Suppression of epithelial mesenchymal transition by a novel Traf2- and Nck-interacting kinase inhibitory compound
ABSTRACT: Recently, we found that a novel Traf2- and Nck-interacting kinase (TNIK) inhibitor, named NCB-0846, was capable of attenuating tumor-initiating cells among human colorectal cancer. The cross link between EMT and cancer stemness has been revealed in several studies and other group showed another TNIK inhibitor named KY-05009 had inhibited the TGF-β-induced EMT. Therefore we evaluated whether this small-molecule compound could have efficacy to inhibit TGF-β-induced EMT. NCB-0846 reduced the expression of mesenchymal markers (Vimentin and N-cadherin) and upregulated the expression of epithelial marker E-cadherin in A549 and H2228 non-small cell lung cancer cells. NCB-0846 suppressed the phosphorylation and nuclear translocation of Smad proteins and also inhibited migration, invasion, and metastasis. NCB-0846 inhibited TGF-β1-induced EMT through the down-regulation of TGF-β receptor-1 (TβRI) in mRNA levels. MiR-186-5p and miR-320 family were identified as candidate miRNAs that could target TβRI and we found that miR-186-5p and miR-320s inhibited TβRI expression. NCB-0846 might be a novel therapeutics drugs that targets the invasion and metastasis through inhibiting TGF-β-induced EMT in lung cancer. Overall design: EMT related markers and ability of migration, invasion, and metastasis were examined after exposure to TGF-B1 only or co-treated with TGF-B1 and NCB-0846 or NCB-970.
INSTRUMENT(S): Agilent-070156 Human_miRNA_V21.0_Microarray 046064 (Feature Number version)
Project description:We have identified a single miRNA, miR-181a, that can modulate TGF-β signaling to induce and maintain EMT, and effect further downstream events of tumour cell survival, altered response to chemotherapy, migration, invasion and dissemination in vivo. Our present study provides an understanding of how enhanced expression of miR-181a can confer malignant and invasive traits through the modulation of a canonical signaling pathway and a consequent maintenance of a mesenchymal state. Furthermore, inhibition of miR-181a led to a reversion of EMT and subsequent events through decreased TGF-β signaling. Our data confirmed Smad7 as a functional target through which TGF-β-mediated EMT occurs; re-expression of Smad7 lacking its 3'UTR was able to rescue miR-181a-mediated phenotypes, deeming Smad7 as a critical mediator of miR-181a-induced EMT. Other recent studies support the crucial role(s) that miRNAs play in mediating EMT and consequent aggressive disease traits. For example, the miR-106b-25 cluster has also been shown to target Smad7 and mediate TGF-β-induced EMT downstream to Six1 in breast cancer34. miR-9 directly targets E-cadherin and inhibition of miR-9 had led to an inhibition of metastasis35. Conversely, the miR-200 and -205 family was shown to target transcriptional repressors of E-cadherin, ZEB1 and SIP1, and re-expression of these miRNAs led to a mesenchymal-to-epithelial transition and prevented TGF-β -induced EMT36. A2780 ovarian cancer cell lines stably expressing either pBABE (control vector), p181a#1( clone 1 expressing miR-181a) or p181a#2( clone 2 expressing miR-181a)
Project description:The role of TGF-β-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination is well established, but the involvement of lncRNAs in TGF-β signaling is still unknown. In this study, we observed that the lncRNA-Activated by TGF-β (lncRNA-ATB) was upregulated in hepatocellular carcinoma (HCC) metastases and associated with poor prognosis. lncRNA-ATB upregulated ZEB1 and ZEB2 by competitively binding the miR-200 family and then induced EMT and invasion. In addition, lncRNA-ATB promoted organ colonization of disseminated tumor cells by binding IL11 mRNA, inducing autocrine of IL11 and triggering STAT3 signaling. Globally, lncRNA-ATB promotes the invasion-metastasis cascade. Thus, these findings suggest that lncRNA-ATB, a mediator of TGF-β signaling, could predispose HCC patients to metastases and may serve as a potential target for anti-metastatic therapies. To identify mRNA species bound by lncRNA-ATB, we performed an RIP to pull down endogenous mRNAs associated with the lncRNA-ATB and sequenced the retrieved RNA.
Project description:Epithelial-mesenchymal transition (EMT) has recently been recognized as a key element of cell invasion, migration, metastasis, and drug resistance in several types of cancer, including non-small cell lung cancer (NSCLC). Our aim was to clarify microRNA (miRNA) -related mechanisms underlying EMT followed by acquired resistance to epidermal growth factor receptor tyrosine-kinase inhibitor (EGFR-TKI) in NSCLC. MiRNA expression profiles were examined before and after transforming growth factor-beta1 (TGF-β1) exposure in four human adenocarcinoma cell lines with or without EMT. Correlation between expressions of EMT-related miRNAs and resistance to EGFR-TKI gefitinib was evaluated. MiRNA array and quantitative RT-PCR revealed that TGF-β1 significantly induced overexpression of miR-134, miR-487b, and miR-655, which belong to the same cluster located on chromosome 14q32, in lung adenocarcinoma cells with EMT. MAGI2 (membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2), a predicted target of these miRNAs and a scaffold protein required for PTEN (phosphatase and tensin homolog), was diminished in A549 cells with EMT after the TGF-β1 stimulation. Overexpression of miR-134 and miR-487b promoted the EMT phenomenon and affected the drug resistance to gefitinib, whereas knockdown of these miRNAs inhibited the EMT process and reversed TGF-β1-induced resistance to gefitinib. Our study demonstrated that the miR-134/487b/655 cluster contributed to the TGF-β1-induced EMT phenomenon and affected the resistance to gefitinib by directly targeting MAGI2, whose suppression subsequently caused loss of PTEN stability in lung cancer cells. The miR-134/miR-487b/miR-655 cluster may be new therapeutic targets in advanced lung adenocarcinoma patients, depending on the EMT phenomenon. miRNA expression profiles before and after TGF-β1 exposure were assessed in the four lung adenocarcinoma cell lines, A549, LC2/ad, PC3, and, PC9 by TaqMan miRNA arrays. Relative ratios of miRNAs in cells after TGF-β1 exposure were calculated when compared with the cells before TGF-β1 exposure.
Project description:Transforming growth factor (TGF)-β is one of the major inducers of epithelial to mesenchymal transition (EMT), a crucial program that plays a critical role in promoting carcinoma’s metastasis formation. MicroRNAs-143 and -145, which are both TGF-β direct transcriptional targets, are essential for the differentiation of vascular smooth muscle cells (VSMC) during embryogenesis, a TGF-β-dependent process reminiscent of EMT. Their role in adult tissues is however less well defined and even ambiguous, since their expression was correlated both positively and negatively with tumor progression. Here we show that high expression of both miRs-143 and -145 in mouse mammary tumor cells expressing constitutively active STAT3 (S3C) is involved in mediating their disrupted cell-cell junctions. Additionally, miR-143 appears to play a unique role in tumorigenesis by enhancing cell migration in vitro and extravasation in vivo while impairing anchorage-independent growth, which may explain the contradictory reports about its role in tumors. Accordingly, we demonstrate that overexpression of either miRNA in the non-transformed mammary epithelial NMuMG cells leads to upregulation of EMT markers and of several endogenous TGF-β targets, downmodulation of several junction proteins and increased motility. This correlates with enhanced basal and TGF-β-induced activity of SMAD transcription factors, suggesting that miR-143 and -145 can help promoting TGF-β mediated EMT via a positive feedback loop. Moreover, pervasive transcriptome perturbation consistent with the described phenotype was observed. In particular, the expression of several transcription factors involved in the mitogenic responses, of MAPK family members and, importantly, of several tight junction proteins and the SMAD co-repressor TGIF was significantly reduced. Our results provide important mechanistic insight into the non-redundant role played by miRs-143 and -145 in EMT-related processes in both transformed and non-transformed cells, and suggest that their expression must be finely coordinated to warrant optimal migration/invasion while not interfering with cell growth. Overall design: Transcriptome profiling by high-throughput sequencing
Project description:The role of TGF-β-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination is well established, but the involvement of lncRNAs in TGF-β signaling is still unknown. In this study, we observed that the lncRNA-Activated by TGF-β (lncRNA-ATB) was upregulated in hepatocellular carcinoma (HCC) metastases and associated with poor prognosis. lncRNA-ATB promotes the invasion-metastasis cascade, which suggest that lncRNA-ATB, a mediator of TGF-β signaling, could predispose HCC patients to metastases and may serve as a potential target for anti-metastatic therapies. Overall design: SMMC-7721 hepatoma cells were continuously treated with 10 ng/ml of recombinant TGF-β1 for 21 days. Total RNA recovered from three untreated cells and three treated cells were used to acquire different expression profiles of mRNAs and lncRNAs.
Project description:The role of TGF-β-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination is well established, but the involvement of lncRNAs in TGF-β signaling is still unknown. In this study, we observed that the lncRNA-Activated by TGF-β (lncRNA-ATB) was upregulated in hepatocellular carcinoma (HCC) metastases and associated with poor prognosis. lncRNA-ATB promotes the invasion-metastasis cascade, which suggest that lncRNA-ATB, a mediator of TGF-β signaling, could predispose HCC patients to metastases and may serve as a potential target for anti-metastatic therapies. SMMC-7721 hepatoma cells were continuously treated with 10 ng/ml of recombinant TGF-β1 for 21 days. Total RNA recovered from three untreated cells and three treated cells were used to acquire different expression profiles of mRNAs and lncRNAs.
Project description:Microarray analysis reveals up-regulation of retinoic acid and hepatocyte growth factor related signaling pathways by pro-insulin C-peptide in kidney proximal tubular cells: Antagonism of the pro-fibrotic effects of TGF-b1 Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameliorate complications of type 1 diabetes. Here we sought to identify new pathways regulated by C-peptide of relevance to the pathophysiology of diabetic nephropathy. Microarray analysis was performed to identify genes regulated by either C-peptide and/or transforming growth factor beta 1 (TGF-β1) in a human proximal tubular cell line, HK-2. Expression of retinoic acid receptor β (RARβ), hepatcoyte growth factor (HGF), cellular retinoic acid binding protein II (CRABPII), vimentin, E-cadherin, Snail and β-catenin was assessed by immunoblotting. The cellular localisation of vimentin and β-catenin was determined by immunocytochemistry. Changes in cell morphology were assessed by phase contrast microscopy. Gene expression profiling demonstrated differential expression of 953 and 1,458 genes after C-peptide exposure for 18h or 48h respectively. From these, members of the anti-fibrotic retinoic acid (RA) and HGF signaling pathways were selected. Immunoblotting demonstrated that C-peptide increased RARβ, CRABPII and HGF. We confirmed a role for RA in reversal of TGF-β1-induced changes associated with epithelial-mesenchymal transition (EMT), including expression changes in Snail, E-cadherin, vimetin and redistribution of β-catenin. Importantly, these TGF-β1-induced changes were inhibited by C-peptide. Further, effects of TGF-β1 on Snail and E-cadherin expression were blocked by HGF and inhibitory effects of C-peptide were removed by blockade of HGF activity. This study identifies a novel role for HGF as an effector of C-peptide, possibly via an RA signaling pathway, highlighting C-peptide as a potential therapy for diabetic nephropathy. Overall design: For microarray analyses, all treatments were performed in triplicate to yield 18 flasks, and RNA from each flask hybridized to a separate chip to give an n of 3 for each of 6 treatments. Flasks were subjected to identical media changes and cells cultured for identical periods in media without supplements. In all experiments, cells were serum-starved overnight before agonist addition. Treatments were initiated such that 18h and 48h incubation periods ended coincidentally and all RNA was prepared at this point.
Project description:Wnt signaling contributes to the reprogramming and maintenance of cancer stem cell (CSC) states that is activated by the epithelial-mesenchymal transition (EMT) program. However, the mechanistic relationship between the EMT and Wnt pathway in CSCs remains unclear. Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) indicated that EMT induces a switch from the β-catenin/E-cadherin/Sox15 complex to the β-catenin/Twist1/TCF4 complex, which then binds to CSC-related gene promoters. In tandem co-IP and re-ChIP experiments using epithelial-type cells, Sox15 associated with the β-catenin/E-cadherin complex and then bound to the proximal promoter region of CASP3, consequently resulting in Twist1 cleavage and negatively regulating the β-catenin–elicited promotion of the CSC phenotype. During the EMT, Twist1 in complex with β-catenin enhanced β-catenin/TCF4 transcriptional activity, which includes binding to the proximal promoter region of ABCG2, a marker of CSCs. For clinical application, the five-gene signature nuclear β-cateninHigh/nuclear Twist1High/E-cadherinLow/Sox15Low/CD133High may be a valuable prognostic marker in patients with human lung cancer. Total cellular RNA was extracted from LM and HM20 cells after treatment with or without Wnt3a for 24hours. Human lung cancer A549 cell–derived spheres were transferred back to adhesive tissue culture plates (LM cells). To establish an EMT/metastasis cell model, LM cells were seeded onto Matrigel-coated Boyden chamber membranes. HM20 cells were serially selected for Matrigel invasion 20 times.
Project description:Snail1 transcriptional factor is essential for triggering epithelial-to-mesenchymal transition (EMT) and inducing tumor cell invasion. We report here that Snail1 plays also a key role in tumor associated fibroblasts since is necessary for enhancement by these cells on epithelial cells tumor invasion. Snail1 expression in fibroblast requires signals derived from tumor cells such as TGF-b; reciprocally, in fibroblasts Snail1 organizes a complex program that favors collective invasion of epithelial cells at least in part by the secretion of diffusible signaling molecules, such as prostaglandin E2. The capability of human or murine tumor-derived cancer associated fibroblasts to promote tumor invasion is associated to Snail1 expression and obliterated by Snail1 depletion. In vivo experiments show that tumor cells co-transplanted with Snail1 depleted fibroblasts show lower invasion than those xenografted with control fibroblasts. Finally Snail1 depletion in mice prevents the formation of breast tumors and decreased their invasion. Therefore, these results demonstrate that the role of Snail1 in tumor invasion is not limited to its effect in EMT but dependent on its expression in stromal fibroblasts where it orchestrates its activation and the crosstalk with epithelial cells. Overall design: 4 samples were analyzed: Snail1-WT MSCs, Snail1-WT MSCs treated with TGF-b1, Snail1-KO MSCs and Snail1-KO MSCs treated with TGF-b1
Project description:Inappropriate activation of developmental pathways is a well-recognized tumor-promoting mechanism. Here we show that overexpression of the homeoprotein Six1, normally a developmentally restricted transcriptional regulator, increases Transforming Growth Factor-beta (TGF-beta) signaling in mammary carcinoma cells and induces an epithelial to mesenchymal transition (EMT) that is in part dependent on its ability to increase TGF-beta signaling. TGF-beta signaling and EMT have been implicated in metastatic dissemination of carcinoma. Using spontaneous and experimental metastasis mouse models, we demonstrate that Six1 overexpression promotes breast cancer metastasis. In addition, we show that, like its induction of EMT, Six1-induced experimental metastasis is dependent on its ability to activate TGF-beta signaling. Importantly, in human breast cancers Six1 significantly correlates with nuclear Smad3, and thus increased TGF-beta signaling. Further, breast cancer patients whose tumors overexpress Six1 have a shortened time to relapse and metastasis, and an overall decrease in survival. Finally, we show that the effects of Six1 on tumor progression likely extend beyond breast cancer, since its overexpression correlates with adverse outcomes in numerous other cancers, including brain, cervical, prostate, colon, kidney, and liver, amongst others. Our findings argue that Six1, acting through TGF-beta signaling and EMT, is a powerful and global promoter of cancer metastasis. Overall design: This study has two parts: the first part compared three Control and three Six1 expressing MCF7 clones. Total RNA was isolated from each clone on two separate occassions and hybridized to Affymetrix arrays. The second part compared MCF7-control and Six1 expressing cell lines expressing either GFP control or TbetaRIIDN. Total RNA was isolated from each cell line on two separate occassions and hybridized to Affymetrix arrays