Epithelial-mesenchymal Transition Related Genes Research Articles

The scRNA-sequencing landscape of pancreatic ductal adenocarcinoma revealed distinct cell populations associated with tumor initiation and progression

Pancreatic ductal adenocarcinoma (PDAC) stands as a formidable malignancy characterized by its profound lethality. The comprehensive analysis of the transcriptional landscape holds immense significance in understanding PDAC development and exploring novel treatment strategies. However, due to the firm consistency of pancreatic cancer samples, the dissociation of single cells and subsequent sequencing can be challenging. Here, we performed single-cell RNA sequencing (scRNA-seq) on 8 PDAC patients with different lymph node metastasis status. We first identified the crucial role of MMP1 in the transition from normal pancreatic cells to cancer cells. The knockdown of MMP1 in pancreatic cancer cell lines decreased the expression of ductal markers such as SOX9 while the overexpression of MMP1 in hTERT-HPNE increased the expression of ductal markers, suggesting its function of maintaining ductal identity. Secondly, we found a S100A2+ tumor subset which fueled lymph node metastasis in PDAC. The knockdown of S100A2 significantly reduced the motility of pancreatic cancer cell lines in both wound healing and transwell migration assays. While overexpression of S100A2 led to increased migratory capability. Moreover, overexpression of S100A2 in KPC1199, a mouse pancreatic cancer cell line, caused a larger tumor burden in a hemi-spleen injection model of liver metastasis. In addition, epithelial-mesenchymal transition-related genes were decreased by S100A2 knockdown revealed by bulk RNA sequencing. We also identified several pivotal contributors to the pro-tumor microenvironment, notably OMD+ fibroblast and CCL2+ macrophage. As a result, our study provides valuable insights for early detection of PDAC and promising therapeutic targets for combatting lymph node metastasis.

Dibutyl phthalate (DBP) promotes Epithelial-Mesenchymal Transition (EMT) to aggravate liver fibrosis into cirrhosis and portal hypertension (PHT) via ROS/TGF-β1/Snail-1 signalling pathway in adult rats

ObjectiveThe primary objective of this study was to investigate the toxicological impact of Dibutyl phthalate (DBP) on the process of liver fibrosis transitioning into cirrhosis and the subsequent development of portal hypertension (PHT) through the mechanism of epithelial-mesenchymal transition (EMT) mediated by the ROS/TGF-β/Snail-1 signaling pathway. MethodCarbon tetrachloride (CCl4) (1 mg/kg) was introduced in adult rats by oral feeding in CCl4 and CCl4+DBP groups twice a week for 8 weeks, and twice for another 8 week in CCl4 group. DBP was introduced by oral feeding in the CCl4+DBP group twice over the following 8 weeks. We subsequently analyzed hemodynamics measurements and liver cirrhosis degree, hepatic inflammation and liver function in the different groups. EMT related genes expression in rats in the groups of Control, DBP, CCl4 and CCl4+DBP were measured by immunohistochemistry (IHC). Enzyme-linked immunosorbent Assay (ELISA), qRT-PCR, western blot were used to detect the EMT related proteins and mRNA gene expression levels in rats and primary hepatocytes (PHCs). Reactive oxygen species (ROS) were examined with a ROS detection kit. ResultsThe results showed that the CCl4+DBP group had higher portal pressure (PP) and lower mean arterial pressure (MAP) than the other groups. Elevated collagen deposition, profibrotic factor, inflammation, EMT levels were detected in DBP and CCl4+DBP groups. ROS, TGF-β1 and Snail-1 were highly expressed after DBP exposure in vitro. TGF-β1 had the potential to regulate Snail-1, and both of them were subject to regulation by ROS. ConclusionDBP could influence the progression of EMT through its toxicological effect by ROS/TGF-β1/Snail-1 signalling pathway, causing cirrhosis and PHT in final. The findings of this research might contribute to a novel comprehension of the underlying toxicological mechanisms and animal model involved in the progression of cirrhosis and PHT, and potentially offered a promising therapeutic target for the treatment of the disease.

Identification of MDK as a Hypoxia- and Epithelial-Mesenchymal Transition-Related Gene Biomarker of Glioblastoma Based on a Novel Risk Model and In Vitro Experiments.

Tumor cells are commonly exposed to a hypoxic environment, which can easily induce the epithelial-mesenchymal transition (EMT) of tumor cells, further affecting tumor proliferation, invasion, metastasis, and drug resistance. However, the predictive role of hypoxia and EMT-related genes in glioblastoma (GBM) has not been investigated. Intersection genes were identified by weighted correlation network analysis (WGCNA) and differential expression analyses, and a risk model was further constructed by LASSO and Cox analyses. Clinical, immune infiltration, tumor mutation, drug treatment, and enrichment profiles were analyzed based on the risk model. The expression level of the MDK gene was tested using RT-PCR, immunohistochemistry, and immunofluorescence. CCK8 and EdU were employed to determine the GBM cells' capacity for proliferation while the migration and invasion ability were detected by a wound healing assay and transwell assay, respectively. Based on the GBM data of the TCGA and GTEx databases, 58 intersection genes were identified, and a risk model was constructed. The model was verified in the CGGA cohort, and its accuracy was confirmed by the ROC curve (AUC = 0.807). After combining clinical subgroups, univariate and multivariate Cox regression analyses showed that risk score and age were independent risk factors for GBM patients. Furthermore, our subsequent analysis of immune infiltration, tumor mutation, and drug treatment showed that risk score and high- and low-risk groups were associated with multiple immune cells, mutated genes, and drugs. Enrichment analysis indicated that the differences between high- and low-risk groups were manifested in tumor-related pathways, including the PI3K-AKT and JAK-STAT pathways. Finally, in vivo experiments proved that the hypoxia environment promoted the expression of MDK, and MDK knockdown reduced the proliferation, migration, and EMT of GBM cells induced by hypoxia. Our novel prognostic correlation model provided more potential treatment strategies for GBM patients.

Determination of In Vitro and In Vivo Effects of Taxifolin and Epirubicin on Epithelial-Mesenchymal Transition in Mouse Breast Cancer Cells.

Background: One of the most significant characteristics of cancer is epithelial-mesenchymal transition and research on the relationship between phenolic compounds and anticancer medications and epithelial-mesenchymal transition is widespread. Methods: In order to investigate the potential effects of Taxifolin on enhancing the effectiveness of Epirubicin in treating breast cancer, specifically in 4T1 cells and an allograft BALB/c model, the effects of Taxifolin and Epirubicin, both individually and in combination, were examined. Cell viability assays and cytotoxicity assays in 4T1 cells were performed. In addition, 4T1 cells were implanted into female BALB/c mice to conduct in vivo studies and evaluate the therapeutic efficacy of Taxifolin and Epirubicin alone or in combination. Tumor volumes and histological analysis were also assessed in mice. To further understand the mechanisms involved, we examined the messenger RNA and protein levels of epithelial-mesenchymal transition-related genes, as well as active Caspase-3/7 levels, using quantitative real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assays, respectively. Results: In vitro results demonstrated that the coadministration of Taxifolin and Epirubicin reduced cell viability and cytotoxicity in 4T1 cell lines. In vivo, coadministration of Taxifolin and Epirubicin suppressed tumor growth in BALB/c mice with 4T1 breast cancer cells. Additionally, this combination treatment significantly increased the levels of active caspase-3/7 and downregulated the messenger RNA and protein levels of N-cadherin, β-catenin, vimentin, snail, and slug, but upregulated the E-cadherin gene. It significantly decreased the messenger RNA levels of the Zeb1 and Zeb2 genes. Conclusion: The in vitro and in vivo results of our study indicate that the concurrent use of Epirubicin with Taxifolin has supportive effects on breast cancer treatment.

EMT-related gene risk model establishment for prognosis and drug treatment efficiency prediction in hepatocellular carcinoma

This study was designed to evaluate the prognosis and pharmacological therapy sensitivity of epithelial mesenchymal transition-related genes (EMTRGs) that obtained from the EMTome database in hepatocellular carcinoma (HCC) using bioinformatical method. The expression status of EMTRGs were also investigated using the clinical information of HCC patients supported by TCGA database and the ICGC database to establish the TCGA cohort as the training set and the ICGC cohort as the validation set. Analyze the EMTRGs between HCC tissue and liver tissue in the TCGA cohort in the order of univariate COX regression, LASSO regression, and multivariate COX regression, and construct a risk model for EMTRGs. In addition, enrichment pathways, gene mutation status, immune infiltration, and response to drugs were also analyzed in the high-risk and low-risk groups of the TCGA cohort, and the protein expression status of EMTRGs was verified. The results showed a total of 286 differentially expressed EMTRGs in the TCGA cohort, and EZH2, S100A9, TNFRSF11B, SPINK5, and CCL21 were used for modeling. The TCGA cohort was found to have a worse outcome in the high-risk group of HCC patients, and the ICGC cohort confirmed this finding. In addition, EMTRGs risk score was shown to be an independent prognostic factor in both cohorts by univariate and multivariate COX regression. The results of GSEA analysis showed that most of the enriched pathways in the high-risk group were associated with tumor, and the pathways enriched in the low-risk group were mainly associated with metabolism. Patients in various risk groups had varying immunological conditions, and the high-risk group might benefit more from targeted treatments. To sum up, the EMTRGs risk model was developed to forecast the prognosis for HCC patients, and the model might be useful in assisting in the choice of treatment drugs for HCC patients.

Epithelial-mesenchymal transition-related gene signature for prognosis of lung squamous cell carcinoma.

Epithelial-mesenchymal transition (EMT) is associated with tumor invasion and progression, and is regulated by DNA methylation. A prognostic signature of lung squamous cell carcinoma (LUSC) with EMT-related gene data has not yet been established. In our study, we constructed a co-expression network using differentially expressed genes (DEGs) obtained from The Cancer Genome Atlas (TCGA) to identify hub genes. We conducted a correlation analysis between the differentially methylated hub genes and differentially expressed EMT-related genes to screen EMT-related differentially methylated genes (ERDMGs). Functional enrichment was performed to annotate the ERDMGs. The least absolute shrinkage and selection operator (LASSO) and stepwise Cox regression analyses were performed to build a survival prognosis prediction model. Additionally, druggability analysis was performed to predict the potential drug targets of ERDMGs. We screened 11 ERDMGs that were enriched in cell adhesion molecules and other signaling pathways. Finally, we constructed a 4-ERDMG model, which showed good ability to predict survival prognosis in the training and validation sets. The model could serve as an independent predictive factor for patients with LUSC. Additionally, our druggability analysis predicted that CC chemokine ligand 23 (CCL23) and Hepatocyte nuclear factor 1b (HNF1B) may be the underlying drug targets of LUSC. We established a new risk score (RS) system as a prognostic indicator to predict the outcome of patients with LUSC, which will help in the improvement of treatment strategies.

Deciphering primary and acquired immunotherapy resistance with whole genome and transcriptome analysis (WGTA).

2602 Background: Most cancer patients (pts) do not respond to antiPD1/PDL1 immune checkpoint inhibitors (ICI) or experience only a temporary benefit. Comprehensive molecular profiling with WGTA could broaden our understanding of the mechanisms contributing to primary (Pr) and acquired resistance (Ar) to ICI and identify novel predictive biomarkers. Methods: The Princess Margaret investigator-initiated Immune Resistance Interrogation Study (NCT04243720) aims to comprehensively characterize ICI-resistant cancers (Genta et. al. ASCO 2021). Fresh tumor biopsies and blood samples were obtained from solid tumor pts at the time of PD on ICI-based treatment. DNA/RNA was extracted from tumor tissue and paired buffy coat and sequenced using an Illumina NovaSeq6000 system (2x150 paired reads for WG and 2X100 paired reads for WT). Copy number alterations (CNA), structural variants (SVs), mutation signatures and genes expression were assessed. Wald test in univariable and multivariable logistic regression model was used for comparison. Results: As of January 2023, 73 pts (45 Pr/28 Ar) were enrolled. WGTA was completed in 22 pts (14 Pr/8 Ar). Key pt characteristics included: median age 60.5 years (range 26-79); male 68%/female 32%; melanoma 50%, squamous cell cancer of the head and neck 36%, and other tumor types 14%. Forty-one percent of the pts were treated with PD-1/PD-L1 inhibitor monotherapy and 59% with PD-1/PD-L1 inhibitor combinations. There were no significant differences in median tumor mutation burden (TMB, coding; 2.8, range 0.6-30.5 for Pr vs 4.4, range 1.6-12.8 for Ar), median percent genome altered by CNA (43% Pr vs 65% Ar) or median number of SVs (144, range 9-775, for Pr vs 76, range 25-924, for Ar pts). After performing RNA sequencing we observed a significantly higher expression of the epithelial mesenchymal transition (EMT) hallmark gene-set from msigDB and of 3 previously reported ICI-resistance gene expression signatures in Pr vs Ar ( p≤0.05) in univariable analysis. The signatures included genes involved in the regulation of EMT, cellular de-differentiation and a cancer associated fibroblast (CAF) signature (IPRES signature, Hugo et al. Cell 2016; melanocytic plasticity signature [MPS], Pérez-Guijarro et al. Nat Med 2020; LRRC15 CAF signature, Dominguez et al. Cancer Discov. 2020). After adjusting for cancer type, the MPS signature was significantly associated with Pr vs Ar resistance in multivariable analysis ( p = 0.048). Conclusions: No significant differences between Ar and Pr were observed in genomic features including TMB, CNAs and number of SVs. Cancers primary resistant to ICI were characterized by a higher expression of EMT, cell de-differentiation and CAF related genes, detected with transcriptome analysis. Our findings might indicate these features as possible driver of early ICI progression. Analysis of additional samples is ongoing.

Transcriptional repression of the MRTF/SRF-cytoskeleton pathway by Rev-erba promotes beigeing via adipogenic progenitor fate switch

The circadian clock exerts temporal control in metabolic processes to maintain homeostasis, with its disruption leading to the development of obesity and insulin resistance. Adipose fibrosis in obese subjects exacerbates insulin resistance by restricting healthy tissue expansion and remodeling. Rev-erbα is a key circadian clock repressor that displays circadian rhythmic expression in adipose tissue, although its role in adipose tissue fibrosis is not known. Here we show that Rev-ebα inhibits the fibrogenic fate of adipocyte progenitors while promoting beige adipogenic potential via transcription repression of the MRTF/SRF-actin cytoskeleton axis. ChIP-seq analysis of Rev-erbα cistrome in adipogenic precursors identified its transcriptional control of pathways involved in cytoskeleton modulation, including established myofibroblast gene markers small muscle α-actin and Tropomysin. Gain-of-function of Rev-erbα in beige adipogenic precursors markedly suppressed MRTF/SRF activity with enhanced beige differentiation, while Rev-erbα silencing suppressed beige adipogenesis. Prrx1-Cre-mediated subcutaneous beige fat-selective Rev-erbα knock-in induced browning with augmented mitochondrial metabolism, resulting in increased energy expenditure, resistance to obesity and insulin sensitivity. Furthermore, single cell RNA-seq of beige progenitors with Rev-erbα overexpression revealed striking loss of preadipcyte-like (PAL) subpopulations with myofibroblastic characteristics that arise from pericyte lineage. PAL beige progenitors displayed enrichment of epithelial mesenchymal transition-related gene signature and contractile program as compared to preadipocytes. Preadipocyte population with Rev-erbα overexpression exhibited activated oxidative phosphorylation pathway with inhibition of myofibrolast characteristics. Taken together, our findings identify a novel regulatory function of Rev-erbα in modulating adipogenic progenitor fate choice that may promote adipose tissue beigeing while suppressing fibrotic complications. NIDDK grant 1R01DK112794, 1R01DK130499 and AR-DMRI 2023 T2D Innovation Grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Knockdown of kinesin family member 4A inhibits cell proliferation, migration, and invasion while promoting apoptosis of urothelial bladder carcinoma cells.

Kinesin family member 4A (KIF4A) is upregulated in a variety of cancers. However, its expression and potential downstream targets in urothelial bladder carcinoma (UBC) remain unclear. Expression data of KIF4A in UBC and noncancerous tissues were downloaded from the GEPIA database. Cell proliferation, migration, invasion, and apoptosis of T24 and 5637 UBC cells were examined using wound healing, transwell, colony formation, CCK-8, and flow cytometry assays. KIF4A and potential downstream genes were analyzed using qRT-PCR, western blot analysis, and immunohistochemistry. In UBC samples, KIF4A expression was significantly higher than in corresponding noncancerous samples. UBC patients with high KIF4A expression had poor cancer-specific survival and overall survival. Knockdown of KIF4A significantly inhibited proliferation and promoted apoptosis of UBC cells, accompanied by dephosphorylation of AKT and increased the protein level of proapoptotic factors. Additionally, knockdown of KIF4A reduced migration and invasion of UBC cells whereas overexpression of KIF4A exhibited opposite effects, along with altered protein level in epithelial-mesenchymal transition-related genes. Furthermore, overexpression of YAP1 promoted KIF4A expression whereas knockdown of YAP1 suppressed KIF4A expression in UBC cells. Alternatively, KIF4A knockdown reduced YAP1 nuclear protein level whereas KIF4A overexpression suppressed YAP1 phosphorylation and facilitated YAP1 nuclear translocation. KIF4A upregulation correlates with poor prognosis of UBC. Knockdown of KIF4A inhibits proliferation, migration, and invasion of UBC cells while inducing apoptosis possibly through dephosphorylation of AKT, changes in EMT-related genes, and interaction with YAP1.

Epithelial-mesenchymal transition-related gene prognostic index and phenotyping clusters for hepatocellular carcinoma patients

Epithelial-mesenchymal transition (EMT) contributes to high tumor heterogeneity and the immunosuppressive environment of the HCC tumor microenvironment (TME). Here, we developed EMT-related genes phenotyping clusters and systematically evaluated their impact on HCC prognosis, the TME, and drug efficacy prediction. We identified HCC specific EMT-related genes using weighted gene co-expression network analysis (WGCNA). An EMT-related genes prognostic index (EMT-RGPI) capable of effectively predicting HCC prognosis was then constructed. Consensus clustering of 12 HCC specific EMT-related hub genes uncovered two molecular clusters C1 and C2. Cluster C2 preferentially associated with unfavorable prognosis, higher stemness index (mRNAsi) value, elevated immune checkpoint expression, and immune cell infiltration. The TGF-β signaling, EMT, glycolysis, Wnt β-catenin signaling, and angiogenesis were markedly enriched in cluster C2. Moreover, cluster C2 exhibited higher TP53 and RB1 mutation rates. The TME subtypes and tumor immune dysfunction and exclusion (TIDE) score showed that cluster C1 patients responded well to immune checkpoint inhibitors (ICIs). Half-maximal inhibitory concentration (IC50) revealed that cluster C2 patients were more sensitive to chemotherapeutic and antiangiogenic agents. These findings may guide risk stratification and precision therapy for HCC patients.

Abstract P6-13-01: EPITHELIAL-MESENCHYMAL TRANSITION AND TUMOR STEM CELLS NETWORK IDENTIFICATION IN BREAST CARCINOMAS: IN SILICO STUDY

Abstract INTRODUCTION: Breast cancer (BC) is the most prevalent malignant tumor in the female population, except for skin tumors. Despite therapeutic advances, over 20% of patients with localized disease will relapse. The research of epithelial-mesenchymal transition (EMT), tumor stem cells (TSC), and the biological mechanisms related to refractoriness and metastasis development is, therefore, an opportunity for new therapeutic strategies and better results. OBJECTIVES: To identify breast cancer cellular markers related to EMT and TSC according to their histological subtypes, stage and to analyze their relationship with clinical outcomes. METHODS: After selecting a public breast cancer patients database with interactome, we identified differentially expressed genes, their associated processes, coexpression networks and interactions with pathways related to the stem phenotype and epithelial-mesenchymal transition. The characterization of key genes and the correlation with histological subtypes and clinical outcome allowed us to determine a group of genes as potential breast cancer EMT/TSC prognostic markers. RESULTS: In the 989 patients studied, 1033 differentially expressed genes (DEGs) were found, categorized according to histological subtype (hormone receptor positive, HER2 positive, triple negative) and stage IV. Seven communities of gene coexpression were found, with the gray community showing greater interaction with hormone positive tumors, the green community with HER2 positive, and the turquoise community with the triple negative one. The hormone positive disease was related to extracellular matrix processes and neuronal communication, the HER2 positive to the extracellular matrix interaction and the triple negative tumors to mitotic processes. Investigation networks with EMT and TSC related genes demonstrated a strong correlation with HER2-positive and triple-negative tumors; being eight genes in HER2 positive subtypes correlated with survival (SYNDIG1, ​​COL10A1, SLC24A2, LINC00922, KLKP1, MMP11 and ECM2); and one of the hormone positive subtype (ITIH5). ECM2 was highlighted in terms of EMT/TSC connectivity and survival. CONCLUSION:The EMT/TSC processes are significant in the various subtypes of breast cancer and impact on survival, especially in the HER2 positive subtype. Citation Format: Vanessa Dybal, Bruno R. Cavalcante, Gisele V. Rocha, Clarissa Gurgel. EPITHELIAL-MESENCHYMAL TRANSITION AND TUMOR STEM CELLS NETWORK IDENTIFICATION IN BREAST CARCINOMAS: IN SILICO STUDY. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-13-01.

Epigenetic Regulation of MAP3K8 in EBV-Associated Gastric Carcinoma.

Super-enhancers (SEs) regulate gene expressions, which are critical for cell type-identity and tumorigenesis. Although genome wide H3K27ac profiling have revealed the presence of SE-associated genes in gastric cancer (GC), their roles remain unclear. In this study, ChIP-seq and HiChIP-seq experiments revealed mitogen-activated protein kinase 8 (MAP3K8) to be an SE-associated gene with chromosome interactions in Epstein-Barr virus-associated gastric carcinoma (EBVaGC) cells. CRISPRi mediated repression of the MAP3K8 SEs attenuated MAP3K8 expression and EBVaGC cell proliferation. The results were validated by treating EBVaGC cells with bromodomain and the extra-terminal motif (BET) inhibitor, OTX015. Further, functional analysis of MAP3K8 in EBVaGC revealed that silencing MAP3K8 could inhibit the cell proliferation, colony formation, and migration of EBVaGC cells. RNA-seq and pathway analysis indicated that knocking down MAP3K8 obstructed the notch signaling pathway and epithelial-mesenchymal transition (EMT) in EBVaGC cells. Further, analysis of the cancer genome atlas (TCGA) and GSE51575 databases exhibited augmented MAP3K8 expression in gastric cancer and it was found to be inversely correlated with the disease-free progression of GC. Moreover, Spearman's correlation revealed that MAP3K8 expression was positively correlated with the expressions of notch pathway and EMT related genes, such as, Notch1, Notch2, C-terminal binding protein 2 (CTBP2), alpha smooth muscle actin isotype 2 (ACTA2), transforming growth factor beta receptor 1 (TGFβR1), and snail family transcriptional repressors 1/2 (SNAI1/SNAI2) in GC. Taken together, we are the first to functionally interrogate the mechanism of SE-mediated regulation of MAP3K8 in EBVaGC cell lines.

A prognostic model based on clusters of molecules related to epithelial-mesenchymal transition for idiopathic pulmonary fibrosis.

Background: Most patients with idiopathic pulmonary fibrosis (IPF) have poor prognosis; Effective predictive models for these patients are currently lacking. Epithelial-mesenchymal transition (EMT) often occurs during idiopathic pulmonary fibrosis development, and is closely related to multiple pathways and biological processes. It is thus necessary for clinicians to find prognostic biomarkers with high accuracy and specificity from the perspective of Epithelial-mesenchymal transition. Methods: Data were obtained from the Gene Expression Omnibus database. Using consensus clustering, patients were grouped based on Epithelial-mesenchymal transition-related genes. Next, functional enrichment analysis was performed on the results of consensus clustering using gene set variation analysis. The gene modules associated with Epithelial-mesenchymal transition were obtained through weighted gene co-expression network analysis. Prognosis-related genes were screened via least absolute shrinkage and selection operator (LASSO) regression analysis. The model was then evaluated and validated using survival analysis and time-dependent receiver operating characteristic (ROC) analysis. Results: A total of 239 Epithelial-mesenchymal transition-related genes were obtained from patients with idiopathic pulmonary fibrosis. Six genes with strong prognostic associations (C-X-C chemokine receptor type 7 [CXCR7], heparan sulfate-glucosamine 3-sulfotransferase 1 [HS3ST1], matrix metallopeptidase 25 [MMP25], murine retrovirus integration site 1 [MRVI1], transmembrane four L6 family member 1 [TM4SF1], and tyrosylprotein sulfotransferase 1 [TPST1]) were identified via least absolute shrinkage and selection operator and Cox regression analyses. A prognostic model was then constructed based on the selected genes. Survival analysis showed that patients with high-risk scores had worse prognosis based on the training set [hazard ratio (HR) = 7.31, p < .001] and validation set (HR = 2.85, p = .017). The time-dependent receiver operating characteristic analysis showed that the area under the curve (AUC) values in the training set were .872, .905, and .868 for 1-, 2-, and 3-year overall survival rates, respectively. Moreover, the area under the curve values in the validation set were .814, .814, and .808 for 1-, 2-, and 3-year overall survival rates, respectively. Conclusion: The independent prognostic model constructed from six Epithelial-mesenchymal transition-related genes provides bioinformatics guidance to identify additional prognostic markers for idiopathic pulmonary fibrosis in the future.

Role of the Circadian Clock and Effect of Time-Restricted Feeding in Adenine-Induced Chronic Kidney Disease

Most physiological functions exhibit circadian rhythmicity that is partly regulated by the molecular circadian clock. Herein, we investigated the relationship between the circadian clock and chronic kidney disease (CKD). The role of the clock gene in adenine-induced CKD and the mechanisms of interaction were investigated in mice in which Bmal1, the master regulator of the clock gene, was knocked out, and Bmal1 knockout (KO) tubule cells. We also determined whether the renoprotective effect of time-restricted feeding (TRF), a dietary strategy to enhance circadian rhythm, is clock gene–dependent. The mice with CKD showed altered expression of the core clock genes with a loss of diurnal variations in renal functions and key tubular transporter gene expression. Bmal1 KO mice developed more severe fibrosis, and transcriptome profiling followed by gene ontology analysis suggested that genes associated with the cell cycle, inflammation, and fatty acid oxidation pathways were significantly affected in the mutant mice. Tubule-specific deletion of BMAL1 in HK-2 cells by CRISPR/Cas9 led to upregulation of p21 and tumor necrosis α and exacerbated epithelial-mesenchymal transition–related gene expression upon transforming growth factor β stimulation. Finally, TRF in the mice with CKD partially restored the disrupted oscillation of the kidney clock genes, accompanied by improved cell cycle arrest and inflammation, leading to decreased fibrosis. However, the renoprotective effect of TRF was abolished in Bmal1 KO mice, suggesting that TRF is partially dependent on the clock gene. Our data demonstrate that the molecular clock system plays an important role in CKD via cell cycle regulation and inflammation. Understanding the role of the circadian clock in kidney diseases can be a new research field for developing novel therapeutic targets.

Construction and validation of a novel signature based on epithelial-mesenchymal transition–related genes to predict prognosis and immunotherapy response in hepatocellular carcinoma by comprehensive analysis of the tumor microenvironment

Immunotherapy has yielded encouraging results in the treatment of advanced hepatocellular carcinoma (HCC). However, the relationship between epithelial-mesenchymal transition (EMT) and immunotherapy for HCC has not been adequately explained. In this study, we comprehensively analyzed a bulk RNA sequence dataset of 365 HCC patients in The Cancer Genome Atlas (TCGA) dataset. Subsequently, we constructed a prognostic signature based on 6 EMT-related genes and divided 365 HCC patients into high- and low-risk groups. The predictive efficacy of the signature was well validated in different clinical subgroups and in two independent external datasets. We further explored the relationship between prognostic signature and immunotherapy response in terms of immune cell infiltration, somatic mutations, tumor mutation burden (TMB), microsatellite instability (MSI), immune checkpoint–associated gene expression, single-nucleotide variants (SNV) neoantigens, cancer testicular antigens (CTA) scores, and tumor immune dysfunction and exclusion (TIDE) scores. We validated the predictive efficacy of prognostic signature for immunotherapy response using external independent immunotherapy data. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to validate EMT-related gene overexpression in HCC tissue samples. Prognostic signature was an independent risk factor affecting the prognosis of HCC patients and has shown superiority in predicting patient survival compared to other clinical factors. Compared with the low-risk group, the proportion of Activated_CD4_T_cell, Type_2_T_helper_cel, and macrophages were higher in the tumor microenvironment of HCC patients in the high-risk group, while the Activated_CD8_T_cell and CD56bright_natural_killer_cell proportions were lower. The prognostic signature was positively correlated with TMB scores, MSI scores, SNV neoantigens scores, expression levels of immune checkpoint–related genes, and TIDE scores, and patients in the high-risk group were more suitable for immunotherapy. qRT-PCR confirms overexpression of 6 EMT-related genes in HCC tissues for the construction of prognostic signature. Our novel prognostic signature can effectively predict the prognosis and immunotherapy response of HCC patients. In the future, it will be an effective tool for physicians to screen suitable immunotherapy populations and improve response rates and overall survival (OS).

Role of Cancer Stem-like Cells in the Process of Invasion and Mesenchymal Transformation by a Reconstituted Triple-negative Breast Cancer Cell Population Resistant to p53-induced Apoptosis.

We investigated the role of cancer stem cells (CSCs) in a population of triple-negative breast cancer (TNBC) cells that are resistant to apoptosis. A human breast cancer cell population capable of inducing p53 expression with doxycycline (Dox) was created and used as an untreated control (UT). After the addition of Dox to UT for 5 days, the cell population reconstituted with cells showing resistance to apoptosis was named RE. Fluorescence-activated cell sorting (FACS) and immunostaining revealed that after the addition of Dox, the ratio of cells in the S and G2/M phases decreased in UT as apoptosis proceeded, but did not markedly change in apoptosis-resistant RE. CSC-like cells in RE exhibited a cell morphology with a larger ratio of the major/minor axis than UT. FACS showed that RE had a higher proportion of CSC-like cells and contained more CD44+CD24- mesenchymal CSCs than ALDH1A3+ epithelial-like CSCs. In a Matrigel invasion assay, UT was more likely to form a three-dimensional cell population, whereas RE exhibited a planar population, higher migration ability, and the up-regulated expression of epithelial-mesenchymal transition-related genes. These results provide insights into the mechanisms by which TNBC cells acquire treatment resistance at the time of recurrence.

Development and validation of an epithelial–mesenchymal transition-related gene signature for predicting prognosis

BACKGROUNDCurrently, there are many therapeutic methods for lung adenocarcinoma (LUAD), but the 5-year survival rate is still only 15% at later stages. Epithelial– mesenchymal transition (EMT) has been shown to be closely associated with local dissemination and subsequent metastasis of solid tumors. However, the role of EMT in the occurrence and development of LUAD remains unclear.AIMTo further elucidate the value of EMT-related genes in LUAD prognosis.METHODSUnivariate, least absolute shrinkage and selection operator, and multivariate Cox regression analyses were applied to establish and validate a new EMT-related gene signature for predicting LUAD prognosis. The risk model was evaluated by Kaplan–Meier survival analysis, principal component analysis, and functional enrichment analysis and was used for nomogram construction. The potential structures of drugs to which LUAD is sensitive were discussed with respect to EMT-related genes in this model.RESULTSThirty-three differentially expressed genes related to EMT were found to be highly associated with overall survival (OS) by using univariate Cox regression analysis (log2FC ≥ 1, false discovery rate < 0.001). A prognostic signature of 7 EMT-associated genes was developed to divide patients into two risk groups by high or low risk scores. Kaplan–Meier survival analysis showed that the OS of patients in the high-risk group was significantly poorer than that of patients in the low-risk group (P < 0.05). Multivariate Cox regression analysis showed that the risk score was an independent risk factor for OS (HR > 1, P < 0.05). The results of receiver operator characteristic curve analysis suggested that the 7-gene signature had a perfect ability to predict prognosis (all area under the curves > 0.5).CONCLUSIONThe EMT-associated gene signature classifier could be used as a feasible indicator for predicting OS.

Conjugated Linoleic Acid Treatment Attenuates Cancerous features in Hepatocellular Carcinoma Cells.

Background A growing number of hepatocellular carcinoma (HCC), and recurrence frequency recently have drawn researchers' attention to alternative approaches. The concept of differentiation therapies (DT) relies on inducing differentiation in HCC cells in order to inhibit recurrence and metastasis. Hepatocyte nuclear factor 4 alpha (HNF4α) is the key hepatogenesis transcription factor and its upregulation may decrease the invasiveness of cancerous cells by suppressing epithelial-mesenchymal transition (EMT). This study aimed to evaluate the effect of conjugated linoleic acid (CLA) treatment, natural ligand of HNF4α, on the proliferation, migration, and invasion capacities of HCC cells in vitro. Materials and Method. Sk-Hep-1 and Hep-3B cells were treated with different doses of CLA or BIM5078 [1-(2′-chloro-5′-nitrobenzenesulfonyl)−2-methylbenzimidazole], an HNF4α antagonist. The expression levels of HNF4a and EMT related genes were evaluated and associated to hepatocytic functionalities, migration, and colony formation capacities, as well as to viability and proliferation rate of HCC cells. Results In both HCC lines, CLA treatment induced HNF4α expression in parallel to significantly decreased EMT marker levels, migration, colony formation capacity, and proliferation rate, whereas BIM5078 treatment resulted in the opposite effects. Moreover, CLA supplementation also upregulated ALB, ZO1, and HNF4α proteins as well as glycogen storage capacity in the treated HCC cells. Conclusion CLA treatment can induce a remarkable hepatocytic differentiation in HCC cells and attenuates cancerous features. This could be as a result of HNF4a induction and EMT inhibition.

EP16.04-022 Prognosis of Epithelial-Mesenchymal Transition-Related lncRNA Profile in LUSC Patients

Epithelial-mesenchymal transition (EMT) process may affect tumor invasion, metastasis and proliferation in lung squamous cell carcinoma (LUSC). Due to this important biological role, EMT related genes and pathways are thought to be associated with clinical outcomes of LUSC patients. Currently, the expression profile of EMT-related lncRNA, the pathological features of LUSC and their prognostic values in LUSC have not been well understood.

Identification of an EMT-related Gene Signature Predicting Recurrence in Stage II/III Colorectal Cancer

To identify a prognostic significant gene signature for predicting colorectal cancer (CRC) recurrence. Traditional prognostic risk assessment in stage II/III CRC patients remains controversial. Epithelial-mesenchymal transition is thought to be closely related to the malignant progression of tumors. Thus, it is promising to establish a prognostic model based on epithelial-mesenchymal transition-related gene (ERG) signature. We retrospectively analyzed transcriptome profiles and clinical information of 1780 stage II/III CRC patients from 15 public datasets. Coefficient variant analysis was used to select reference genes for normalizing gene expression levels. Univariate, LASSO, and multivariate Cox regression analyses were combined to develop the ERG signature predicting disease-free survival (DFS). The patients were divided into high-risk and low-risk based on the ERG signature recurrence risk score. The survival analysis was performed in different CRC cohorts. The proposed ERG signature contained 7 cancer-related ERGs and 3 reference genes. The ERG signature recurrence risk score was prognostically relevant in all cohorts ( P <0.05) and proved as an independent prognostic factor in the training cohort. In the pooled cohort, high-risk CRC patients exhibited worse DFS ( P <0.0001) and overall survival ( P =0.0058) than low-risk patients. The predictive performance of the ERG signature was superior to Oncotype DX colon cancer. An integrated decision tree and nomogram were developed to improve prognosis evaluation. The identified ERG signature is a promising and powerful biomarker predicting recurrence in CRC patients. Moreover, the presented ERG signature might help to stratify patients according to their tumor biology and contribute to personalized treatment.