POLR3G promotes EMT via PI3K/AKT signaling pathway in bladder cancer.

RNA Polymerase III Subunit G (POLR3G) promotes tumorigenesis, metastasis, cancer stemness, and chemoresistance of breast cancer and lung cancer; however, its biological function in bladder cancer (BLCA) remains unclear. Through bioinformatic analyses, we found that POLR3G expression was significantly elevated in BLCA tumor tissues and was associated with decreased survival. Multivariate Cox analysis indicated that POLR3G could serve as an independent prognostic risk factor. Our functional investigations revealed that POLR3G deficiency resulted in reduced migration and invasion of BLCA cells both in vitro and in vivo. Additionally, the expressions of EMT-related mesenchymal markers were also downregulated in POLR3G knockdown cells. Mechanistically, we showed that POLR3G could activate the PI3K/AKT signaling pathway. Inhibition of this pathway with LY294002 reduced the enhanced migration and invasion of BLCA cells induced by POLR3G overexpression, whereas the activation of this pathway using 740Y-P restored the abilities that were inhibited by POLR3G knockdown. Taken together, our findings suggested that POLR3G is a prognostic predictor for BLCA and promotes EMT of BLCA through activation of the PI3K/AKT signaling pathway.

Chen H ，Ma L ，Yang W ，Li Y ，Ji Z ... -  《-》

TEAD4 functions as a prognostic biomarker and triggers EMT via PI3K/AKT pathway in bladder cancer.

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. 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. 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. 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.

Chi M ，Liu J ，Mei C ，Shi Y ，Liu N ，Jiang X ，Liu C ，Xue N ，Hong H ，Xie J ，Sun X ，Yin B ，Meng X ，Wang B ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》

CAV-1 contributes to bladder cancer progression by inducing epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) is a critical step in tumor metastasis. CAV-1 has been shown to be an oncogene in bladder cancer. However, little is known about the relationship between CAV-1 and EMT in bladder cancer metastasis. Immunohistochemical analysis was carried out retrospectively on tumor samples from patients treated for bladder cancer. CAV-1 and E-cadherin expression levels were measured and correlated with clinical features of tumor grade and metastasis. EMT was assessed in bladder cancer cell lines (T24, UMUC3, HT1376, and 5637) using wound healing and in vitro transwell invasion assays, and small interfering RNA was used to knock down CAV-1 and Slug expression. In this study, we show that increased CAV-1 expression induces bladder cancer cell migration and promotes the EMT, which was determined by the reduction of E-cadherin expression and the induction of N-cadherin and vimentin expression. Knockdown of CAV-1 expression with specific small interfering RNA reduced cell migration and EMT. Mechanistically, CAV-1-induced expression of the transcription factor, Slug. Slug knockdown abolished the CAV-1-induced EMT in bladder cancer cells. We further show that Slug expression is mediated by the CAV-1 regulation of the PI3K/AKT signaling. In addition, positive CAV-1 expression was significantly correlated with negative E-cadherin expression, as determined by immunohistochemistry analysis in bladder cancer tissues. Our results suggest that CAV-1 promotes invasive phenotypes in bladder cancer cells by inducing EMT through up-regulation of Slug expression, which occurs through activation of the PI3K/AKT signaling pathway. This new role for CAV-1 in promoting bladder cancer metastasis presents CAV-1 and related pathways as potential therapeutic targets in invasive bladder cancer.

Liang W ，Hao Z ，Han JL ，Zhu DJ ，Jin ZF ，Xie WL ... -  《-》

A-kinase-interacting protein 1 promotes EMT and metastasis via PI3K/Akt/IKKβ pathway in cervical cancer.

Overexpression of A-kinase-interacting protein 1 (AKIP1) has been reported in prostate and breast cancers. Nevertheless, the clinical potential of AKIP1 during the development of cervical cancer (CC) remains unclear. A series of experiments involving BdU, colony formation, wound healing and cell invasion assays were performed to determine cell proliferation, migration and invasion, respectively. Gene expression changes were validated by qRT-PCR, Western blotting and immunocytochemistry. We found that AKIP1 expression is increased in CC cell lines and tissue specimens from CC patients. The elevated AKIP1 expression in primary tumours was related to lymph node metastasis in CC patients. In addition, we observed that overexpression of AKIP1 promotes CC cell proliferation. Enhanced expression of AKIP1 facilitated the migration and invasion of CC cells by inducing NF-κB-dependent epithelial-mesenchymal transition (EMT). Moreover, mechanistic investigations revealed that AKIP1 induced nuclear translocation and phosphorylation of the p65 NF-κB subunit through the PI3K/Akt/IKKβ pathway. Conversely, enhanced expression of phosphatase and tensin homologue (PTEN) inhibited this signalling pathway and restored an epithelial phenotype to CC cells in the presence of overexpressed AKIP1. Our results indicate that AKIP1 promotes the EMT and metastasis in CC by activating NF-κB signalling through the PI3K/Akt/IKKβ pathway, suggesting that AKIP1 could be a pivotal regulator of an EMT axis in CC. SIGNIFICANCE OF THE STUDY: AKIP1 expression in the samples of CC patients and in in vitro tumour cell lines provides evidence that expression of AKIP1 in CC contributes to cancer progression. Our findings indicate that AKIP1 promotes the epithelial-mesenchymal transition and metastasis in CC by activating NF-κB signalling through the PI3K/Akt/IKKβ pathway, suggesting that AKIP1 is a pivotal regulator of an EMT axis in CC. AKIP1 could be implicated as a potential therapeutic target as well as a valuable biomarker for evaluating prognosis for patients with CC.

Zhang X ，Liu S ，Zhu Y 《-》

WSTF promotes proliferation and invasion of lung cancer cells by inducing EMT via PI3K/Akt and IL-6/STAT3 signaling pathways.

Williams syndrome transcription factor (WSTF), which is encoded by the BAZ1B gene, was first identified as a hemizygously deleted gene in patients with Williams syndrome. WSTF protein has been reported to be involved in transcription, replication, chromatin remodeling and DNA damage response, and also functions as a tyrosine protein kinase. However, the function of WSTF in cancer is not known. Here, we show that WSTF overexpression promotes proliferation, colony formation, migration and invasion of lung cancer A549 and H1299 cells. WSTF overexpression also promotes tumor growth and invasive abilities of lung cancer cells in mouse xenograft models. cDNA microarray and subsequent qRT-PCR validation revealed that WSTF overexpression significantly upregulated the expression of EMT (epithelial to mesenchymal transition) marker fibronectin (FN1) and EMT-inducing genes Fos and CEACAM6. The changes of EMT markers including downregulated E-cadherin and upregulated N-cadherin and FN1 were further confirmed at both mRNA and protein levels upon WSTF overexpression, with typical morphological changes of EMT. Furthermore, WSTF activates both PI3K/Akt and IL-6/STAT3 oncogenic signaling pathways. Treatment with PI3K inhibitor ZSTK474 or STAT3 inhibitor niclosamide reversed the effects of WSTF overexpression by inhibiting cell proliferation, migration and invasion, with decreased level of p-Akt, p-STAT3 and IL-6. ZSTK474 and niclosamide also reversed EMT markers and EMT-inducing proteins including Snail, Slug, Twist and CEACAM6 in WSTF-overexpressing A549 cells. Taken together, these results demonstrate that WSTF may act as an oncoprotein in lung cancer to accelerate tumor aggressiveness by promoting EMT via activation of PI3K/Akt and IL-6/STAT3 pathways.

Meng J ，Zhang XT ，Liu XL ，Fan L ，Li C ，Sun Y ，Liang XH ，Wang JB ，Mei QB ，Zhang F ，Zhang T ... -  《-》