Changes In Epithelial-mesenchymal Transition Markers Research Articles

Increased UBD Is a Potential Diagnostic and Prognostic Biomarker in Glioma.

Various studies have demonstrated that ubiquitin D (UBD) is overexpressed in different cancer types and may serve as a potential prognostic factor. However, additional research is necessary to establish the prognostic significance and possible role of UBD in glioma. Transcriptomic expression data from The Cancer Genome Atlas database (TCGA) and Chinese Glioma Genome Atlas (CGGA) were analyzed to identify UBD expression differences in tumor and normal tissues. The relative levels of UBD in glioma and normal tissues were determined using qRT-PCR and WB. Logistic regression analysis was performed to investigate the association between UBD expression and clinicopathological characteristics of glioma patients. To evaluate the diagnostic and prognostic predictive values of UBD, we used Kaplan-Meier survival curves, Cox regression analysis, diagnostic receiver operating characteristic (ROC) curves, and nomogram model. We also conducted wound healing assays, transwell assays, EdU assays, and colony formation assays to verify the UBD function. Gene ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, as well as gene set enrichment analysis (GSEA), were employed to determine the functions of UBD. Finally, we performed the western blot assays to assess changes in EMT markers as well as p-PI3K, p-AKT, and p-mTOR expressions. Our study revealed a remarkable increase of UBD expression in glioma samples. Cox regression analysis demonstrated that high expression of UBD mRNA was an independent prognostic factor for overall survival (OS) in TCGA. ROC curve analysis showed that UBD expression levels could differentiate glioma from adjacent normal tissues accurately. Additionally, knockdown of UBD reduced the migration, invasion, and proliferation ability of glioma cells while UBD overexpression had the opposite effect. GSEA showed that the expression of UBD involved with various pathways including epithelial-mesenchymal transition (EMT), PI3K-AKT-mTOR signaling, P53 pathway, angiogenesis, inflammatory response, KRAS signaling, hypoxia, as well as TGF-β signaling. Furthermore, our findings suggest that UBD accelerates the activation of EMT and PI3K/AKT/mTOR pathway.

Vitamin A Status Modulates Epithelial Mesenchymal Transition in the Lung: The Role of Furin.

Vitamin A deficiency (VAD) induced TGF-β hyperactivation and reduced expression of cell adhesion proteins in the lung, suggesting that the disruption of retinoic acid (RA) signaling leads to epithelial-mesenchymal transition (EMT). To elucidate the role of lung vitamin A status in EMT, several EMT markers and the expression of the proprotein convertase furin, which activates TGF-β, were analyzed in two experimental models. Our in vivo model included control rats, VAD rats, and both control rats and VAD rats, treated with RA. For the in vitro studies, human bronchoalveolar epithelial cells treated with RA were used. Our data show that EMT and furin are induced in VAD rats. Furthermore, furin expression continues to increase much more markedly after treatment of VAD rats with RA. In control rats and cell lines, an acute RA treatment induced a significant increase in furin expression, concomitant with changes in EMT markers. A ChIP assay demonstrated that RA directly regulates furin transcription. These results emphasize the importance of maintaining vitamin A levels within the physiological range since both levels below and above this range can cause adverse effects that, paradoxically, could be similar. The role of furin in EMT is discussed.

Abstract 992: The impact of tamoxifen on autophagic and epigenetic regulation in endometrial cancer

Abstract Background: Endometrial cancer, predominantly endometrioid adenocarcinoma, ranks as the fourth most common cancer in women globally, with an increasing incidence over the last two decades. Tamoxifen, well-known for its antiestrogenic characteristics in the treatment of breast cancer, paradoxically increases the risk of endometrial cancer. Understanding this dual role of tamoxifen is crucial for optimizing breast cancer therapy while mitigating endometrial cancer risk. This study aims to elucidate the molecular alterations induced by tamoxifen in endometrial thickening and its potential to induce cancer, particularly focusing on autophagy, epithelial-mesenchymal transition (EMT), and cancer stem-like cells (CSCs). Methods: In this study, female BALB/c mice were administered tamoxifen (20 mg/ml) orally for five consecutive days. We monitored changes in EMT markers, autophagy-related genes, and stem cell markers over 1 to 5 months. Furthermore, we investigated the epigenetic modulation of key genes involved in these processes. Results: Tamoxifen treatment led to altered expression of both epithelial and mesenchymal EMT markers, autophagy-related genes (p62, LC3B, Beclin-1, mTOR, ATG12, ATG16), and stem cell markers (Nanog, SOX2, ALDH1). Notably, we observed significant epigenetic changes, including increased methylation of E-cadherin and decreased methylation of Twist. Furthermore, we observed hypermethylation (a process often linked to the silencing of tumor-suppressing genes) in MGMT (Methylguanine methyltransferase), a DNA repair enzyme involved in chemoresistance, and DAPK (Death-associated protein kinase), a kinase associated with apoptosis, autophagy, and inflammation. We also observed hypermethylation in the CpG promoter regions of autophagy-related genes (Beclin-1 and LC3B) highlighting a potential mechanism for tamoxifen-induced tumorigenic changes. Conclusion: Our findings demonstrate tamoxifen's influence on cellular and molecular pathways, highlighting the complex interplay between drug treatment and cancer progression. The observed epigenetic changes provide insight into the mechanisms by which tamoxifen may contribute to endometrial cancer risk, suggesting potential targets for therapeutic intervention. Citation Format: Shilpa Thota, Rizwana Begum, Naveen Chintala, Abhishek Pandit, Biplov Sapkota, Joseph Francis. The impact of tamoxifen on autophagic and epigenetic regulation in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 992.

Targeting Mitochondrial ROS Production to Reverse the Epithelial-Mesenchymal Transition in Breast Cancer Cells.

Experimental evidence implicates reactive oxygen species (ROS) generation in the hypoxic stabilization of hypoxia-inducible factor (HIF)-1α and in the subsequent expression of promoters of tumor invasiveness and metastatic spread. However, the role played by mitochondrial ROS in hypoxia-induced Epithelial-Mesenchymal Transition (EMT) activation is still unclear. This study was aimed at testing the hypothesis that the inhibition of hypoxia-induced mitochondrial ROS production, mainly at the mitochondrial Complex III UQCRB site, could result in the reversion of EMT, in addition to decreased HIF-1α stabilization. The role of hypoxia-induced ROS increase in HIF-1α stabilization and the ability of antioxidants, some of which directly targeting mitochondrial Complex III, to block ROS production and HIF-1α stabilization and prevent changes in EMT markers were assessed by evaluating ROS, HIF-1α and EMT markers on breast cancer cells, following 48 h treatment with the antioxidants. The specific role of UQCRB in hypoxia-induced EMT was also evaluated by silencing its expression through RNA interference and by assessing the effects of its downregulation on ROS production, HIF-1α levels, and EMT markers. Our results confirm the pivotal role of UQCRB in hypoxic signaling inducing EMT. Thus, UQCRB might be a new therapeutic target for the development of drugs able to reverse EMT by blocking mitochondrial ROS production.

SPP1 facilitates cell migration and invasion by targeting COL11A1 in lung adenocarcinoma

BackgroundSecreted phosphoprotein 1 (SPP1), an extracellular secreted glycol phosphoprotein, is closely related to tumor biologies, such as proliferation, migration, and invasion. However, the role and biological function of SPP1 in lung adenocarcinoma (LUAD) was still ambiguous.MethodsSPP1 expression in LUAD tissues and its associations with clinical features and prognosis was investigated using meta-analysis, immunohistochemistry (IHC) staining methods, and quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, the potential mechanism related to SPP1 was identified by using the Gene Set Enrichment Analysis (GSEA) method. A series of function assays were conducted to determine the biological role of SPP1 in LUAD cell migration and invasion in vitro and vivo. The co-expressed genes of SPP1 were obtained and verified by western blot assays. The influence of SPP1 on Collagen type XI alpha 1 (COL11A1) expression and epithelial-mesenchymal transition (EMT) markers was analyzed using western blot assays.ResultsThe expression of SPP1 in LUAD tissues and cells was significantly higher than that in normal tissues and cells. And positively associations of SPP1 expression with TNM stage, lymph node metastasis, and invasion depth were observed. Patients with high SPP1 expression had unfavorable survival. The multivariable Cox regression analysis revealed that SPP1 expression was an independent prognostic factor of LUAD patients. Furthermore, downregulation of SPP1 could inhibit cell migration and invasion both in vitro and vivo, reduce the expression of epithelial marker (E-cadherin), and increase the expression of mesenchymal markers (N-cadherin and vimentin). Using bioinformatics and western blot assays, we confirmed that COL11A1 acted as the downstream of SPP1, and SPP1 knockdown could significantly downregulate the COL11A1 expression. Importantly, suppression of cell migration and invasion and the expression changes of EMT markers induced by SPP1 downregulation could be reversed by COL11A1 overexpression.ConclusionsSPP1 facilitates cell migration and invasion by upregulating COL11A1 expression and that acts as a potential biomarker of metastasis and prognosis for LUAD.

Oleanolic Acid (OA) Targeting UNC5B Inhibits Proliferation and EMT of Ovarian Cancer Cell and Increases Chemotherapy Sensitivity of Niraparib

Objective To investigate the effect of OA on proliferation, migration, and epithelial-mesenchymal transition (EMT) of ovarian cancer cells by inhibiting UNC5B and to study its mechanism. Methods TCGA database was used to analyze the expression of UNC5B in ovarian cancer and its relationship with prognosis. The expression of UNC5B in ovarian cancer cells was detected by qPCR assay. qRT-PCR was used to detect the changes of EMT markers after different treatments. CCK-8 assay was used to detect cell proliferation, transwell assay was used to evaluate cell migration, and clonogenesis assay was used to evaluate the effect of UNC5B on ovarian cancer cell proliferation. Meanwhile, the synergistic effect of OA on niraparib was evaluated. Results UNC5B was highly expressed in ovarian cancer, and its expression was negatively correlated with the prognosis of ovarian cancer patients. UNC5B was highly expressed in ovarian cancer cells SKOV3 and OVCA420 compared with normal ovarian epithelial cells. In addition, silencing UNC5B inhibits the proliferation, invasion, clonogenesis, and EMT processes of ovarian cancer cells. OA inhibits proliferation, invasion, and clonogenesis of ovarian cancer cells by inhibiting UNC5B and increases the antitumor activity of niraparib. Conclusion UNC5B acts as an oncogenic gene in ovarian cancer. OA inhibits ovarian cancer cell proliferation, migration, and EMT by targeting UNC5B and increases the antitumor effect of niraparib. UNC5B is expected to be a new potential therapeutic target for ovarian cancer. OA may be used as an antitumor drug and deserves further study.

Cysteine-Rich Intestinal Protein 1 Served as an Epithelial Ovarian Cancer Marker via Promoting Wnt/β-Catenin-Mediated EMT and Tumour Metastasis.

Objective To explore the expression, functions, and the possible mechanisms of cysteine-rich intestinal protein 1 (CRIP1) in epithelial ovarian cancer. Methods Using open microarray datasets from The Cancer Genome Atlas (TCGA), we identified the tumorigenic genes in ovarian cancer. Then, we detected CRIP1 expression in 26 pairs of epithelial ovarian cancer tissue samples by immunohistochemistry (IHC) and performed a correlation analysis between CRIP1 and the clinicopathological features. In addition, epithelial ovarian cancer cell lines A2780 and OVCAR3 were used to examine CRIP1 expression by western blot and qRT-PCR. Various cell function experiments related to tumorigenesis were performed including the CCK8 assay, EdU, Annexin V-FITC/PI apoptosis assay, wound healing, and Transwell assay. In addition, the expression of epithelial-mesenchymal transition (EMT) markers was detected by western blot to illustrate the relationship between CRIP1 and EMT. Furthermore, KEGG pathway enrichment analysis and western blot were conducted to reveal the signaling pathways in which CRIP1 is involved in ovarian cancer pathogenesis. Results CRIP1 was identified as an oncogene from the TCGA database. The IHC score demonstrated that the CRIP1 protein was expressed at a higher level in tumours than in tumour-adjacent tissues and was associated with a higher pathological stage, grade, and positive lymphatic metastasis. In cell models, CRIP1 was overexpressed in serous epithelial ovarian cancer. Cell function experiments showed that the knockdown of CRIP1 did not significantly affect cell proliferation or apoptosis but could exert an inhibitory effect on cell migration and invasion, and also induce changes in EMT markers. Furthermore, KEGG pathway enrichment analysis and western blot showed that CRIP1 could induce ovarian cancer cell metastasis through activation of the Wnt/β-catenin pathway. Conclusion This study is the first to demonstrate that CRIP1 acts as an oncogene and may promote tumour metastasis by regulating the EMT-related Wnt/β-catenin signaling pathway, suggesting that CRIP1 may be an important biomarker for ovarian cancer metastasis and progression.

Silencing of the lncRNA TUG1 attenuates the epithelial-mesenchymal transition of renal tubular epithelial cells by sponging miR-141-3p via regulating β-catenin.

Renal interstitial fibrosis (RIF) is characterized by excessive extracellular matrix deposition and involves epithelial-mesenchymal transition (EMT). The lncRNA taurine-upregulated gene 1 (TUG1) participates in EMT in several cancers; however, the effect and underlying mechanism of TUG1 in RIF-related EMT remain unclear. Here, we explored the mechanisms by which TUG1 modulates RIF. An in vivo model of renal fibrosis was established by unilateral ureteral obstruction in Balb/c mice. Human renal proximal tubular epithelial (HK-2) cells treated with transforming growth factor (TGF)-β1 were used to induce the in vitro model. Morphological changes and TUG1 expression were assessed. HK-2 cells were transfected with siRNA to silence TUG1. Western blot analysis, immunofluorescence staining, cell proliferation, and migration assays were performed to examine TGF-β1-induced changes in EMT markers and EMT-like cell behaviors. TUG1 and β-catenin (CTNNB1) levels were significantly upregulated, whereas miR-141-3p was significantly downregulated, during EMT in vitro and in vivo. TUG1 knockdown or miR-141-3p overexpression supported the epithelioid morphology of HK-2 cells while enhancing the downregulation of E-cadherin and upregulation of vimentin, α-smooth muscle actin, and β-catenin levels in TGF-β1-treated HK-2 cells. TUG1 knockdown promoted the proliferation and decreased the migration of HK-2 cells and enhanced the downregulation of miR-141-3p levels in TGF-β1-treated HK-2 cells. TUG1 directly targeted miR-141-3p, and miR-141-3p was directly bound to CTNNB1. Downregulation of miR-141-3p inhibited TUG1 silencing-induced suppression of EMT. In conclusion, TUG1 promotes EMT in TGF-β1-induced HK-2 cells via upregulation of β-catenin levels by sponging miR-141-3p, suggesting a novel therapeutic candidate for RIF.

Sirtuin 6 attenuates epithelial–mesenchymal transition by suppressing the TGF-β1/Smad3 pathway and c-Jun in asthma models

Allergic asthma is a chronic inflammatory airway disease involving airway remodeling. The histone deacetylase sirtuin6 (SIRT6) has protective effects in cardiac and liver fibrosis; however, its role in airway remodeling is unclear. In this study, we investigated the expression of SIRT6 in a rat model of airway remodeling and observed its effects on the epithelial–mesenchymal transition (EMT) in human bronchial epithelial 16HBE cells. Sprague–Dawley rats were sensitized and challenged with ovalbumin to induce airway remodeling or with phosphate-buffered saline as a control for different periods. Morphological changes, cell counts in the bronchoalveolar lavage fluid, and SIRT6 expression were assessed. 16HBE cells were transfected with plasmids to silence or overexpress SIRT6. Western blotting, quantitative polymerase chain reaction, Transwell assays, and cell proliferation assays were performed to examine the transforming growth factor (TGF)-β1-induced changes in EMT indicators and EMT-related cell behaviors. SIRT6 expression was upregulated in bronchial epithelial cells from rats with airway remodeling and in TGF-β1-treated 16HBE cells. SIRT6 overexpression affected TGF-β1-induced changes in EMT markers and EMT-like cell behaviors. In particular, SIRT6 overexpression alleviated the reduction in E-cadherin and the increases in N-cadherin, vimentin, alpha-smooth muscle actin, and metalloproteinase-9 levels in TGF-β1-treated 16HBE cells. Forced expression of SIRT6 also decreased the rates of cell migration and proliferation, reduced activation of phosphorylated Smad3 induced by TGF-β1 treatment, suppressed the acetylation level at histone H3K9, and inhibited the transcriptional activity of the c-Jun promotor. These results suggested that SIRT6 expression is upregulated during airway remodeling and modulates EMT in bronchial epithelial cells targeting Smad3 and c-Jun, highlighting a new therapeutic candidate for improving airway remodeling in asthma.

Sanguinarine inhibits epithelial\u2013mesenchymal transition via targeting HIF-1\u03b1/TGF-\u03b2 feed-forward loop in hepatocellular carcinoma

Epithelial–mesenchymal transition (EMT) plays a crucial role in hepatocellular carcinoma (HCC) progression. Hypoxia and excessive transforming growth factor-β (TGF-β) have been identified as inducers and target for EMT in HCC. Here, we show hypoxia inducible factor-1α (HIF-1α) and TGF-β form a feed-forward loop to induce EMT in HCC cells. Further mechanistic study indicates under both hypoxia and TGF-β stimulation, Smad and PI3K-AKT pathways are activated. We show sanguinarine, a natural benzophenanthridine alkaloid, impairs the proliferation of nine kinds of HCC cell lines and the colony formation of HCC cells. In hypoxic and TGF-β cell models, sanguinarine inhibits HIF-1α signaling and the expression of EMT markers, translocation of Snail and activation of both Smad and PI3K-AKT pathways. Sanguinarine could also inhibit TGF-β-induced cell migration in HCC cells. In vivo studies reveal that the administration of sanguinarine inhibits tumor growth and HIF-1α signaling, inhibits the expression changes of EMT markers as well as Smad and PI3K-AKT pathway proteins. Our findings suggest that sanguinarine is a promising candidate targeting HIF-1α/TGF-β signaling to improve the treatment for HCC patients.

Insulin-like Growth Factor-1-Induced Epithelial-Mesenchymal Transition Promotes Multiple Myeloma Progression

MM is an incurable B cell neoplasm, which is characterized by the clonal proliferation of plasma cells in the bone marrow (BM), accounts for approximately 10% of hematological malignancies. Insulin-like growth factor-1 (IGF-1) is a vital proliferation factor in myeloma cells, recent researches demonstrated that it may be involved in MM cell metastasis. In last decade, studies have demonstrated that IGF-1 induces epithelial-mesenchymal transition (EMT), which has been tightly correlated with increased motility and invasive capacity in several cancers including gastric cancer, hepatocellular carcinoma, and melanoma. However, it remains unclear whether IGF-1 induces EMT and promote myeloma progression. In this study, we firstly observed the morphology of the myeloma cell line Karpas707 changed from tight polygon to loose spindle, a mesenchymal phenotype after treated with IGF-1 (50 ng/ml) after 72h. We supposed IGF-1 may induced EMT in myeloma cells. Furthermore, when serum-starved MM cell lines RPMI8226 and MM1.S were stimulated with 50ng/mL of IGF-1, we found that the expression of mesenchymal features was significantly upregulated in a time-dependent manner, as determined by both flow cytometry analysis and western blotting. Next we confirmed that IGF-1 can activate the PI3K/Akt signaling pathway by inducing the phosphorylation of Akt in MM cells. To investigated whether PI3K/Akt signaling pathway involved in the IGF-1 mediated EMT, we blocked PI3K/Akt signaling pathway using Akt inhibitor MK2206 in IGF-1 treated MM.1S cells. We found MK2206 reversed these changes of EMT markers. Moreover, we found the IGF-1-induced EMT promotes myeloma progression, including migration, invasion, adhesion and clone formation in MM cells, using transwell migration and invasion assay, cell adhesion assay, colony formation assay and flow cytometry. Furthermore, to further validate IGF-1-induced EMT promote MM progression, we knocked down mesenchymal marker vimentin using siRNA in MM.1S cells (VIM k/d cells). We detected that these function IGF-1 plays in myeloma was impaired in Vim k/d cells, indicating that IGF-1-induced EMT promotes MM progression. Finally, we validated that IGF1 expression is aberrantly upregulated and correlated with the expression of the EMT markers by analyzing the mRNA data of BM samples from normal donors and patients in different stages of plasma cell neoplasm from the GEO database. Taken together, these data demonstrate that the mesenchymal phenotype induced by IGF1 contributes to MM progression via PI3K/Akt pathway. Disclosures No relevant conflicts of interest to declare.

RhoA-Dependent HGF and c-Met Mediate Gas6-Induced Inhibition of Epithelial-Mesenchymal Transition, Migration, and Invasion of Lung Alveolar Epithelial Cells.

Previously, we demonstrated that growth arrest-specific protein 6 (Gas6)/Axl or Mer signaling inhibited the transforming growth factor (TGF)-β1-induced epithelial–mesenchymal transition (EMT) in lung epithelial cells. Hepatocyte growth factor (HGF) has also been shown to inhibit TGF-β1-induced changes in EMT markers. Here, we examined whether Gas6 signaling can induce the production of HGF and c-Met in lung alveolar epithelial cells to mediate the inhibition of EMT and to inhibit the migration and invasion of epithelial cells. The inhibition of the RhoA/Rho kinase pathway, using either a RhoA-targeted small interfering RNA (siRNA) or the Rho kinase pharmacologic inhibitor Y27362, prevented the inhibition of TGF-β1-induced EMT in LA-4 cells and primary alveolar type II (AT II) epithelial cells. The c-Met antagonist PHA-665752 also blocked the anti-EMT effects associated with Gas6. Moreover, treatment with Y27362 or PHA-665752 prevented the Gas6-mediated inhibition of TGF-β1-induced migration and invasion. Our data provided evidence that the RhoA-dependent production of HGF and c-Met mediated the Gas6-induced inhibition of EMT, migration and invasion in lung alveolar epithelial cells. Thus, Gas6/Axl and Mer/RhoA signaling may be necessary for the maintenance of homeostasis in the alveolar epithelium, via HGF and c-Met.

Mechanisms of doxorubicin-induced drug resistance and drug resistant tumour growth in a murine breast tumour model

BackgroundDoxorubicin is currently the most effective chemotherapeutic drug used to treat breast cancer. It has, however, been shown that doxorubicin can induce drug resistance resulting in poor patient prognosis and survival. Studies reported that the interaction between signalling pathways can promote drug resistance through the induction of proliferation, cell cycle progression and prevention of apoptosis. The aim of this study was therefore to determine the effects of doxorubicin on apoptosis signalling, autophagy, the mitogen-activated protein kinase (MAPK)- and phosphoinositide 3-kinase (PI3K)/Akt signalling pathway, cell cycle control, and regulators of the epithelial-mesenchymal transition (EMT) process in murine breast cancer tumours.MethodsA tumour-bearing mouse model was established by injecting murine E0771 breast cancer cells, suspended in Hank’s Balances Salt Solution and Corning® Matrigel® Basement Membrane Matrix, into female C57BL/6 mice. Fourty-seven mice were randomly divided into three groups, namely tumour control (received Hank’s Balances Salt Solution), low dose doxorubicin (received total of 6 mg/ml doxorubicin) and high dose doxorubicin (received total of 15 mg/ml doxorubicin) groups. A higher tumour growth rate was, however, observed in doxorubicin-treated mice compared to the untreated controls. We therefore compared the expression levels of markers involved in cell death and survival signalling pathways, by means of western blotting and fluorescence-based immunohistochemistry.ResultsDoxorubicin failed to induce cell death, by means of apoptosis or autophagy, and cell cycle arrest, indicating the occurrence of drug resistance and uncontrolled proliferation. Activation of the MAPK/ extracellular-signal-regulated kinase (ERK) pathway contributed to the resistance observed in treated mice, while no significant changes were found with the PI3K/Akt pathway and other MAPK pathways. Significant changes were also observed in cell cycle p21 and DNA replication minichromosome maintenance 2 proteins. No significant changes in EMT markers were observed after doxorubicin treatment.ConclusionsOur results suggest that doxorubicin-induced drug resistance and tumour growth can occur through the adaptive role of the MAPK/ERK pathway in an effort to protect tumour cells. Previous studies have shown that the efficacy of doxorubicin can be improved by inhibition of the ERK signalling pathway and thereby treatment failure can be overcome.

OSM-induced CD44 contributes to breast cancer metastatic potential through cell detachment but not epithelial-mesenchymal transition.

BackgroundHormone receptor status in human breast cancer cells is a strong indicator of the aggressiveness of a tumor. Triple negative breast cancers (TNBC) are aggressive, difficult to treat, and contribute to high incidences of metastasis by possessing characteristics such as increased tumor cell migration and a large presence of the transmembrane protein, cluster of differentiation 44 (CD44) on the cell membrane. Estrogen receptor-positive (ER+) cells are less aggressive and do not migrate until undergoing an epithelial-mesenchymal transition (EMT).MethodsThe relationship between EMT and CD44 during metastatic events is assessed by observing changes in EMT markers, tumor cell detachment, and migration following cytokine treatment on both parental and CD44 knockdown human breast tumor cells.ResultsER+ T47D and MCF-7 human breast cancer cells treated with OSM demonstrate increased CD44 expression and CD44 cleavage. Conversely, ER- MDA-MB-231 human breast cancer cells do not show a change in CD44 expression nor undergo EMT in the presence of OSM. In ER+ cells, knockdown expression of CD44 by shRNA did not prevent EMT but did change metastatic processes such as cellular detachment and migration. OSM-induced migration was decreased in both ER+ and ER- cells with shCD44 cells compared to control cells, while the promotion of tumor cell detachment by OSM was decreased in ER+ MCF7-shCD44 cells, as compared to control cells. Interestingly, OSM-induced detachment in ER- MDA-MB-231-shCD44 cells that normally don’t detach at significant rates.ConclusionOSM promotes both EMT and tumor cell detachment in ER+ breast cancer cells. Yet, CD44 knockdown did not affect OSM-induced EMT in these cells, while independently decreasing OSM-induced cell detachment. These results suggest that regulation of CD44 by OSM is important for at least part of the metastatic cascade in ER+ breast cancer.

Inhibition of LONP1 Suppresses Pancreatic Cancer Progression Via c-Jun N-Terminal Kinase Pathway-Meditated Epithelial-Mesenchymal Transition.

The aim of this study was to investigate the role of LONP1 in the progression of pancreatic cancer. Lentivirus was used to silence LONP1 in PANC-1 cells. Colony formation assay, cell counting kit (CCK8) assay, cell scratch-wound assay, and transwell assay were used to assess the effects of our strategy on inhibiting cancer growth, migration, and invasion. Protein expression was detected by Western blot analysis. The expression of LONP1 in pancreatic carcinoma tissues was higher than that in adjacent normal pancreatic tissues. Downregulation of LONP1 suppressed the proliferation, migration, and invasion of PANC-1 cells. Knockdown of LONP1 in PANC-1 cells inhibited epithelial-mesenchymal transition and matrix metalloprotein (MMP) 2/9 by downregulation of vimentin, snail, slug, MMP2, and MMP9 and upregulation of claudin-1. The c-Jun N-terminal kinase pathway was inactivated in LONP1 knockdown PANC-1 cells. Activation of the c-Jun N-terminal kinase pathway by anisomycin treatment significantly reversed the changes in epithelial-mesenchymal transition markers and MMP2/9 induced by ablation of LONP1 in PANC-1 cells. LONP1 plays a vital role in the proliferation and metastasis of pancreatic cancer, which provides a potential therapeutic target for the treatment of pancreatic cancer.

Mitochondrial pyruvate carrier 1 expression controls cancer epithelial-mesenchymal transition and radioresistance.

Mitochondrial pyruvate carrier (MPC) is known to cause different expressions in normal and cancer cells. We observed a change in phenotype with the suppression of MPC expression. We knocked down MPC1 and/or MPC2 using siRNA or shRNA. We observed its cell morphology and accompanying molecular marker. Furthermore, the radioresistance of the MPC knockdown cell line was examined using a colony formation assay. MPC1‐suppressed cells changed their morphology to a spindle shape. Epithelial‐mesenchymal transition (EMT) was suspected, and examination of the EMT marker by PCR showed a decrease in E‐cadherin and an increase in fibronectin. Focusing on glutamine metabolism as the mechanism of this phenomenon, we knocked down the glutamine‐metabolizing enzyme glutaminase (GLS). EMT was also observed in GLS‐suppressed cells. Furthermore, when MPC1‐suppressed cells were cultured in a glutamine‐deficient medium, changes in EMT markers were suppressed. In addition, MPC1‐suppressed cells also increased with a significant difference in radioresistance. Decreased MPC1 expression favorably affects EMT and radioresistance of cancer.

CD147 mediates transforming growth factor-β1-induced epithelial-mesenchymal transition and cell invasion in squamous cell carcinoma of the tongue.

Epithelial-mesenchymal transition (EMT) is a physiological process in which epithelial cells attain the motile and invasive characteristics of mesenchymal cells, which results in the development of increased migratory and invasive cell behavior, serving as a vital mechanism of cancer progression. Hence, controlling the EMT for cancer treatment, including head and neck squamous cell carcinoma (HNSCC), is imperative. Among EMT-associated factors, transforming growth factor-β (TGF-β) is a well-established potent inducer. Recent research has revealed that CD147, a member of the immunoglobulin superfamily, promotes the EMT. However, the role of CD147 in the EMT and the following tumorigenicity in HNSCC has not been completely elucidated. This study aims to investigate the role of CD147 in the EMT and related tumorigenicity in HNSCC. The present study used two HNSCC cell lines, SAS and FaDu, for in vitro studies. In HNSCC cells, TGF-β1 induced spindle-shaped morphological changes, and western blot analysis revealed that TGF-β1 induced changes in EMT markers, downregulation of vimentin, and upregulation of E-cadherin, yet increased CD147. In addition, TGF-β1 increased cell migration in HNSCC cells. However, a TGF-β1-induced alteration in EMT makers was attenuated with CD147 silencing by small interfering RNA (siRNA) in SAS cells. In addition, the TGF-β1-induced cell invasion of SAS was attenuated with CD147 silencing. In conclusion, the present study suggests that CD147 mediates TGF-β1-induced EMT and tumorigenicity in HNSCC. Hence, CD147 may serve as a vital therapeutic target in HNSCC.

Epigenetic reprogramming of epithelial mesenchymal transition in triple negative breast cancer cells with DNA methyltransferase and histone deacetylase inhibitors

BackgroundTriple negative breast cancer (TNBC) is an aggressive neoplasia with no effective therapy. Our laboratory has developed a unique TNBC cell model presenting epithelial mesenchymal transition (EMT) a process known to be important for tumor progression and metastasis. There is increasing evidence showing that epigenetic mechanisms are involved in the activation of EMT. The objective of this study is to epigenetically reverse the process of EMT in TNBC by using DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi).MethodsWe evaluated the antitumor effect of three DNMTi and six HDACi using our TNBC cell model by MTT assay, migration and invasion assay, three dimensional culture, and colony formation assay. We then performed the combined treatment both in vitro and in vivo using the most potent DNMTi and HDACi, and tested the combined treatment in a panel of breast cancer cell lines. We investigated changes of EMT markers and potential signaling pathways associated with the antitumor effects.ResultsWe showed that DNMTi and HDACi can reprogram highly aggressive TNBC cells that have undergone EMT to a less aggressive phenotype. SGI-110 and MS275 are superior to other seven compounds being tested. The combination of SGI with MS275 exerts a greater effect than single agent alone in inhibiting cell proliferation, motility, colony formation, and stemness of cancer cells. We also demonstrated that MS275 and the combination of SGI with MS275 exert in vivo antitumor effect. We revealed that the combined treatment synergistically reverses EMT through inhibiting EpCAM cleavage and WNT signaling, suppressing mutant p53, ZEB1, and EZH2, and inducing E-cadherin, apoptosis, as well as histone H3 tri-methylation.ConclusionsOur study showed that DNMTi and HDACi exert antitumor activity in TNBC cells partially by epigenetically reprograming EMT. Our findings strongly suggest that TNBC is sensitive to epigenetic therapies. Therefore, we propose a new strategy to treat TNBC by using the combination of SGI-110 with MS275, which exerts superior antitumor effects by simultaneously targeting multiple pathways.

Membrane rafts-redox signalling pathway contributes to renal fibrosis via modulation of the renal tubular epithelial-mesenchymal transition.

Membrane rafts (MRs)-redox signalling pathway is activated in response to transforming growth factor-β1 (TGF-β1) stimulation in renal tubular cells. This pathway contributes to TGF-1β-induced epithelial-mesenchymal transition (EMT) in renal tubular cells. The the MRs-redox signalling pathway is activated in renal tubular cells isolated from angiotensin II (AngII)-induced hypertensive rats. Inhibition of this pathway attenuated renal inflammation and fibrosis in AngII-induced hypertension. The membrane rafts (MRs)-redox pathway is characterized by NADPH oxidase subunit clustering and activation through lysosome fusion, V-type proton ATPase subunit E2 (encoded by the Atp6v1e2 gene) translocation and sphingomyelin phosphodiesterase 1 (SMPD1, encoded by the SMPD1 gene) activation. In the present study, we hypothesized that the MRs-redox-derived reactive oxygen species (ROS) are involved in renal inflammation and fibrosis by promoting renal tubular epithelial-mesenchymal transition (EMT). Results show that transforming growth factor-β1 (TGF-β1) acutely induced MR formation and ROS production in NRK-52E cells, a rat renal tubular cell line. In addition, transfection of Atp6v1e2 small hairpin RNAs (shRNA) and SMPD1 shRNA attenuated TGF-β1-induced changes in EMT markers, including E-cadherin, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) in NRK-52E cells. Moreover, Erk1/2 activation may be a downstream regulator of the MRs-redox-derived ROS, because both shRNAs significantly inhibited TGF-β1-induced Erk1/2 phosphorylation. Further in vivo study shows that the renal tubular the MRs-redox signalling pathway was activated in angiotensin II (AngII)-induced hypertension, as indicated by the increased NADPH oxidase subunit Nox4 fraction in the MR domain, SMPD1 activation and increased ROS content in isolated renal tubular cells. Finally, renal transfection of Atp6v1e2 shRNA and SMPD1 shRNA significantly prevented renal fibrosis and inflammation, as indicated by the decrease of α-SMA, fibronectin, collagen I, monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1) and tumour necrosis factor-α (TNF-α) in kidneys from AngII-infused rats. It was concluded that the the MRs-redox signalling pathway is involved in TGF-β1-induced renal tubular EMT and renal inflammation/fibrosis in AngII-induced hypertension.

CTEN induces epithelial-mesenchymal transition (EMT) and metastasis in non small cell lung cancer cells.

To explore the effects and mechanism of CTEN (COOH-terminus tensin-like molecule) on EMT, cell migration and invasion of Human lung adenocarcinoma cells. The pCMV-vector, pCMV-CTEN, Control-shRNA, and CTEN-shRNA were transfected into A549 and NCI-H1299 cells by Lipofectamine 2000. Transforming growth factor-β1(TGF-β1)and epithelial-mesenchymal transition (EMT) -related biomarkers were detected by eliseand western blot. The migration and invasion ability of A549 cells and NCI-H1299 were examined by scratch-wound assay and transwell assay respectively. We found compare with control group, the expression of TGF-β and mesenchymal markers in CTEN overexpression group were increased, and the epithelial marker was decreased, which induced the EMT process. Meanwhile, scratch-woundassay showed that the migration efficiency of A549 and NCI-H1299 cells in CTEN overexpression group were higher than that in control group.Transwell assay demonstrated that the number of cells that migrated and invaded through the membrane were obviously more than those in control group.Furthermore, Knockdown of CTEN partially reversed transforming growth factor-β1(TGF-β1)-induced changes in EMT markers. In conclusion, CTEN activated the expression of TGF-β1, thereby prompting EMT in lung adenocareinma cancer cells.