TEAD4 在膀胱癌中作为预后生物标志物，通过 PI3K/AKT 通路触发 EMT。

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.

背景：远处转移是膀胱癌（BLCA）患者发病和死亡的主要原因。所有推荐的治疗方法在很大程度上取决于癌症的侵袭程度。已经证实上皮间质转化（EMT）是膀胱癌转移的主要原因，这使得膀胱癌难以治愈。本研究的目的是确定可作为新的预后生物标志物和治疗靶点的膀胱癌相关基因，并探讨 TEAD4 在 EMT 失调中的功能机制。

方法：使用“limma”R 包鉴定 TCGA BLCA 和 GTEx 数据库中正常和肿瘤样本之间的差异表达基因（DEGs）。Kaplan-Meier 和 UniCox 分析用于筛选在 BLCA 中具有预后价值的 DEGs。逐步 Cox 分析用于基于临床表型特征构建预后风险评分模型。基因集富集分析（GSEA）用于探索影响 BLCA 预后的可能分子机制。无监督层次聚类分析用于评估 EMT 过程对预后的影响。单样本 GSEA（ssGSEA）用于计算 DEGs 的表达与 EMT 富集评分之间的相关性。在 TCGA 数据集和 BLCA 组织微阵列样本中评估 TEAD4 表达及其与病理分级和生存的关系。通过下调或上调 BLCA 细胞中的 TEAD4 水平，进行集落形成实验和 CCK8 实验，研究 BLCA 细胞增殖的变化。通过 Transwell 和划痕愈合实验，分析 TEAD4 对 BLCA 细胞侵袭和转移的影响。通过 Western blot 检测 EMT 相关标志物和 PI3K/AKT 信号通路中涉及的活性分子的变化。对与 TEAD4 表达相关的基因进行京都基因与基因组百科全书（KEGG）富集分析。使用 740Y-P（PI3K/AKT 通路激活剂）和 LY294002（PI3K/AKT 通路抑制剂）评估 PI3K/AKT 信号通路在 BLCA 细胞 EMT 中的作用。通过尾静脉注射 TEAD4 敲低 BLCA 细胞设计转移性裸鼠模型，检查 TEAD4 在体内对肿瘤转移的影响。并使用 PET/CT 成像评估肺转移的程度。

结果：共鉴定出 1592 个 DEGs，其中 4 个 DEGs 已被确定为 BLCA 的独立预后因素，如 FASN、IGFL2、PLOD1 和 TEAD4。根据中位风险评分，TCGA BLCA 样本被分为显著不同的低风险和高风险组；GSEA 分析显示，当比较高风险和低风险组时，HALLMARK EMT 通路是最富集的基因特征，这也通过无监督聚类分析得到了验证。EMT 特征衍生的 ssGSEA 评分表明，TEAD4 与 EMT 过程具有最正的相关性。此外，TEAD4 在 TCGA BLCA 样本中表达上调，并与 pT 分期、肿瘤分期和肿瘤分级相关。功能研究表明，通过慢病毒 TEAD4 shRNA 下调 TEAD4 抑制了 BLCA 细胞系体外和体内的迁移和侵袭，降低了 EMT 相关标志物的表达；TEAD4 敲低细胞的迁移和侵袭可以通过异位表达 TEAD4 恢复。同时，与 TEAD4 表达相关的基因的 KEGG 富集分析表明，富集与 PI3K/AKT 通路显著相关。通路抑制剂 LY294002 阻断了 TEAD4 诱导的迁移和侵袭增强以及 EMT 相关标志物的表达，而激动剂 740Y-P 挽救了由 TEAD4 敲低引起的迁移、侵袭和 EMT 减弱。

结论：TEAD4 与 BLCA 的不良预后密切相关，通过调节 EMT 介导其转移，通过 PI3K/AKT 通路，证明 TEAD4 不仅是预测预后的有效生物标志物，也是治疗转移性 BLCA 的有潜力的靶点。