MiR-23a regulates TGF-β-induced epithelial-mesenchymal transition by targeting E-cadherin in lung cancer cells

Transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition (EMT) has been shown to be related to the pathogenesis of various diseases including lung cancer. Recently, microRNAs (miRNA) have been recognized as a new class of genes involved in human tumorigenesis. MiR-23a/24/27a is a miRNA cluster located in chromosome 19p13.12, which can function as an oncogene in several human cancers. In this study, we analyzed miR-23a/24/27a expression in 10 non-small cell cancer (NSCLC) cell lines by real-time PCR analysis. Correlation between expression of these miRNAs and TGF-β/Smad signaling was evaluated. We found that miR-23a could be regulated by TGF-β1 in a Smad-dependent manner in A549 lung adenocarcinoma cells showing the EMT phenomenon. Knockdown of miR-23a partially restored E-cadherin expression under conditions of TGF-β1 stimulation. In contrast, overexpression of miR-23a could suppress E-cadherin expression and stimulate EMT. Furthermore, A549 cells with overexpressed miR-23a were more resistant to gefitinib compared to the parental cells. These findings suggest that miR-23a regulates TGF-β-induced EMT by targeting E-cadherin in lung cancer cells and may be useful as a new therapeutic target in NSCLC.     

TGF-β-induced epithelial-mesenchymal transition: A link between cancer and inflammation

Metastatic spread of tumor cells to vital organs is the major cause of death in cancer. Accumulating data support an important role of infiltrating immune cells in promoting carcinoma progression into metastatic disease. Tumor-infiltrating immune cells produce and secrete cytokines, growth factors and proteases that re-activate latent developmental processes including epithelial-mesenchymal transition (EMT). EMT provides tumor cells with invasive, migratory and stem cell properties allowing them to disseminate and propagate at distant sites. Induction of EMT requires two criteria to be fulfilled: (i) cells are competent to undergo EMT (ii) an EMT-permissive microenvironment exists. The cytokine TGF-β, which is expressed by tumor-infiltrating immune cells, stands out as a master regulator of the pro-invasive tumor microenvironment. TGF-β cooperates with stem cell pathways, such as Wnt and Ras signaling, to induce EMT. In addition, TGF-β contributes to an EMT-permissive microenvironment by switching the phenotypes of tumor-infiltrating immune cells, which thereby mount pro-invasive and pro-metastatic immune responses. In this review, we discuss the role of TGF-β-induced EMT as a link between cancer and inflammation in the context of questions, which from our point of view are key to answer in order to understand the functionality of EMT in tumors.     

DJ-1 promotes proliferation and epithelial-mesenchymal transition of colorectal cancer cells by regulating p53 signaling pathway北大核心CSCD

Objective: To investigate the effects of up-regulating or silencing DJ-1 gene expression on the apoptosis, migration and invasion of colorectal cancer (CRC), and to explore the possible molecular mechanism. Methods: The expression level of DJ-1 in CRC tissues and cells was detected by immunohistochemistry, Western blotting and real-time fluorescent quantitative PCR, respectively. The SW480 and HCT116 cells were transfected with the recombinant lentiviral vector carrying human DJ-1 gene to obtain DJ-1 overexpressed SW480/OE-DJ-1 and HCT116/OE-DJ-1 cells, while the cells transfected with the empty vector was as the negative control (OE-NC). The SW480 and HCT116 cells were transfected with the recombinant lentiviral vector carrying the specific shRNA targeting DJ-1 gene to generate the SW480/shDJ-1 and HCT116/sh-DJ-1 cells with stable knockdown of DJ-1, while the cells transfected with the empty vector was as the negative control (sh-NC). Subsequently, the expressions of DJ-1 and p53 protein and mRNA were detected by immunohistochemistry and real-time fluorescent quantitative PCR, respectively; and their relationship was analyzed. The expressions of p53 and its downstream apoptosis-related proteins Bax and Bcl-2 in SW480 and HCT116 cells with DJ-1 over-expression or knockdown were detected by Western blotting. The effects of overexpressing and silencing DJ-1 gene expression on the invasion and migration abilities of SW480 and HCT116 cells were detected by Transwell chamber assay. The epithelial-mesenchymal transition (EMT) of CRC cells was induced by transforming growth factor-β1 (TGF-β1), then the expression levels of DJ-1 and EMT-related markers (N-cadherin, β-catenin, vimentin, E-cadherin) were analyzed by Western blotting. Results: DJ-1 was highly expressed in 34 CRC tissues (24/34, 70.59%) (P < 0.001). The overall survival time of the patients with DJ-1 high expression was significantly shorter than that of the patients with DJ-1 low expression (P < 0.001). The high expression of DJ-1 was correlated with TNM stage, tumor location, lymph node metastasis, and degree of differentiation (all P < 0.05). There was a negative correlation between DJ-1 and p53 expressions (r =-0.428, P = 0.015). Silencing DJ-1 increased the expression level of p53 and its downstream apoptotic protein Bax, decreased the expression of anti-apoptotic protein Bcl-2 (all P < 0.05), and decreased the invasion and migration capacities of SW480 and HCT116 cells (both P < 0.01); Conversely, overexpressing DJ-1 decreased the expression level of p53 and Bax, increased the expression of Bcl-2 (all P < 0.05), and increased the invasion and migration capacities of SW480 and HCT116 cells (both P < 0.01). Overexpression of DJ-1 induced by TGF-β1 increased the expressions of N-cadherin, β-catenin and vimentin, and decreased the expression of E-cadherin in the process of EMT (P < 0.05). Conclusion: DJ-1 promotes the apoptosis and invasion of CRC cells by negatively regulating the p53 signaling pathway. © 2019 by TUMOR All rights reserved.     

Thioredoxin 1 mediates TGF-β-induced epithelial-mesenchymal transition in salivary adenoid cystic carcinoma

Epithelial-mesenchymal transition (EMT) plays an important role in the invasion and metastasis of salivary adenoid cystic carcinoma (SACC) which is characterized by wide local infiltration, perineural spread, a propensity to local recurrence and late distant metastasis. Our recent studies have disclosed that TGF-β is a crucial factor for EMT in metastatic SACC. In this study, we further uncovered small redox protein thioredoxin 1 (TXN) as a critical mediator of TGF-β induced EMT. Immunohistochemistry analysis revealed significantly higher expressions of TXN, thioredoxin reductase 1 (TXNRD1) and N-cadherin, and lower expression of E-cadherin in human metastatic SACC compared to non-metastatic SACC tissues. Consistently, cultured SACC cells with stable TXN overexpression had decreased E-cadherin and increased N-cadherin as well as Snail and Slug expressions. The enhanced migration and invasion potential of these cells was abrogated by Akt or TXNRD1 inhibitors. Expression of N-cadherin and Akt p-Akt decreased, whereas E-cadherin expression increased in a BBSKE (TXNRD1 inhibitor)-dose-dependent manner. In a xenograft mouse model, TXN overexpression facilitated the metastatic potential of SACC-83 cells to the lung. Our results indicate that TXN plays a key role in SACC invasion and metastasis through the modulation of TGF-β-Akt/GSK-3β on EMT. TXN could be a potential therapeutic target for SACC.     

PKCα-induced drug resistance in pancreatic cancer cells is associated with transforming growth factor-β1

Background

Drug resistance remains a great challenge in the treatment of pancreatic cancer. The goal of this study was to determine whether TGF-β1 is associated with drug resistance in pancreatic cancer.

Methods

Pancreatic cancer BxPC3 cells were stably transfected with TGF-β1 cDNA. Cellular morphology and cell cycle were determined and the suppressive subtracted hybridization (SSH) assay was performed to identify differentially expressed genes induced by TGF-β1. Western blotting and immunohistochemistry were used to detect expression of TGF-β1-related genes in the cells and tissue samples. After that, the cells were further treated with an anti-cancer drug (e.g., cisplatin) after pre-incubated with the recombinant TGF-β1 plus PKCα inhibitor Gö6976. TGF-β1 type II receptor, TβRII was also knocked down using TβRII siRNA to assess the effects of these drugs in the cells. Cell viability was assessed by MTT assay.

Results

Overexpression of TGF-β1 leads to a markedly increased invasion potential but a reduced growth rate in BxPC3 cells. Recombinant TGF-β1 protein increases expression of PKCα in BxPC3 cells, a result that we confirmed by SSH. Moreover, TGF-β1 reduced the sensitivity of BxPC3 cells to cisplatin treatment, and this was mediated by upregulation of PKCα. However, blockage of PKCα with Gö6976 and TβRII with siRNA reversed the resistance of BxPC3 cells to gemcitabine, even in the presence of TGF-β1. Immunohistochemical data show that pancreatic cancers overexpress TGF-β1 and P-gp relative to normal tissues. In addition, TGF-β1 expression is associated with P-gp and membranous PKCα expression in pancreatic cancer.

Conclusions

TGF-β1-induced drug resistance in pancreatic cancer cells was associated with PKCα expression. The PKCα inhibitor Gö6976 could be a promising agent to sensitize pancreatic cancer cells to chemotherapy.     

CCR7 enhances TGF-β1-induced epithelial-mesenchymal transition and is associated with lymph node metastasis and poor overall survival in gastric cancer

CCR7 is a G protein-coupled chemokine receptor. In this study, we used immunohistochemistry with tissue microarrays to measure CCR7 expression in tumor specimens from 122 patients with gastric cancer. We show that CCR7 expression is associated with lymph node metastasis (P = 0.022) and overall survival (OS; P = 0.025), and is an independent factor associated with poorer overall survival (P = 0.032). The CCR7 mechanism was predicted based on bioinformatic analysis and verified in gastric cancer cell lines and primary tumor samples. The data show that CCR7 contributes to TGF-β1-induced epithelial-mesenchymal transition (EMT) and that the effects of TGF-β1 are inhibited by a CCR7 neutralizing antibody or a NF-κB inhibitor. Increased TGF-β1 expression was accompanied by nuclear localization of NF-κB-p65 and higher levels of the mesenchymal marker vimentin in human gastric cancer samples. We conclude that the CCR7 axis mediates TGF-β1-induced EMT via crosstalk with NF-κB signaling, facilitating lymph node metastasis and poorer overall survival in patients with gastric cancer. These findings suggest CCR7 is a novel prognostic indicator and a potential target for gastric cancer therapy.     

Mesenchymal stem cells play a potential role in regulating the establishment and maintenance of epithelial-mesenchymal transition in MCF7 human breast cancer cells by paracrine and induced autocrine TGF-β

Although the epithelial-mesenchymal transition (EMT) is a normal process that occurs during development, it is thought to be associated with cancer progression and metastasis. Emerging evidence links mesenchymal stem cells (MSCs) in the tumor microenvironment with the occurrence of EMT in cancer progression. In this study, the human breast cancer cell line MCF7 was co-cultured with human adipose-derived MSCs (hAD-MSCs) in a transwell system. Co-cultured cells were analyzed for changes in cellular morphology, EMT markers, protein expression and tumor characteristics. We found that co-cultured MCF7 cells underwent EMT and established a stable mesenchymal phenotype after prolonged co-culturing. Here, we demonstrate that paracrine transforming growth factor-β1 (TGF-β1) secreted by hAD-MSCs regulated the establishment of EMT in MCF7 cells by targeting the ZEB/miR-200 regulatory loop. The downregulation of paracrine TGF-β1 levels can inhibit and reverse the EMT progress by downregulating ZEB1/2 and upregulating miR-200b and miR-200c. The maintenance of a stable mesenchymal state by MCF7 cells required the establishment of autocrine TGF-β signaling to drive and sustain ZEB expression, which had been initiated by the prolonged co-culturing with hAD-MSCs. These results suggest that MSCs may promote breast cancer metastasis by stimulating and facilitating the EMT process.     

乳腺癌MCF-7细胞通过TGF-β诱导的上皮-间质转化获得耐药北大核心CSCD

目的:通过研究高侵袭转移性乳腺癌细胞株MDA-MB-231和低侵袭转移性细胞株MCF-7中上皮-间质转化(epithelial-mesenchymal transition,EMT)和耐药的相关性,以探讨乳腺癌耐药的机制。方法:采用实时荧光定量PCR法检测MDA-MB-231和MCF-7细胞中EMT标志物E-钙黏蛋白(E-cadherin)、波形蛋白(vimentin)和纤黏蛋白(bronectin)以及转录因子Snail、Slug和锌指E-box同源结合框1(Zincnger E-box binding homeobox 1,ZEB1)mRNA的表达水平;划痕愈合实验和Transwell小室实验分别检测MDA-MB-231和MCF-7细胞的迁移和侵袭能力;CCK-8法检测MDA-MB-231和MCF-7细胞对化疗药物5-氟尿嘧啶、顺铂和紫杉醇的敏感性,并采用实时荧光定量PCR法检测耐药基因多重耐药相关蛋白1(multidrug resistance-associated protein 1,MDR1)和MDR相关蛋白1(MDR-associated protein,MRP1)mRNA的表达水平。用转化生长因子β(transforming growth factor-β,TGF-β)诱导MCF-7细胞发生EMT,或用靶向E-cadherin基因的E-cadherin-siRNA沉默EMT相关标志物E-cadherin的表达,再用CCK-8法检测MCF-7细胞对5-氟尿嘧啶敏感性的变化。结果:MDA-MB-231细胞中vimentin、fibronectin、Slug和ZEB1 mRNA的表达水平均高于MCF-7细胞(P值均<0.000 1),E-cadherin mRNA的表达水平明显低于MCF-7细胞(P=0.000 2),Snail mRNA的表达水平无明显差异。与MCF-7细胞相比,MDA-MB-231细胞的迁移和侵袭能力明显更强(P值均<0.000 1);5-氟尿嘧啶、顺铂和紫杉醇对MDAMB-231细胞的半数抑制浓度(half maximal inhibitory concentration,IC50)值明显高于MCF-7细胞(P值均<0.05);MDA-MB-231细胞中耐药相关基因MDR1和MRP1 mRNA表达水平明显更高(P值均<0.000 1)。TGF-β诱导MCF-7细胞发生EMT后,5-氟尿嘧啶对MCF-7细胞的IC50值明显上升(P<0.05);沉默E-cadherin表达后,MCF-7细胞对5-氟尿嘧啶的耐药性明显增强(P<0.05)。结论:乳腺癌中EMT和耐药存在相关性,诱导EMT发生可导致细胞耐药。     

Disulfiram inhibits TGF-β-induced epithelial-mesenchymal transition and stem-like features in breast cancer via ERK/NF-κB/Snail pathway

Disulfiram (DSF), an anti-alcoholism drug, has been reported as an inhibitor of NF-κB. NF-κB is involved in epithelial-mesenchymal transition (EMT) and self-renewal of breast cancer stem cells (CSCs). In this study, we treated MCF-7 and MDA-MB-231 breast cancer cells with TGF-β to induce EMT and cancer stem-like features and studied whether DSF can reverse this process. We found that DSF inhibited TGF-β induced EMT in breast cancer cells in a dose-dependent manner. Also, DSF inhibited EMT-associated stem-like features, migration and invasion of tumor cells as well as tumor growth in xenograft model. The activation of NF-κB was linked with EMT and stem-like cells. We conclude that DSF can suppress NF-κB activity and downregulate ERK/NF-κB/Snail pathway, leading to reverse EMT and stem-like features. Our data suggest that DSF inhibits EMT and stem-like properties in breast cancer cells associated with inhibition of the ERK/NF-κB/Snail pathway.     

The effects of TGF-α and 17β-estradiol on polyphosphoinositide metabolism in MCF-7 breast cancer cells

Summary The mechanism by which transforming growth factor-alpha (TGF-) stimulates breast cancer cell proliferation is largely unknown. Furthermore, its potential role as an autocrine effector of estradiol-17 (E₂)-stimulated growth of hormone-dependent mammary tumors remains controversial. Transient changes in phosphatidylinositol (PI) turnover have been demonstrated in several tissues in response to growth factors. In these experiments, we tested the effects of TGF- and E₂ on PI metabolism in three MCF-7 breast cancer cell sublines (MCF-7B, MCF-7I, and MCF-7J). Although TGF- was mitogenic in MCF-7I and MCF-7J cells, PI hydrolysis was stimulated by the growth factor only in the MCF-7I cells. In addition, the TGF- effect was relatively modest, ranging from 23% to 42%. E₂ effects on PI turnover were tested in the MCF-7B cells, which were the most sensitive to the proliferative effect of the hormone. E₂ did not stimulate PI hydrolysis, whether or not the cells were labelled in the presence of the hormone. On the other hand, E₂ did seem to stimulatede novo synthesis of phosphatidylinositol and induce activation of PI kinases. These results demonstrate that TGF--stimulated PI hydrolysis is modest and cell type dependent. At least under certain conditions, PI metabolism is not involved in the proliferative effects of TGF- (MCF-7J) or E₂ (MCF-7B). The role of increased PI synthesis in E₂-stimulated MCF-7 cell growth remains to be established.This work is supported by a grant from the National Cancer Institute, POI CA40011.     

E/N-cadherin switch mediates cancer progression via TGF-β-induced epithelial-to-mesenchymal transition in extrahepatic cholangiocarcinoma

Background:

Epithelial-to-mesenchymal transition (EMT) is a fundamental process governing not only morphogenesis in multicellular organisms, but also cancer progression. During EMT, epithelial cadherin (E-cadherin) is downregulated while neural cadherin (N-cadherin) is upregulated, referred to as ‘cadherin switch''. This study aimed to investigate whether cadherin switch promotes cancer progression in cholangiocarcinoma (CC).

Methods:

CC cell lines were examined for migration, invasion, and morphological changes with typical EMT-induced model using recombinant TGF-β1. The changes in E-cadherin and N-cadherin expression were investigated during EMT. We also examined E-cadherin and N-cadherin expression in resected specimens from extrahepatic CC patients (n=38), and the associations with clinicopathological factors and survival rates.

Results:

TGF-β1 treatment activated cell migration, invasion, and fibroblastic morphological changes, especially in extrahepatic CC HuCCT-1 cells. These changes occurred with E-cadherin downregulation and N-cadherin upregulation, that is, cadherin switch. Patients with low E-cadherin expression had a significantly lower survival rate than patients with high E-cadherin expression (P=0.0059). Patients with decreasing E-cadherin and increasing N-cadherin expression had a significantly lower survival rate than patients with increasing E-cadherin and decreasing N-cadherin expression (P=0.017).

Conclusion:

Cadherin switch promotes cancer progression via TGF-β-induced EMT in extrahepatic CC, suggesting a target for elucidating the mechanisms of invasion and metastasis in extrahepatic CC.     

BMP-7 inhibits TGF-β-induced invasion of breast cancer cells through inhibition of integrin β(3) expression

Background

The transforming growth factor (TGF)-?? superfamily comprises cytokines such as TGF-?? and Bone Morphogenetic Proteins (BMPs), which have a critical role in a multitude of biological processes. In breast cancer, high levels of TGF-?? are associated with poor outcome, whereas inhibition of TGF-??-signaling reduces metastasis. In contrast, BMP-7 inhibits bone metastasis of breast cancer cells.

Methods

In this study, we investigated the effect of BMP-7 on TGF-??-induced invasion in a 3 dimensional invasion assay.

Results

BMP-7 inhibited TGF-??-induced invasion of the metastatic breast cancer cell line MCF10CA1a, but not of its premalignant precursor MCF10AT in a spheroid invasion model. The inhibitory effect appears to be specific for BMP-7, as its closest homolog, BMP-6, did not alter the invasion of MCF10CA1a spheroids. To elucidate the mechanism by which BMP-7 inhibits TGF-??-induced invasion, we analyzed invasion-related genes. BMP-7 inhibited TGF-??-induced expression of integrin ??_v??₃ in the spheroids. Moreover, targeting of integrins by a chemical inhibitor or knockdown of integrin ??₃ negatively affected TGF-??-induced invasion. On the other hand, overexpression of integrin ??₃ counteracted the inhibitory effect of BMP7 on TGF-??-induced invasion.

Conclusion

Thus, BMP-7 may exert anti-invasive actions by inhibiting TGF-??-induced expression of integrin ??₃.     

TGF-β1-induced epithelial–mesenchymal transition and acetylation of Smad2 and Smad3 are negatively regulated by EGCG in Human A549 lung cancer cells

Transforming growth factor-β1, the key ligand of Smad-dependent signaling pathway, is critical for epithelial–mesenchymal transition during embryo-morphogenesis, fibrotic diseases, and tumor metastasis. In this study, we found that activation of p300/CBP, a histone acetyltransferase, by TGF-β1 mediates Epithelial–mesenchymal transition (EMT) via acetylating Smad2 and Smad3 in TGF-β1 signaling pathway. We demonstrated that treatment with EGCG inhibited p300/CBP activity in human lung cancer cells. Also, we observed that EGCG potently inhibited TGF-β1-induced EMT and reversed the up-regulation of various genes during EMT. Our findings suggest that EGCG inhibits the induction of p300/CBP activity by TGF-β1. Therefore, EGCG inhibits TGF-β1-mediated EMT by suppressing the acetylation of Smad2 and Smad3 in human lung cancer cells.     

A83-01 inhibits TGF-β-induced upregulation of Wnt3 and epithelial to mesenchymal transition in HER2-overexpressing breast cancer cells

Purpose

The aim of this study is to investigate the mechanisms of interactions between TGF-β and Wnt/β-catenin pathways that induce and regulate EMT and promote breast cancer cells to become resistant to treatment.

Methods

The effect of TGF-β on Wnt/β-catenin signaling pathway was examined by using a human Wnt/β-catenin-regulated cDNA plate array and western blot analysis. The interaction of Twist at promoter of Wnt3 was examined by chromatin immunoprecipitation (ChIP) assay. Secreted Wnt3 level was determined by ELISA assay.

Results

HER2-overexpressing breast cancer cells treated with TGF-β have a reduced response to trastuzumab and exhibited EMT-like phenotype. The TGF-β-induced EMT in HER2-cells was concordant with upregulation of Wnt3 and β-catenin pathways. The TGF-β-induced induction of Wnt3 during EMT was found to be Smad3-dependent. ChIP analysis identified occupancy of Twist at promoter region of Wnt3. Knock-down of Twist by shRNA confirmed the significance of Twist in response to TGF-β regulating Wnt3 during EMT. Subsequently, TGF-β-induced matrix metalloproteinases, MMP1, MMP7, MMP9, MMP26, Vascular endothelial growth factors (VEGF), and activation of Wnt/β-catenin signaling were repressed by the shRNA treatment. TGF-βR1 ALK5 kinase inhibitor, A83-01 can effectively prevent the TGF-β-induced Twist and Wnt3. Co-treating A83-01 and trastuzumab inhibited TGF-β-induced cell invasion significantly in both trastuzumab responsive and resistant cells.

Conclusions

Our data demonstrated an important interdependence between TGF-β and Wnt/β-catenin pathways inducing EMT in HER2-overexpressing breast cancer cells. Twist served as a linkage between the two pathways during TGF-β-induced EMT. A83-01 could inhibit the TGF-β-initiated pathway interactions and enhance HER2-cells response to trastuzumab treatment.

     

Effect of hyperthermia on invasion ability and TGF-β1 expression of breast carcinoma MCF-7 cells

The 5-year survival rate of nasopharyngeal carcinoma (NPC) is still disappointing despite the much improved technologies in its treatment. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) can selectively induce apoptosis in most tumor cells while sparing normal cells. However, its potential in the treatment of NPC has been limited by the eventual emergence of drug resistance. Latent membrane protein 1 (LMP1) is a major oncogene of the human DNA tumor virus Epstein-Barr virus (EBV) and is associated with the development of NPC and the emergence of chemo-resistance in NPC. In this study, we investigated the potential role of LMP1 in TRAIL resistance in CNE-1 NPC cells. Results show that overexpression of LMP1 could induce TRAIL resistance in NPC cells without influencing death receptors. The LMP1-induced TRAIL resistance is associated with increased expression of FLIP and elevated cleavage of caspase-8 without altering the TRAIL-mediated mitochondrial events and Bid cleavage. Knockdown of the FLIP gene with siRNA prevented the LMP1-induced TRAIL resistance. Furthermore, we found that overexpression of LMP1 activated Akt. Inhibition of Akt with LY294002 completely prevented the LMP1-induced FLIP expression and TRAIL resistance. Together, these results show that LMP1 can inhibit the TRAIL-mediated apoptosis through activation of PI3K/Akt and expression of FLIP in CNE-1 NPC cells, and may provide new methods to prevent and reverse drug resistance in NPC.