling and bioinformatics analysis, we identify the FAK upstream gene, thrombospondin 1, as a direct transcriptional target of YAP-TEAD. Silencing THBS1 could reverse the YAP-induced FAK activation and focal adhesion.

Conclusion: Our results unveil a new signal axis, YAP/THBS1/FAK, in the modulation of cell adhesion and invasiveness, and provides new insights into the crosstalk between Hippo signalling and focal adhesion.

Keywords: Breast cancer; FAK; Focal adhesion; THBS1; YAP.

Fig. 1

YAP overexpression and activation were associated with lymphatic metastasis and poor prognosis in breast cancer patients. (a) Immunohistochemistry staining of YAP protein in paired primary and lymphatic metastatic specimens from one breast cancer patient. Lymphatic metastasis revealed a higher expression level of YAP. Scale bar: 20 μm. (b) Immunohistochemistry staining score (IHC score) of YAP expression in 101 paired primary/lymphatic metastatic breast cancer specimens from a breast cancer tissue microarray. The expression level of YAP was significantly higher in lymphatic metastases than in primary lesions (**p < 0.01 by paired Student’s t-test). Primary: primary lesion; Metastasis: lymphatic metastasis. (c) Immunohistochemistry cytoplasm expression (cytoplasmic score, left panel) and nucleus accommodation (nucleus score, right panel) of YAP in the 101 paired primary/lymphatic metastatic breast cancer specimens. The cytoplasm expression and nucleus accumulation of YAP was significantly higher in lymphatic metastases than in primary lesions (*p < 0.05 by paired Student’s t-test). Primary: primary lesion; Metastasis: lymphatic metastasis. (d) Analysis of TCGA breast invasive carcinoma dataset (n = 962) via SurvExpress program. Left: Heat map summarizing the expression values of YAP and its target genes (CTGF, CYR61, AXL and MYC) in breast cancer specimens from the TCGA dataset. Patients were sorted by prognostic index and divided into “Low Risk” and “High Risk” groups, according to the “Maximized Risk Groups” algorithm (see reference [32]). Middle: patients in the “High Risk” group presented a significantly higher expression level of YAP and its downstream genes (p < 0.01). Right: Kaplan-Meier analysis revealed that patients in the “High Risk” group suffered from poor prognosis (p < 0.01)

Fig. 2

YAP was able to induced cell migration, invasion and focal adhesion in breast cancer cell lines. (a) Western blot verified the overexpression of YAP in MCF7 cells. EV: empty vector; o/e: overexpression. (b) Western blot verified the knockdown of YAP in MDA-MB-231 cells via a collection of siRNAs; siYAP-#2 and siYAP-#3 has relatively high knockdown efficiency, thus these two siRNAs were used in this research. (c, d) Transwell assay showing that overexpression of YAP induced cell migration and invasion ability in MCF7 cells. The experiment was performed in triplicate. **p < 0.01 by Student’s t-test. Scale bar: 100 μm. (e, f) Transwell assay showing that knockdown of YAP significantly inhibited cell migration and invasion ability in MDA-MB-231 cells. The experiment was performed in triplicate. ** p < 0.01 by ANOVA test. Scale bar: 100 μm. (g, h) Overexpression of YAP induced MCF7 cell adhesion to gelatin. The attached cells were stained with Wright’s-Giemsa and are shown in (g). The experiment was performed in triplicate. ** p < 0.01 by Student’s t-test. Scale bar: 100 μm. (i, j) Knockdown of YAP significantly inhibited MDA-MB-231 cell adhesion to gelatin. The attached cells were stained with Wright’s-Giemsa and are shown in (i). The experiment was performed in triplicate. ** p < 0.01 by Student’s t-test. Scale bar: 100 μm. (k) Overexpression of YAP induced focal adhesions in MCF7 cells. Focal adhesions were visualized by co-localization of paxilin (green) and F-actin (stained with phalloidin, red). Nuclei were counterstained with DAPI (blue). Scale bar: 20 μm. (l) Knockdown of YAP expression inhibited focal adhesions in MDA-MB-231 cells. Scale bar: 20 μm. (m) Quantification of the membrane-localized paxilin in (k). The experiment was performed in triplicate. ** p < 0.01 by Student’s t-test. (n) Quantification of the membrane-localized paxilin in (l). The experiment was performed in triplicate. ** p < 0.01 by ANOVA test

Fig. 3

YAP-TEAD interaction was essential for tumour cell invasiveness and focal adhesion formation. (a) Western blot verified the overexpression of two YAP mutants, YAP-S127A (FLAG-tagged) and YAP-S94A (GFP-tagged) in MCF7 cells. EV: empty vector; S127A: YAP constitutively activated mutant (YAP1-S127A); S94A: YAP TEAD-binding domain mutant (YAP-S94A). (b) (c) Cell adhesion assays showed that ectopic expression of YAP-S127A, rather than YAP-S94A, induced MCF7 cell adhesion to gelatin. The experiment was performed in triplicate. ** p < 0.01 by ANOVA test. Scale bar: 100 μm. (d, e, f) Transwell assays showed that compared with the YAP-S94A mutant, YAP-S127A could significantly induce cell migration and invasion ability in MCF7 cells. The experiment was performed in triplicate. ** p < 0.01 by ANOVA test. Scale bar: 100 μm. (g) Ectopic expression of YAP-S127A, rather than YAP-S94A, induced focal adhesions in MCF7 cells. Focal adhesions were visualized by co-localization of paxilin (stained with Dylight 649, violet) and F-actin (stained with phalloidin, red). Nuclei were counterstained with DAPI (blue). GFP is represented as green. Scale bar: 20 μm. (h, i) Representative images of MCF7-YAP-S127A cell adhesion to gelatin after treatment with verteporfin at a dose of 10 μM for 24 h (DMSO was used as negative control). Verteporfin significantly inhibited cell adhesion ability of MCF7 cells expressing YAP-S127A mutant. The experiment was performed in triplicate. **p < 0.01 by ANOVA test. Scale bar: 100 μm. (j, k) Transwell assays showed that verteporfin significantly inhibited invasion ability of MCF7-YAP-S127A cells. MCF7-YAP-S127A cells were treated with verteporfin at a dose of 10 μM for 24 h (DMSO was used as negative control) before transwell assays were performed. The experiment was performed in triplicate. **p < 0.01 by ANOVA test. Scale bar: 100 μm. (l) Verteporfin inhibited focal adhesions in MCF7-YAP-S127A cells. Cells were exposed to verteporfin (10 μM) or DMSO (negative control) for 24 h and then stained with paxilin (green). F-actin was stained with phalloidin (red). Nuclei were counterstained with DAPI (blue). Scale bar: 20 μm. (m, n) Verteporfin significantly inhibited cell adhesion ability in MDA-MB-231 cells. Cells were treated with verteporfin at a dose of 10 μM for 24 h before cell adhesion assays were performed. DMSO was used as negative control. The experiment was performed in triplicate. **p < 0.01 by Student’s t-test. Scale bar: 100 μm. (o, p) Verteporfin significantly inhibited invasion abilities in MDA-MB-231 cells. Cells were treated with verteporfin at a dose of 10 μM for 24 h (DMSO was used as negative control) before transwell assays were performed. The experiment was performed in triplicate. **p < 0.01 by Student’s t-test. Scale bar: 100 μm. (q) Treatment with verteporfin (10 μM) for 24 h decreased focal adhesions in MDA-MB-231 cells. Paxilin (green), F-actin (stained with phalloidin, red). Nuclei (blue). Scale bar: 20 μm

Fig. 4

YAP-TEAD promoted focal adhesion formation in breast cancer cell lines through inducing FAK phosphorylation. (a) Western blot revealed that compared with the YAP-S94A mutant, overexpression of the YAP-S127A mutant could promote FAK Y397 phosphorylation. (b) Verteporfin reversed YAP-S127A-induced FAK phosphorylation in MCF7 cells. MCF7-YAP-S127A was treated with verteporfin at a dose of 10 μM for 24 h before the Western blot assay was performed. DMSO was used as a negative control. (c) Knockdown of endogenous YAP expression inhibited FAK Y397 phosphorylation in MDA-MB-231 cells. (d) Verteporfin inhibited FAK phosphorylation in MDA-MB-231 cells. Cells were treated with verteporfin at a dose of 10 μM for 24 h before Western blot assays were performed. DMSO was used as a negative control. (e, f) Western blot verified the inhibition of FAK Y397 phosphorylation via defactinib in MCF7-YAP-S127A (e) and MDA-MB-231 (f) cells. Cells were exposed to defactinib at a dose of 10 μM for 8 h before Western blot assays were performed. DMSO was used as a negative control. (g, h) Treatment with defactinib (10 μM) for 8 h decreased focal adhesions, both in MCF7-YAP-S127A (g) and MDA-MB-231 (h). DMSO was used as a negative control. Paxilin (green), F-actin (stained with phalloidin, red). Nuclei (blue). Scale bar: 20 μm. (i) Transwell invasion assays showed that exposure to defactinib (10 μM) could significantly reverse YAP-S127A-induced cell invasion in MCF7 cells. DMSO was used as a negative control. The experiment was performed in triplicate. Scale bar: 100 μm. (j) Exposure to defactinib (10 μM) significantly decreased cell invasion ability in MDA-MD-231 cells. DMSO was used as a negative control. The experiment was performed in triplicate. Scale bar: 100 μm. (k) Quantification of the relative invasion ability in (i). ** p < 0.01 by ANOVA test. (l) Quantification of the relative invasion ability in (j). ** p < 0.01 by Student’s t-test

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

Fig. 6

YAP triggered FAK phosphorylation and focal adhesion through THBS1. (a) Western blot assays revealed that knockdown of THBS1 expression in MCF7-YAP-S127A cells could significantly reverse FAK Y397 phosphorylation. (b) Cell adhesion assays showed that knockdown of THBS1 could significantly reverse YAP-S127A-induced cell adhesion in MCF7 cells. The experiments were performed in triplicate. Scale bar: 100 μm. (c) Transwell invasion assays showed that knockdown of THBS1 could significantly reverse YAP-S127A-induced cell invasion in MCF7 cells. The experiments were performed in triplicate. Scale bar: 100 μm. (d) Quantification of the cell adhesion ability in (b). * p < 0.05 and ** p < 0.01 by ANOVA test. (e) Quantification of the cell invasion ability in (c). ** p < 0.01 by ANOVA test. (f) Knockdown of THBS1 inhibited focal adhesion in MCF7-YAP-S127A cells. Red: F-actin (stained with phalloidin); Green: paxilin; Blue: nucleus (stained with DAPI). Scale bar: 20 μm. (g) Knockdown of THBS1 reduced FAK Y397 phosphorylation in MDA-MB-231 cells. (h) Knockdown of THBS1 expression reduced cell adhesion to gelatin in MDA-MB-231 cells. The experiments were performed in triplicate. Scale bar: 100 μm. (i) Transwell invasion assays showed that knockdown of THBS1 expression reduced cell invasion in MDA-MB-231 cells. The experiments were performed in triplicate. Scale bar: 100 μm. (j) Quantification of the cell adhesion ability in (h). ** p < 0.01 by ANOVA test. (k) Quantification of the cell invasion ability in (i). ** p < 0.01 by ANOVA test. (l) Knockdown of THBS1 reduced focal adhesion in MDA-MB-231 cells. Red: F-actin (stained with phalloidin); Green: paxilin; Blue: nucleus (stained with DAPI). Scale bar: 20 μm. (m) Model for how YAP regulates THBS1 expression and induces focal adhesion