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.


Introduction
Prostate cancer (PCa) is the second most commonly diagnosed cancer and the fifth leading cause of cancer deaths in men in the Western world. [1]PCa progression is accompanied by gradual loss of epithelial organization and reorganization of the associated extracellular matrix (ECM) in the affected prostate glands.Heterodimeric integrins are the major family of ECM receptors.Integrin ligation to the ECM not only mediates adhesion but also leads to assembly of large signaling platforms that convey the microenvironmental cues to regulate cell growth, survival and migration. [2]everal studies have implicated altered integrin functions in PCa pathogenesis. [3,4]7] PCa progression is associated with increased expression of collagen that is the major constituent of stromal ECM.11and 21-integrins are collagen-binding integrins that may also interact with laminins and are expressed in prostate epithelial cells. [8,9]21integrins have been reported to promote tumorigenesis, particularly metastatic invasion to the bone. [4]However, other studies have observed tumor suppressive activity and/or loss of 21-integrin expression during PCa progression. [8,10,11]To our knowledge, the potential role of 11-integrins or synergistic role of the neighboring ITGA1 and ITGA2 genes, both encoding for collagen-binding integrin -subunits, have not been addressed in human prostate tumorigenesis and PCa progression.
Here we have performed a meta-analysis consisting of several large cohorts of PCa patients and revealed integrin signaling as the most significantly affected pathway associated with PCa progression.Remarkably, genomic co-deletion or downregulation of ITGA1 and ITGA2 in PCa tumors was significantly associated with disease progression.We hence sought to investigate the potential role of 11and 21-integrins in promoting tumorigenicity of PCa.Functional studies showed that both 1and 2-integrins independently contribute to collagen adhesion and glandular morphogenesis of prostate epithelial cells.Simultaneous loss of 1and 2-integrins induced transformation of benign prostate epithelial cells, leading to increased invasive properties in vitro and metastatic capacity in vivo.Mechanistically, this effect was mediated by induced secretion and activation of autocrine TGF1 that promoted epithelial-tomesenchymal transition (EMT) and nuclear translocation of YAP1.YAP1 transcriptional co-regulator and binding partner of TEAD1 was found to control ITGA1 and ITGA2 expression.Strikingly, loss of TEAD1 phenocopied 12-dKO cells as they were transformed with increased metastatic capacity in vitro and in vivo.Therefore, our work has deciphered a novel mechanism by which TEAD1 regulates 1and 2-integrin expression and thereby exerts tumor suppressive functions while loss of TEAD1 and/or ITGA1/ITGA2 in PCa genomes were found to correlate with disease progression and aggressiveness.

A Meta-Analysis Reveals Prevalent Downregulation of Integrin Signaling Pathway Genes in PCa Development
The roles of androgens and AR-signaling are well-documented in metastatic PCa.However, several other cellular signaling cascades may contribute to PCa development and progression.To comprehensively dissect major signaling pathways involved in PCa, we performed a meta-analysis by incorporating nine clinical PCa expression profiling data.The meta-analysis identified a total of 1284 downregulated and 610 upregulated genes over PCa tumor progression (Figure 1a).Differentially expressed genes (DEGs) were subsequently subjected to PANTHER Pathway analysis for an over-representation test, which remarkably, revealed integrin signaling pathway, consisting of 191 genes, as the most significant cellular pathway deregulated during PCa progression (Figure 1b).Notably, the meta-analysis identified a total of 55 DEGs from the integrin signaling pathway and most of them were downregulated upon PCa development (Figure S1a, Supporting Information).

𝜶1and 𝜶2-Integrins Genomic Loss or Downregulation Correlates with PCa Progression
Previous studies have shown that genes contributing to tumorigenesis are expected to manifest association between DNA copy number alterations and RNA expression levels. [12]We therefore sought to identify potential causal genes from the Integrin signaling pathway by the joint analysis of gene copy number alterations and expression levels.We compared the copy number profiles of differentially expressed genes from the Integrin signaling pathway in a pan-cancer analysis from the TCGA (n = 10967), including PCa.The result pinpointed ITGA1 and ITGA2 as the two genes with the highest proportions of deep loss in PCa compared to other cancer types (Figure 1c).
We assessed whether the genomic alterations of ITGA1/ITGA2 display any clinical impacts on PCa, and thus investigated potential correlation between ITGA1/ITGA2 copy number loss and their expression levels in human prostate tumors.The results showed that ITGA1/ITGA2 expression levels were dramatically decreased in PCa patient tumors with ITGA1/ITGA2 copy number loss/deletion (loss/del) in two independent clinical PCa cohorts (Figure 1d,e; Figure S1b,c, Supporting Information).Next, we examined whether ITGA1/ITGA2 copy loss directly correlates with PCa metastasis in two independent cohorts of PCa patients with primary and metastatic tumors.This analysis revealed that the proportion of ITGA1/ITGA2 copy number loss/del was substantially higher in PCa patients with metastasis compared to patients with localized tumors (Figure 1f; Figure S1d, Supporting Information).These findings suggest a potential functional relevance of ITGA1/ITGA2 copy loss/del in PCa progression.We hence conducted a gene set enrichment analysis (GSEA) using the genome-wide expression profiling data in the TCGA cohort to identify gene sets that are enriched in phenotypes of diploid and ITGA1/ITGA2 loss/del, respectively.These results demonstrated that "CHANDRAN Metastasis DN", a gene signature representing genes downregulated in metastatic versus nonmetastatic prostate carcinoma, was identified as the top enriched gene set across all gene sets depleted in PCa tumors with ITGA1/ITGA2 loss/del compared to diploid tumors (Figure 1g,h).Interestingly, we also found that several cell cycle-related pathways including E2F targets and G2M checkpoint were highly enriched in PCa tumors with ITGA1/ITGA2 loss/del (Figure S1e-h, Supporting Information), further implicating the functional role of ITGA1/ITGA2 loss/del in PCa tumorigenesis and metastasis.
We further evaluated the clinical impact of copy number alterations of ITGA1/ITGA2 on PCa progression and therefore conducted the Kaplan-Meier survival analysis to examine the association of ITGA1/ITGA2 copy number loss/del and patient prognosis in multiple independent PCa cohorts.The results displayed that PCa patients with ITGA1/ITGA2 copy number loss/del were associated with shorter biochemical recurrencefree survival in three distinct PCa cohorts (Figure S1i-n, Supporting Information).To validate the robustness of the association, we additionally carried out a meta-analysis to systematically review, interpret and estimate the overall association between ITGA1/ITGA2 copy number loss/del and PCa patient survival outcomes.This analysis demonstrated that PCa patients with ITGA1 copy number loss/del was significantly associated with shorter biochemical recurrence-free survival (P = 3.07e-05, ln(HR) = 0.74; 95%, ln(CI): 0.39-1.09)(Figure 1i).Similar association was observed for PCa tumor with ITGA2 copy number loss/del (P = 1.26e-05, ln(HR) = 0.77; 95% ln(CI): 0.42-1.11)(Figure 1j).Having demonstrated the high proportion of ITGA1/ITGA2 copy loss/del in metastatic PCa tumors overrepresenting metastasis pathways (Figure 1f-h), we proceeded to investigate the effect of ITGA1/ITGA2 copy number loss/del on patient metastatic progression outcomes.The result strongly indicated that ITGA1/ITGA2 copy number loss/del in PCa patients was associated with increased metastatic risk (Figure 1k,l).Taken together, these data demonstrated that genomic loss/depletion of ITGA1/ITGA2 is accompanied with their decreased expression and is significantly associated with metastatic PCa progression, thereby suggesting a potential prognostic value for the ITGA1/ITGA2-status in PCa risk prediction.
Given that the expression levels of ITGA1/ITGA2 were downregulated in PCa tumors with copy number loss/del (Figure 1d,e; Figure S1b,c, Supporting Information), and that ITGA1/ITGA2 genomic alterations in turn demonstrated clinical impacts on PCa development and progression (Figure 1i-l; Figure S1i-n, Supporting Information), we next explored whether lower mRNA levels of ITGA1/ITGA2 correlated with human PCa progression in the clinical setting and thus analyzed multiple independent PCa cohorts.The results indicated that ITGA1/ITGA2 were reproducibly found to be downregulated in prostate tumor tissues compared to normal prostate glands (Figure 2a-f; Figure S2a,b,d,e, Supporting Information).In particular, we observed that the mRNA levels of ITGA1/ITGA2 were significantly decreased in metastatic PCa samples (Figure 2c,f; Figure S2a-f, Supporting Information).To further validate this finding, we analyzed a tissue microarray consisting of matched normal versus cancer samples from 242 local PCa patients (Table S6, Supporting Information).Four tissue cylinders were picked from each patient sample (two from normal/benign areas and two from carcinoma regions).In normal/benign tissue, the most intense 1-integrin expression was observed in the stromal cells of the prostate and low levels were detected in the basal epithelium whereas 2-integrin was highly expressed by basal epithelial cells and moderately by luminal epithelial cells (Figure 2g).This analysis confirmed a significant downregulation of 2-integrins in tumor lesions (Figure S3a,c, Supporting Information).Curiously, 1-integrin expression was significantly reduced in the tumor stroma (Figure S3a,b, Supporting Information).These results suggest a potential functional role of ITGA1/ITGA2 in prostate tumors.Thus, we investigated the association of ITGA1/ITGA2 expression with PCa patient prognosis by conducting the Kaplan-Meier analysis.The results showed that the patient group with tumors expressing lower levels of ITGA1/ITGA2 had increased risk of biochemical relapse and metastasis in multiple independent PCa cohorts (Figure 2h-k; Figure S2g,h,j,k, Supporting Information).Notably, we also observed that PCa patients with lower ITGA1/ITGA2 mRNA levels experienced shorter time trends for overall survival (Figure S2i,l, Supporting Information).Additionally, we analyzed PCa expression data with clinical features, and found that ITGA1/ITGA2 mRNA levels were significantly lower in tumors with high tumor stage, Gleason score, lymph node and elevated PSA levels in multiple distinct patient cohorts (Figure 2l-s; Figure S2m-q, Supporting Information).Taken together, these results demonstrate prognostic values of ITGA1/ITGA2 loss/del and downregulation upon PCa progression to metastatic stage, suggesting that ITGA1/ITGA2 are new biomarkers that could distinguish aggressive disease and might possess tumor suppressive functions in PCa development.

Loss of 𝜶1and 𝜶2-Integrins Synergistically Promote Prostate Epithelial Cell Invasiveness
Given the strong correlation of 1and 2-integrin depletion and downregulation with PCa progression we analyzed the expression levels of 1and 2-integrin levels in benign prostate epithelial cells (RWPE1) and the malignant PCa cell lines (DuCaP, PC3, VCaP, DU145 and 22Rv1) by Western blotting (Figure 3a; Figure S12, Supporting Information).As expected, HPV18-immortalized benign RWPE1 cells expressed high levels of both 1and 2-integrins.In contrast, brain metastasis-derived DuCaP and DU145 and prostate carcinoma cell line 22Rv1 displayed reduced levels of 1and especially of 2-integrins.Two bone metastasis-derived cell lines showed reduced levels of either 1-(PC3) or 2integrins (VCaP).In summary, cancer cell lines displayed downregulated levels of either one or both collagen-binding integrins.
To study the potential functional role of these integrins in prostate epithelial cells, we took benign RWPE1 cells, expressing high levels of both 1and 2-integrins and generated 1-integrin knockout (1KO) and 2KO RWPE1 cells using 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.
CRISPR/Cas9-mediated genome editing. [13]Two independent gRNAs targeting different exons were designed for each gene and used as independent biological replicates in the subsequent experiments to confirm the specificity of the KO-phenotype and to exclude possible off-target effects (Table S2, Supporting Information).To address the potential redundance or synergistic functions of the two collagen receptors we also established a double knockout cell line (12dKO).Specific loss of the target proteins was confirmed by Western blotting (Figure 3b; Figures S4a and S12, Supporting Information).In our culture conditions, parental RWPE1 cells and RWPE1 cells transduced with a control virus form a cobblestone-like cell monolayer upon confluency (Figure 3c).Individual RWPE1-1KO cells appeared somewhat rounder than control cells and while it took longer for RWPE1-1KO cultures to reach confluency, they eventually formed a monolayer resembling that of the controls.RWPE1-2KO cells, and especially the RWPE1-12dKO cells, displayed an elongated shape reminiscent of mesenchymal or actively migrating cells (Figure 3c).Curiously, RWPE1-2KO cells reached confluency significantly faster than the other cell lines.To study cell proliferation in more detail, we performed an XTT assay.As expected, RWPE1-2KO cells grew significantly faster whereas RWPE1-1KO cells proliferated with slower kinetics when compared with control or 12dKO RWPE1 variants (Figure 3d).
Given that both 1and 2-integrins are collagen receptors, we examined their respective functional roles in collagen adhesion.RWPE1 control, -1KO, -2KO and -12dKO cell variants were seeded onto collagen I-coated substrates and cell spreading in each sample was monitored.RWPE1 control, -1KO and -2KO cells all efficiently spread on collagen whereas the RWPE1-12dKO cells lacking both 1and 2-integrins failed to adhere and spread even after long (up to 12 hours) incubation periods (Figure S4b,c, Supporting Information).Thus, collagen-binding and cell spreading can be mediated by either 1or 2-integrins but in the absence of both collagen adhesion is severely impaired in prostate epithelial cells.Next, we studied the migratory properties of the different RWPE1 variants using the IncuCyte S3 Scratch wound module.Confluent monolayers were scratched, and wound closure was monitored over a period of 30 hours.In line with the observed elongated morphology, wounds in the RWPE1-2KO and RWPE1-12dKO monolayers recovered significantly faster than in RWPE1 control monolayers (Figure 3e,f).In contrast, RWPE1-1KO cells showed significantly impaired wound closure capacity (Figure 3e,f).Since 12dKO cells are incapable of spreading on collagen these results suggest that wound closure is mediated by cell migration on another cell secreted ECM, presumably fibronectin or laminin.

Loss of 𝜶1𝜷1and 𝜶2𝜷1-Integrins Leads to Impaired Acinar Morphogenesis in 3D
PCa progression is associated with gradual loss of cell polarity leading to aberrant glandular morphology and function.Benign RWPE1 cells form hollow cysts resembling acinar topology in vivo when cultured in 3D gels. [6]To assess morphology of control, 1-, 2and 12-depleted RWPE1 cells, the different variants were seeded into 3D basement membrane extract (BME) gels and cultured for up to 7 days.The vast majority of control cells formed hollow cysts with smooth basal surface and an apical lumen occasionally containing few individual cells inside the lumen (Figure 3g,h; Figure S4d, Supporting Information).Only half of the RWPE1-1KO cells formed a central lumen while basal surfaces had a grape-like appearance possibly due to reduced cell-cell interactions (Figure 3g,h).In contrast, 80% of RWPE1-2KO cysts were smooth-surfaced solid clusters lacking visible apical lumens (Figure 3g,h).Surprisingly, the 3D morphology of RWPE1-12dKO cells could not be properly quantitated as they hardly formed any cysts and instead grew as solid cell clusters connected to each other via chains of collectively migrating cells (Figure 3g).This phenotype suggests that 1/2dual depleted RWPE1 cells possess invasive properties in 3D cultures.
To investigate potential functional categories of ITGA1/ITGA2 KO target genes, we performed a gene set enrichment analysis (GSEA) that revealed the epithelial-to-mesenchymal transition (EMT) pathway to be highly enriched in 12-dKO upregulated genes in the Hallmark gene sets (Figure 4b,c).The interferon response pathways and EMT pathway were top enriched in the 1or 2-KO upregulated genes, respectively (Figure S5e,f, Supporting Information).Given the strongest morphological  d,e).g) Representative images of 1and 2-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. phenotype and invasive behavior was observed in the RWPE1-12dKO cells, we focused our analysis on these variants.To further consolidate these findings, we performed additional enrichment analysis in multiple collections of annotated gene sets.The results constantly revealed the strong enrichment of genes upregulated upon 12-dKO for pathways relevant with cancer metastasis and EMT (Figure 4d).Further expression profiling analysis of the EMT pathway revealed that the majority of genes in the EMT pathway were upregulated upon 12-dKO (Figure 4e), which is in line with our above observations on the association of ITGA1/ITGA2 copy number loss/del and downregulation with PCa tumor progression to advanced stages and metastasis (Figure 1f-l, Figure 2; Figures S1d-n, S2, and S3a-c, Supporting Information).We next sought to evaluate whether the copy loss/del or expression levels of ITGA1/ITGA2 directly correlates with PCa progression, and thus further performed correlation analysis with the EMT score that was reported to correlate with known EMT markers in PCa patient tumors (see Experimental Section/Methods).The result showed that the EMT score was markedly upregulated in PCa patients with ITGA1/ITGA2 copy loss/del (Figure 4f).Consistently, ITGA1/ITGA2 expression levels showed negative correlation with the EMT score (Figure 4g).We further stratified PCa patients by the median expression value of ITGA1/ITGA2, and found that in line with previous findings, the EMT score was elevated in patient group with ITGA1/ITGA2 low expression levels (Figure 4h).To further validate these findings, we checked the expression levels of selected key EMT markers in the different cell lines by quantitative PCR.Consistent with the in vitro phenotype, RWPE1-12dKO cells showed significant upregulation of SLUG, TWIST, SNAII, CDH2, ITGA5 and ITGAV and downregulation of CDH1 expression levels indicative of EMT (Figure 4i).Of note, CDH1 was strongly downregulated also in RWPE1-1KO cells whereas the RWPE1-2KO cells showed curious downregulation of SNAIL and CDH2 and neither of the single KOs displayed activation of the full EMT program relevant genes (Figure 4i).Western blot analysis in the different cell lines showed robust upregulation of vimentin, another key EMT-marker (Figure S5g, Supporting Information).Interestingly, the GSEA analysis in the Hallmark gene sets also revealed the TGF signaling pathway, representing a subset of the EMT pathway, significantly enriched in the upregulated genes identified by RNA-seq of 12dKO in the RWPE1 cells (Figure 4j,k).Taken together, our transcriptome profiling analysis highlighted activation of the EMT pathway and the associated TGF-signaling pathway as possible cellular transcriptional programs that underlie the potential higher tumorigenic potential of ITGA1/ITGA2 loss/del in PCa patients and the invasive behavior in 1-/2-integrin dual depleted prostate epithelial cells.

RWPE1-𝜶1𝜶2dKO Cells have Elevated Levels of TGF𝜷1 Secretion Leading to YAP1 Activation
To directly assess activation of the TGF1 pathway in RWPE1-12dKO cells we harvested culture medium from confluent cultures and analyzed levels of TGF1 from concentrated medium.
TGF1 is secreted as a latent complex that can associate with several ECM proteins, such as fibronectin, and that needs to be mechanically or proteolytically activated prior to binding to TGF-receptor proteins at the cell surface. [14]RWPE1-1KO and RWPE1-12dKO cells both secreted higher levels of TGF1 but only in the latter it was found in an activated form (Figure 5a,b; Figure S6a,b, Supporting Information).We subsequently applied two independent approaches to experimentally confirm the role of TGF1 in invasive migration.First, we treated 3D cultures of control RWPE1 cells with recombinant TGF1 and found that this treatment induced similar invasive structures as were seen in RWPE1-12dKO cells (Figure 5c).Second, RWPE1-12dKO cells grown in 3D BME gels were treated with TGF inhibitor, SB431542, that strongly inhibited the branching capacity of these cultures (Figure 5c).
The transcriptional coactivator YAP1 is a common oncogenic factor that promotes proliferation and migration in several cancer types including PCa. [15] Integrins are central regulators of YAP1 that is activated by the Hippo pathway and has been shown to crosstalk with the TGF pathway. [16,17]We therefore analyzed YAP1 expression and subcellular localization in the different RWPE1 variants.In RWPE1 cells YAP1 is mainly cytoplasmic, while in 12dKO cells it was observed in the nucleus (Figure 5d; Figure S6c,d, Supporting Information).
To study if TGF activation regulates nuclear translocation of YAP1, we treated RWPE1 cells with TGF.This resulted in robust nuclear accumulation of YAP1 (Figure 5d).In contrast, inhibition of TGF signaling prevented nuclear targeting of YAP1 in 12dKO RWPE1 cells (Figure 5d).Thus, TGF activation was shown to drive nuclear translocation of YAP1.To investigate the functional role of YAP1, we generated lentiviral shRNA-mediated knockdown of YAP1.Depletion of YAP1 in RWPE1 (Figure S7a,b, Supporting Information) and RWPE1 cells with 12dKO (Figure S7c,d, Supporting Information) was confirmed at both mRNA and protein levels by RT-qPCR and Western blotting, respectively.Interestingly, YAP1-KD in RWPE1 and PC3 cells led to reduced expression levels of ITGA1 and ITGA2 (Figure S7e,f, Supporting Information).We next investigated the correlation between ITGA1/ITGA2 and YAP1 expression in the human prostate tumors and observed significant positive correlation in multiple independent PCa cohorts (Figure S7g, Supporting Information).These findings suggest that YAP1 might contribute to the regulation of ITGA1/ITGA2 transcription.Given that YAP1 has been reported to regulate transcription by interacting with TEAD-family members, [18] we Figure 4. Loss of both 1and 2-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-12dKO (left panel), -1KO (middle panel) or -2KO (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 RWPE1-12dKO cells.Categories were ranked by the Normalized Enrichment Score (NES).c) GSEA enrichment plot displaying the EMT pathway enriched in upregulated genes in RWPE1-12dKO 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 12-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 TGF signaling pathway was significantly enriched in the upregulated genes with 12dKO in the RWPE1 cells in multiple gene set resources.k) GSEA enrichment plot demonstrating the TGF signaling pathway enriched in the upregulated genes in RWPE1 cells with 12dKO.explored a large collection of genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) data derived from PCa and other tissues including breast and lung to study the regulatory mechanisms at the ITGA1/ITGA2 loci.We found an enrichment of chromatin binding of TEAD1 and YAP1 transcription factors in the proximity of ITGA1 and ITGA2 promoter regions (Figure S7h, Supporting Information).To study whether YAP1 is required for the activation of TGF signaling in 12dKO cells, we generated YAP1-depleted 12dKO RWPE1 cells (12dKO-YAP1KD).While 12dKO-YAP1KD cells showed reduced YAP1 levels, no significant effect was seen on their ability for secreting and activating TGF1 (Figure S7i, Supporting Information).However, downregulation of YAP1 indeed led to reduced proliferation of 12dKO cells, suggesting that YAP1 activation contributes to the proliferative capacity of 1and 2-integrin depleted PCa cells (Figure S7j, Supporting Information).

𝜶1𝜶2KO Cells have Enhanced Metastatic Capacity In Vivo
The parental RWPE1 cells are benign and do not form tumors in vivo. [6]Given the apparent invasive properties of 1and 2-depleted RWPE1 cells in vitro, we next assessed the metastatic capacity of control, 1KO, 2KO and 12dKO RWPE1 cells using the tail-vein injection model in immunocompromised SCID mice.All the cell lines were transduced to express luciferase and GFP to allow in vivo visualization.After 4 weeks the mice were sacrificed, and the lungs were imaged using an in vivo imaging system (IVIS).Significant tumor foci were seen only for the 12dKO-injected group and weak signals were observed in some of the lungs in the 2KO group (Figure 6a).Interestingly, detailed histological analysis of the lungs revealed several micrometastases in all but one mouse of the 12dKO group and in two 2KO samples.A single very small lesion was found in one of the 1KO samples (Figure 6b).As expected, no lesions were found in the RWPE1 control samples.The sizes of micrometastases in 12dKO samples were significantly larger than those found in 2KO cells (Figure 6c).Given that the capacity of tumor cells to survive in circulation is crucial for their metastatic capacity, we collected the blood from each mouse and quantitated the number of circulating tumor cells (CTCs).Blood could not be collected from one mouse injected with 12dKO cells.This analysis revealed that significantly more CTCs were detected in 12dKO and 2KO groups (Figure 6d).Collectively, these results provide supporting evidence that the benign prostatic epithelial cells with co-deletion of 1and 2-integrins showed enhanced metastatic potential in vivo.

TEAD1 Transcriptionally Correlates with ITGA1 and ITGA2 and Directly Regulates their Expression via Chromatin Binding
Next, we studied the mechanisms of transcriptional regulation of ITGA1 and ITGA2 expression in PCa.We performed a genomewide co-expression analysis of ITGA1/ITGA2 in multiple PCa datasets to prioritize potential transcription factors that regulate ITGA1/ITGA2.Interestingly, TEAD1 emerged as the top candidate gene that was co-expressed with ITGA1/ITGA2 in the TCGA cohort consisting of 498 localized PCa tumor samples (Figure 7a-d).Consistently, this finding was verified in another PCa cohort comprised of 150 tumor samples (Figure S8a-d, Supporting Information).In contrast, we observed that the expression levels of the other members of the TEAD gene family, TEAD2-4, were not correlated with either ITGA1 or ITGA2 (Figure 7a,b; Figure S8a,b, Supporting Information).This find-ing suggested that TEAD1 could be a key transcriptional driver of ITGA1 and ITGA2.
To experimentally validate this, we performed TEAD1 ChIPseq in the PC-3 cell line as previously described, [19,20] (Figure S8e, Supporting Information).A follow-up analysis showed topenriched DNA-binding motif of the TEAD family transcription factors (Figure S8f, Supporting Information), demonstrating the success and reliability of the ChIP-seq experiment.The subsequent genomic annotation analysis of ChIP-seq peaks illustrated that TEAD1 chromatin occupancies were greatly enriched in the gene proximal promoter regions compared to the genome background (Figure S8g, Supporting Information).Notably, among TEAD1 chromatin binding regions, we observed an enrichment of TEAD1 binding sites at the bi-directional promoter of ITGA1 and ITGA2 genes and within the ITGA1 gene body (Figure 7e).To further validate the binding of TEAD1 at ITGA1 and ITGA2 gene regulatory regions, we performed ChIP-qPCR analysis in PC-3 cells as described previously. [21]This analysis confirmed TEAD1 chromatin binding at the three regions determined by ChIP-seq (Figure 7f).To further evaluate TEAD1-mediated regulation of ITGA1/ITGA2 expression, we established TEAD1-KO cells of RWPE1 and PC3 and observed reduction of ITGA1 and ITGA2 mRNA levels in TEAD1-KO RWPE1 cells (Figure 7g).Furthermore, Western blot analysis confirmed robust downregulation of 1and 2-integrins in RWPE1-and PC3-TEAD1-KO cells (Figure 7h; Figure S12, Supporting Information).Collectively, these results demonstrate that TEAD1 directly regulates the expression of ITGA1/ITGA2, and this gene expression control might also hold true in the clinical setting due to their robust co-expression rates across multiple independent cohorts of PCa.

TEAD1 Expression Correlates with ITGA1 and ITGA2 Expression and is Lost during PCa Progression
Given that ITGA1/ITGA2 copy loss and downregulation correlated with PCa severity and TEAD1 regulated ITGA1 and ITGA2, we next studied whether TEAD1 genomic alterations and expression levels directly correlate with PCa clinical characteristics.We found that TEAD1 genomic loss/del correlated with lower expression levels (Figure 8a; Figure S9a,b, Supporting Information).Notably, PCa patients with TEAD1 copy loss/del showed significantly higher risk for biochemical recurrence (Figure 8b).In line with the association of TEAD1 copy loss/del with PCa clinical severity, we observed that TEAD1 expression levels were gradually decreased upon PCa development and progression compared to normal prostates (Figure 8c,d; Figure S9c-e, Supporting Information).Moreover, we also observed that decreased TEAD1 expression levels were significantly correlated with PCa severity, including advanced tumor stage (Figure 8e), higher Gleason score (Figure 8f), lymph node metastasis (Figure 8g) and PSA levels (Figure 8h).Given that TEAD1 was downregulated upon disease progression, we studied whether TEAD1 expression may possess prognostic value.We conducted the Kaplan-Meier survival analysis and found that low TEAD1 expression significantly correlated with an increased risk of biochemical recurrence (Figure 8i,j; Figure S9f, Supporting Information) and metastasis (Figure 8k) in PCa patients.To further validate in silico analysis, we utilized our Tissue Micro Array (TMA) samples to investigate TEAD1 expression in PCa patient tissues.We found that in benign samples TEAD1 was highly expressed in the nucleus of basal epithelial cells (Figure S9g, Supporting Information).Some nuclear TEAD1 expression was also seen in stromal cells (Figures S9g and S3a, d, Supporting Information).In contrast, we observed little or no TEAD1 staining in cancer tissues (Figure 8l; Figure S3a, d, Supporting Information).In line with these observations, western blot analysis revealed high levels of TEAD1 in benign RWPE1 cells whereas some malignant PCa cell lines exhibited lower TEAD1 levels (Figure 8l; Figure S12, Supporting Information).Curiously, TEAD1 levels in the different PCa cell lines appeared to correlate with 2-integrin levels (Figure 3a).Given the strong correlation of TEAD1 and ITGA1/ITGA2 expression and the observed regulatory circuit of TEAD1-ITGA1/ITGA2, we next examined whether the RWPE1-TEAD1-KO cells display similar transformed phenotypes observed in RWPE1-12dKO cells.While not as striking as seen in 12dKO cells, confluent RWPE1-TEAD1-KO cells had slightly more elongated shape (Figure 8m).Like 12-dKO cells, TEAD1-KOs showed similar proliferative capacity when compared with the controls (Figure 8n) but migrated significantly faster than the controls in the scratch wound assay (Figure 8o).Collectively, these results indicate that dampened regulatory circuit of TEAD1 and ITGA1/ITGA2 could contribute to PCa progression.

Loss of TEAD1 Phenocopies the 𝜶1𝜶2KO Cells
To further understand the mechanisms by which TEAD1 promotes PCa progression, we conducted RNA-seq in PC3 cells treated with either negative control sgRNA or two different sgR-NAs targeting TEAD1.Two biological replicates were included in each group and high correlations were determined between the replicates (Figure S10a-c, Supporting Information; r = 0.9994 for control, r = 0.99996 for tEAD1 KOa, and r = 0.9995 for tEAD1 KOb).We then performed differential gene expression analysis and identified 549 and 345 genes significantly upregulated and downregulated by TEAD1, respectively (Figure S10d, Supporting Information).To better understand how these TEAD1 deregulated genes may impact biological processes underlying specific phenotypes, we carried out GSEA analysis in five distinct pathway gene sets, including Hallmark, Chemical and genetic perturbations (CGP), Reactome, WikiPathways (WP), and BioCarta from the MSigDB database.Intriguingly, multiple cancer-promoting pathways, including TGF signaling, EMT, and metastasis, were repeatedly found highly enriched in upregulated genes upon TEAD KO (Figure 9a; Figure S10e, Supporting Information).GSEA enrichment plots further indicated that these functional pathways were highly enriched in upregulated genes targeted by TEAD1 in PC3 cells (Figure 9b,c).To observe how TEAD1 affects normal prostate cells, we conducted similar RNA-seq with TEAD1 KO in RWPE1 cells.High sample correlations were obtained between two biological replicates (Figure S10f,g, Supporting Information).Follow-up differential gene expression analysis identified 1368 and 929 upregulated and downregulated genes, respectively (Figure S10h, Supporting Information).Remarkably, TGF signaling, required for tumor invasiveness and metastasis, was repetitively found enriched in five different pathway gene sets (Figure 9d; Figure S10i, Supporting Information).GSEA plots indicated upregulation of TGF signaling pathway in TEAD1-KO RWPE1 cells (Figure 9e,f).Vimentin was upregulated in RWPE1-TEAD1-KO cells (Figure S5g, Supporting Information).These results further support a functional role for TEAD1 loss in PCa tumorigenesis.To directly study the activation of TGF1 pathway, 3D cultures of control or TEAD1KO RWPE1 cells were treated with or without addition of recombinant TGF1.Intriguingly, the 3D BME-grown RWPE1-TEAD1-KO cells displayed branching morphogenesis in the absence of TGF, suggesting an invasive phenotype (Figure 9g).The branching of TEAD1-KO RWPE1 cells was shown to be TGF-1-dependent as TGF-1i completely blocked invasive migration in 3D (Figure 9h).TEAD1-KO RWPE1 cells also displayed autocrine secretion and activation of TGF1 (Figure 9i).The tumorigenicity of RWPE1-KO cells was analyzed using the tail vein injection model as described for the integrin mutants above.RWPE1-TEAD1-KO cells formed micrometastases in all the injected mice and the size of these lesions was similar as was seen for 12dKO lesions (Figure 9j,k).Moreover, CTCs were recovered from the blood of every mouse injected with TEAD1-KO RWPE1 cells (Figure 9l).

Synergistic Effects of ITGA1, ITGA2 and TEAD1 Downregulation Promotes PCa Progression and Severity
Given downregulation of ITGA1, ITGA2 or TEAD1 apparently associated with poor PCa prognosis (Figure 2h-k, Figure 8i-k; Figures S2g-l and S9f, Supporting Information), we sought to explore whether ITGA1, ITGA2 or TEAD1 expression levels may hold predictive value for stratifying PCa patients with low-and high-risks.We hence stratified several cohorts of PCa patients based on Gleason scores and investigated the potential correlation between gene expression and patient prognosis.The result suggested an explicit predictive value of ITGA1, ITGA2 or TEAD1 mRNA levels for patient prognosis in PCa group with a Gleason score of 7 (intermediate risk, Figure 10a-f), but not for the low-risk cases with Gleason score ≤6 (Figure S11a-f, Supporting Information) nor high-risk group with Gleason score ≥8 (Figure S11g-l, Supporting Information).These results indicate ITGA1, ITGA2 or TEAD1 as a potential independent prognostic marker in distinguishing PCa patients who are classified with the intermediate risks, representing the most difficult patient group in clinic to avoid the overtreatment.
Although we found that decreased expression of ITGA1, ITGA2, or TEAD1 correlated with PCa severity, it remains unclear whether loss of these three genes have synergistic effects on tumor severity compared with loss of either individual gene.Therefore, we calculated the expression sum of the three genes in pairwise or triple-wise manner and examined the impact on patient prognosis.Intriguingly, the results showed that triple low expression of the three genes demonstrated highest hazard ratio for the overall survival (Figure 10g) and biochemical recurrence (Figure 10h) of PCa patients.To solidify our findings, we stratified PCa patients with triple low or -high expression levels of ITGA1, ITGA2 and TEAD1 and conducted the Kaplan-Meier analysis in multiple PCa cohorts.We found that patients with simultaneous triple low expression of ITGA1, ITGA2 and TEAD1 demonstrated significantly shorter overall survival (Figure 10i), higher risk of biochemical recurrence (Figure 10j; Figure S11m, Supporting Information), and metastasis (Figure 10k).Moreover, upon stratification of our inhouse TMA patients with expression data of all the three proteins combined, we found that patients with triple negative/low expression have an increased risk to develop bone metastasis when compared with patients with a loss of expression of only one or two of the markers.The biggest difference was found when the triple-negative/low patient groups were compared with triple-positive patient group (Figure 10l).To further explore the synergistic effects of the loss of the three genes in clinical settings, we examined the correlation with several clinical features, including Gleason score, PSA, tumor stage, and lymph nodes, in PCa cases.The results constantly showed that PCa patients with triple low expression of ITGA1, ITGA2 and TEAD1 demonstrated higher Gleason score (Figure 10m), PSA levels (Figure 10n; Figure S11n, Supporting Information), advanced tumor stages (Figure 10o) and lymph node metastasis (Figure 10p).Taken together, these findings suggested that identification of the ITGA1/ITGA2/TEAD1-triple negative PCa cases may have potential in detecting aggressive forms, especially in the most problematic Gleason score 7 PCa cases with uncertain PCa clinical consequences.Despite the usefulness of PSA screening of potential PCa cases, it alone is not a reliable diagnostic tool. [22]Our data showed that PCa patient group with triple low expression of ITGA1/ITGA2/TEAD1 was markedly associated with poor prognosis, increased disease severity and PSA levels, suggesting the ITGA1/ITGA2/TEAD1-axis as a potential biomarker for early de-tection of PCa.Taken together, ITGA1/ITGA2/TEAD1 expression level in the PCa specimens could be considered as novel additional biomarkers helping to make more accurate treatment decisions in the clinic for PCa patients with high PSA levels and intermediate Gleason score 7.

Discussion
PCa progression involves drastic changes in cell-ECM contacts.][25] Our meta-analysis performed here revealed a general downregulation of the integrin signaling pathway.While it is thought that invasive PCa cells need to interact with the stromal ECM rich in collagen, we found that the subunits of the main collagen-binding integrins, 1 and 2, were also strongly downregulated.Importantly, we demonstrated that loss of both 1and 2-integrins not only correlated with PCa progression but also contributed to tumorigenesis by inducing autocrine secretion and activation of TGF, thereby leading to EMT.ITGA1 and ITGA2 are neighboring genes in the integrin collagen receptor locus on human chromosome 5q11.2.Certain single nucleotide polymorphisms (SNPs) within this region have been found to be associated with increased PCa risk. [26]We report here that deletion of this locus is a relatively common (>2%) event in prostate, ovarian and esophageal cancers and deletion and loss as well as downregulation of both ITGA1 and ITGA2 correlate with development of an aggressive form of PCa.
Here we identified TEAD1/Tef-1, encoded by TEAD1 gene, as the key regulator of ITGA1/ITGA2 transcription.Out of the four different TEAD family members, only TEAD1 expression was strongly correlated with ITGA1 and ITGA2 expression.All the four TEAD proteins have been implicated in coordination of especially the Hippo signaling pathway, but TEADs regulate also Wnt-, TGF-and epidermal growth factor receptor (EGFR) signaling pathways.Surprisingly, while TEAD overexpression has frequently been linked to tumorigenesis, we observed here that TEAD1 downregulation or deletion was strongly associated with aggressive PCa.Our data suggest that deletion or downregulation of TEAD1 contributes to PCa progression by abrogating 1and 2-integrin expression.In contrast to our data, a previous study reported that upregulation of TEAD1 correlated with worse PCa prognosis. [27]The reasons for these discrepant results are unclear but the specificity of the TEAD1/Tef-1 antibodies used for immunohistology could perhaps be one contributing factor.In addition to histological studies, our current study included analyses of TEAD1 mRNA levels and genomic deletion or loss 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.
of TEAD1 with all the data supporting a tumor suppressive function for TEAD1 in PCa.Curiously, in agreement with our data, Knight et al. also reported aberrant 3D morphogenesis in TEAD1depleted RWPE1 cells.Thus, it seems clear that the role of TEADs in PCa needs further scrutiny.It is also noteworthy, that despite early promise, recent studies addressing the pharmacological potential of targeting the TEAD/YAP1-axis have raised concerns related to activation of alternative oncogenic pathways as seen for TEAD1-KO cells in our study. [28]hile TEAD1 loss mediates some of its tumorigenic effects by influencing 1and 2-integrin levels there were also apparent differences in the mechanisms.First, we found that 12dKO led to TGF-driven EMT and nuclear translocation of YAP1 to promote proliferation.[31] Importantly, despite their avid DNA-binding capacity, TEAD family members have limited transcriptional activity by themselves.Transcriptional activation depends on their ability to recruit cofactors, namely YAP1 and its paralog Taz. [18]lthough the nuclear YAP1 translocation could be mediated by the other TEAD/Tef-family proteins in TEAD1-KO cells we did not find evidence supporting this in our model.Second, the transcriptomic analysis of RWPE1-12-dKO and -TEAD1-KO cells revealed significant differences with 12-dKO cells displaying robust activation of the EMT signaling pathway while TEAD1-KO cells showed activation of myc-, TNF and interferon pathways (data not shown).Given the similarity between the different TEAD proteins, depletion of TEAD1 might promote aberrant activation of the others potentially contributing to PCa oncogenesis.Indeed, TEAD2/Tef-4 has been shown to promote TGF-driven EMT. [29]GF signaling is thought to play a dualistic role in solid tumors.While it initially limits cancer growth by inhibiting cell cycle progression and inducing apoptosis at later stages of tumorigenesis TGF signaling promotes proliferation, invasion and drug resistance. [14]Uncoupling of the proapoptotic signaling arm of TGF cascade in pretumorigenic cells is thought to convert TGF from a tumor suppressor into an oncogene.Moreover, TGF is a strong immune suppressant.Thus, autocrine TGF signaling has potentially strong protumorigenic effects.TGF is secreted in an inactive latent form that can be stored in the ECM within the tumor microenvironment.Activation of TGF is a complex process that can occur via multiple mechanisms many of which involve integrin functions. [14]Interestingly, we found that cells lacking 1-integrin activated TGF production and secretion, but it remained mostly in inactive latent form.However, in 12-dKO cells lacking both collagen receptors, upregulated TGF was efficiently secreted and activated in autocrine manner.One potential mechanism underlying TGF activation could be the observed upregulation of V-integrins in 12dKO cells.In particular, V6and V8-integrins are critical activators of latent TGF. [32,33]Moreover, fibronectin expression is also upregulated in 12-dKO cells.Fibronectin is not only a ligand for Vclass integrins, but it also sequestrates latent TGF complex and is essential for TGF activation. [34]Whether an integrin switch from laminin-(64) and collagen-binding (11 and 21) integrins to RGD-binding integrins (V-class integrins and 51) is indeed driving PCa tumorigenesis merits further studies.Interestingly, a bispecific antibody targeting Vand 51-integrins, both of which were upregulated in 12-dKO cells, was shown to inhibit PCa tumorigenicity. [35]In addition, high expression levels of V5and V3 integrins have been detected in metastatic PCa. [36,37]

Conclusions
Taken together, our data identifies downregulation of 1and 2-integrins as a risk factor in PCa.Genomic loss or deletion of ITGA1/ITGA2 locus was found to be highly frequent in PCa cases and equally prevalent in ovarian and esophageal cancers, suggesting that the tumor suppressor functions of 1and 2integrins might not be limited to PCa.Loss of these collagen receptors triggered autocrine TGF-signaling and subsequent EMT leading to increased invasive and metastatic potential of 1/2-integrin deficient PCa cells in vitro and in vivo.1/2depleted cells also activated YAP1 signaling, that contributed to cell proliferation in these cells.Moreover, TEAD1 was identified as a key transcriptional regulator of ITGA1 and ITGA2 whose downregulation also contributed to PCa progression.The TEAD1/ITGA1/ITGA2-axis can potentially be exploited for both diagnostics purposes and for the development of more potent therapies targeting malignant forms of PCa.

Experimental Section
Cell Culture: Benign RWPE-1 prostate epithelial cells (ATCC CRL-11609) were cultured in in K-SFM + Human recombinant Epidermal Growth Factor (rEGF) and Bovine Pituitary Extract (BPE) supplemented Figure 9. Loss of TEAD1 phenocopies the dual loss of 1and 2-integrins in vitro and in vivo.a) Multiple functional categories, including TGF 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 TGF signaling, EMT, and metastasis enriched in upregulated genes upon TEAD KO in PC3 cells.d) TGF 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 TGF 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 TGF1 inhibitor (TGF1i) as described in (d).Scale bar is 50 μm.i) Culture medium was harvested and concentrated from RWPE1-WT, RWPE1-12dKO 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.with 1% penicillin and streptomycin (all from Thermo Fisher Scientific).PCa cell line PC-3 (CRl-1435), VCap (CRL-2876), DuCap (CVCL_2025), 22Rv1 (CRL-2505), LNCaP (CRL-1740) and DU145 (HTB-81) were purchased from ATCC.VCaP and 293T-D10 (a kind gift from Dr. Peppi Karppinen, University of Oulu) were grown in Dulbecco's Modified Eagle's Medium (DMEM) (11 965 092, Thermo Fisher Invitrogen), for DU145 Eagle's Minimum Essential Medium (EMEM) (30-2003, ATCC) was used.LNCaP, VCaP and 22Rv1 were grown in Roswell Park Memorial Institute Medium (RPMI 1640) (R8758, Sigma).PC-3 were grown in F12-K (30-2004,  Invitrogen).DMEM, EMEM and RPMI were supplied with a final concentration of 10% fetal bovine serum (16 000 044, Thermo Fisher) and 1% of penicillin and streptomycin (15 140 122, Thermo Fisher).All cell lines were cultured in humidified incubators at 37 °C and 5% CO 2 .
Antibodies and Reagents: Primary antibodies were listed in Table S1 (Supporting Information).All secondary antibodies, nonspecific sheep, rabbit, and mouse IgG isotype controls were purchased from Jackson Im-munoResearch.TRITC-phalloidin and DAPI were from Merck.
Lentivirus-Mediated Gene Knockout: Gene knockouts using the lentiviral CRISPR/Cas9-methodology were generated as described previously. [13,38]In short, two separate target sequences from constitutive early exons were designed for each gene (Table S2, Supporting Information).Target sequences with no predicted off-target sites (a minimum of three mismatches with any other site in the human genome (GRCh38.p13))were selected based on FASTA similarity search tool (EMBL-EBI).For ITGA1, ITGA2 and TEAD1-KO1 constructs gRNA oligos with BsmBI (New England Biolabs) overhangs were ordered from Sigma-Aldrich and subcloned into lentiCRISPRv2-puro (Addgene plasmid # 52 961, a gift from Feng Zhang) or lentiCRISPRv2-neo (Addgene plasmid #98 292, a gift from Feng Zhang) and used for lentivirus preparation.The TEAD1-KO2 lentiviral construct was purchased from the Biomedicum Functional Genomics Unit at the Helsinki Institute of Life Science and Biocenter Finland at the University of Helsinki.To produce lentiviruses, 70%-80% confluent 293T-D10 on CellBind 10 cm Ø tissue culture dishes (Corning) were co-transfected with lentiCRISPR (Addgene plasmid 52 961), pPAX2 and VSVg plasmids using Lipofectamine 2000 reagent (Thermo Fisher Scientific) in Opti-MEM (Thermo Fisher Scientific).For infection, subconfluent cultures were incubated with virus-containing supernatants in the presence of 4 μg mL −1 polybrene (Sigma-Aldrich, TR-1003) for 24 h, virus was removed, and culture continued for 24 h, as described before. [13]Cells were then trypsinized and reseeded into medium containing 3 μg mL −1 of puromycin to select transduced cells.Clonal cell lines were established from resistant populations.To generate double 1/2-KO cells, clonal 1KO cells were transduced with 2-targeting lentiCRISPRv2-neo vector as described above, expanded and selected by different antibiotic selection, puromycin for 1-targeting lenti-CRISPR vector and 500 μg mL −1 of neomycin for 2-targeting lentiCRISPR vector.
Lentivirus-Mediated Gene Knock-Down: The lentiviral pLKO.1-basedshRNA expressing vectors targeting YAP1, were purchased from the Biomedicum Functional Genomics Unit at the Helsinki Institute of Life Science and Biocenter Finland at the University of Helsinki.Second generation lentiviral vectors were packaged using 293T-D10 cells, as described above.Target cells were transduced sequentially two times, and the transduced cell population was selected using 1 μg mL −1 puromycin (Sigma) as described previously. [39] Cell Culture: One hundred μls of Matrigel (Corning, #354 230) was layered onto a 3.5 cm high glass bottom cell culture-dish (IBIDI, #81 156) and allowed to solidify for 30 min at + 37 °C (5% CO 2 ).Control and 1KO, 2KO or 12dKO RWPE1 cells grown to confluency were trypsinized and counted.Five thousand cells per sample were resuspended into 100 μl of ice-cold Keratinocyte-SFM containing 2% of FBS and 2% (v/v) of Matrigel.Resuspended cells were seeded onto Matrigel-coated dishes and incubated for 10 min at + 37 °C after which 1 ml of Keratinocyte-SFM medium containing and 2% Matrigel was added.Matrigel-containing medium was subsequently refreshed every two days.One week later, cells were fixed, stained using DAPI (Sigma, #D9542-10) and filamentous actin (Sigma, #A5441), and analyzed by using an Olympus FluoView FV1000 confocal microscope or Olympus Cellsense both equipped with 20x UPLSAPO objective (NA = 0.75).
Immunofluorescence Staining and Microscopy: Cells were seeded on slides (2D culture) or on IBIDI glass bottom dishes (3D culture) and were cultured until desired confluency.Cells were fixed with 4% PFA in PBS+/+ (PBS with 0.9 mM CaCl 2 and 0.5 mM MgCl 2 ) for 15 min at room temperature for slide and 30 min for IBIDI.Immunofluorescence staining was performed as previously described 7 .Confocal images were acquired with the Zeiss LSM 780 laser scanning confocal microscope using 40x Plan-Apochromat objective (NA = 1.4) or with Olympus FluoView FV100 confocal microscope using 20x and 40x objective (NA = 0.75).Image acquisition software was ZEN (black edition, LSM 780; blue edition Cell Observer) and with Olympus FluoView viewer software, respectively.
SDS-PAGE and Western Blotting: Confluent cultures were washed in PBS-/-(Gibco) and scraped into RIPA buffer: 0.5% SDS,150 mM NaCl, 10 mM Tris-HCl pH 8.0, 1% IGEPAL, 1% sodium deoxycholate with 2 mM PMSF (phenylmethylsulfonyl fluoride), 10 μg mL −1 aprotinin, and 10 μg mL −1 leupeptin.Protein concentration was calculated using BCA Protein Assay Kit (Pierce).30 ug of protein lysate were separated on 7,5% SDS-PAGE gels and transferred onto a Protran pure 0.2 micron nitrocellulose (Perkin Elmer) for 1 h 30 min in 20% ethanol, 0.192 M glycine and 0.025 M Tris.The membranes were blocked for 1 h in 5% skimmed milk and probed with specific primary antibodies (Table S1, Supporting Information) overnight at 4 °C.Secondary antibodies conjugated with HRP and Lumi-Light Western Blotting Substrate (Roche) were used to visualize specific protein bands.The bands were detected using Fujifilm LAS-3000 bioimaging or AZURE 600 and scientific research imaging equipment (Fuji photo film co., LTD).
Measurement of Secreted TGF-b: Ten milliliters of K-SFM with supplements was added onto confluent cultures of the different RWPE1 variants on Ø 10 cm tissue culture dishes.Forty-eight hours later conditioned medium (CM) was collected and centrifuged at 168xg for 5 min to remove cells and debris.CMs were concentrated using Amicon Ultra (Merk Millipore UFC503096) columns centrifuged at 101xg for 10 min.The 100x concentrated CM was analyzed for TGF by Western blotting.
Adhesion Assay: Control, 1KO, 2KO and 12dKO RWPE1 cells were seeded onto 6-well plates (Corning-Sigma) coated with 20 μg mL −1 collagen-I (Advanced BioMatrix, 5153-A).After 30 min, plates were placed into Incucyte S3 automated imaging system (Essen Bioscience) and phase contrast image were taken for 24 with 3 h intervals using Brightfield 20x objective.Posterior analysis of the attached cells was performed by counting the cells that were properly attached and the average cell count were subjected for statistical analysis.).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 1-integrin, 2-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.
XTT Assay: 4000 cells were seeded on 96-wells plate and allowed to grow overnight.The Cell Proliferation Kit II XTT (11 465 015 001, Roche) was used according to the manufacturer.Cell proliferation was examined at day 1, 3, 5 and 7 by XTT colorimetric assay (absorbance at 450 nm).At least three replicate wells were prepared per condition and the data were statistically analyzed with a two-tailed Student's t test.
Scratch Wound Assay: Cells were seeded into 96 well plates and allowed to grow to confluence.The scratch wound was done using the Incucyte Cell Migration Kit (Essen Bioscience 4493) and the wells were washed twice with PBS to remove detached cells.Fresh culture medium was added, and the wound areas were imaged at 3 h intervals using Incucyte Zoom (Essen Bioscience) equipped with a 20x objective.The wound area closure was analyzed using the Incucyte Zoom software (Essen Bioscience).
Chromatin Immunoprecipitation (ChIP): The cells on tissue culture plates were cross-linked in final concentration of 1% formaldehyde for 10 min at room temperature.Glycine was added to a final concentration of 125 mM to stop the reaction.Cell pellets were collected and snapped frozen in liquid nitrogen.Pellets were then suspended in hypotonic lysis buffer (20 mM Tris-Cl, pH 8.0, with 10 mM KCl, 10% glycerol, 2 mM DTT, and Complete protease inhibitor cocktail (Roche)) and incubated for up to 50 min to isolate the nuclei.The nuclei were washed twice with cold PBS and lysed in SDS lysis buffer (50 mM Tris-HCl, pH 8.1, with 0.5% SDS, 10 mM EDTA, and cOmplete Protease Inhibitor).An average size of 400 bp of chromatin was prepared by sonication (Q800R sonicator, Q Sonica).Seventy μl of Dynabead protein G (Invitrogen) slurry per each reaction was washed twice with blocking buffer (0.5% BSA in IP buffer), followed by 10 h incubation with 7 μg of TEAD1/Tef-1 antibody against the target protein or control IgG in 1000 μl of 0.5% BSA in IP buffer (20 mM Tris-HCl, pH8.0, with 2 mM EDTA, 150 mM NaCl, 1%Triton X-100, and Protease inhibitor cocktail).Sonicated chromatin lysate (200-250 μg) was diluted into 1.3 ml of IP buffer and added onto bead/antibody complexes followed by incubation at 4°C for at least 12 h.Next, the complex was washed once with wash buffer I (20 mM Tris-HCl, pH 8.0, with 2 mM EDTA, 0.1%SDS, 1% Triton X-100, and 150 mM NaCl) and once with buffer II (20 mM Tris-HCl pH, 8.0, with 2 mM EDTA, 0.1% SDS, 1% Triton X-100, and 500 mM NaCl), followed by two washes each with buffer III (10 mM Tris-HCl, pH 8.0, with 1 mM EDTA, 250 mM LiCl, 1% Deoxycholate, and 1% NP-40) and buffer IV (10 mM TrisHCl, pH 8.0, and 1 mM EDTA), respectively.A hundred ul of extraction buffer (10 mM TrisHCl, pH 8.0, 1 mM EDTA, and 1% SDS) was added to extract the DNA-protein complexes from the beads.Proteinase K (5 μl from 20 mg mL −1 stock) and NaCl final 0.3 M were added into the complexes incubating overnight at 65 °C to reverse the cross-links of protein-DNA interactions.Finally, DNA was purified with MinElute PCR Purification Kit (Qiagen) and the target DNA fragments were analyzed by qPCR (Table S5, Supporting Information).
Quantitative RT-PCR: RNA was isolated from cultured cell lines using RNeasy Mini Kit (QIAGEN), while DNA in these samples were removed by RNase-Free DNase (QIAGEN).The RevertAid reverse transcriptase Kit (Thermo Scientific) was used to synthesize cDNA from 2 ug RNA.The SYBR Select Master Mix (Applied Biosystems) was used in the Quantitative RT-PCR reactions.High specificity primers were selected for each target (Table S4, Supporting Information).Primer sequences are listed in Table S3 (Supporting Information).For the analysis of mRNA levels, each gene was analyzed at least in triplicate and the data was normalized against an endogenous GAPDH control.For ChIP-qPCR, all target primers had three technical replicates and the data were normalized to the control regions, then the relative enrichment of the target antibodies at target DNA fragment were determined by compared with the background (IgG control).
Immunohistochemical Staining: The tissue slides were deparaffinized by incubation for 1 h at 55 °C.To rehydrate, they were washed twice in xylene preceded 100% ethanol, 94% ethanol and 70% ethanol washes (5 min in each).Heat mediated antigen retrieval was executed by incubating the slides in boiling citric acid buffer (pH 6.0) for 10 min, then washed in PBS-/-, incubated with 3% H 2 O 2 for 10 min and then blocked with 5% BSA in PBS-/-for 1 h.After overnight incubation with primary antibodies at 4 °C, the slides were washed in PBS-/-and probed for 1 h with HRP conju-gated secondary antibody.The positive antibody signal was revealed using DAB Substrate Kit (Abcam, ab64238).Slides were stained for 2 min in Harris hematoxylin solution followed by dehydration steps in 70%, 94% and 100% ethanol and two 5 min treatments in xylene and then imaged using Zeiss Axio Imager.M2 microscope.
Cancer metastasis In Vivo Model: All animal experiments were approved by the Finnish National Animal Experiment Board (permissions ESAVI/3901/2021) and conducted in the Laboratory Animal Centre of the University of Oulu according to the principles of 3R (reduction, refinement, replacement).Intravenous tail injections were performed on 7 weeks old male IcrTac:ICR-Prkdc (SCID) mice (Taconic Biosciences A/S) as described in. [6]In short, 1 × 10 5 RWPE1 cell variants expressing luciferase and GFP were injected into the lateral tail vein (WT (n = 6), 1KO (n = 6), 2KO (n = 6), 12dKO (n = 6) and TEAD1KO (n = 6)).One mouse from each group except the TEAD1KO-group died from unrelated causes during the experiment and were omitted from the analysis.Mice were monitored on a weekly basis using the in vivo imaging system (IVIS) Spectrum CT in vivo bioluminescence imaging system (PerkinElmer).Body weight was monitored every 3 days and no significant differences in body weights between the groups were observed.For analysis, the mice were injected with D-luciferin (Caliper Lifesciences) at 150 mg k −1 g intraperitoneally (IP) and imaged 4 times at 5 min intervals.Four weeks post injection, mice were euthanized, the blood was collected for analysis of circulating cancer cells and the lungs were dissected for analysis of luminescence and GFP signals using the IVIS system.The lungs were divided in two, one was embedded for cryosection (Fisher Healthcare Tissue-Plus O.C.T Compound, 23-730-571, Fisher & Paykel Healthcare AB, Helsinki, Finland) and one for formalin-fixed paraffin embedded (FFPE) sections.FFPE sections covering the entire lung were cut and stained with hematoxylin and eosin (HE).Slides were screened and metastatic lesions were captured using Zeiss Axio Imager M2 microscope with 40x objective.The size of micrometastatic lesions was determined based on sections where the individual lesions covered the largest area.The areas were measured using ImageJ.
The Recovery of Circulating Cancer Cells: 0,5-0,6 ml of blood from mice was immediately diluted in RBC lysis buffer (0.8% NH4Cl, 0.084% NaHCO3, 0.037% EDTA), mixed for 5 min, washed in PBS-/-and the recovered cells were seeded onto 10 cm dishes in K-SFM.After 2 days the cells were harvested by trypsin-treatment, washed in PBS-/-and GFP-positive cells were sorted using BD FACS Aria II (BD Biosciences).
META Analysis and Pathway Enrichment Analysis: We incorporated a total of nine PCa data sets from the GEO repository for conducting the meta-analysis to identify differentially expressed genes among normal prostate tissues, primary and metastatic tumors.The nine PCa data sets (GSE3325, GSE3933, GSE6099, GSE8511, GSE21034, GSE27616, GSE35988-GPL6480, GSE35988-GPL6848 and GSE62872, [40][41][42][43][44][45][46][47] ) were downloaded by R package "GEOquery" with respective GEO IDs.Microarray expression data from each study was first normalized by logarithm transformation.Differential gene expression analysis was conducted on normalized expression data by R package "limma" . [48]We applied adjustment to raw P values by using Benjamini & Hochberg method for multiple testing.Gene symbols were added with respective GEO Platform (GPL) identifiers.Each gene list was further filtered by removing probes corresponding to more than one gene, and unannotated probes and incomplete data was filtered out.Genes with multiple probes were selected based on the lowest P-value.We used R package″ MetaVolcanoR″ [49] to incorporate multiple PCa cohorts with microarray data measured from different platforms for conducting the meta-analysis analysis.We applied both the vote counting approach that identifies differential expressed genes (DEG) for each study, and the Combining-approach that summarizes the fold change of a gene in different studies by the mean and calculates the gene differential expression P values using the Fisher method.We integrated results from both methods, applied a stringent cutoff by selecting differentially expressed genes with significant P values, and followed the same direction of being up or downregulated in at least 6 out of 8 data sets.We finally generated a comprehensive set of 1519 differentially expressed genes with 403 and 1116 up-and downregulated genes, respectively.We next used PAN-THER Pathway, [50] to investigate the underlying biological mechanisms of this DEG set in PCa and identified the integrin signaling pathway as the top altered cellular signaling cascade.
Survival Analysis: The Kaplan-Meier survival analysis was conducted to evaluate the impact of the copy number loss& deletion or low expression level of ITGA1, ITGA2 or TEAD1 on patient prognosis in multiple independent PCa data sets.Patients were stratified based on the copy number loss & deletion or the median expression level of ITGA1, ITGA2 or TEAD1.For the investigation of the synergistic effect of ITGA1, ITGA2 or TEAD1 on patient survival, we included PCa patients with consensus triple high or low expression levels of ITGA1, ITGA2 and TEAD1.To investigate whether ITGA1, ITGA2 or TEAD1 possess prognostic value for PCa patients with intermediate risk, we pre-stratified patients by Gleason score > = 6, 7 or > = 8 that represents low-, intermediate-and -high risks, respectively.Kaplan-Meier survival analysis were conducted using R package "Survival" (v.3.2.13), [51,52]and assessed by using log-rank test.Cox proportional hazards model, [53] was applied to calculate the hazard ratio (HR) for assessing the relative risk between different patient groups.
RNA-Sequencing (RNA-seq) and Differential Gene Expression Analysis: The RNA-seq library of double knockout of ITGA1 and ITGA2, single knockout of ITGA1, ITGA2, or TEAD1 generated paired-end raw sequence reads of 150 bp.Fastq files were first processed by FastQC for quality evaluation.Trimmomatic [54] was subsequently used on raw sequence reads for quality control.A second run of FastQC was applied on cleaned reads to guarantee high read quality.STAR (v2.7.2a) [55] was applied to align processed reads to the GRCh38 human genome reference by default settings.Uniquely mapped reads were then quantitated with parameters "-s no, -I gene_name" using HTSeq-count, [56] with gene annotation information in gene transfer format (GTF) from Encode.Genes with low expression counts (<2 cumulative read count across samples) were excluded before conducting the differential expression analysis by DESeq2 (1.26.0).Genes with FDR <0.05 were identified as differentially expressed.Data normalization was processed by the Variance Stabilizing Transformation (VST) method from DESeq2.Heatmaps comparing gene expression levels across samples were generated by R package "pheatmap" (1.0.12).

Chromatin Immunoprecipitation Sequencing (ChIP-seq):
The ChIPseq library was sequenced, which generated 150 bp long single end reads.FastQC was used to check the quality of raw sequence reads.Trimmomatic, [54] was then applied for the quality control process with following parameters: TruSeq3-SE.fa:2:30:10SLIDINGWINDOW:5:20.Read length less than 10 bp were filtered out.Bowtie2 [61] was then used to map the processed reads to the human genome hg38 with default settings.MACS2 [62] was applied for calling peaks with default settings.UCSC tools were applied to create ChIP-seq coverage signals and Integrative Genomics Viewer (IGV), [63] was used for visualization.
Univariate Analysis: For the univariate analysis, we investigated the association of the PCa patients' overall survival, biochemical recurrence and metastasis with single, pairwise, and triple-wise combinations of gene expression levels of ITGA1, ITGA2 and TEAD1.The z-score sum of gene expression was calculated and PCa patients were then stratified into higher and lower groups by the median expression values of the cumulative expression levels.The analysis was conducted by using R packages "tidyverse", [64] "tidytidbits" [65] and "survivalAnalysis" [66] Statistics were summarized and presented in the forest plot.
Meta-Analysis for Time-to-Event Outcomes: The meta-analysis for investigating the association between the ITGA1 or ITGA2 copy number loss/del and patient prognosis across multiple PCa cohorts was conducted using the R package "metafor" (v.3.4.0) in the R environment v.4.2.0. [67]The pooled HR was calculated by a fixed effect model, [68] provided the I 2 statistic was less than 30% or the fixed effects P value for the I 2 statistic was greater than 0.10, indicating insignificant heterogeneity across studies. [69]tatistical Analysis: The RNA-seq, microarray or clinical data was acquired from the cBioPortal for Cancer Genomics, [70,71] NCBI Gene Expression Omnibus (GEO) repository 49 , Oncomine, [72] and literature.Microarray data for the Meta analysis was uniformly normalized by logarithm transformation (log2).Statistical tests applied across normal prostate tissues, primary and/or metastatic tumors were evaluated by meta-analysis, Mann-Whitney U test or Kruskal-Wallis H test depending on the context and the number of groups.The correction between gene expression data and clinical variables were assessed by Mann-Whitney U test or Kruskal-Wallis H test.The proportion of PCa patients with primary or metastatic tumors presenting the degree of number loss and deletion of ITGA1 or ITGA2 was examined by fisher-exact test.For investigating the synergistic effect of ITGA1, ITGA2 and TEAD1, PCa patients were stratified to high and low groups by the median expression values of ITGA1, ITGA2 and TEAD1, respectively.Patients with consensus high or low expressions of ITGA1, ITGA2 and TEAD1 were labeled as triple high or low group, respectively.The correlations between the synergic expression of ITGA1, ITGA2 and TEAD1 with different clinical variables including PSA, lymph node, tumor stage, and Gleason score were investigated by fisher-exact test.For results from microarray-based expression profiling, gene probes with lowest P values were selected.Samples with missing gene expression or patient survival data were excluded from analyses.P value <0.05 was considered to be statistically significant.For boxplots, the interquartile range (IQR) is depicted by the box with the median represented by the center line.Whiskers maximally extend to 1.5 × IQR (with outliers shown).The above-mentioned statistical analyses were carried out by R (v. 4.1.0)and RStudio (v.1.4.1106).Sample size from each analysis was mentioned in relevant figure or figure legend.Data were expressed as means ± SD of at least three independent experiments.Comparative data were analysed with the unpaired or paired Student's t-test or one-way ANOVA as indicated using GraphPad Prism 8 software.The results were considered statistically significant when the P value was less than 0.05 (*), 0.01 (**) or 0.001 (***).
Ethical Approval Statement: The Oulu PCa cohort samples derived from radical prostatectomy patients were obtained under written informed consent with approval of the Regional Ethics Committee of the Northern Ostrobothnia Hospital District (EETTMK 4/2015) in compliance with the Declaration of Helsinki.Mouse cancer models and protocols were approved by the National Animal Experiment Board (ESAVI/3901/2021).In all animal work, the principle of 3R (reduction, refinement, replacement) was respected.

Figure 2 .
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 1and 2-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) risksfor 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 .
Figure 3. Loss of 1or 2-integrins leads to different but synergistic phenotypes in prostate epithelial cells.a) Level of 1and 2-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, RWPE1-1KO, RWPE1-2KO and RWPE112dKO cell lysates were analyzed for the expression levels of 1and 2-integrins.-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, RWPE1-1KO, RWPE1-2KO and RWPE1-12dKO 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, -1KO and 2KO cell lines.RWPE1-12dKO 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 5 .
Figure 5. TGF activation drives invasive potential in 1and 2-integrin-deficient prostate epithelial cells.a) The culture medium from RWPE1-WT and RWPE1-12dKO cells was harvested and concentrated as described in Experimental Section/Methods.Secreted latent TGF and activated TGF were visualized by western blotting.The data is representative of three independent experiments with similar results.b) Quantitative analysis of the secreted TGF.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 TGF1 for 7 days while RWPE1-12dKO cells were grown in the presence or absence of 5 μM TGF1 inhibitor (TGF1i).Scale bar: 50 μm.d) RWPE1-WT and RWPE1-12dKO cells were grown on glass coverslips in the absence or presence of 10 ng TGF1 or 5 μM of TGF1i, respectively in coverslips for 2 days, fixed and stained for nuclei (blue), actin (red) and YAP1 (green).Scale bar:10 μm.

Figure 6 .
Figure 6.Deletion of 1and 2-integrins in benign RWPE1 prostate epithelial cell line promotes formation of lung micro-metastases.a) 1 × 10 5 RWPE1-WT, -1KO, -2KO and -12dKO 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 RWPE1-12dKO 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 .
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 1and 2-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 10 .
Figure10.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 recurrencefree 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 1-integrin, 2-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.