Expression Of EMT-associated Proteins Research Articles

Targeting SALL4 by Entinostat Inhibits the Malignant Phenotype of Gastric Cancer Cells by Reducing EMT Signaling.

Spalt-like transcription factor 4 (SALL4), as a proto-oncogene, is expressed in various tumors and correlates with poor prognosis of patients. Entinostat, a histone deacetylase (HDAC) inhibitor, has emerged as a potentially promising anti-cancer drug. This study aims to explore the biological role and underlying mechanism of SALL4 targeting by entinostat in gastric cancer. Online databases were used to exam the link between SALL4 and prognosis. We tested the biological roles of SALL4 in gastric cancer cells. Cell viability and growth were analyzed using the Cell Counting Kit-8 (CCK-8) assay and clone formation assay. Cell migration was assessed using the wound healing assay. The effects of entinostat on gastric cancer cell lines were measured by the CCK-8 assay, clone formation assay, wound healing assay and transwell assay. Epithelial-mesenchymal transition (EMT) signaling pathways were detected by western blot. SALL4 expression was upregulated in gastric cancer tissues and positively correlated with tumor stage and prognosis of patients by TCGA dataset analysis. Knockdown of SALL4 by siRNA inhibited the proliferation and migration of gastric cancer cells. In contrast, SALL4 overexpression by stably transfecting a SALL4-expressing plasmid promoted the proliferation and invasiveness of gastric cancer cells in vitro through alteration of EMT-related genes. In addition, entinostat, a HDAC inhibitor targeting SALL4, could suppress the proliferation, migration, and invasion of gastric cancer cells via regulating expression of EMT-associated proteins. SALL4 may be a new therapeutic target for the treatment of gastric cancer, and entinostat is a potential novel agent for the treatment of gastric cancer partially by targeting SALL4.

Исследование молекулярно-клеточных механизмов фиброзирования печени крыс при посттоксическом гепатозе смешанной этиологии и при применении окисленного декстрана

Introduction. The development of liver cirrhosis, regardless of etiology, is based on the process of fibrosis and structural alteration of the organ. Regulation of this process is associated with a high level of TGF-β expression and suppression of apoptosis in hepatocytes. Oxidized dextran (OD) has a high antifibrotic activity and is able to change the functional state of the phagocytic cell, thus preventing the development of fibrosis and stimulating reparative processes in organs with post-toxic hepatosis and liver cirrhosis. Aim. To study the molecular and cellular mechanisms of the effect of OD on the expression of epithelial-mesenchymal transition (EMT)-associated proteins during fibrosis and the development of liver cirrhosis in rats with post-toxic hepatosis. Materials and methods. In the experiment, 30 male Wistar rats weighing 280–320 g were used. The animals were divided into 2 groups. In group 1 rats (hepatosis), the post-toxic hepatosis was modeled by administration of a solution of CCl4 and ethyl alcohol. In rats from group 2, the post-toxic hepatosis was modeled in the same way, and OD was administered. The numerical density (Nai) of liver nonparenchymal cells expressing TGF-β (Kupffer cells, endothelial cells, fibroblasts) was calculated. The expression of E-cadherin, vimentin, SNAIL + SLUG by fibroblasts and hepatocytes was evaluated. Results. The numerical density (Nai) of hepatocytes expressing vimentin prevailed in the liver of group 1 rats (hepatosis), compared with that of group 2 animals (hepatosis + OD) on the 30th and 60th days. In animals of the 1st (hepatosis) group on the 60th day, a 3-fold lower numerical density of hepatocytes expressing E-cadherin was noted in comparison with that in rats treated with OD (group 2). In animals of the 1st (hepatosis) group on the 30th and 60th days, a 2-fold and 5-fold higher numerical density of hepatocytes expressing SNAIL + SLUG was noted in comparison with that in rats of the 2nd group. The numerical density of fibroblasts expressing vimentin prevailed in the liver of group 2 rats (hepatosis + OD), compared with that of group 1 animals (hepatosis) on day 30. In animals of the 1st (hepatosis) group on the 60th day, a 6-fold higher numerical density of fibroblasts expressing E-cadherin was noted in comparison with that in rats treated with OD (group 2). In animals of the 1st (hepatosis) group on the 30th and 60th days, the numerical density of fibroblasts expressing SNAIL + SLUG was 6 and 7 times higher than in rats treated with OD (group 2). In the liver of animals of the 2nd group (hepatosis + OD), the numerical density of cells expressing TGF-β was lower in comparison with that of animals of the 1st group (hepatosis) by 2.5 times on the 30th day and 3.6 times on the 60th day of the experiment. Conclusion. In post-toxic hepatosis, the expression of TGF-β, SNAIL + SLUG and vimentin proteins increases in liver parenchymal and nonparenchymal cells, contributing to the acquisition of a mesenchymal immunophenotype by cells, which leads to increased profibrotic activity and the development of liver cirrhosis. The use of OD in post-toxic hepatosis reduces the expression of vimentin, TGF-β and EMT-associated proteins in liver parenchymal and nonparenchymal cells, which decreases the severity of fibroplastic processes and prevents the development of liver cirrhosis.

Expression of epithelial–mesenchymal transition-associated proteins and proliferating cell nuclear antigen in dihydropyridine-induced gingival overgrowth fibroblasts: A preliminary study

Background/purposeThe clinical features of dihydropyridine-induced gingival overgrowth (DIGO), including extracellular matrix accumulation and cell hyperplasia, are regulated by inflammatory factors (e.g., Interleukin-1β [IL-1β]) in combination with calcium channel blockers (e.g., nifedipine [Nif]). We speculated that IL-1β and Nif (IL-1β/Nif) may be the main factor modulating the proliferative potential and turnover of fibroblasts in DIGO. Materials and methodsWe cultured four DIGO fibroblast strains and analysed the possible effects of IL-1β/Nif treatments on epithelial–mesenchymal transition (EMT)-associated proteins. We developed short hairpin ribonucleic acids (shRNAs) and used them to explore the role of IL-1β/Nif in regulating proliferating cell nuclear antigen (PCNA) levels in DIGO tissues. ResultsOur results revealed that compared with control cells, DIGO cells stimulated with IL-1β/Nif had higher levels of the EMT-associated proteins Snail, Slug, and Twist. Moreover, both drugs enhanced androgen receptor (AR), Slug, and PCNA expression. ConclusionTaken together, our data indicate that proinflammatory cytokines in combination with calcium channel blockers can regulate the expression of EMT-associated proteins and increase the proliferative potential of DIGO fibroblasts.

GATA binding protein 5-mediated transcriptional activation of transmembrane protein 100 suppresses cell proliferation, migration and epithelial-to-mesenchymal transition in prostate cancer DU145 cells

ABSTRACT It has been reported that transmembrane protein 100 (TMEM100) acts as a tumor regulator in several types of cancers. However, whether the expression of TMEM100 is associated with the development and prognosis of prostate cancer (PCa) remains elusive. Therefore, the present study aimed to uncover the role of GATA binding protein 5 (GATA5)-mediated activation of TMEM100 in the proliferation, migration and epithelial-to-mesenchymal transition (EMT) of PCa cells. The expressions of TMEM100 and GATA5 in PCa patients were analyzed by the GEPIA database. The binding site of GATA5 and TMEM100 promoter was predicted by the JASPAR database. Expressions of TMEM100 and GATA5 in PCa cells were detected by qRT-PCR and Western blot analysis. Cell Counting Kit 8 and colony formation assays were performed to measure cell proliferation. In addition, cell migration, invasion and the expression of EMT-associated proteins were evaluated using wound healing, transwell assay and Western blotting assays, respectively. The bioinformatics analysis revealed that TMEM100 was downregulated in PCa and was associated with overall survival of PCa. In addition, TMEM10 overexpression attenuated cell proliferation, migration, invasion and EMT in PCa cells. The interaction between TMEM100 and GATA5 was verified using dual luciferase reporter and chromatin immunoprecipitation assays. Furthermore, the results showed that GATA5 was downregulated and GATA5 silencing reversed the inhibitory effects of TMEM10 on PCa cells. Overall, the current study suggested that the GATA5-mediated transcriptional activation of TMEM100 could affect the behavior of PCa cells and was associated with poor prognosis in PCa.

Bone Marrow Mesenchymal Stem Cells Derived Discoidin Domain-Containing Receptor 2 (DDR2) as a Communication Mediator to Strengthen the Invasiveness and Metastasis of Papillary Thyroid Carcinoma

Our current study plans to dissect the impacts and its underlying mechanisms of bone marrow mesenchymal stem cells (BMSCs) on the invasive and metastatic features of PTC. Clinical specimens from distantly metastatic PTC were collected to measure DRR2 level. After being identified via tri-lineage differentiation and flow cytometry, BMSCs were co-cultured with PTC cells followed by analysis of cell proliferation and migration by CCK-8 and Transwell assays, expression of DDR2 and EMT-associated proteins by Western blot. Eventually, shDDR2-transfected BMSCs were infused with PTC cells into the abdominal cavity of mice to establish a mouse model assess their effect on tumor growth and distant metastasis. DDR2 was upregulated in BMSCs and malignant cells located in the metastatic sites. Co-culture with BMSCs enhanced DRR2 expression in PTC cells, which was simultaneously accompanied by the escalated mesenchymalization process. In vivo experiments exhibited that co-injection with BMSCs facilitated disease progression and distant metastasis of malignancies. Instead, DDR2 knockdown significantly impeded BMSCs-triggered migrative and proliferative behaviors of malignant cells. In conclusion, DDR2 derived from BMSCs can function as a communication mediator to strengthen the invasiveness and metastasis of PTC.

MiRNA-223 expression in patient-derived eutopic and ectopic endometrial stromal cells and its effect on epithelial-to-mesenchymal transition in endometriosis

ObjectiveThis study was designed to evaluate the expression of microRNA-223 (miRNA-223) in patient-derived eutopic and ectopic endometrial stromal cells (SCs). Given the fact that miRNA-223 was previously shown to be upregulated in these cells and that this upregulation has been linked to epithelial-to-mesenchymal transition (EMT) during endometriosis, this study aimed to further explore the expression of miRNA-223, its effect in endometriosis, and the mechanisms underlying its effects. MethodsEndometrial tissue was collected from 26 patients with endometriosis and 14 patients with hysteromyoma (control group). Primary endometrial SCs were isolated and cultured from several endometrial samples and miRNA-223 expression was evaluated using qRT-PCR. Cells were then transfected with a miRNA-223 overexpression lentiviral vector (sh-miR-223 cells) or an empty control (sh-NC cells) and then used to monitor the effects of miRNA-223 on the expression of several EMT-associated proteins, including N-cadherin, vimentin, and Slug, using western blot. Cellular migration, invasion, and proliferation were then evaluated using a wound healing, Transwell, and CCK-8 assay, respectively. Flow cytometry was used to detect apoptosis. ResultsThere was a significant decrease in the expression of miRNA-223 in both eutopic and ectopic endometrial SCs (p < 0.05) whereas upregulation of miRNA-223 inhibited the expression of EMT-related molecules and reduced cell migration, invasion, and proliferation. High levels of miRNA-223 also promoted apoptosis. ConclusionmiRNA-223 expression decreased in endometrial SCs from endometriosis patients, which may facilitate the differential regulation of EMT during endometriosis. Clinical Trial registration numberSWYX2020-211.

Rabeprazole has efficacy per se and reduces resistance to temozolomide in glioma via EMT inhibition.

Epithelial to mesenchymal transition (EMT) is pivotal in embryonic development and wound healing, whereas in cancer it inflicts malignancy and drug resistance. The recognition of an EMT-like process in glioma is relatively new and its clinical and therapeutic significance has, as yet, not been fully elucidated. Here, we aimed to delineate the clinical significance of the EMT-like process in glioma and its therapeutic relevance to rabeprazole. We investigated the expression profiles of EMT-associated proteins in primary glioma biopsies through Western blotting and immunohistochemistry, and correlatedthem with various clinicopathological features and data listed in the cancer genome atlas (TCGA). In addition, the anticancer efficacy of rabeprazole and its therapeutic relevance to EMT along with temozolomide chemo-sensitization were assessed using multiple cell-based assays, Western blotting and confocal imaging. For in vivo assessment, we used a stereotaxic C6-rat glioma model. Expression analysis of EMT-associated proteins in glioma biopsies, in conjunction with clinicopathological and TCGA dataset analyses, revealed non-canonical expression of E/N-cadherin and upregulation of GFAP, vimentin and β-catenin. The increased expression of EMT-associated proteins may attribute to glioma malignancy and a poor patient prognosis. Subsequent in vitro studies revealed that rabeprazole treatment attenuated glioma cell growth and migration, and induced apoptosis. Rabeprazole suppressed EMT by impeding AKT/GSK3β phosphorylation and/or NF-κB signaling and sensitized temozolomide resistance. Additional in vivo studies showed restricted tumor growth and inhibited expression of EMT-associated proteins after rabeprazole treatment. Our data revealed (i) a clinical association of the EMT-like process with glioma malignancy and a poor survival and (ii) an anticancer and temozolomide sensitizing effect of rabeprazole by repressing EMT.

SATB1 protein is associated with the epithelial‑mesenchymal transition process in non‑small cell lung cancers.

Lung cancer is one of the most frequently diagnosed neoplasms and the leading cause of cancer-related mortality worldwide. Its predominant subtype is non-small cell lung cancer (NSCLC), which accounts for over 80% of the cases. Surprisingly, the majority of lung cancer-related deaths are caused not by a primary tumour itself, but by its metastasis to distant organs. Therefore, it becomes especially important to identify the factors involved in lung cancer metastatic spread. Special AT-rich binding protein 1 (SATB1) is a nuclear matrix protein that mediates chromatin looping and plays the role of global transcriptional regulator. During the past decade, it has received much attention as a factor promoting tumour invasion. In breast, colorectal and prostate cancers, SATB1 has been shown to influence the epithelial-mesenchymal transition (EMT) process, which is thought to be crucial for cancer metastasis. The aim of this study was to analyse the possible correlations between the expression of SATB1 and major EMT-associated proteins in NSCLC clinical samples. Additionally, the impact of EMT induction in NSCLC cell lines on SATB1 mRNA expression was also investigated. Immunohistochemistry was used to assess the expression of SATB1, SNAIL, SLUG, Twist1, E-cadherin, and N-cadherin in 242 lung cancer clinical samples. EMT was induced by TGF-β1 treatment in the A549 and NCI-H1703 lung cancer cell lines. Changes in gene expression profiles were analyzed using real-time PCR and Droplet Digital PCR. SATB1 expression was positively correlated with the expression of SNAIL (R=0.129; P=0.045), SLUG (R=0.449; P<0.0001), and Twist1 (R=0.264; P<0.0001). Moreover, SATB1 expression significantly increased after in vitro EMT induction in A549 and NCI-H1703 cell lines. The results obtained may point to the role of SATB1 as one of the regulators of EMT in NSCLC.

The lncRNA NEAT1 promotes the epithelial-mesenchymal transition and metastasis of osteosarcoma cells by sponging miR-483 to upregulate STAT3 expression

BackgroundOsteosarcoma is one of the most prevalent primary bone tumours in adolescents. Accumulating evidence shows that aberrant expression of the long non-coding RNA (lncRNA) NEAT1 and microRNA-483 (miR-483) contribute to the epithelial-mesenchymal transition (EMT), invasion and metastasis of tumour cells. However, the potential regulatory effects of NEAT1 and miR-483 on the EMT of osteosarcoma remain elusive.MethodsThe expression of the NEAT1, miR-483, signal transducer and activator of transcription-1 (STAT1), STAT3, and EMT-associated markers was measured using qRT-PCR or western blotting. NEAT1 overexpression or knockdown was induced by lentivirus-mediated transfection. A luciferase reporter assay was employed to confirm the association between NEAT1/miR-483 and miR-483/STAT3. RNA immunoprecipitation (RIP) was also performed to verify the NEAT1 and miR-483 interaction. Wound healing and transwell assays were implemented to assess the migration and invasion of U2OS cells. Unilateral subcutaneous injection of U2OS into nude mice was performed to investigate tumour metastasis in vivo.ResultsThe expression of miR-483 was downregulated in both osteosarcoma cell lines and osteosarcoma tissues. The overexpression of miR-483 suppressed the migration, invasion, and expression of EMT-associated proteins in U2OS cells, while simultaneous overexpression of STAT3 partially relieved this suppression. Mechanistically, miR-483 specifically targeted the 3′ untranslated region (3′UTR) of STAT3 and repressed its expression. However, NEAT1 sponged miR-438, increased STAT3 expression, and repressed STAT1 expression, subsequently increasing the EMT of osteosarcoma cells. The knockdown of NEAT1 in transplanted U2OS cells impaired the liver and lung metastases of osteosarcoma in nude mice. Moreover, NEAT1 silencing inhibited the mesenchymal- epithelial transition (MET) of osteosarcoma at metastasis sites.ConclusionsThe lncRNA NEAT1/miR-483/STAT3 axis plays a crucial role in regulating the metastasis of osteosarcoma and potentially represents one appealing therapeutic target for osteosarcoma treatment in the future.

MicroRNA-199a-5p regulates epithelial-to-mesenchymal transition in diabetic cataract by targeting SP1 gene

BackgroundAs a common ocular complication of diabetes mellitus, diabetic cataract is becoming a leading cause of visual impairment. The progression of diabetic cataract progression involves epithelial-to-mesenchymal transition (EMT), the precise role of which remains to be investigated. As microRNAs (miRNAs) are suggested to be involved in the pathogenesis of many diseases, identification of aberrantly expressed miRNAs in diabetic lens epithelial cells (LECs) and their targets may provide insights into our understanding of diabetic cataract and potential therapeutic targets.MethodsDiabetic cataract capsules and LECs exposed to high glucose (25 mmol/L, 1–5 days) were used to mimic the model. Quantitative RT-PCR was performed to evaluate the differential expression of miRNA. Dual luciferase reporter assay was used to identify the binding target of miR-199a-5p. The expression of EMT-associated proteins was determined by immunofluorescence and Western blot analysis.ResultsOur results showed the differential expression of miR-9, -16, -22, -199a and -204. MiR-199a was downregulated in diabetic cataract capsule and hyperglycemia-conditioned human LECs. Specific protein 1 could be directly targeted and regulated by miR-199a in LECs and inhibit EMT in diabetic LECs.ConclusionOur findings implied miR-199a could be a therapeutic target by regulating SP1 directly to affect EMT in diabetic cataract and provided novel insights into the pathogenesis of diabetic cataract.

Role of TGF-β1-mediated epithelial-mesenchymal transition in the pathogenesis of tympanosclerosis.

The present study aimed to explore the role of TGF-β1-mediated epithelial-mesenchymal transition (EMT) in the pathogenesis of tympanosclerosis. Sprague Dawley rats were injected with inactivated Streptococcus pneumoniae suspension to establish a rat model of tympanosclerosis. The rats were sacrificed 8 weeks after the model was established. H&E and von Kossa staining was used to observe the morphological changes of middle ear mucosa. Western blotting was used to detect the expression of TGF-β1 and EMT-associated proteins in the mucosa samples. Middle ear mucosal epithelial cells of rats were collected to establish a primary culture. The cultured cells were stimulated with TGF-β1 and the expression of EMT-associated proteins was detected by western blotting and immunofluorescence. In addition, the cells were treated with TGF-β receptor type I/II inhibitor and the expression level of EMT-associated proteins was detected by western blotting. Sclerotic lesions appeared on 72.4% of tympanic membranes, and marked inflammation, inflammatory cell infiltration and fibrosis were found in the middle ear mucosa of rat models of tympanosclerosis. In middle ear mucosa of rats with tympanosclerosis, the expression of mesenchymal cell markers increased and that of epithelial cell markers decreased compared with the control group. TGF-β1 stimulated the activation of the EMT pathway in middle ear mucosal epithelial cells, resulting in an increased expression of fibronectin and N-cadherin. In addition, a decreased expression level of EMT-associated proteins was observed when TGF-β1 was inhibited. In conclusion, the present study indicated that TGF-β1-mediated EMT may play an important role in the pathogenesis of tympanosclerosis.

CD147 promotes proliferation and migration of oral cancer cells by inhibiting junctions between E-cadherin and β-catenin.

Although association between oral squamous cell carcinoma (OSCC) with epithelial-mesenchymal transition (EMT) has been demonstrated, we found CD147, one transmembrane protein we previously studied in oral submucous fibrosis, was correlated with E-cadherin, one marker of EMT. Here, we investigated CD147 expression in the different stages of OSCC and assessed its association with epithelial-mesenchymal transition (EMT). CD147 and E-cadherin expression in tissue microarrays containing 48 OSCC specimens and matched adjacent tissues was analysed using immunohistochemistry. CD147 was overexpressed or knocked down using exogenous cloning vector and RNA interference, respectively, in OSCC cell lines. Cell proliferation and migration were measured using the CCK8 assay and scratch test, respectively. The expression and localization of EMT-associated proteins was analysed by Western blotting and immunofluorescence. CD147 expression in OSCC tissues was significantly higher than that in adjacent tissues and was markedly higher in cancer tissues with metastasis (P<.05). CD147 expression showed significant negative correlation with E-cadherin expression. CD147 overexpression downregulated E-cadherin and inhibited its complex with β-catenin and then upregulated N-cadherin and vimentin. Additionally, alterations in CD147 protein expression affected proliferation and migration ability in OSCC cells and were related to β-catenin nuclear translocation. CD147 plays an important role in tumorigenesis and metastasis by promoting EMT progression in OSCC. It may be considered as a novel potential diagnostic and therapeutic target for OSCC.

Characterization of Triple-Negative Breast Cancer MDA-MB-231 Cell Spheroid Model.

BackgroundThe tumor three-dimensional (3D) spheroid model in vitro is effective on detecting malignant cells and tumorigenesis, and assessing drug resistance. Compared with two-dimensional (2D) monolayer culture, breast cancer (BC) spheroids more accurately reflect the complex microenvironment in vivo, which have been extensively reported in BC research. MDA-MB-231 cells, the triple-negative breast cancer (TNBC) cell line, display representative epithelial to mesenchymal transition (EMT) associated with BC metastasis. However, the characterization of MDA-MB-231 spheroids has been largely unknown at present, which requires further attention.Materials and MethodsMicrowell array was conducted for the formation of MDA-MB-231 spheroids. In addition, H&E staining, immunohistochemistry (IHC), CellTiter-Glo® 3D cell viability assay, and flow cytometry were performed to investigate the structure and growth characteristics. Besides, Transwell and scratch healing assays were carried out to detect the migratory capacities compared with 2D culture. Western blotting and confocal fluorescence were selected to detect the expression of EMT-associated proteins. Additionally, the half maximal inhibitory concentration (IC50) values of antitumor compounds Carboplatin and Doxorubicin were measured to assess drug resistance.ResultsThe MDA-MB-231 spheroids were viable, which maintained a compact structure with zonation features for up to 9 days. Moreover, those spheroids had a slower growth rate than those cultured as a monolayer and differential zones of proliferation. The migratory capacities were significantly enhanced by transferring the spheroids to 2D adherent culture. Compared with 2D culture, the levels of EMT-associated proteins were significantly up-regulated in spheroids. Furthermore, toxicity assessment showed that spheroids exhibited an increased resistance to the antitumor compounds.ConclusionThis study develops the simple spheroids and demonstrates their structure, growth and proliferation characteristics. According to our results, the spheroids are associated with superior EMT and high resistance to toxicological response compared with the standard 2D monocultures.

Presence of circulating tumor cells is more significant than tumor stage in characterizing cellular local immunity in colorectal cancer.

e16073 Background: The purpose of the study was a correlation analysis of parameters of local immunity and markers of the epithelial-mesenchymal transition (EMT) with the number of circulating tumor cells (CTCs) in patients with stage II-IV colorectal cancer (CRC). Methods: The study included 60 patients newly diagnosed with stage II-IV CRC. CTCs were detected with the Cell Search System (Janssen Diagnostics, LLC) prior to surgery. CTC≤3 were considered negative, CTC &gt; 3 – positive. After surgery, IHC analysis of tumor infiltration with immune system cells was performed with monoclonal antibodies for human CD3, CD4, CD8, CD20 and CD56; expression of EMT-associated proteins (E-cadherin, N-cadherin) was measured. All patients were informed of the goal of the study and provided informed consent. Results: CTC-negative patients with later cancer stages showed the loss of connection between the local levels of CD4+ and CD8+, and in stage IV - between CD3+ and CD4+. Starting from stage III, a negative correlation was observed between the expressions of E- and N-cadherin, not associated with the studied indices of local cellular immunity. The presence of CTCs in all 3 investigated stages of CRC, apparently, causes the loss of many connections between the indicators of the 3 units of local cellular immunity. While negative correlations were revealed between T-, B- and NK-cells in CTC-negative status, in CTC+ their number decreased to 1. An important positive correlation was between local levels of CD3+ and CD8+, indicating the predominance of cytotoxic T cells in the tumor and detected at all stages in CTC-negative patients, losing significance in the presence of CTCs regardless of the stage. In stage II, the only correlation (inverse one) was observed between the parameters of local (CD4+) immunity and the expression of cadherins (E-cadherin), which probably reflected the presence of immunosuppressive and growth-stimulating Tregs fraction among these cells. Conclusions: In general, the presence of CTCs was more significant for the number and nature of interconnections of cellular parameters of the local immunity than the tumor stage.

Ascofuranone inhibits epidermal growth factor-induced cell migration by blocking epithelial-mesenchymal transition in lung cancer cells

Ascofuranone, an isoprenoid antibiotic initially purified from a culture broth of Ascochyta viciae, has multiple anticancer effects. However, the impacts of ascofuranone on the epithelial–mesenchymal transition (EMT) and epidermal growth factor (EGF)-induced effects on human lung cancer cell lines have not been previously reported. Here, we show that ascofuranone exerts its anticancer effects by inhibiting the EGF-induced EMT and cell migration in human lung cancer cell lines. Ascofuranone significantly inhibited EGF-induced migration and invasion by lung cancer cells, and suppressed EGF-induced morphologic changes by regulating the expression of EMT-associated proteins. In addition, ascofuranone upregulated E-cadherin, and downregulated fibronectin, vimentin, Slug, Snail, and Twist. Inhibition of ERK/AKT/mTOR promoted EGF-induced E-cadherin downregulation and inhibited EGF-induced vimentin upregulation in response to ascofuranone, implying that inhibition of the EGF-induced EMT by ascofuranone was mediated by the ERK and AKT/mTOR pathways. Inhibition of c-Myc suppressed EGF-induced vimentin upregulation, suggesting the involvement of c-Myc. Collectively, these findings suggest that ascofuranone inhibits tumor growth by blocking the EGF-induced EMT through a regulatory mechanism involving ERK, AKT/mTOR, and c-Myc in lung cancer cells.

LncRNA TUG1 contributes to ESCC progression via regulating miR-148a-3p/MCL-1/Wnt/β-catenin axis in vitro.

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies. Latest studies report that long noncoding RNAs (LncRNAs) play an essential role in diversified pathological processes of ESCC, although the mechanism by which they do so remains unknown. This study aimed to explore the parts of lncRNA taurine upregulated gene 1 (TUG1) in ESCC tissues and cells, its biofunctional effect and its underlying regulatory mechanism in ESCC.MethodsThe levels of TUG1 and miR‐148a‐3p were detected by quantitative real‐time polymerase chain reaction (qRT‐PCR) in ESCC cells and tissues. The biofunctional effects were examined by MTT, flow cytometry, and transwell assay. The protein expression levels of epithelial‐mesenchymal transition (EMT)‐related proteins and MCL‐1 were determined by western blot analysis. The binding sites between miR‐148a‐3p and TUG1 or MCL‐1 were predicted by online software starBase and confirmed by dual luciferase reporter assay.ResultsThe mRNA expression of TUG1 was significantly upregulated in ESCC tissues or cells, and was negatively correlated to miR‐148a‐3p expression in tissues. Knockdown of TUG1 inhibited the proliferation, migration, and invasion, promoted apoptosis, and relieved the EMT progression in EC9706 and OE19 cells. Besides, knockdown of miR‐148a‐3p inverted positive effects from TUG1 deletion on ESCC cells. Besides, MCL‐1 reversed the inhibitive effects from TUG1 deletion on expression of EMT‐associated proteins (Wnt1, C‐myc, CyclinD1, and β‐catenin) above subsequently.ConclusionTUG1 regulated the biofunction and EMT progression of ESCC by mediating miR‐148a‐3p/MCL‐1/Wnt/β‐catenin axis in vitro.

Prognostic value of multiple epithelial mesenchymal transition‑associated proteins in intrahepatic cholangiocarcinoma

The aim of the present study was to investigate the expression of epithelial mesenchymal transition (EMT)-associated proteins and their prognostic value in intrahepatic cholangiocarcinoma (ICC). The expression of six EMT-associated proteins, including E-cadherin, N-cadherin, Vimentin, Snail family transcriptional repressor 1 (Snail), Snail family transcriptional repressor 2 (Slug) and S100 calcium binding protein A4 (S100A4) was determined by immunohistochemistry in 109 patients with ICC who had received surgery. Survival analysis showed that patients with low E-cadherin expression (P<0.001) or high S100A4 (P<0.001) or Snail (P<0.001) expression had a reduced survival time. Based on the numbers of alterations in the expression of EMT-associated proteins as determined by immunohistochemical analysis, the patients were categorized as low (score, 0-3; n=75) or high (score, ≥4; n=34) EMT expression groups. The high EMT expression group was significantly associated with positive lymph node metastasis (P=0.023) and late Tumor-Node-Metastasis (TNM) stage (P<0.001). Furthermore, patients in the high EMT expression group had a significantly poorer overall survival time than those in the low EMT expression group (P<0.001). Multivariate analysis indicated that EMT status was a significant independent predictor for overall survival time (P=0.004), and was linked to surgical margin (P=0.013) and TNM stage (P<0.001). In conclusion, the reduced expression of E-cadherin and high expression of Snail and S100A4 were significantly associated with the poor survival of patients with ICC after surgery. The EMT protein expression status was associated with ICC progression, and may be considered as an independent prognostic indicator for patients with ICC.

SOX9 drives the epithelial\u2013mesenchymal transition in non-small-cell lung cancer through the Wnt/\u03b2-catenin pathway

BackgroundThe distant metastasis of cancer cells is a risk factor for tumor lethality and poor prognosis in non-small-cell lung carcinoma (NSCLC). Increased SOX9 expression has been associated with clinical stage and poor prognosis in NSCLC, but the molecular mechanisms by which SOX9 promotes metastasis in NSCLC are still unknown.MethodsThe relationship between SOX9 expression and T, N, M classification was assessed using the χ2 test and Spearman’s analysis in 142 immunohistochemically diagnosed specimens of NSCLC. We also generated SOX9-overexpression and SOX9-knockdown cells lines and their corresponding control cell lines by transfection with lentiviral constructs. In vivo assay, SOX9-overexpressing and SOX9-knockdown NSCLC cells were injected in zebrafish to examine distance metastasis. Gene set enrichment analysis (GSEA) was applied to analysis the correlation between SOX9 overexpression and Wnt/β-catenin pathway. Luciferase assay was used to check transcriptional activity of TCF/LEF and western blot and immunofluorescence was employed to detect β-catenin translocation in SOX9-overexpression, SOX9-knockdown and their corresponding control cell lines.ResultsWe found that SOX9 overexpression correlates with the T, N and M stage significantly (p = 0.03, 0.000, and 0.032 respectively) in 142 immunohistochemically diagnosed specimens of NSCLC. SOX9 overexpression was found to decrease the expression of the epithelial cell markers E-cadherin and γ-catenin and increase the expression of the mesenchymal cell markers N-cadherin and vimentin. An in vivo assay showed distant metastasis of the SOX9-overexpressing cells, which was not observed in the SOX9-knockdown cells. These findings indicate that SOX9 promotes distant metastasis by promoting EMT in NSCLC cells. GSEA showed that SOX9 overexpression was significantly correlated with the Wnt/β-catenin pathway which was corroborated by the expression of EMT-associated proteins in this pathway and its downstream target genes. SOX9 overexpression was also found to enhance the transcriptional activity of TCF/LEF, promote the nuclear translocation of β-catenin and increase the phosphorylation of GSK3β at Ser9. Further, inhibition of β-catenin suppressed the metastasis-promoting effects of SOX9 overexpression.ConclusionsThis study is the first to report that SOX9 is associated with clinical TNM stage and indicates that SOX9 promotes migration, invasion and the EMT process through the Wnt/β-catenin pathway.

MTORC1 inhibitor RAD001 (everolimus) enhances non-small cell lung cancer cell radiosensitivity in vitro via suppressing epithelial-mesenchymal transition.

Resistance to radiotherapy causes non-small cell lung cancer (NSCLC) treatment failure associated with local recurrence and metastasis. Thus, understanding the radiosensitization of NSCLC cells is crucial for developing new treatments and improving prognostics. mTORC1 has been shown to regulate tumor cell radiosensitivity, but the underlying mechanisms are unclear. Moreover, mTORC1 also regulates epithelial-mesenchymal transition (EMT) that is important to metastasis and recurrence. In this study we explored whether mTORC1 regulated NSCLC cell radiosensitivity by altering EMT. We performed immunohistichemical analysis using tumor, adjacent and normal tissues from 50 NSCLC patients, which confirmed significantly elevated mTOR protein expression in NSCLC tissue. Then we used NCI-H460 and NCI-H661 cell lines to examine the effects of the mTORC1 inhibitor RAD001 (everolimus) on in vitro radiosensitivity, protein expression and dose-survival curves. RAD001 (10 nmol/L) significantly inhibited the mTORC1 pathway in both the cell lines. Pretreatment with RAD001 (0.1 nmol/L) enhanced the radiosensitivity in NCI-H661 cells with wild-type PIK3CA and KRAS but not in NCI-H460 cells with mutant PIK3CA and KRAS; the sensitivity enhancement ratios in the two NSCLC cell lines were 1.40 and 1.03, respectively. Furthermore, pretreatment with RAD001 (0.1 nmol/L) significantly decreased the migration and invasion with altered expression of several EMT-associated proteins (significantly increased E-cadherin and decreased vimentin expression) in irradiated NCI-H661 cells. Publicly available expression data confirmed that irradiation affected mTOR and EMT-associated genes at the transcript level in NSCLC cells. These results suggest that mTORC1 inhibition enhances the in vitro radiosensitivity of NSCLC cells with wild-type PIK3CA and KRAS by affecting EMT. Our preclinical data may provide a potential new strategy for NSCLC treatment.

ZEB2, a master regulator of the epithelial-mesenchymal transition, mediates trophoblast differentiation.

Does the upregulation of the zinc finger E-box binding homeobox 2 (ZEB2) transcription factor in human trophoblast cells lead to alterations in gene expression consistent with an epithelial-mesenchymal transition (EMT) and a consequent increase in invasiveness? Overexpression of ZEB2 results in an epithelial-mesenchymal shift in gene expression accompanied by a substantial increase in the invasive capacity of human trophoblast cells. In-vivo results have shown that cytotrophoblast differentiation into extravillous trophoblast involves an epithelial-mesenchymal transition. The only EMT master regulatory factor which shows changes consistent with extravillous trophoblast EMT status and invasive capacity is the ZEB2 transcription factor. This study is a mechanistic investigation of the role of ZEB2 in trophoblast differentiation. We generated stable ZEB2 overexpression clones using the epithelial BeWo and JEG3 choriocarcinoma lines. Using these clones, we investigated the effects of ZEB2 overexpression on the expression of EMT-associated genes and proteins, cell morphology and invasive capability. We used lentiviral transduction to overexpress ZEB2 in BeWo and JEG3 cells. Stable clones were selected based on ZEB2 expression and morphology. A PCR array of EMT-associated genes was used to probe gene expression. Protein measurements were performed by western blotting. Gain-of-function was assessed by quantitatively measuring cell invasion rates using a Transwell assay, a 3D bioprinted placenta model and the xCelligenceTM platform. The four selected clones (2 × BeWo, 2 × JEG3, based on ZEB2 expression and morphology) all showed gene expression changes indicative of an EMT. The two clones (1 × BeWo, 1 × JEG3) showing >40-fold increase in ZEB2 expression also displayed increased ZEB2 protein; the others, with increases in ZEB2 expression <14-fold did not. The two high ZEB2-expressing clones demonstrated robust increases in invasive capacity, as assessed by three types of invasion assay. These data identify ZEB2-mediated transcription as a key mechanism transforming the epithelial-like trophoblast into cells with a mesenchymal, invasive phenotype. PCR array data have been deposited in the GEO database under accession number GSE116532. These are in-vitro studies using choriocarcinoma cells and so the results should be interpreted in view of these limitations. Nevertheless, the data are consistent with in-vivo findings and are replicated in two different cell lines. The combination of these data with the in-vivo findings clearly identify ZEB2-mediated EMT as the mechanism for cytotrophoblast differentiation into extravillous trophoblast. Having characterized these cellular mechanisms, it will now be possible to identify the intracellular and extracellular regulatory components which control ZEB2 and trophoblast differentiation. It will also be possible to identify the aberrant factors which alter differentiation in invasive pathologies such as preeclampsia and abnormally invasive placenta (AKA accreta, increta, percreta). Funding was provided by the Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine and Surgery at Hackensack Meridian Health, Hackensack, NJ. The 3D bioprinted placental model work done in Drs Kim and Fisher's labs was supported by the Children's National Medical Center. The xCELLigence work done in Dr Birge's lab was supported by NIH CA165077. The authors declare no competing interests.