Esophageal Squamous Cell Carcinoma Cell Lines Research Articles

Methionine oxidation of CLK4 promotes the metabolic switch and redox homeostasis in esophageal carcinoma via inhibiting MITF selective autophagy.

BackgroundMetabolic reprogramming and redox homeostasis contribute to esophageal squamous cell carcinoma (ESCC). CDC‐like kinase 4 (CLK4) is a dual‐specificity kinase that can phosphorylate substrates’ tyrosine or serine/threonine residue. However, the role and mechanism of CLK4 in ESCC remain unknown.MethodsCLK4 expression was analysed using publicly available datasets and confirmed in ESCC tissues and cell lines. The biological roles of CLK4 were studied with gain and loss‐of‐function experiments. Mass spectrometry was employed to examine the effects of CLK4 on metabolic profiling. In vitro kinase assay, co‐immunoprecipitation, glutathione S‐transferase pulldown, chromatin immunoprecipitation and luciferase reporter were used to elucidate the relationship among CLK4, microphthalmia‐associated transcription factor (MITF), COP1 and ZRANB1.ResultsCLK4 down‐regulation was observed in ESCC cell lines and clinical samples and associated with the methylation of its promoter. Low levels of CLK4 promoted ESCC development by affecting the purine synthesis pathway and nicotinamide adenine dinucleotide phosphate (NADPH)/nicotinamide adenine dinucleotide phosphate (NADP+) ratio. Interestingly, CLK4 inhibited ESCC development by blocking MITF‐enhanced de novo purine synthesis and redox balance. Mechanistically, wild type CLK4 (WT‐CLK4) but not kinase‐dead CLK4‐K189R mutant phosphorylated MITF at Y360. This modification promoted its interaction with E3 ligase COP1 and its K63‐linked ubiquitination at K308/K372, leading to sequestosome 1 recognition and autophagic degradation. However, the deubiquitinase ZRANB1 rescued MITF ubiquitination and degradation. In turn, MITF bound to E‐ rather than M‐boxes in CLK4 promoter and transcriptionally down‐regulated its expression in ESCC. Clinically, the negative correlations were observed between CLK4, MITF, and purine metabolic markers, which predicts a poor clinical outcome of ESCC patients. Notably, CLK4 itself was a redox‐sensitive kinase, and its methionine oxidation at M307 impaired kinase activity, enhanced mitochondria length and inhibited lipid peroxidation, contributing to ESCC.ConclusionsOur data highlight the potential role of CLK4 in modulating redox status and nucleotide metabolism, suggesting potential therapeutic targets in ESCC treatment.

Weighted Correlation Network Analysis of Cancer Stem Cell-Related Prognostic Biomarkers in Esophageal Squamous Cell Carcinoma.

Background: The role of cancer stem cells in esophageal squamous cell carcinoma (ESCC) remains unclear. Methods: The mRNA stemness index (mRNAsi) of 179 ESCC patients (GSE53625) was calculated using a machine learning algorithm based on their mRNA expression. Stemness-related genes were identified by weighted correlation network analysis (WGCNA) and LASSO regression, whose associations with mutation status, immune cell infiltrations, and potential compounds were also analyzed. The role of these genes in proliferation and their expressions was assessed in ESCC cell lines and 112 samples from our center. Results: The ESCC samples had significantly higher mRNAsi than the normal tissues. Patients with high mRNAsi exhibited higher worse OS. Seven stemness-related genes were identified by WGCNA and LASSO regression, based on which a risk-predicted score model was constructed. Among them, CST1, CILP, PITX2, F2RL2, and RIOX1 were favorable for OS, which were adverse for DPP4 and ZFHX4 in the GSE53625 dataset. However, RIOX1 was unfavorable for OS in patients from our center. In vitro assays showed that CST1, CILP, PITX2, F2RL2, and RIOX1 were pro-proliferated, which were opposite for DDP4 and ZFHX4. In addition, SMARCA4, NOTCH3, DNAH5, and KALRN were more mutated in the low-score group. The low-score group had significantly more memory B cells, monocytes, activated NK cells, and Tregs and less macrophages M2, resting mast cells, and resting dendritic cells. Conclusions: Seven stemness-related genes are significantly related to the prognosis, gene mutations, and immune cell infiltration of ESCC. Some potential anticancer compounds may be favorable for OS.

METTL3 Facilitates Tumor Progression by COL12A1/MAPK Signaling Pathway in Esophageal Squamous Cell Carcinoma.

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common aggressive tumors in the world. m6A modification has been implicated to play an important role in many biological progressions. METTL3 as the main methyltransferase has been found in many cancers, including ESCC. Here, we investigated the underlying mechanism of METTL3 in the development of ESCC.Methods: Quantitative real-time PCR (qRT-PCR), immunohistochemical (IHC) and western blot were used to detect METTL3 expression. To evaluate the function of METTL3, MTS, colony formation, scratch wound healing assay, and transwell and invasion assays were performed. To find out the downstream target of METTL3, mRNA sequencing (mRNA-seq) was conducted. GO and KEGG functional enrichment analyses were carried out to predict possible biological processes and signaling pathways. qRT-PCR and western blot were performed to identify the expression of COL12A1 and the phosphorylation status of RAF, MRK and ERK. Cotransfection of small interfering RNA (for METTL3 silence) with plasmid (for overexpression of COL12A1) and the following gain- and loss-of-function experiments were performed to detect the target gene function of COL12A1 in progression of ESCC mediated by METTL3.Results: Using TCGA database, higher METTL3 expression was found in ESCC tissues. Moreover, we found that METTL3 was significantly increased in ESCC patient tissues compared with normal tissues and correlated with poor prognosis. The expression of METTL3 in ESCC cell lines was assessed. The gain- and loss-of-function indicates that METTL3 promotes cell proliferation, migration and invasion. Additionally, we confirmed that METTL3 can promote the expression of COL12A1 and upregulate the phosphorylation of RAF, MER and ERK, and moreover COL12A1 can restrain siMETTL3-mediated inhibition of proliferation, migration and invasion in ESCC.Conclusion: Our study revealed that METTL3 may have an oncogenic role, facilitating the ESCC progression and metastasis by COL12A1/MAPK signaling pathway.

Targeting the LSD1-G9a-ER Stress Pathway as a Novel Therapeutic Strategy for Esophageal Squamous Cell Carcinoma.

Despite recent advances in the management and treatment of esophageal squamous cell carcinoma (ESCC), the prognosis remains extremely poor, and current nonsurgical treatment options are limited. To identify new therapeutic targets, we screened a curated library of epigenetic compounds using a panel of cancer cell lines and found that coinhibiting the histone demethylase LSD1 and the histone methyltransferase G9a potently suppresses cell growth; similar results were obtained by knocking down both LSD1 and G9a expression. Importantly, we also found that inhibiting LSD1 and G9a significantly decreased tumor growth in a xenograft mouse model with ESCC cell lines. To examine the clinical relevance of these findings, we performed immunohistochemical analyses of microarray profiling data obtained from human esophageal squamous cancer tissues and found that both LSD1 and G9a are upregulated in cancer tissues compared to healthy tissues, and this increased expression was significantly correlated with poor prognosis. Mechanistically, we discovered that inhibiting LSD1 and G9a induces cell death via S-phase arrest and apoptosis, and cotargeting ER stress pathways increased this effect both in vitro and in vivo. Taken together, these findings provide compelling evidence that targeting LSD1, G9a, and ER stress-related pathways may serve as a viable therapeutic strategy for ESCC.

Regulation of IncRNA ZNF667-AS1 in Proliferation and Invasion of Esophageal Squamous Cell Carcinoma Cells via Mediating ceRNA Network.

Esophageal squamous cell carcinoma (ESCC), classified as a primary histological subtype of esophageal cancer (EC), dominates approximately 90% of the newly diagnosed EC. Long non-coding RNAs (lncRNAs) are frequently related to the course of ESCC. The current study aimed to investigate whether lncRNA zinc finger protein 667-antisense RNA 1 (ZNF667-AS1) modulates the proliferation and invasion of ESCC cells. ESCC tissues and cell lines, para-carcinoma tissues, and human esophageal epithelial cells (HEEpiCs) were collected. lncRNA ZNF667-AS1 expression in the above tissues and cells was detected. The effect of lncRNA ZNF667-AS1 on proliferation and invasion of Eca109 cells was detected using cell counting kit-8, colony formation, and Transwell assays. lncRNA ZNF667-AS1 subcellular localization was determined via the nuclear/cytosol fractionation assay. The binding relationships between miR-18b-5p and lncRNA ZNF667-AS1 and RAS p21 protein activator 1 (RASA1) were verified using dual-luciferase reporter gene experiment and RNA immunoprecipitation experiment. The expressions of miR-18b-5p and RASA1 in the tissues and cells were identified. The roles of miR-18b-5p overexpression or silencing RASA1 in proliferation and invasion of ESCC cells were examined through rescue experiments. lncRNA ZNF667-AS1 was underexpressed in ESCC tissues and cells, and lncRNA ZNF667-AS1 overexpression hampered ESCC cell proliferation and invasiveness. miR-18b-5p targeted RASA1 while lncRNA ZNF667-AS1 promoted RASA1 transcription via binding to miR-18b-5p. Over-expression miR-18b-5p or silencing RASA1 reversed the inhibitory effects of lncRNA ZNF667-AS1 overexpression on ESCC cell proliferation and invasion. lncRNA ZNF667-AS1 overexpression accelerated RASA1 transcription by competitively binding to miR-18b-5p, thus suppressing ESCC cell proliferation and invasion.

MiR-204-5p inhibits cell proliferation and induces cell apoptosis in esophageal squamous cell carcinoma by regulating Nestin.

Esophageal cancer (EC) is a highly malignant gastrointestinal tumor, and esophageal squamous cell carcinoma (ESCC) is one of the most common histological types of EC. MicroRNAs (miRNAs) are small noncoding RNAs closely related to tumorigenesis and tumor progression. In addition, Nestin is an intermediate filament protein (class VI) and contributes to the progression of numerous tumors. However, the correlation between miRNAs and Nestin in ESCC remains unclear. This study aimed to investigate the relationship between miR-204-5p and Nestin in ESCC. First, Nestin-related miRNAs in ESCC were explored using RNA sequencing. In ESCC tissues and cell lines, the expression of miR-204-5p was decreased detected by quantitative real-time polymerase chain reaction (qPCR), whereas Nestin protein level was upregulated identified by Western blotting (WB). Besides, Nestin was the direct target of miR-204-5p in ESCC determined via the luciferase reported assay. Moreover, miR-204-5p regulated Nestin to inhibit ESCC cell proliferation detected by the colony formation assay and promote ESCC cell apoptosis identified using the flow cytometry and TUNEL assay. Furthermore, miR-204-5p suppressed tumorigenesis in vivo evaluated by the murine xenograft tumor model. In conclusion, these results indicated that miR-204-5p inhibited cell proliferation and induced cell apoptosis in ESCC through targeting Nestin, which might provide novel therapeutic targets for ESCC therapy.

Long Noncoding RNA PCAT6 Regulates Cell Proliferation and Migration in Human Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the sixth most common cancer type in East Asian countries. Mounting evidences illustrated that long noncoding RNAs (lncRNAs) play important roles in a variety of human cancers, including ESCC. LncRNA PCAT6 has been identified as a tumor promoter in multiple cancers. However, the roles and underlying mechanism of PCAT6 in ESCC remain largely unclear. In the current study, we discovered that lncRNA PCAT6, which was aberrantly upregulated in ESCC tumor tissues, significantly promoted cell proliferation and migration in ESCC cell lines Eca-109 and Kyse-30 cells. Flow cytometry assays showed that PCAT6 knockdown promoted the apoptosis of ESCC cells. Mechanistically, RNA-seq and Gene Ontology analyses indicated that PCAT6 knockdown influenced the expression of genes that participated in cell proliferation and migration. Furthermore, real-time PCR and western blot assays validated that knockdown of PCAT6 could increase the levels of GDF15 and DUSP4 in Eca-109 and Kyse-30 cells. In brief, our findings reveal that lncRNA PCAT6 plays an oncogenic role in the progression of ESCC by inhibiting the expression of genes related to cell proliferation and migration.

Mechanism and Role of the Neuropeptide LGI1 Receptor ADAM23 in Regulating Biomarkers of Ferroptosis and Progression of Esophageal Cancer.

Background According to recent studies, ferroptosis is closely related to the efficacy and prognosis of tumour treatment. However, the role of ferroptosis in esophageal squamous cell carcinoma (ESCC) has not been explored comprehensively. Materials and Methods The esophageal cancer (EC) transcriptome data was downloaded from The Cancer Genome Atlas (TCGA), then analyzed, to obtain the differentially expressed messenger RNA (mRNA), microRNA (miRNA), and long noncoding RNA (lncRNA) between groups with the low and high Ferroptosis Potential Index (FPI) and construct a ferroptosis-associated ceRNA network. In addition, the expression of ARHGEF26-AS1 and miR-372-3p in ESCC cell lines was assessed, and the appropriate cell lines were selected. The interaction between ARHGEF26-AS1, miR-372-3p, and ADAM23 was also determined through a dual-luciferase reporter assay. Moreover, the Western blot, Cell Counting Kit-8 (CCK-8), wound healing, cell viability, and cell death assays were conducted to establish the biological functions of the ARHGEF26-AS1/miR-372-3p/ADAM23 pathway in ESCCs. Results An FPI scoring model reflecting the activity of the ferroptosis pathway was constructed, and a ferroptosis-associated ceRNA network was established. The findings revealed that low expression of ADAM23 and ARHGEF26-AS1 as well as high expression of miR-372-3p was associated with poor prognosis and a lower FPI score in EC patients. Functionally, overexpression of ADAM23 and ARHGEF26-AS1 and the miR-372-3p inhibitor not only promoted ferroptosis in ESCC cells in vitro but also inhibited the proliferation and migration of cells. Mechanistically, ARHGEF26-AS1 upregulated the expression of ADAM23 by competitively binding to miR-372-3p. Conclusions The study showed that the lncRNA, ARHGEF26-AS1 acts as a miR-372-3p sponge that regulates the neuropeptide LGI1 receptor ADAM23 expression. This in turn not only inhibits the proliferation and migration of ESCC cells but also upregulates the ferroptosis pathway. A neuropeptide-related ferroptosis regulatory pathway was identified in this study.

LncRNA FOXP4-AS1 Promotes the Progression of Esophageal Squamous Cell Carcinoma by Interacting With MLL2/H3K4me3 to Upregulate FOXP4.

Malignant tumors are a grave threat to human health. Esophageal squamous cell carcinoma (ESCC) is a common gastrointestinal malignant tumor. China has a high incidence of ESCC, and its morbidity and mortality are higher than the global average. Increasingly, studies have shown that long noncoding RNAs (lncRNAs) play a vital function in the occurrence and development of tumors. Although the biological function of FOXP4-AS1 has been demonstrated in various tumors, the potential molecular mechanism of FOXP4-AS1 in ESCC is still poorly understood. The expression of FOXP4 and FOXP4-AS1 was detected in ESCC by quantitative real-time PCR (qRT–PCR) or SP immunohistochemistry (IHC). shRNA was used to silence gene expression. Apoptosis, cell cycle, MTS, colony formation, invasion and migration assays were employed to explore the biological functions of FOXP4 and FOXP4-AS1. The potential molecular mechanism of FOXP4-AS1 in ESCC was determined by dual-luciferase reporter, RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP). Here, we demonstrated that FOXP4-AS1 was significantly increased in ESCC tissues and cell lines, associated with lymph node metastasis and TNM staging. Cell function experiments showed that FOXP4-AS1 promoted the proliferation, invasion and migration ability of ESCC cells. The expression of FOXP4-AS1 and FOXP4 in ESCC tissues was positively correlated. Further research found that FOXP4-AS1, upregulated in ESCC, promotes FOXP4 expression by enriching MLL2 and H3K4me3 in the FOXP4 promoter through a “molecular scaffold”. Moreover, FOXP4, a transcription factor of β-catenin, promotes the transcription of β-catenin and ultimately leads to the malignant progression of ESCC. Finally, FOXP4-AS1 may be a new therapeutic target for ESCC.

CtBP2 interacts with TGIF to promote the progression of esophageal squamous cell cancer through the Wnt/β‑catenin pathway.

C-terminal-binding protein 2 (CtBP2), a transcriptional co-repressor, plays a main role in tumorigenesis and in the development of multiple tumors. Transforming growth interacting factor (TGIF) is involved in a number of cellular signal transduction pathways and is related to tumor occurrence and development. In the present study, the proteins interacting with CtBP2 were identified and the mechanisms underlying the biological activity of CtBP2 in esophageal squamous cell carcinoma (ESCC) were investigated. The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to search for known proteins interacting with CtBP2, and co-immunoprecipitation (Co-IP) assay was performed to validate the interactions. Reverse transcription-quantitative PCR (RT-qPCR), immunohistochemistry (IHC) and western blot analysis were performed to examine the expression levels of CtBP2 and TGIF in ESCC. The correlation between CtBP2 and TGIF was analyzed using Gene Expression Profiling Interactive Analysis (GEPIA) by Pearson's correlation analysis, and the co-localization of CtBP2 with TGIF in the ECA109 cells was identified using immunofluorescence staining. XAV939 treatment, CCK-8, 5-ethynyl-2′-deoxyuridine (EdU) staining, wound healing and Transwell assays were performed to investigate the signaling pathways involved in the biological activity of CtBP2 in ECA109 cells. According to the results obtained from STRING and Co-IP analysis, an interaction between CtBP2 and TGIF was indicated, and these proteins were co-localized in the nucleus. CtBP2 and TGIF mRNA and protein expression levels were robustly and simultaneously increased in both ESCC tissues and cell lines. There was a direct correlation between CtBP2 and TGIF expression levels in ESCC tissues, and both were significantly associated with metastasis and survival. The TGIF and CtBP2 expression levels were significantly increased or decreased simultaneously, in ECA109 cells transfected with LV-CtBP2 or sh-CtBP2, and vice versa. According to the results of CCK-8 assay, EdU staining and Transwell assay, CtBP2 promoted the proliferation, migration and invasion of ECA109 cells through the Wnt/β-catenin pathway. On the whole, the present study demonstrates that CtBP2 interacts with TGIF and promotes the malignant progression of ESCC through the Wnt/β-catenin pathway.

PAR1 and PAR4 exert opposite effects on tumor growth and metastasis of esophageal squamous cell carcinoma via STAT3 and NF-\u03baB signaling pathways

BackgroundEsophageal carcinogenesis is a multifactorial process in which genetic and environmental factors interact to activate intracellular signals, leading to the uncontrolled survival and growth of esophageal squamous cell carcinoma (ESCC) cells. The intracellular pathways of ESCC cells could be regulated by proteinase activated-receptors (PARs), which are comprised of four receptors (i.e., PAR-1, PAR-2, PAR-3, and PAR-4). Therefore, the function and possible mechanism of PAR1 and PAR4 in the progression of ECSS were explored in our study.MethodsFirst, we detected the expression levels of PAR1 and PAR4 in 27 cases of ESCC specimens and cell lines by RT-qPCR, IHC and western blot. Meanwhile, the correlation between PAR1/PAR4 expression levels, clinicopathological characteristics, and disease free survival was analyzed. Then, we constructed PAR1/PAR4 knockdown cell models and investigated the role of PAR1/PAR4 knockdown on the proliferation, apoptosis, changes of calcium flow, and metastasis of ESCC cells via MTT, flow cytometry, transwell and wound healing assays in vitro. Further, an experimental metastasis model in vivo was established to explore the role of stable PAR1/PAR4 knockdown on the growth and metastasis of ESCC cells. Finally, the role of nSMase2 in the activation of NF-κB induced by PAR4 and the role of NF-κB and STAT3 signaling pathways in the PAR1/PAR4-mediated tumor promoting or suppressive functions were measured by immunoprecipitation, western blot and immunofluorescence assays.ResultsFirst, the integrated results demonstrated the expression levels of PAR1 and PAR4 are inversely proportional in ESCC. PAR1 potently enhanced tumor growth and metastasis, while PAR4 had an inhibitory effect. Further, the co-activation of STAT3 and NF-κB was involved in the PAR1 activation-induced tumor promoting effect, while only NF-κB participated in the PAR4 activation-induced tumor inhibitory effect in ESCC. To be specific, FAK/PI3K/AKT/STAT3/NF-κB signaling mediated PAR1 activation-induced tumor promoting effect and nSMase2/MAPK/NF-κB signaling mediated PAR4 activation-induced tumor inhibitory effect.ConclusionsOverall, the study has provided new insights into the potential implication of PAR1 and PAR4 in the pathogenesis of ESCC. Besides, FAK/PI3K/AKT/STAT3/NF-κB and nSMase2/MAPK/NF-κB pathways may be novel targets for regulating tumor growth and metastasis in ESCC patients.

Gene Expression in Barrett's Esophagus Cell Lines Resemble Esophageal Squamous Cell Carcinoma Instead of Esophageal Adenocarcinoma.

Simple SummaryStable cell lines derived from primary tissues and tumors are widely used in medical research. This study presents interesting findings from evaluation of publicly available gene expression profiles (GEPs) of primary tissues derived from the normal esophagus, Barrett’s esophagus (BE), esophageal adenocarcinoma (EAC), esophageal squamous cell carcinoma (ESCC), as well as available esophageal cell lines. We observed that the GEPs of currently available BE cell lines deviate from the GEPs of primary BE tissues (columnar) and EAC tumors (glandular), and are unexpectedly similar to the GEPs of normal esophageal mucosa and ESCC tumors. In vitro exposure to an acid and bile environment was not sufficient to reverse this “squamous like” GEP adopted by a BE cell line, BAR-T. This incomprehensible change in the GEP may result in ambiguous changes in the phenotype of the BE cell lines, and needs careful consideration during experimental design.Esophageal adenocarcinoma (EAC) is strongly associated with Barrett’s esophagus (BE), a pre-malignant condition resulting from gastric reflux. Esophageal squamous cell carcinoma (ESCC), the other major subtype of esophageal cancer, shows strong association with smoking and alcohol intake and no association with gastric reflux. In this study, we constructed and validated gene expression signatures of EAC vs. ESCC tumors using publicly available datasets, and subsequently assessed the enrichment levels of these signatures in commonly used EAC and ESCC cell lines, normal esophageal tissues and normal esophageal cell lines, and primary BE tissues and BE cell lines. We found that unlike ESCC cell lines which were quite similar to primary ESCC tumors, EAC cell lines were considerably different from primary EAC tumors but still more similar to EAC tumors than ESCC tumors, as the genes up in EAC vs. ESCC (EAChi) had considerably lower expression in EAC cell lines than EAC tumors. However, more surprisingly, unlike various normal cell lines (EPC2, Het-1A) which were very similar to various tissues from normal esophagus, BE cell lines (BAR-T, CP-A) were extremely different from primary BE tissues, as BE cell lines had substantially lower levels of EAChi and substantially higher levels of ESCChi gene expression. This ESCC-like profile of the BAR-T remained unaltered even after prolonged exposure to an acidic bile mixture in vitro resulting in malignant transformation of this cell line. However, primary BE tissues had EAC-like gene expression profiles as expected. Only one EAC case from the Cancer Genome Atlas resembled BE cell lines, and while it had the clinical profile and some mutational features of EAC, it had some mutational features, the copy number alteration profile, and the gene expression profile of ESCC instead. These incomprehensible changes in gene expression patterns may result in ambiguous changes in the phenotype and warrants careful evaluation to inform selection of appropriate in vitro tools for future studies on esophageal adenocarcinoma.

Metformin Downregulates PD-L1 Expression in Esophageal Squamous Cell Catrcinoma by Inhibiting IL-6 Signaling Pathway.

PurposeTo characterize the mechanism by which metformin inhibits PD-L1 expression in esophageal squamous cell carcinoma (ESCC) and to evaluate the effect of metformin on the antitumor immune response.MethodsThe Cancer Genome Atlas (TCGA) database was used to analyze the correlations between IL-6 and prognosis and between IL-6 and PD-L1 gene expression in esophageal cancer. Reverse transcription-quantitative polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence were used to study the mechanism by which metformin affects PD-L1 expression. Additionally, T cell function was assessed in a coculture system containing ESCC cells and peripheral blood mononuclear cells (PBMCs) treated with metformin or IL-6. In an in vivo assay, we used a model established with NPIdKO™ mice, which have a reconstituted immune system generated by transplanting PBMCs through intravenous injection, to evaluate the effect of metformin on tumors.ResultsThe TCGA esophageal cancer data showed that IL-6 expression was positively correlated with PD-L1 expression and that patients with high IL-6 expression had a significantly lower overall survival rate than patients with low IL-6 expression. PD-L1 expression in ESCC cell lines was significantly inhibited by metformin via the IL-6/JAK2/STAT3 signaling pathway but was not correlated with the canonical AMPK pathway. In the coculture system, the metformin pretreatment group showed higher T cell activation and better T cell killing function than the control group. Animal experiments confirmed that metformin downregulated PD-L1 expression and that combination treatment with metformin and PD-1 inhibitors synergistically enhanced the antitumor response.ConclusionsMetformin downregulated PD-L1 expression by blocking the IL-6/JAK2/STAT3 signaling pathway in ESCC, which enhanced the antitumor immune response.

Identification of KIF4A as a prognostic biomarker for esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the most common and aggressive tumor worldwide, and the long-term survival of these patients remains poor. Three databases (GSE17351, GSE20347, and GSE100942) were obtained from Gene Expression Omnibus, and 193 differentially expressed genes including 56 upregulated and 137 downregulated genes were identified by paired test using limma R package. Then, functional enrichments by gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed these genes were mainly related protein digestion and absorption, and IL-17 signaling pathway. We then constructed a protein-protein interaction network and cytoHubba module to determine the six hub genes and overall survival analysis of the six hub genes were evaluated by UALCAN and GEPIA2 analysis. Ultimately, the experimental results confirmed the KIF4A was overexpressed in the ESCC tissues and cell lines compared with the normal esophageal mucosal tissues and was linked to poor prognosis. Moreover, we also revealed that KIF4A facilitates proliferation, cell cycle, migration, and invasion of ESCC in vivo and in vitro. Overall, these findings demonstrated that KIF4A could serve as diagnostic and prognostic biomarkers and may help facilitate therapeutic targets in ESCC patients.

SLC7A9 as a Potential Biomarker for Lymph Node Metastasis of Esophageal Squamous Cell Carcinoma.

The expression of solute carrier (SLC) 7 family genes is reportedly associated with several malignancies. Here, we focused on SLC7A9 and investigated its expression, function, and clinical significance in esophageal squamous cell carcinoma (ESCC). SLC7A9 transcription levels were evaluated in 13 ESCC cell lines, and polymerase chain reaction (PCR) array analysis was conducted to detect coordinately expressed genes with SLC7A9. SLC7A9 contributions to proliferation, invasion, and migration were evaluated in ESCC cells subjected to siRNA-mediated gene knockdown and pCMV6-entry plasmid-mediated overexpression. SLC7A9 expression was detected in 189 ESCC tissues by quantitative reverse-transcription (qRT)-PCR and correlated with clinicopathological parameters. The expression levels of SLC7A9 varied widely in ESCC cell lines and correlated with FGFBP1 expression. Knockdown of SLC7A9 significantly suppressed the proliferation, invasion, and migration of the ESCC cell lines. Moreover, overexpression of SLC7A9 enhanced cell proliferation and migration. In analyses of clinical specimens, SLC7A9 mRNA was overexpressed in the ESCC tissues compared with the adjacent normal esophageal tissues. High mRNA expression was significantly associated with high levels of squamous cell carcinoma-related antigen and carcinoembryonic antigen, advanced disease stage, and lymph node metastasis. High SLC7A9 expression was also significantly associated with poor disease-specific and disease-free survival, and lymph node recurrence after radical surgery, but not with the other recurrence patterns. On multivariate analysis, high SLC7A9 expression was an independent predictor of lymph node recurrence. SLC7A9 influences the malignant behavior of ESCC cells. Tumor SLC7A9 expression may serve as a novel biomarker for predicting lymph node metastasis and recurrence in ESCC patients.

SNHG17, as an EMT-related lncRNA, promotes the expression of c-Myc by binding to c-Jun in esophageal squamous cell carcinoma.

Dysregulation of long noncoding RNA SNHG17 is associated with the occurrence of several tumors; however, its role in esophageal squamous cell carcinoma (ESCC) remains obscure. The present study demonstrated that SNHG17 was upregulated in ESCC tissues and cell lines, induced by TGF‐β1, and associated with poor survival. It is also involved in the epithelial‐to‐mesenchymal transition (EMT) process. The mechanism underlying SNHG17‐regulated c‐Myc was detected by RNA immunoprecipitation, RNA pull‐down, chromatin immunoprecipitation, and luciferase reporter assays. SNHG17 was found to directly regulate c‐Myc transcription by binding to c‐Jun protein and recruiting the complex to specific sequences of the c‐Myc promoter region, thereby increasing its expression. Moreover, SNHG17 hyperactivation induced by TGF‐β1 results in PI3K/AKT pathway activation, promoting cells EMT, forming a positive feedback loop. Furthermore, SNHG17 facilitated ESCC tumor growth in vivo. Overall, this study demonstrated that the SNHG17/c‐Jun/c‐Myc axis aggravates ESCC progression and EMT induction by TGF‐β1 and may serve as a new therapeutic target for ESCC.

Ixazomib Induces Apoptosis and Suppresses Proliferation in Esophageal Squamous Cell Carcinoma through Activation of the c-Myc/NOXA Pathway.

Esophageal squamous cell carcinoma (ESCC) is one of the major subtypes of esophageal cancer. More than half of the patients with ESCC in the world are in China, and the 5-year survival rate is less than 10%. As a new oral proteasome inhibitor, ixazomib has shown strong therapeutic effect in many solid tumors. In this study, we aimed to investigate the effects of ixazomib on the proliferation inhibition and apoptosis of ESCC cells. We used four human ESCC cell lines, cell viability assay, cell cycle and apoptosis assay, reverse-transcription polymerase chain reaction (RT-PCR), Western blot, immunohistochemistry, and ESCC xenografts model to clarify the roles of the therapeutic effect and mechanism of ixazomib in ESCC. Ixazomib significantly inhibited the proliferation and induced apoptosis in ESCC cells. RT-PCR results showed that the expressions of endoplasmic reticulum stress-related gene phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) and MYC proto-oncogene (c-Myc) significantly increase after treatment with ixazomib in ESCC cells. When we knocked down the NOXA and c-Myc by small interfering RNA, the therapeutic effect of ixazomib markedly decreased, which confirmed that c-Myc/NOXA pathway played a key role in the treatment of ESCC with ixazomib. In vivo, the xenograft ESCC model mice were given 10 mg/kg of ixazomib every other day for 30 days. The results showed that the tumor size in the treatment group was significantly smaller than the control group. These results suggested that ixazomib is known to suppress proliferation and induce apoptosis in ESCC cell lines, and this effect was likely mediated by increased activation of the c-Myc/NOXA signaling pathways. SIGNIFICANCE STATEMENT: Esophageal squamous cell carcinoma (ESCC) is the common worldwide malignant tumor, but conventional chemotherapeutics suffer from a number of limitations. In this study, the results suggested that ixazomib suppresses proliferation and induces apoptosis in ESCC cell lines. Therefore, ixazomib may be a potential new strategy for ESCC therapy.

The promoter hypermethylation of SULT2B1 accelerates esophagus tumorigenesis via downregulated PER1.

BackgroundEsophageal cancer is currently the eighth most common tumor in the world and a leading cause of cancer death. SULT2B1 plays crucial roles in tumorigenesis. The purpose of this study is to explore the role of SULT2B1 in esophageal squamous cell carcinoma (ESCC).MethodsThe expression of SULT2B1 and its clinicopathological characteristics were evaluated in ESCC cohorts. Bisulfite genomic sequencing and methylation specific PCR were used to detect the promoter hypermethylation of the SULT2B1 gene. The effects of SULT2B1 on the biological characters of ESCC cells were identified on functional assays. Subcutaneous xenograft models revealed the role of SULT2B1 in vivo with tumor growth. RNA‐Seq analysis and qRT‐PCR were performed to recognize the targeted effect of SULT2B1 on PER1.ResultsSULT2B1 was not expressed or at a low level in most patients with ESCC or in ESCC cell lines, and this was accompanied by poor clinical prognosis. Furthermore, the downregulation of SULT2B1 occurred in promoter hypermethylation. According to the functional results, overexpression of SULT2B1 could inhibit tumoral proliferation in vitro and retard tumor growth in vivo, whereas SULT2B1 knockdown could accelerate ESCC progression. Mechanistically, SULT2B1 targeted PER1 at the mRNA level during post‐transcriptional regulation. Finally, PER1 was verified as a suppressor and poor‐prognosis factor in ESCC.ConclusionsSULT2B1 loss is a consequence owing to its ability to promote hypermethylation. In addition, it serves as a suppressor and poor‐prognosis factor because of the post‐transcriptional regulation of PER1 in ESCC.

Anterior gradient 2 regulates cancer progression in TP53‑wild‑type esophageal squamous cell carcinoma.

Anterior gradient 2 (AGR2) reportedly promotes tumor growth and has an unfavorable impact on survival in several cancers. However, no comprehensive functional analysis of AGR2 in esophageal squamous cell carcinoma(ESCC) has been performed. In the present study, the function and clinical significance of AGR2 were examined using ESCC cell lines and clinical samples. AGR2 was upregulated in EC tissue and ESCC cell lines. The downregulation of AGR2 suppressed cell proliferation and increased the proportion of G2/M‑phase cells and phosphorylation of p53 in TP53‑wild‑type ESCC and osteosarcoma cells. However, these changes were not observed in TP53‑mutant ESCC cells. In addition, immunohistochemistry results demonstrated that high AGR2 and low p53 expression levels in ESCC tissues were correlated with a worse prognosis. These results suggested that although AGR2 enhanced cell proliferation by inhibiting p53 phosphorylation in TP53‑wild‑type ESCC, the same mechanism did not regulate cell functions in TP53‑mutant ESCC. Thus, AGR2 served an important role in ESCC progression and might be a useful prognostic marker in patients with TP53‑wild‑type ESCC.

WISP3 suppresses ESCC progression by inhibiting the IGF-2-IGF1R-AKT signaling cascade

Esophageal squamous cell carcinoma (ESCC) is a major health problem worldwide, especially in the Chinese population. However, the intrinsic molecular mechanisms of ESCC progression are largely unclear, thus there is an unmet need to identify essential genes governing this disease. Here, we discovered WISP3, an important member of the CCN family, is markedly downregulated in ESCC tissues compared to the normal esophageal epithelium. Downregulation of WISP3 in cancer tissue correlates with worse overall survival of ESCC patients. Using ESCC cell lines as models, we found that forced expression of WISP3 not only suppressed proliferation and migration of cancer cells in vitro, but also inhibited ESCC tumor growth and metastasis in vivo. On the contrary, WISP3 depletion strongly promoted the tumorigenicity of ESCC cells. Mechanistically, we found that WISP3 negates the activity of AKT via inhibiting the IGF-2-IGF1R signaling cascade, which mediates the tumor-suppressive function of WISP3 in esophageal cancers. Together, we identified a novel factor driving the development of ESCC, and revealed a potential therapeutic target for ESCC treatment.