Dampened Regulatory Circuitry of TEAD1/ITGA1/ITGA2 Promotes TGFβ1 Signaling to Orchestrate Prostate Cancer Progression

Abstract

The extracellular matrix (ECM) undergoes substantial changes during prostate cancer (PCa) progression, thereby regulating PCa growth and invasion. Herein, a meta-analysis of multiple PCa cohorts is performed which revealed that downregulation or genomic loss of ITGA1 and ITGA2 integrin genes is associated with tumor progression and worse prognosis. Genomic deletion of both ITGA1 and ITGA2 activated epithelial-to-mesenchymal transition (EMT) in benign prostate epithelial cells, thereby enhancing their invasive potential in vitro and converting them into tumorigenic cells in vivo. Mechanistically, EMT is induced by enhanced secretion and autocrine activation of TGFβ1 and nuclear targeting of YAP1. An unbiased genome-wide co-expression analysis of large PCa cohort datasets identified the transcription factor TEAD1 as a key regulator of ITGA1 and ITGA2 expression in PCa cells while TEAD1 loss phenocopied the dual loss of α1- and α2-integrins in vitro and in vivo. Remarkably, clinical data analysis revealed that TEAD1 downregulation or genomic loss is associated with aggressive PCa and together with low ITGA1 and ITGA2 expression synergistically impacted PCa prognosis and progression. This study thus demonstrated that loss of α1- and α2-integrins, either via deletion/inactivation of the ITGA1/ITGA2 locus or via loss of TEAD1, contributes to PCa progression by inducing TGFβ1-driven EMT.

Keywords: ECM; EMT; epithelium; integrin; prostate.

Figure 1

Integrin signaling pathway and the copy number loss/del of ITGA1 or ITGA2 are associated with PCa severity and prognosis. a) Differentially expressed genes identified from a meta‐analysis consisting of nine independent PCa cohorts. Up‐ and downregulated genes were highlighted in orange and blue, respectively. b) PANTHER pathway ontology analysis identified the Integrin signaling pathway as the most affected pathway in PCa. c) ITGA1 and ITGA2 were determined with the highest frequencies of deep copy loss in PCa among other cancer types (ovarian – ov, esophageal – esca, bladder – blca, stomach – stca, cervical – cesc, head and neck – hnsc, breast – brca, lung – lnsc and luad, pheochromocytoma and paraganglioma – pcpg, colorectal ‐coadread, uterine corpus endometrial – ucec and brain – gbm). d) ITGA1 or e) ITGA2 expression in prostate tumors with diploid or copy number loss/del of ITGA1 or ITGA2 (left panel), and Pearson correlation between ITGA1 or ITGA2 mRNA expression and copy number changes (right panel). P values determined by the Mann‐Whitney U test or from the Pearson correlation coefficient. f) The fraction of PCa tumors harboring ITGA1 or ITGA2 copy number loss/del was elevated in metastasis when compared with primary PCa. P values were examined by the Fisher's exact test. g,h) Top gene set depleted in PCa tumors with ITGA1 g) or ITGA2 h) copy number loss/del versus diploid in the TCGA PCa cohort. NES, normalized enrichment score. i,j) Forest plots displaying the meta‐analysis of the hazard ratio estimates of ITGA1 i) or ITGA2 j) for the biochemical recurrence in multiple PCa cohorts. The horizontal error bars represent the 95% CI with the measure of center as HR. The HR and 95% CI were presented in the form of natural logarithm (ln). P values were calculated by the two‐way Fixed‐Effects Model. k,l) Kaplan Meier plots indicate increased risk for metastasis in PCa patients with tumors harboring ITGA1k) or ITGA2 l) copy number loss/del. P‐values were assessed by log‐rank tests.

Figure 2

Downregulation of ITGA1 or ITGA2 correlates with PCa severity and progression. a–f) Boxplots displaying expression levels of ITGA1 a–c) or ITGA2 d–f) downregulated during PCa development and progression. P‐values were determined by the Kruskal‐Wallis H test for comparing three or more groups c and f) or the Mann‐Whitney U test for comparing two groups a,b and d,e). g) Representative images of α1‐ and α2‐integrin expression and localization in normal and PCa tissue. Scale bar = 100 µm. h,i) Kaplan Meier plots indicate increased biochemical recurrence h), and metastatic i) risks for PCa patients with tumors expressing lower ITGA1 levels. j,k) PCa patients with decreased ITGA2 expression level are associated with increased risks for biochemical recurrence j), and metastasis k). PCa patients were stratified into lower and higher expression groups by the median value of ITGA1 or ITGA2 expression levels. P‐values were assessed by log‐rank test. l–o) Decreased ITGA1 expression level correlates with higher tumor stage l), Gleason score m), lymph node metastasis n) and PSA levels o). (p‐s) ITGA2 downregulation is associated with higher tumor stage p), Gleason score q), lymph node metastasis r) and PSA levels s). P values were determined by Kruskal‐Wallis H test.

Figure 3

Loss of α1‐ or α2‐integrins leads to different but synergistic phenotypes in prostate epithelial cells. a) Level of α1‐ and α2‐integrins in normal (RWPE1) and PCa (DuCaP, PC‐3, VCaP, DU145, 22Rv1) epithelial cells. The data shows the mean value from three independent experiments, analyzed with One‐way ANOVA. b) RWPE1‐WT, RWPE1‐α1KO, RWPE1‐α2KO and RWPE1α1α2dKO cell lysates were analyzed for the expression levels of α1‐ and α2‐integrins. β‐tubulin was used as a loading control. The data shows the mean value from three independent experiments, analyzed with One‐way ANOVA. c) RWPE1‐WT, RWPE1‐α1KO, RWPE1‐α2KO and RWPE1‐α1α2dKO cells grown on glass coverslips for 2 days were imaged using phase contrast microscopy. Scale bar = 10 µm. d) Proliferation of the indicated RWPE1 cell lines were analyzed using an XTT assay, analyzed with Two‐way ANOVA. The data shows mean ±SD from three independent experiments performed in triplicates. e) Migration of the different RWPE1 variants was analyzed using the IncuCyteS3 Scratch Wound module. f) A plot showing the wound‐closure dynamics of the indicated RWPE1 cell lines. The mean ± SD from a representative assay with 3 replicates is plotted in the graph, analyzed with Two‐way ANOVA. The assay was repeated thrice with similar results. g) Phase contrast microscopy images of the indicated RWPE1 variants grown in 3D Matrigel for 7 days. Scale bar = 50 µm. h) Quantitation of the 3D morphology analysis of RWPE1‐WT, ‐α1KO and α2KO cell lines. RWPE1‐α1α2dKO cells formed interconnected multicellular networks and could not be analyzed. Cysts were classified into 4 categories: cysts with a central hollow lumen, multilumen cysts, cysts with individual cells in the lumen and solid cysts with no visible lumen. The data shows the mean ± SD from three independent experiments in which at least 100 cysts per sample was analyzed. Statistical significance is indicated by asterisks: ∗ = p < 0.05; ∗∗ = p < 0.01; ∗∗∗ = p < 0.001.

Figure 4

Loss of both α1‐ and α2‐integrins activates EMT‐associated pathways in prostate epithelial cells. a) Heatmap representation of differentially expressed genes (FDR < 0.05) identified from the RNA‐seq analysis in RWPE1‐WT (control) and RWPE‐α1α2dKO (left panel), ‐α1KO (middle panel) or ‐α2KO (right panel) cells. P values were evaluated by the Wald test and adjusted for multiple comparisons. b) Top‐ranked pathways enriched in the upregulated genes form the GSEA analysis in RWPE1‐ α1α2dKO cells. Categories were ranked by the Normalized Enrichment Score (NES). c) GSEA enrichment plot displaying the EMT pathway enriched in upregulated genes in RWPE1‐α1α2dKO cells. d) Validation of the metastasis/EMT‐related pathway signatures in additional resources. e) Heatmap representation of genes in the EMT pathways measured by RNA‐seq in the RWPE1 cells with α1α2‐dKO. f) The EMT score is significantly elevated in PCa tumors with copy loss/del of both ITGA1 and ITGA2. g) The EMT score is negatively correlated with the expression levels of ITGA1 and ITGA2 in PCa. P value was assessed by the two‐sided Pearson's product‐moment correlation test. h) The EMT score is downregulated in PCa tumors with higher expression levels of ITGA1/ITGA2. The EMT score and the ITGA1/ITGA2 expression levels are calculated as the z‐score sum. In f and h, P values were assessed by two‐sided Mann–Whitney U test. i) Quantitative PCR analysis confirming regulation of the EMT pathway genes, analyzed with One‐way ANOVA. The data is presented as mean ± SD of triplicate experiments. ∗ = p < 0.05; ∗∗ = p < 0.01; ∗∗∗ = p < 0.001, analyzed with One‐way ANOVA. j) The TGFβ signaling pathway was significantly enriched in the upregulated genes with α1α2dKO in the RWPE1 cells in multiple gene set resources. k) GSEA enrichment plot demonstrating the TGFβ signaling pathway enriched in the upregulated genes in RWPE1 cells with α1α2dKO.

Figure 5

TGFβ activation drives invasive potential in α1‐ and α2‐integrin‐deficient prostate epithelial cells. a) The culture medium from RWPE1‐WT and RWPE1‐α1α2dKO cells was harvested and concentrated as described in Experimental Section/Methods. Secreted latent TGFβ and activated TGFβ were visualized by western blotting. The data is representative of three independent experiments with similar results. b) Quantitative analysis of the secreted TGFβ. The data is presented as mean ± SD of triplicate experiments. ∗ = p < 0.05; ∗∗ = p < 0.01; ∗∗∗ = p < 0.001, analyzed with One‐way ANOVA. c) RWPE‐1‐WT cells were grown in 3D BME gel with or without 10 ng of TGFβ1 for 7 days while RWPE1‐α1α2dKO cells were grown in the presence or absence of 5 µM TGFβ1 inhibitor (TGFβ1i). Scale bar: 50 µm. d) RWPE1‐WT and RWPE1‐α1α2dKO cells were grown on glass coverslips in the absence or presence of 10 ng TGFβ1 or 5 µM of TGFβ1i, respectively in coverslips for 2 days, fixed and stained for nuclei (blue), actin (red) and YAP1 (green). Scale bar:10 µm.

Figure 6

Deletion of α1‐ and α2‐integrins in benign RWPE1 prostate epithelial cell line promotes formation of lung micro‐metastases. a) 1 × 10⁵ RWPE1‐WT, ‐α1KO, ‐α2KO and ‐α1α2dKO cells expressing GFP and luciferase were injected into the tail veins of 6 immunodeficient SCID‐mice per group. After four weeks, mice were sacrificed and the luciferase signals originating from cancer cells were measured using IVIS imaging system. b) HE‐stained lungs sections were analyzed for presence of metastatic lesions. Tumor areas are highlighted by a dotted line. Scale bar is 50 µm. c) The area covered by individual lung metastatic lesions were determined for each cell variant from HE‐stained lung sections as described in methods. The data shows mean ± SD. d) FACS‐analysis of the number of circulating GFP‐positive RWPE‐1 cell variants recovered from the blood of sacrificed SCID‐mice. For RWPE1‐α1α2dKO cells, blood samples were obtained from only 4 mice. Data shows mean ± SD. ∗ = p < 0.05; ∗∗ = p < 0.01; ∗∗∗ = p < 0.001, analyzed with One‐way ANOVA.

Figure 7

TEAD1 regulates expression of ITGA1 and ITGA2. a,b) A genome‐wide co‐expression analysis identifies strong positive expression correlation of ITGA1a) or ITGA2b) with TEAD1, but not with TEAD2, TEAD3 or TEAD4 in the TCGA cohort. The X‐axis demonstrates Pearson coefficient while Y‐axis represents –log10 (P value). c,d) Expression correlation analysis revealed significant positive correlation between TEAD1 and ITGA1 c) or ITGA2 d) in the TCGA data set. The color bars on the right side of figures indicate the expression level of ITGA2 and ITGA1, respectively. In a‐d, P‐values were assessed by the Pearson's product‐moment correlation test. I Genome browser representation of ChIP‐seq signals of transcription factor TEAD1 enriched in the promoters and surrounding regulatory regions of ITGA1 and ITGA2 in PC3 cells. f) ChIP‐qPCR validation of TEAD1 at the three binding sites in PC3 cells, analyzed with One‐way ANOVA. g) Knockdown of TEAD1 in the RWPE1 cell line downregulates expression of ITGA1 and ITGA2, analyzed with One‐way ANOVA. The data is presented as mean ± SD of triplicate experiments each with duplicates. h) Western blotting of α1‐ and α2‐integrins and TEAD1 in the indicated RWPE1 and PC3 cell variants. The blot is representative of three independent experiments with similar results. ∗ = p < 0.05; ∗∗ = p < 0.01; ∗∗∗ = p < 0.001, analyzed with One‐way ANOVA.

Figure 8

TEAD1 is highly co‐expressed with ITGA1 and ITGA2 and is downregulated during PCa development and progression. a) TEAD1 expression levels are downregulated upon copy loss/del in PCa. The P value was examined by the Mann‐Whitney U test. b) PCa patients with TEAD1 copy number loss/del show higher risks for biochemical recurrence. P‐value was assessed by the log‐rank test. c,d) TEAD1 expression levels are significantly decreased upon PCa tumor progression to metastasis. P values were determined by Kruskal‐Wallis H test. e–h) TEAD1 downregulation correlates with PCa tumor progression to high tumor stage e), Gleason score f), lymph node metastasis g) and PSA levels h). P values were determined by Kruskal‐Wallis H test or Mann‐Whitney U test g). i–k) Lower TEAD1 expression levels in PCa patients are associated with higher risks for biochemical recurrence i,j) and metastasis k). P‐values were assessed by the log‐rank test. l) Western blot analysis of the TEAD1 expression in a benign (RWPE‐1) and malignant (DU145, PC3, LnCap, VCap) epithelial prostate cell lines. The blot is representative of three independent experiments with similar results, analyzed with One‐way ANOVA. m) RWPE1‐WT and RWPE1‐TEAD1KO cells grown on glass coverslips for 2 days were imaged using phase contrast microscopy. Scale bar = 10 µm. n) Cell proliferation analysis of RWPE1 and RWPE1‐TEAD1KO cells was done using XTT assay. The data shows mean ±SD from three independent experiments performed in triplicates, analyzed with Two‐way ANOVA. o) Cell migration analysis using the IncuCyte S3 scratch wound module. The plot shows the mean ± SD from a representative assay done in triplicate, analyzed with Two‐way ANOVA. The assay was done three times with similar results. ∗ = p < 0.05; ∗∗ = p < 0.01; ∗∗∗ = p < 0.001.

Figure 9

Loss of TEAD1 phenocopies the dual loss of α1‐ and α2‐integrins in vitro and in vivo. a) Multiple functional categories, including TGFβ signaling, EMT, and metastasis derived from five different pathway gene sets from MSigDB database, were repeatedly upregulated upon TEAD1 KO profiled by RNA‐seq in PC3 cells. b,c) GSEA plots demonstrating pathways relevant to TGFβ signaling, EMT, and metastasis enriched in upregulated genes upon TEAD KO in PC3 cells. d) TGFβ signaling were commonly found upregulated from five distinct pathway gene sets from MSigDB database upon TEAD1 KO profiled by RNA‐seq in RWPE1 cells. PID: the Pathway Interaction Database. e,f) GSEA enrichment plots display enrichment of TGFβ signaling pathway among upregulated genes following TEAD1 KO in RWPE1 cells. g) Phase contrast microscopy images of RWPE1‐WT and RWPE1‐TEAD1KO cells grown in 3D BME gels for 7 days. The scale bar is 50 µm. h) RWPE1‐ TEAD1KO cells were grown in the presence or absence of 5 µM TGFβ1 inhibitor (TGFβ1i) as described in (d). Scale bar is 50 µm. i) Culture medium was harvested and concentrated from RWPE1‐WT, RWPE1‐α1α2dKO and RWPE1‐TEAD1KO cells. The blot is representative of three independent experiments with similar results, analyzed with One‐way ANOVA. j) Luciferase expressing RWPE1‐WT and ‐TEAD1KO cells were imaged using the IVIS imaging system. Tumor areas were highlighted by dotted lines. Scale bar: 50 µm. k) The area covered by individual lung metastatic lesions was determined for each cell variant from HE‐stained lung sections as described in methods. The data shows mean ± SD and was analyzed with One‐way ANOVA l) FACS analysis of the circulating WT and TEAD1‐KO RWPE1 cells collected from the blood of sacrificed SCID mice. Total GFP‐positive cell count from the sample of each mouse and the mean for each group is shown in the plot, analyzed with One‐way ANOVA. Data shows mean ± SD. ∗ = p < 0.05; ∗∗ = p < 0.01; ∗∗∗ = p < 0.001.

Figure 10

The prognostic value of ITGA1, ITGA2 and TEAD1 for PCa patient risk stratification and their synergistic impact on PCa in the clinical settings a–c) Lower expression levels of ITGA1 exhibit predictive values for overall survival (a), biochemical recurrence b), and metastasis c) in PCa patient group with an intermediate risk (Gleason Score 7). d) PCa tumors with lower expression levels of ITGA2 are associated with decreased biochemical recurrence‐free survival in patient group with Gleason score 7. e,f) Lower expression levels of TEAD1 holds predictive values for increased risks of biochemical recurrence e) and metastasis f) in PCa patients with an intermediate risk (Gleason score 7). g,h) Forest plots demonstrating triple‐low expression of ITGA1, ITGA2 and TEAD1 with high hazard ratio for the overall survival g) and biochemical recurrence‐free survival h) of PCa patients. i–k) PCa patients with triple‐low expression of ITGA1, ITGA2 and TEAD1 are associated with shorter overall survival i), increased risks of biochemical recurrence j) and metastasis k). In a‐k, P‐values were assessed by log‐rank tests. l) Bone metastasis ratio for patients stratified based on high or low α1‐integrin, α2‐integrin and TEAD1 expression status. m–p) PCa patients with triple‐low expression of ITGA1, ITGA2 and TEAD1 are associated with advanced tumors including Gleason Score m), PSA levels n), tumor stage o), and lymph node metastasis p). P values were evaluated by two‐sided Fisher's exact test.