TEAD4 functions as a prognostic biomarker and triggers EMT via PI3K/AKT pathway in bladder cancer
Authors: Chi Ming , Liu Jiao , Mei Chenxue , Shi Yaxing , Liu Nanqi , Jiang Xuefeng , Liu Chang , Xue Nan , Hong Hong , Xie Jisheng , Sun Xun , Yin Bo , Meng Xin , Wang Biao 
Published: May 2022

Abstract
Background
The distant metastasis is the primary cause of cancer morbidity and mortality for bladder cancer (BLCA) paitents. All the recommended therapy for it largely depends on how far the cancer has invaded. It has been confirmed that epithelial to mesenchymal transition (EMT) is the leading reason for the BLCA metastasis which makes BLCA difficult to cure. The aim of the present study is to identify the BLCA-related genes that can be used as the new prognostic biomarker and treatment target, and to investigate the functional mechanisms of TEAD4 in EMT dysregulation.

Methods
The "limma" R package was used to identify the differentially expressed genes (DEGs) between the normal and the tumor samples from TCGA BLCA and GTEx databases. Kaplan–Meier and UniCox analysis were used to filter DEGs with prognostic value in BLCA. Step muti-Cox analysis was used to construct a prognostic risk score model based on clinical phenotype characters. Gene set enrichment analysis (GSEA) was performed to explore the possible molecular mechanisms affecting the prognosis in BLCA. Unsupervised hierarchical clustering analysis was performed to evaluate the effects of EMT process on the prognosis. Single-sample GSEA (ssGSEA) was used to calculate the correlation betweeen the expression of DEGs and EMT enrichment scores. TEAD4 expression and its association with pathological grading and survival were appraised in samples from TCGA dataset and BLCA tissue microarray. Colony formation assays and CCK8 assays were performed to study the changes in BLCA cell proliferation when the TEAD4 levels was down- or up-regulated in BLCA cells. Transwell and wound healing assays were utilized to analyze the impact of TEAD4 on the invasion and metastasis of the BLCA cells. Western Blot was carried out to detect the changes of EMT-related markers and the active molecules involved in PI3K/AKT signaling in BLCA cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was conducted on the genes related to TEAD4 expression. 740Y-P (activator of PI3K/AKT pathway) and LY294002 (inhibitor of PI3K/AKT pathway) were applied to evaluate the contribution of PI3K/AKT signaling pathway in the EMT of BLCA cells. To examine the in vivo effect of TEAD4 on tumor metastasis, we designed a metastatic nude-mouse model by tail vein injection of TEAD4-knockdown BLCA cells. And PET/CT imaging was used to assess the extent of lung metastases.

Results
A total of 1592 DEGs were recognized, among which 4 DEGs have been identified as independent prognostic factors for BLCA, such as FASN, IGFL2, PLOD1 and TEAD4. TCGA BLCA samples were divided into significantly different low- and high-risk groups according to the median risk score; GSEA analysis showed that HALLMARK EMT pathway was the top enriched gene signature when compared high-risk and low-risk groups, which was also verified by unsupervised cluster analysis. EMT signature-derived ssGSEA scores demonstrated that TEAD4 had the most positive correlation with EMT process. In addition, TEAD4 expression was upregulated in TCGA BLCA samples and correlated with pT stage, tumor stage and tumor grade. Functional studies showed that TEAD4 knockdown via lentiviral TEAD4 shRNA inhibited cell migration and invasion in vitro and in vivo, with the reduced expression of EMT related markers in BLCA cell lines; the migration and invasion of TEAD4 knockdown cells could be restored by ectopic expression of TEAD4. Meanwhile, KEGG enrichment analysis of genes related to TEAD4 expression showed that enrichment was significantly related to PI3K/AKT pathway. The pathway inhibitor LY294002 blocked the TEAD4-induced enhancement of migration and invasion as well as the expression EMT-related markers, whereas the agonist 740Y-P rescued the decreased migration, invasion and EMT induced by TEAD4 knockdown.

Conclusions
TEAD4 is closely correlated with poor prognosis in BLCA and mediates its metastasis through regulating EMT via PI3K/AKT pathway, proving that TEAD4 is not only an effective biomarker for predicting the prognosis but also a great potential target for treatment of metastatic BLCA.