Twist-related protein 1 (TWIST1) plays an essential role in the carcinogenesis and metastasis of NSCLC. Our aims were to identify the molecule at the downstream of TWIST1 and to evaluate its potential as a diagnostic and a prognostic marker in NSCLC.

We have demonstrated that complement 3 (C3) is upregulated and induces epithelial-mesenchymal transition (EMT) phenomenon and renal fibrosis in unilateral ureteral obstruction (UUO) kidney. We investigated roles of twist-related protein 1 (TWIST1) in EMT phenomenon and renal fibrosis through C3 upregulation in a mouse UUO model with gene silencer pyrrole-imidazole (PI) polyamides targeting TWIST1. We designed and synthesized PI polyamides targeting TWIST1 binding site on mouse pre-pro C3 promoter. Increased expression C3 mRNA with interferon-γ was significantly inhibited with PI polyamide in nephrotubular epithelial cells. Immunofluorescence showed suppression of E-cadherin and enhancement of α-smooth muscle actin (α-SMA) stainings as EMT phenomena in UUO kidney. TWIST1 and C3 expression was significantly increased in UUO kidney versus contralateral unobstructed kidney (CUK). Expression of transforming growth factor-β1 (TGF-β1), α-SMA and renin mRNAs was increased in UUO kidney versus CUK. Systemic administration of TWIST1 PI polyamide significantly suppressed increased C3 expression in UUO kidney versus CUK. PI polyamide administration also suppressed the increased expression of TGF-β1, α-SMA and renin mRNAs and histologically improved renal fibrosis in UUO kidney. These findings indicate that TWIST1 induces EMT phenomenon and renal fibrosis by TGF-β1 upregulation of C3 in mouse UUO model and that TWIST1 PI polyamide may be a novel medicine for renal fibrosis.

This article has been withdrawn at the request of the publisher in December 2020.

Ajuba is a multiple LIM domain-containing protein and functions as a transcriptional coregulator to modulate many gene expressions in various cellular processes. Here, we describe that the LIM domain of Ajuba interacts with Twist, and the Twist box is a pivotal motif for the interaction. Biologically, Ajuba enhances transcription of target gene N-cadherin as an obligate coactivator of Twist. The enhancement is achieved by binding to the E-box element within N-cadherin promoter as revealed by luciferase reporter and chromatin immunoprecipitation assays. Mechanistic investigation demonstrates that Ajuba recruits CBP and Twist to form a ternary complex at the Twist target promoter region and concomitantly enhances histone acetylation at these sites. These findings identify that Twist is a new interacting protein of Ajuba and Ajuba/Twist/CBP ternary complex may be a potential treatment strategy for Twist-related tumour metastasis.

Periostin (PN) (also known as osteoblast‑specific factor OSF‑2) is a protein that in humans is encoded by the POSTN gene and has been correlated with a reduced survival of cholangiocarcinoma (CCA) patients, with the well‑known effect of inducing epithelial‑to‑mesenchymal transition (EMT). The present study investigated the effect of PN, through integrin (ITG)α5β1, in EMT‑mediated CCA aggressiveness. The alterations in EMT‑related gene and protein expression were investigated by real‑time PCR, western blot analysis and zymogram. The effects of PN on migration and the level of TWIST‑2 were assessed in CCA cells with and without siITGα5 transfection. PN was found to induce CCA cell migration and EMT features, including increments in Twist‑related protein 2 (TWIST‑2), zinc finger protein SNAI1 (SNAIL‑1), α-smooth muscle actin (ASMA), vimentin (VIM) and matrix metallopeptidase 9 (MMP‑9), and a reduction in cytokeratin 19 (CK‑19) together with cytoplasmic translocation of E-cadherin (CDH‑1). Additionally, PN markedly induced MMP‑9 activity. TWIST‑2 was significantly induced in PN‑treated CCA cells; this effect was attenuated in the ITGα5β1‑knockdown cells and corresponded to reduced migration of the cancer cells. These results indicated that PN induced CCA migration through ITGα5β1/TWIST-2‑mediated EMT. Moreover, clinical samples from CCA patients showed that higher levels of TWIST‑2 were significantly correlated with shorter survival time. In conclusion, the ITGα5β1‑mediated TWIST‑2 signaling pathway regulates PN‑induced EMT in CCA progression, and TWIST‑2 is a prognostic marker of poor survival in CCA patients.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. It has been reported that HCC has a poor prognosis. In the majority of cases, once metastatic, HCC is incurable. To identify an effective treatment for HCC, it is important to understand the underlying molecular mechanisms of HCC‑associated occurrence, proliferation, metastasis and carcinogenesis. In the present study, the role of Up‑frameshift 1 (UPF1), a potential tumor suppressor, was investigated in the HCC cell lines. The expression levels of UPF1 in an HCC cell line were examined by reverse transcription‑quantitative polymerase chain reaction. The expression levels of 19 key proteins in numerous signaling pathways were detected via protein array analysis in the presence of UPF1 overexpression. The present study further investigated the effects of UPF1 expression levels on the epithelial‑mesenchymal transition (EMT) process by targeting E‑cadherin, N‑cadherin, Vimentin and Twist‑related protein 1 (Twist). The results of the present study revealed that UPF1 was significantly downregulated in an HCC cell line. The majority of the proteins exhibited upregulated expression levels in the presence of UPF1 overexpression in the HCC cell line, Huh‑7. Key proteins, including cluster of differentiation (CD)31 (platelet endothelial cell adhesion molecule‑1), Vimentin, CD44, PCNA, Ki‑67, N‑Cadherin, Survivin, P53, Met and retinoblastoma exhibited a significant association with UPF1. Furthermore, western blotting indicated that the expression levels of N‑cadherin, Vimentin and Twist were notably upregulated while UPF1 was overexpressed; however, E‑cadherin was downregulated and opposing observations were reported with protein array analysis. In summary, E‑cadherin expression levels were regulated by the manifold, and UPF1, a potential tumor suppressor, may promote the EMT process in Huh‑7 HCC cells. The findings of the present study suggested that UPF1 expression levels affected the EMT process by targeting E‑cadherin, N‑cadherin, Vimentin and Twist.

Hepatocellular carcinoma is one of the most common cancer types worldwide. In cases of advanced-stage disease, sorafenib is considered the treatment of choice. However, resistance to sorafenib remains a major obstacle for effective clinical application. Based on integrated phosphoproteomic and The Cancer Genome Atlas (TCGA) data, we identified a transcription factor, Y-box binding protein-1 (YB-1), with elevated phosphorylation of Ser102 in sorafenib-resistant HuH-7R cells. Phosphoinositide-3-kinase (PI3K) and protein kinase B (AKT) were activated by sorafenib, which, in turn, increased the phosphorylation level of YB-1. In functional analyses, knockdown of YB-1 led to decreased cell migration and invasion in vitro. At the molecular level, inhibition of YB-1 induced suppression of zinc-finger protein SNAI1 (Snail), twist-related protein 1 (Twist1), zinc-finger E-box-binding homeobox 1 (Zeb1), matrix metalloproteinase-2 (MMP-2) and vimentin levels, implying a role of YB-1 in the epithelial-mesenchymal transition (EMT) process in HuH-7R cells. Additionally, YB-1 contributes to morphological alterations resulting from F-actin rearrangement through Cdc42 activation. Mutation analyses revealed that phosphorylation at S102 affects the migratory and invasive potential of HuH-7R cells. Our collective findings suggest that sorafenib promotes YB-1 phosphorylation through effect from the EGFR/PI3K/AKT pathway, leading to significant enhancement of hepatocellular carcinoma (HCC) cell metastasis. Elucidation of the specific mechanisms of action of YB-1 may aid in the development of effective strategies to suppress metastasis and overcome resistance.

Cancer-associated fibroblasts (CAFs) are a prominent component in the tumor microenvironment (TME), which plays an important role in lung carcinogenesis. Here, we investigated microenvironmental markers expressed by CAFs, including α-smooth muscle actin, CD10, podoplanin, fibroblast-specific protein 1, platelet-derived growth factor α and β, fibroblast-associated protein, tenascin-C, zinc finger E-box binding homeobox 1 (ZEB1), and twist-related protein 1 expression levels. We evaluated samples from 257 patients with lung adenocarcinoma (LAD) to assess the associations of CAF-related protein expression patterns with prognosis. LAD cases were stratified using cluster analysis. To determine the utility of prognostic markers in LAD, univariate and multivariate analyses were performed. LAD cases were classified into subgroups 1 and 2. Subgroup 2 was shown to be significantly correlated with disease-free and overall survival using univariate and multivariate analyses in this group. Upregulation of podoplanin was identified as a single prognostic marker in this study by univariate and multivariate analyses. In addition, ZEB1 overexpression was correlated with disease-free survival. Our current results suggested that the specific CAF phenotype (e.g., the expression pattern of CAF-related proteins) could predict outcomes in patients with LAD. In addition, podoplanin upregulation may predict outcomes in these patients.

Objective: Recent studies have shown that cancer-associated fibroblasts (CAFs) and the epithelial-mesenchymal transition (EMT) play important roles in the progression and metastasis of CRC. Although prediction of lymph node metastasis in submucosal invasive colorectal cancer (SiCRC) is important, the relationships of CAF and EMT with lymph node metastasis of SiCRC have not yet been examined. Here, we aimed to analyze the expression patterns of CAF- and EMT-related proteins in SiCRC. Materials and Methods: The expression of CAF-related markers, including α-smooth muscle actin, CD10, podoplanin, fibroblast specific protein 1, and adipocyte enhancer-binding protein 1, and EMT-related proteins [zinc finger protein SNAI2 (ZEB1) and twist-related protein 1 (TWIST1) in SiCRC with (n = 29) or without (n = 80) lymph node metastasis was examined by immunohistochemistry. We examined the expression patterns of biomarkers using hierarchical cluster analysis. Consequently, four subgroups were established based on the expression patterns of CAF- and EMT-related markers, and the associations of these subgroups with clinicopathological variables. Results: In multivariate analysis, subgroup 2, which was characterized by high expression of all markers, was correlated with lymph node metastasis (p < 0.01). Next, we examined the associations of individual biomarkers with lymph node metastasis. Multivariate analysis showed that moderately differentiated adenocarcinoma was significantly associated with lymph node metastasis (p < 0.05). Conclusions: Our findings showed that expression patterns of CAF markers and EMT-related proteins may allow for stratification of patients into risk categories for lymph node metastasis in SiCRC.

Bone morphogenetic protein 9 (BMP9) belongs to the transforming growth factor‑β (TGF‑β) superfamily, and has been reported to promote cancer cell proliferation and epithelial‑mesenchymal transition (EMT). Cisplatin (DDP) is the first line treatment for ovarian cancer. However, initiation of EMT confers insensitivity to chemotherapy. The present study aimed to verify and examine the mechanisms underlying the effects of BMP9 on treatment with DDP for ovarian cancer. Prior to treatment with DDP, ovarian cancer cells were exposed to BMP9 for 3 days. Following this, cell viability, apoptosis rate and the extent of DNA damage were evaluated to compare the effects of DDP on BMP9‑pretreated and non‑pretreated ovarian cancer cells. In addition, EMT marker expression was evaluated by western blotting and immunofluorescence. The results demonstrated that BMP9 pretreatment inhibited the cytotoxicity of DDP on ovarian cancer cells. Additionally, BMP9‑pretreated ovarian cancer cells had downregulated expression of the epithelial marker E‑cadherin, which was accompanied by an upregulation of the mesenchymal markers N‑cadherin, Snail, Slug, and Twist. Taken together, the findings of the present study indicated that BMP9 conferred resistance to DDP in ovarian cancer cells by inducing EMT. The present study provided valuable insight into the mechanisms of chemotherapy in ovarian cancer and highlighted the potential of BMP9 as a novel therapeutic target for improving cisplatin chemosensitivity.

Promyelocytic leukemia protein (PML) modulates diverse cell functions that contribute to both tumor suppressor and pro-oncogenic effects, depending on the cellular context. We show here that PML knockdown (KD) in MDA-MB-231, but not MCF7, breast cancer cells, prolonged stem-cell-like survival, and increased cell proliferation and migration, which is in line with gene-enrichment results from their RNA sequencing analysis. Of note, increased migration was accompanied by higher levels of the epithelial-mesenchymal transition (EMT) regulator Twist-related protein 2 (TWIST2). We showed here that PML binds to TWIST2 via its basic helix-loop-helix (bHLH) region and functionally interferes with the suppression of the epithelial target of TWIST2, CD24. In addition, PML ablation in MDA-MB-231 cells led to higher protein levels of hypoxia-inducible factor 1-alpha (HIF1a), resulting in a higher cell hypoxic response. Functionally, PML directly suppressed the induction of the HIF1a target gene vascular endothelial growth factor A (VEGFa). In line with these results, tumor xenografts of MDA-MB-231 PML-KD cells had enhanced aggressive properties, including higher microvessel density, faster local growth, and higher metastatic ability, with a preference for lung. Collectively, PML suppresses the cancer aggressive behavior by multiple mechanisms that impede both the HIF-hypoxia-angiogenic and EMT pathways.

Gastric carcinoma is one of the most common human malignancies and remains the second leading cause of cancer-associated mortality worldwide. Gastric carcinoma is characterized by early-stage metastasis and is typically diagnosed in the advanced stage. Previous results have indicated that bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) overexpression has been demonstrated to inhibit growth and metastasis of gastric cancer cells. However, the molecular mechanisms of the BAMBI-mediated signaling pathway in the progression of gastric cancer are poorly understood. In the present study, to assess whether BAMBI overexpression inhibited the growth and aggressiveness of gastric carcinoma cells through regulation of transforming growth factor (TGF)-β/epithelial-mesenchymal transition (EMT) signaling pathway, the growth and metastasis of gastric carcinoma cells were analyzed following BAMBI overexpression and knockdown in vitro and in vivo. Molecular changes in the TGF-β/EMT signaling pathway were studied in gastric carcinoma cells following BAMBI overexpression and knockdown. DNA methylation of the gene regions encoding the TGF-β/EMT signaling pathway was investigated in gastric carcinoma cells. Tumor growth in tumor-bearing mice was analyzed after mice were subjected to endogenous overexpression of BAMBI. Results indicated that BAMBI overexpression significantly inhibited gastric carcinoma cell growth and aggressiveness, whereas knockdown of BAMBI significantly promoted its growth and metastasis compared with the control (P<0.01). The TGF-β/EMT signaling pathway was downregulated in BAMBI-overexpressed gastric carcinoma cells; however, signaling was promoted following BAMBI knockdown. In addition, it was observed that BAMBI overexpression significantly downregulated the DNA methylation of the gene regions encoding the TGF-β/EMT signaling pathway (P<0.01). Furthermore, RNA interference-mediated BAMBI overexpression also promoted apoptosis in gastric cancer cells and significantly inhibited growth of gastric tumors in murine xenografts (P<0.01). In conclusion, the present findings suggest that BAMBI overexpression inhibited the TGF-β/EMT signaling pathway and suppressed the invasiveness of gastric tumors, suggesting BAMBI may be a potential target for the treatment of gastric carcinoma via regulation of the TGF-β/EMT signaling pathway.

No abstract available

The relationships of Paired Like Homeodomain 2 (PITX2), Ninjurin 2 (NINJ2), TWIST-Related Protein 1 (TWIST1), Ras Interacting Protein 1 (Rasip1), Solute Carrier Family 17 Member 3 (SLC17A3), Methylmalonyl Co-A Mutase (MUT) and Fer3 Like BHLH Transcription Factor (FERD3L) polymorphisms and gene expression with ischemic stroke have yet to be determined in Malaysia. Hence, this study aimed to explore the associations of single nucleotide polymorphisms (SNPs) and gene expression with ischemic stroke risk among population who resided at the Northern region of Malaysia.

Epithelial cells spontaneously form acini (also known as cysts or spheroids) with a single, fluid-filled central lumen when grown in 3D matrices. The size of the lumen is dependent on apical secretion of chloride ions, most notably by the CFTR channel, which has been suggested to establish pressure in the lumen due to water influx. To study the cellular biomechanics of acini morphogenesis and homeostasis, we used MDCK-2 cells. Using FRET-force biosensors for E-cadherin, we observed significant increases in the average tension per molecule for each protein in mature 3D acini as compared to 2D monolayers. Increases in CFTR activity resulted in increased E-cadherin forces, indicating that ionic gradients affect cellular tension. Direct measurements of pressure revealed that mature acini experience significant internal hydrostatic pressure (37 ± 10.9 Pa). Changes in CFTR activity resulted in pressure and/or volume changes, both of which affect E-cadherin tension. Increases in CFTR chloride secretion also induced YAP signaling and cellular proliferation. In order to recapitulate disruption of acinar homeostasis, we induced epithelial-to-mesenchymal transition (EMT). During the initial stages of EMT, there was a gradual decrease in E-cadherin force and lumen pressure that correlated with lumen infilling. Strikingly, increasing CFTR activity was sufficient to block EMT. Our results show that ion secretion is an important regulator of morphogenesis and homeostasis in epithelial acini. Furthermore, this work demonstrates that, for closed 3D cellular systems, ion gradients can generate osmotic pressure or volume changes, both of which result in increased cellular tension.

Twist-related protein 1 (Twist1), also known as class A basic helix-loop-helix protein 38 (bHLHa38), has been implicated in cell lineage determination and differentiation. Previous studies demonstrate that Twist1 expression is up-regulated in gastric cancer with poor clinical outcomes. Besides, Twist1 is suggested to be involved in progression of human gastric cancer. However, its biological functions remain largely unexplored. In the present study, we show that Twist 1 overexpression leads to a significant up-regulation of FoxM1, which plays a key role in cell cycle progression in gastric cancer cells. In contrast, knockdown of Twist 1 reduces FoxM1 expression, suggesting that FoxM1 might be a direct transcriptional target of Twist 1. At the molecular level, we further reveal that Twist 1 could bind to the promoter region of FoxM1, and subsequently recruit p300 to induce FoxM1 mRNA transcription. Therefore, our results uncover a previous unknown Twist 1/FoxM1 regulatory pathway, which may help to understand the mechanisms of gastric cancer proliferation.

In many cancer types, integrin-mediated signaling regulates proliferation, survival and invasion of tumorigenic cells. However, it is still unclear how integrins crosstalk with oncogenes to regulate tumorigenesis and metastasis. Here we show that oncogenic K-RasV12 upregulates α6-integrin expression in Madin-Darby canine kidney (MDCK) cells via activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)/Fos-related antigen 1-signaling cascade. Activated α6-integrins promoted metastatic capacity and anoikis resistance, and led to perturbed growth of MDCK cysts. Transcriptomic analysis of K-RasV12-transformed MDCK cells also revealed robust downregulation of αV-class integrins. Re-expression of αV-integrin in K-RasV12-transformed MDCK cells synergistically upregulated the expression of Zinc finger E-box-binding homeobox 1 and Twist-related protein 1 and triggered epithelial-mesenchymal transition leading to induced cell motility and invasion. These results delineate the signaling cascades connecting oncogenic K-RasV12 with α6- and αV-integrin functions to modulate cancer cell survival and tumorigenesis, and reveal new possible strategies to target highly oncogenic K-RasV12 mutants.

Lung cancer is the leading cause of global cancer-associated mortality, therefore it is important to reveal the molecular mechanisms of lung cancer progression and to develop novel therapeutic targets. The results of the present study identified that manganese-12 acetate (Mn12Ac) was able to significantly inhibit the migration and invasion of A549 cells. Western blotting demonstrated that treatment with Mn12Ac was able to upregulate E-cadherin, and downregulate N-cadherin and vimentin. It was also identified by a quantitative polymerase chain reaction analysis that Mn12Ac was able to reduce the mRNA expression levels of EMT-associated transcription factors Snail, Slug, Twist-related protein 1 and zinc finger E-box-binding homeobox 1. It was also demonstrated that Mn12Ac was able to reduce the expression levels of Wnt and β-catenin proteins, and suppress the phosphorylation of phosphoinositide 3-kinase (PI3K) and AKT in A549 cells. Notably, it was revealed that Mn12Ac was able to decrease the mRNA and protein expression levels of programmed death ligand-1. Taken together, the results suggested that Mn12Ac is able to inhibit cell migration, invasion and EMT in lung cancer cells by regulating the Wnt/β-catenin and PI3K/AKT signaling pathways.

Asbestos exposure increases the risk of asbestosis and malignant mesothelioma (MM). Both fibrosis and cancer have been correlated with the Epithelial to Mesenchymal Transition (EMT)-an event involved in fibrotic development and cancer progression. During EMT, epithelial cells acquire a mesenchymal phenotype by modulating some proteins. Different factors can induce EMT, but Transforming Growth Factor β (TGF-β) plays a crucial role in promoting EMT. In this work, we verified if EMT could be associated with MM development. We explored EMT in human mesothelial cells (MeT-5A) exposed to chrysotile asbestos: we demonstrated that asbestos induces EMT in MeT-5A cells by downregulating epithelial markers E-cadherin, β-catenin, and occludin, and contemporarily, by upregulating mesenchymal markers fibronectin, α-SMA, and vimentin, thus promoting EMT. In these cells, this mechanism is mediated by increased TGF-β secretion, which in turn downregulates E-cadherin and increases fibronectin. These events are reverted in the presence of TGF-β antibody, via a Small Mother Against Decapentaplegic (SMAD)-dependent pathway and its downstream effectors, such as Zinc finger protein SNAI1 (SNAIL-1), Twist-related protein (Twist), and Zinc Finger E-Box Binding Homeobox 1 (ZEB-1), which downregulate the E-cadherin gene. Since SNAIL-1, Twist, and ZEB-1 have been shown to be overexpressed in MM, these genes could be considered possible predictive or diagnostic markers of MM development.

Platelets promote tumor metastasis by inducing promalignant phenotypes in cancer cells and directly contributing to cancer-related thrombotic complications. Platelet-derived extracellular vesicles (EVs) can promote epithelial-mesenchymal transition (EMT) in cancer cells, which confers high-grade malignancy. 12S-hydroxyeicosatetraenoic acid (12-HETE) generated by platelet-type 12-lipoxygenase (12-LOX) is considered a key modulator of cancer metastasis through unknown mechanisms. In platelets, 12-HETE can be esterified into plasma membrane phospholipids (PLs), which drive thrombosis. Using cocultures of human platelets and human colon adenocarcinoma cells (line HT29) and LC-MS/MS, we investigated the impact of platelets on cancer cell biosynthesis of 12S-HETE and its esterification into PLs and whether platelet ability to transfer its molecular cargo might play a role. To this aim, we performed coculture experiments with CFSE[5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester]-loaded platelets. HT29 cells did not generate 12S-HETE or express 12-LOX. However, they acquired the capacity to produce 12S-HETE mainly esterified in plasmalogen phospholipid forms following the uptake of platelet-derived medium-sized EVs (mEVs) expressing 12-LOX. 12-LOX was detected in plasma mEV of patients with adenomas/adenocarcinomas, implying their potential to deliver the protein to cancer cells in vivo. In cancer cells exposed to platelets, endogenous but not exogenous 12S-HETE contributed to changes in EMT gene expression, mitigated by three structurally unrelated 12-LOX inhibitors. In conclusion, we showed that platelets induce the generation of primarily esterified 12-HETE in colon cancer cells following mEV-mediated delivery of 12-LOX. The modification of cancer cell phospholipids by 12-HETE may functionally impact cancer cell biology and represent a novel target for anticancer agent development.