Lysophosphatidic acid promotes ESCC progression by increasing the level of CCL2 secreted by esophageal epithelial cells.

BACKGROUND: Lysophosphatidic acid (LPA) is a small bioactive lipid which acts as a potent regulator in various tumor progressions through six G-protein-coupled receptors (LPA1-LPA6). Our previous study demonstrated that the LPA-producing enzyme, autotaxin (ATX), was upregulated in esophageal squamous cell carcinoma (ESCC) and ATX high expression levels indicated a poor prognosis. Esophageal squamous cell carcinoma is a type of malignant tumor which originates from epithelial cells. Its progression can be affected by the interaction between cancer cells and normal cells. However, the impact of LPA on the interaction between esophageal epithelial cells and cancer cells in the development of ESCC remains uncertain. METHODS: MTS and Edu assays were performed to determine ESCC cell proliferation in culture medium (CM) derived from LPA-stimulated esophageal epithelial cells (Het-1a). A wound healing assay, transwell migration and an invasion assay were performed to assess the metastatic ability of ESCC cells. Cytokine array analysis was conducted to detect the differentially secreted cytokines in CM. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were utilized to uncover the pathways and cytokines that are influenced by LPA in ESCC. Immunohistochemical staining was employed to measure the expression of ATX and CCL2 in early-stage ESCC. Quantitative real-time PCR, western blot, enzyme-linked immunosorbent assay and an antibody neutralization assay were employed to measure the mechanism of LPA-mediated communication between epithelial cells and cancer cells. RESULTS: Functional experiments showed that exposing ESCC cancer cells to CM from LPA-treated Het-1a results in promoting proliferation, migration, invasion and epithelial-mesenchymal transition processes. Using cytokine array analysis, we discovered that LPA triggers the release of multiple cytokines from epithelial cells. After screening of the TCGA and GEO databases, CCL2 was identified and found to be correlated with ATX expression in ESCC. Furthermore, CCL2 levels in both mRNA expression and secretion were observed to be upregulated in epithelial cells upon stimulation with LPA. Blocking CCL2 effectively reduced the pro-migration influence of CM derived from LPA-treated Het-1a. Mechanism studies have demonstrated that LPA activated the NF-κB signaling pathway through LPA1/3, ultimately causing an increase in CCL2 expression and secretion in Het-1a. CONCLUSIONS: Our findings, taken together, demonstrate that CM from LPA-treated esophageal epithelial cells plays a significant role in promoting the progression of ESCC, with CCL2 acting as the primary regulator.

Prognostic value of genomic mutation signature associated with immune microenvironment in southern Chinese patients with esophageal squamous cell carcinoma.

The genomic landscape of esophageal squamous cell cancer (ESCC), as well as its impact on the regulation of immune microenvironment, is not well understood. Thus, tumor samples from 92 patients were collected from two centers and subjected to targeted-gene sequencing. We identified frequently mutated genes, including TP53, KMT2C, KMT2D, LRP1B, and FAT1. The most frequent mutation sites were ALOX12B (c.1565C > T), SLX4 (c.2786C > T), LRIG1 (c.746A > G), and SPEN (c.6915_6917del) (6.5%). Pathway analysis revealed dysregulation of cell cycle regulation, epigenetic regulation, PI3K/AKT signaling, and NOTCH signaling. A 17-mutated gene-related risk model was constructed using random survival forest analysis and showed significant prognostic value in both our cohort and the validation cohort. Based on the Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression (ESTIMATE) algorithm, the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm, and the MCPcounter algorithm, we found that the risk score calculated by the risk model was significantly correlated with stimulatory immune checkpoints (TNFSF4, ITGB2, CXCL10, CXCL9, and BTN3A1; p < 0.05). Additionally, it was significantly associated with markers that are important in predicting response to immunotherapy (CD274, IFNG, and TAMM2; p < 0.05). Furthermore, the results of immunofluorescence double staining showed that patients with high risk scores had a significantly higher level of M2 macrophage than those with low risk scores (p < 0.05). In conclusion, our study provides insights into the genomic landscape of ESCC and highlights the prognostic value of a genomic mutation signature associated with the immune microenvironment in southern Chinese patients with ESCC.

HMGA1 sensitizes esophageal squamous cell carcinoma to mTOR inhibitors through the ETS1-FKBP12 axis.

Esophageal carcinoma is amongst the prevalent malignancies worldwide, characterized by unclear molecular classifications and varying clinical outcomes. The PI3K/AKT/mTOR signaling, one of the frequently perturbed dysregulated pathways in human malignancies, has instigated the development of various inhibitory agents targeting this pathway, but many ESCC patients exhibit intrinsic or adaptive resistance to these inhibitors. Here, we aim to explore the reasons for the insensitivity of ESCC patients to mTOR inhibitors. We assessed the sensitivity to rapamycin in various ESCC cell lines by determining their respective IC50 values and found that cells with a low level of HMGA1 were more tolerant to rapamycin. Subsequent experiments have supported this finding. Through a transcriptome sequencing, we identified a crucial downstream effector of HMGA1, FKBP12, and found that FKBP12 was necessary for HMGA1-induced cell sensitivity to rapamycin. HMGA1 interacted with ETS1, and facilitated the transcription of FKBP12. Finally, we validated this regulatory axis in in vivo experiments, where HMGA1 deficiency in transplanted tumors rendered them resistance to rapamycin. Therefore, we speculate that mTOR inhibitor therapy for individuals exhibiting a reduced level of HMGA1 or FKBP12 may not work. Conversely, individuals exhibiting an elevated level of HMGA1 or FKBP12 are more suitable candidates for mTOR inhibitor treatment.

CD276 regulates the immune escape of esophageal squamous cell carcinoma through CXCL1-CXCR2 induced NETs.

BACKGROUND: CD276 (B7-H3), a pivotal immune checkpoint, facilitates tumorigenicity, invasiveness, and metastasis by escaping immune surveillance in a variety of tumors; however, the underlying mechanisms facilitating immune escape in esophageal squamous cell carcinoma (ESCC) remain enigmatic. METHODS: We investigated the expression of CD276 in ESCC tissues from patients by using immunohistochemistry (IHC) assays. In vivo, we established a 4-nitroquinoline 1-oxide (4NQO)-induced CD276 knockout (CD276wKO) and K14cre; CD276 conditional knockout (CD276cKO) mouse model of ESCC to study the functional role of CD276 in ESCC. Furthermore, we used the 4NQO-induced mouse model to evaluate the effects of anti-CXCL1 antibodies, anti-Ly6G antibodies, anti-NK1.1 antibodies, and GSK484 inhibitors on tumor growth. Moreover, IHC, flow cytometry, and immunofluorescence techniques were employed to measure immune cell proportions in ESCC. In addition, we conducted single-cell RNA sequencing analysis to examine the alterations in tumor microenvironment following CD276 depletion. RESULTS: In this study, we elucidate that CD276 is markedly upregulated in ESCC, correlating with poor prognosis. In vivo, our results indicate that depletion of CD276 inhibits tumorigenesis and progression of ESCC. Furthermore, conditional knockout of CD276 in epithelial cells engenders a significant downregulation of CXCL1, consequently reducing the formation of neutrophil extracellular trap networks (NETs) via the CXCL1-CXCR2 signaling axis, while simultaneously augmenting natural killer (NK) cells. In addition, overexpression of CD276 promotes tumorigenesis via increasing NETs' formation and reducing NK cells in vivo. CONCLUSIONS: This study successfully elucidates the functional role of CD276 in ESCC. Our comprehensive analysis uncovers the significant role of CD276 in modulating immune surveillance mechanisms in ESCC, thereby suggesting that targeting CD276 might serve as a potential therapeutic approach for ESCC treatment.

Jag1/2 maintain esophageal homeostasis and suppress foregut tumorigenesis by restricting the basal progenitor cell pool.

Basal progenitor cells are crucial for maintaining foregut (the esophagus and forestomach) homeostasis. When their function is dysregulated, it can promote inflammation and tumorigenesis. However, the mechanisms underlying these processes remain largely unclear. Here, we employ genetic mouse models to reveal that Jag1/2 regulate esophageal homeostasis and foregut tumorigenesis by modulating the function of basal progenitor cells. Deletion of Jag1/2 in mice disrupts esophageal and forestomach epithelial homeostasis. Mechanistically, Jag1/2 deficiency impairs activation of Notch signaling, leading to reduced squamous epithelial differentiation and expansion of basal progenitor cells. Moreover, Jag1/2 deficiency exacerbates the deoxycholic acid (DCA)-induced squamous epithelial injury and accelerates the initiation of squamous cell carcinoma (SCC) in the forestomach. Importantly, expression levels of JAG1/2 are lower in the early stages of human esophageal squamous cell carcinoma (ESCC) carcinogenesis. Collectively, our study demonstrates that Jag1/2 are important for maintaining esophageal and forestomach homeostasis and the onset of foregut SCC.

Antitumour effects of artesunate via cell cycle checkpoint controls in human oesophageal squamous carcinoma cells.

Artesunate (ART), an effective antimalarial semisynthetic derivative of artemisinin, exhibits antitumour properties, but the mechanism(s) involved remain elusive. In this study, we investigated the antitumour effects of ART on human oesophageal squamous cell carcinoma (ESCC) cell lines. Treatment of ESCC cell lines with ART resulted in the production of excessive reactive oxygen species (ROS) that induced DNA damage, reduced cell proliferation and inhibited clonogenicity via G1-S cell cycle arrest and/or apoptosis in vitro. The administration of ART to nude mice with ESCC cell xenografts inhibited tumour formation in vivo. However, the cytotoxicity of ART strongly differed among the ESCC cell lines tested. Transcriptomic profiling revealed that although the expression of large numbers of genes in ESCC cell lines was affected by ART treatment, these genes could be functionally clustered into pathways involved in regulating cell cycle progression, DNA metabolism and apoptosis. We revealed that p53 and Cdk4/6-p16-Rb cell cycle checkpoint controls were critical determinants required for mediating ART cytotoxicity in ESCC cell lines. Specifically, KYSE30 cells with p53Mut/p16Mut were the most sensitive to ART, KYSE150 and KYSE180 cells with p53Mut/p16Nor exhibited intermediate responses to ART, and Eca109 cells with p53Nor/p16Nor exhibited the most resistance to ATR. Consistently, perturbation of p53 expression using RNA interference (RNAi) and/or Cdk4/6 activity using the inhibitor palbociclib altered ART cytotoxicity in KYSE30 cells. Given that the p53 and Cdk4/6-cyclin D1-p16-Rb genes are commonly mutated in ESCC, our results potentially shed new light on neoadjuvant chemotherapy strategies for ESCC.

circ_0000592 facilitates the progression of esophageal squamous cell carcinoma via miR-155-5p/FZD5 axis.

Esophageal squamous cell carcinoma (ESCC) is one of the gastrointestinal malignancies with high prevalence and poor prognosis. Previous reports suggested that circular ribose nucleic acids might exert regulatory functions in ESCC. This study aims to explore the role of circ_0000592 in ESCC progression, providing novel insights into the diagnosis and therapeutic avenues for ESCC. The GSE131969 data set was utilized to assess circ_0000592 expression in ESCC. The validation was performed in the tumorous tissues of ESCC patients (n = 80) and human-immortalized ESCC cell lines. The correlation between circ_0000592 expression and prognosis was analyzed. The impact of circ_0000592 on ESCC cell activity was evaluated through downregulating circ_0000592, as well as encompassing cell viability, migration, and invasion abilities. The downstream pathway of circ_0000592 was explored by binding site prediction from the TargetScan database, followed by in vitro and in vivo experiments. An in vivo xenograft tumor model was established to highlight the role of circ_0000592 in ESCC. Patients with ESCC exhibited higher circ_0000592 expression levels compared to noncancerous patients, which were associated with reduced survival time, higher TNM stage, and increased lymph node metastasis. The circ_0000592 downregulation suppressed cell viability, migration, and invasion abilities in vitro. Mechanistically, circ_0000592 countered the inhibitory effects on the target gene Frizzled 5 (FZD5) through interactions with miR-155-5p. The overexpression of miR-155-5p curtailed the luciferase activity of circ_0000592 in ESCC cells, inhibiting downstream molecule FZD5 protein expression and subsequently mitigating the detrimental consequences of escalated circ_0000592 expression in ESCC cells. Consistently, circ_0000592 downregulation curbed proliferation and metastasis of ESCC tumors in vivo. In summary, circ_0000592 promoted the progress of ESCC by counteracting the inhibitory impact on FZD5 through its interaction with miR-155-5p. Together, our findings highlighted circ_0000592 as a prospective therapeutic target for ESCC.

Tripartite Motif-containing Protein 11 Silencing Inhibits Proliferation and Glycolysis and Promotes Apoptosis of Esophageal Squamous Cell Carcinoma Cells by Inactivating Signal Transduction and Activation of Transcription Factor 3/c-Myc Signaling.

Esophageal squamous cell carcinoma (ESCC) is a common type of human digestive tract cancer with poor survival. Tripartite motif-containing protein 11 (TRIM11) is an oncogene in certain cancers that can regulate glycolysis and signal transduction and activation of transcription factor 3 (STAT3) signaling. This study was designed to investigate the role and the mechanism of TRIM11 in ESCC. First, TRIM11 expression in ESCC tissues and the correlation between TRIM11 expression and prognosis were analyzed using bioinformatics tools. After TRIM11 expression was detected by Western blot in ESCC cells, TRIM11 was silenced to evaluate its effect on the malignant phenotypes of ESCC cells. Cell proliferation and apoptosis were assessed by cell counting kit-8 assay, ethynyl-2'- deoxyuridine staining, and flow cytometry, respectively. The glucose uptake and lactate secretion were detected to examine glycolysis. In addition, Western blot was employed to detect the expression of proteins related to apoptosis, glycolysis, and STAT3/c-Myc signaling. Then, ESCC cells were treated with STAT3 activator further to clarify the regulatory effect of TRIM11 on STAT3/c-Myc signaling. TRIM11 was upregulated in ESCC tissues and cells, and high expression of TRIM11 was associated with a poor prognosis. TRIM11 knockdown inhibited the proliferation and glycolysis while facilitating apoptosis of ESCC cells. Besides, the expression of p-STAT3 and c-Myc was significantly downregulated by TRIM11 silencing. Of note, the STAT3 activator partially reversed the effects of TRIM11 depletion on the proliferation, apoptosis, and glycolysis in ESCC cells. Collectively, TRIM11 loss-of-function affects the proliferation, apoptosis, and glycolysis in ESCC cells by inactivating STAT3/c-Myc signaling.

Selenium May Be Involved in Esophageal Squamous Cancer Prevention by Affecting GPx3 and FABP1 Expression: A Case-Control Study Based on Bioinformatic Analysis.

The role of selenium in the developmental process of esophageal cancer (EC) requires further investigation. To explore the relationship between selenium-related factors and EC through bioinformatic analysis, a case-control study was conducted to verify the results. Utilizing the GEPIA and TCGA databases, we delineated the differential expression of glutathione peroxidase 3 (GPx3) in EC and normal tissues, identified differentially expressed genes (DEGs), and a performed visualization analysis. Additionally, 100 pairs of dietary and plasma samples from esophageal precancerous lesions (EPLs) of esophageal squamous cancer (ESCC) cases and healthy controls from Huai'an district, Jiangsu, were screened. The levels of dietary selenium, plasma selenium, and related enzymes were analyzed using inductively coupled plasma mass spectrometry (ICP-MS) or ELISA kits. The results showed lower GPx3 expression in tumor tissues compared to normal tissues. Further analysis revealed that DEGs were mainly involved in the fat digestion and absorption pathway, and the core protein fatty acid binding protein 1 (FABP1) was significantly upregulated and negatively correlated with GPx3 expression. Our case-control study found that selenium itself was not associated with EPLs risk. However, both the decreased concentration of GPx3 and the increase in FABP1 were positively correlated with the EPLs risk (p for trend = 0.035 and 0.046, respectively). The different expressions of GPx3 and FABP1 reflect the potential of selenium for preventing ESCC at the EPLs stage. GPx3 may affect myocardial infarction through FABP1, which remains to be further studied.

Molecular and immunological characteristics of postoperative relapse in lymph node-positive esophageal squamous cell cancer.

BACKGROUND: The molecular and immunological characteristics of primary tumors and positive lymph nodes in esophageal squamous cell carcinoma (ESCC) are unknown and the relationship with recurrence is unclear, which this study attempted to explore. METHODS: A total of 30 ESCC patients with lymph node positive (IIB-IVA) were enrolled. Among them, primary tumor and lymph node specimens were collected from each patient, and subjected to 551-tumor-targeted DNA sequencing and 289-immuno-oncology RNA panel sequencing to identify the different molecular basis and immunological features, respectively. RESULTS: The primary tumors exhibited a higher mutation burden than lymph nodes (p < 0.001). One-year recurrent ESCC exhibited a higher Mucin16 (MUC16) mutation rate (p = 0.038), as well as univariate and multivariate analysis revealed that MUC16 mutation is independent genetic factor associated with reduced relapse-free survival (univariate, HR: 5.39, 95% CI: 1.67-17.4, p = 0.005; multivariate, HR: 7.36, 95% CI: 1.79-30.23, p = 0.006). Transcriptomic results showed non-relapse group had higher cytolytic activity (CYT) score (p = 0.025), and was enriched in the IFN-α pathway (p = 0.036), while those in the relapsed group were enriched in the TNF-α/NF-κB (p = 0.001) and PI3K/Akt pathway (p = 0.014). CONCLUSION: The difference in molecular characteristics between primary lesions and lymph nodes may be the cause of the inconsistent clinical outcomes. Mutations of MUC16 and poor immune infiltration are associated with rapid relapse of nodes-positive ESCC.

Bioinformatics Analysis Reveals a Novel Prognostic Model for Esophageal Squamous Cell Carcinoma.

Background: Esophageal squamous cell carcinoma (ESCC), a gastrointestinal cancer, is associated with poor prognosis. Prognostic models predict the likelihood of disease progression and are important for the management of patients with ESCC. The objective of this study was to develop a prognostic model for ESCC using bioinformatics analysis. Methods: Two transcriptome microarray Gene Expression Omnibus ESCC datasets (GSE53624 and GSE53622) were analyzed using bioinformatics methods. Differentially expressed genes (DEGs) were identified using the R package limma, and genes associated with survival outcomes in both datasets were identified by Kaplan-Meier analysis. Genes with diagnostic or prognostic value were selected for further analysis, and hazard ratios and their relationship with pathological TNM (pTNM) staging were investigated using univariate and multivariate Cox analysis. After selecting the independent factors from pTNM staging, Cox analysis and nomogram plotting were performed. The ability of the model to stratify risk and predict survival was evaluated and compared with the pTNM staging system to determine its potential clinical value. Key genes were analyzed by immunohistochemistry and RT-PCR. Results: Four candidate genes (B3GNT3, MACC1, NELL2, and USH1G) with prognostic value were identified from the two transcriptome microarray datasets. Age, pTNM stage, and B3GNT3, MACC1, and NELL2 were identified as independent factors associated with survival in the multivariate Cox analysis and used to establish a prognostic model. The model demonstrated significantly higher accuracy in predicting 3-year survival than the pTNM staging system and was useful for further risk stratification in patients with ESCC. B3GNT3 was significantly downregulated in ESCC tumor tissues and negatively associated with lymph node metastasis. Bioinformatics analysis indicated that B3GNT3 may play a role in immune regulation by regulating M2 macrophages. Conclusion: This study developed a new prognostic model for ESCC and identified B3GNT3 as a potential biomarker negatively associated with lymph node metastasis, which warrants further validation.

A novel fatty acid metabolism-related signature identifies MUC4 as a novel therapy target for esophageal squamous cell carcinoma.

Fatty acid metabolism has been identified as an emerging hallmark of cancer, which was closely associated with cancer prognosis. Whether fatty acid metabolism-related genes (FMGs) signature play a more crucial role in biological behavior of esophageal squamous cell carcinoma (ESCC) prognosis remains unknown. Thus, we aimed to identify a reliable FMGs signature for assisting treatment decisions and prognosis evaluation of ESCC. In the present study, we conducted consensus clustering analysis on 259 publicly available ESCC samples. The clinical information was downloaded from The Cancer Genome Atlas (TCGA, 80 ESCC samples) and Gene Expression Omnibus (GEO) database (GSE53625, 179 ESCC samples). A consensus clustering arithmetic was used to determine the FMGs molecular subtypes, and survival outcomes and immune features were evaluated among the different subtypes. Kaplan-Meier analysis and the receiver operating characteristic (ROC) was applied to evaluate the reliability of the risk model in training cohort, validation cohort and all cohorts. A nomogram to predict patients' 1-year, 3-year and 5-year survival rate was also studied. Finally, CCK-8 assay, wound healing assay, and transwell assay were implemented to evaluate the inherent mechanisms of FMGs for tumorigenesis in ESCC. Two subtypes were identified by consensus clustering, of which cluster 2 is preferentially associated with poor prognosis, lower immune cell infiltration. A fatty acid (FA) metabolism-related risk model containing eight genes (FZD10, TACSTD2, MUC4, PDLIM1, PRSS12, BAALC, DNAJA2 and ALOX12B) was established. High-risk group patients displayed worse survival, higher stromal, immune and ESTIMATE scores than in the low-risk group. Moreover, a nomogram revealed good predictive ability of clinical outcomes in ESCC patients. The results of qRT-PCR analysis revealed that the MUC4 and BAALC had high expression level, and FZD10, PDLIM1, TACSTD2, ALOX12B had low expression level in ESCC cells. In vitro, silencing MUC4 remarkably inhibited ESCC cell proliferation, invasion and migration. Our study fills the gap of FMGs signature in predicting the prognosis of ESCC patients. These findings revealed that cluster subtypes and risk model of FMGs had effects on survival prediction, and were expected to be the potential promising targets for ESCC.

EnsemBic: An effective ensemble of biclustering to identify potential biomarkers of esophageal squamous cell carcinoma.

The development of functionally enriched and biologically competent biclustering algorithm is essential for extracting hidden information from massive biological datasets. This paper presents a novel biclustering ensemble called EnsemBic based on p-value, which calculates the functional similarity of genetic associations. To validate the effectiveness and robustness of EnsemBic, we apply three well-known biclustering techniques, viz. Laplace Prior, iBBiG, and xMotif to implement EnsemBic and have been compared using different leading parameters. It is observed that the EnsemBic outperforms its competing algorithms in several prominent functional and biological measures. Next, the biclusters obtained from EnsemBic are used to identify potential biomarkers of Esophageal Squamous Cell Carcinoma (ESCC) by exploring topological and biological relevance with reference to the elite genes, attained from genecards. Finally, we discover that the genes F2RL3, APPL1, CALM1, IFNGR1, LPAR1, ANGPT2, ARPC2, CGN, CLDN7, ATP6V1C2, CEACAM1, FTL, PLAU,PSMB4, and EPHB2 carry both the topological and biological significance of previously established ESCC elite genes. Therefore, we declare the aforementioned genes as potential biomarkers of ESCC.

Construction of a Novel Mitochondria-Associated Gene Model for Assessing ESCC Immune Microenvironment and Predicting Survival.

Esophageal squamous cell carcinoma (ESCC) is among the most common malignant tumors of the digestive tract, with the sixth highest fatality rate worldwide. The ESCC-related dataset, GSE20347, was downloaded from the Gene Expression Omnibus (GEO) database, and weighted gene co-expression network analysis was performed to identify genes that are highly correlated with ESCC. A total of 91 transcriptome expression profiles and their corresponding clinical information were obtained from The Cancer Genome Atlas database. A mitochondria-associated risk (MAR) model was constructed using the least absolute shrinkage and selection operator Cox regression analysis and validated using GSE161533. The tumor microenvironment and drug sensitivity were explored using the MAR model. Finally, in vitro experiments were performed to analyze the effects of hub genes on the proliferation and invasion abilities of ESCC cells. To confirm the predictive ability of the MAR model, we constructed a prognostic model and assessed its predictive accuracy. The MAR model revealed substantial differences in immune infiltration and tumor microenvironment characteristics between high- and low-risk populations and a substantial correlation between the risk scores and some common immunological checkpoints. AZD1332 and AZD7762 were more effective for patients in the low-risk group, whereas Entinostat, Nilotinib, Ruxolutinib, and Wnt.c59 were more effective for patients in the high-risk group. Knockdown of TYMS significantly inhibited the proliferation and invasive ability of ESCC cells in vitro. Overall, our MAR model provides stable and reliable results and may be used as a prognostic biomarker for personalized treatment of patients with ESCC.

Involvement of circRNA Regulators MBNL1 and QKI in the Progression of Esophageal Squamous Cell Carcinoma.

OBJECTIVES: To investigate the role of circRNA regulators MBNL1 and QKI in the progression of esophageal squamous cell carcinoma. BACKGROUND: MBNL1 and QKI are pivotal regulators of pre-mRNA alternative splicing, crucial for controlling circRNA production - an emerging biomarker and functional regulator of tumor progression. Despite their recognized roles, their involvement in ESCC progression remains unexplored. METHODS: The expression levels of MBNL1 and QKI were examined in 28 tissue pairs from ESCC and adjacent normal tissues using data from the GEO database. Additionally, a total of 151 ESCC tissue samples, from stage T1 to T4, consisting of 13, 43, 87, and 8 cases per stage, respectively, were utilized for immunohistochemical (IHC) analysis. RNA sequencing was utilized to examine the expression profiles of circRNAs, lncRNAs, and mRNAs across 3 normal tissues, 3 ESCC tissues, and 3 pairs of KYSE150 cells in both wildtype (WT) and those with MBNL1 or QKI knockouts. Transwell, colony formation, and subcutaneous tumorigenesis assays assessed the impact of MBNL1 or QKI knockout on ESCC cell migration, invasion, and proliferation. RESULTS: ESCC onset significantly altered MBNL1 and QKI expression levels, influencing diverse RNA species. Elevated MBNL1 or QKI expression correlated with patient age or tumor invasion depth, respectively. MBNL1 or QKI knockout markedly enhanced cancer cell migration, invasion, proliferation, and tumor growth. Moreover, the absence of either MBNL1 or QKI modulated the expression profiles of multiple circRNAs, causing extensive downstream alterations in the expression of numerous lncRNAs and mRNAs. While the functions of circRNA and lncRNA among the top 20 differentially expressed genes remain unclear, mRNAs like SLCO4C1, TMPRSS15, and MAGEB2 have reported associations with tumor progression. CONCLUSIONS: This study underscores the tumor-suppressive roles of MBNL1 and QKI in ESCC, proposing them as potential biomarkers and therapeutic targets for ESCC diagnosis and treatment.

LINC00330/CCL2 axis-mediated ESCC TAM reprogramming affects tumor progression.

BACKGROUND: Tumor-associated macrophages (TAMs) significantly influence the progression, metastasis, and recurrence of esophageal squamous cell carcinoma (ESCC). The aberrant expression of long noncoding RNAs (lncRNAs) in ESCC has been established, yet the role of lncRNAs in TAM reprogramming during ESCC progression remains largely unexplored. METHODS: ESCC TAM-related lncRNAs were identified by intersecting differentially expressed lncRNAs with immune-related lncRNAs and performing immune cell infiltration analysis. The expression profile and clinical relevance of LINC00330 were examined using the TCGA database and clinical samples. The LINC00330 overexpression and interference sequences were constructed to evaluate the effect of LINC00330 on ESCC progression. Single-cell sequencing data, CIBERSORTx, and GEPIA were utilized to analyze immune cell infiltration within the ESCC tumor microenvironment and to assess the correlation between LINC00330 and TAM infiltration. ESCC-macrophage coculture experiments were conducted to investigate the influence of LINC00330 on TAM reprogramming and its subsequent effect on ESCC progression. The interaction between LINC00330 and C-C motif ligand 2 (CCL2) was confirmed through transcriptomic sequencing, subcellular localization analysis, RNA pulldown, silver staining, RNA immunoprecipitation, and other experiments. RESULTS: LINC00330 is significantly downregulated in ESCC tissues and strongly associated with poor patient outcomes. Overexpression of LINC00330 inhibits ESCC progression, including proliferation, invasion, epithelial-mesenchymal transition, and tumorigenicity in vivo. LINC00330 promotes TAM reprogramming, and LINC00330-mediated TAM reprogramming inhibits ESCC progression. LINC00330 binds to the CCL2 protein and inhibits the expression of CCL2 and downstream signaling pathways. CCL2 is critical for LINC00330-mediated TAM reprogramming and ESCC progression. CONCLUSIONS: LINC00330 inhibited ESCC progression by disrupting the CCL2/CCR2 axis and its downstream signaling pathways in an autocrine fashion; and by impeding CCL2-mediated TAM reprogramming in a paracrine manner. The new mechanism of TAM reprogramming mediated by the LINC00330/CCL2 axis may provide potential strategies for targeted and immunocombination therapies for patients with ESCC.

Radiation-induced upregulation of FGL1 promotes esophageal squamous cell carcinoma metastasis via IMPDH1.

BACKGROUND: While radiation therapy remains pivotal in esophageal squamous cell carcinoma (ESCC) treatment, the perplexing phenomenon of post-radiation metastasis presents a formidable clinical challenge. This study investigates the role of fibrinogen-like protein 1 (FGL1) in driving ESCC metastasis following radiation exposure. METHODS: FGL1 expression in post-radiation ESCC cells was meticulously examined using qRT-PCR, western blotting, and immunofluorescence. The impact of FGL1 on ESCC cell invasion and migration was assessed through Transwell and wound healing assays. In vivo, the metastatic potential of ESCC in response to FGL1 was scrutinized using nude mice models. Comprehensive RNA sequencing and functional experiments elucidated the intricate mechanism associated with FGL1. RESULTS: Radiation induced upregulation of FGL1 in ESCC cells through FOXO4, intensifying ESCC cell invasion and migration. Targeted knockdown of FGL1 effectively alleviated these characteristics both in vitro and in vivo. FGL1 depletion concurrently suppressed IMPDH1 expression. Rescue experiments underscored that IMPDH1 knockdown robustly reversed the pro-invasive effects induced by FGL1 in ESCC cells. ESCC tissues exhibited heightened IMPDH1 mRNA levels, demonstrating a correlation with patient survival. CONCLUSIONS: Radiation-induced upregulation of FGL1 propels ESCC metastasis through IMPDH1, proposing a potential therapeutic target to mitigate post-radiotherapy metastasis in ESCC patients.

PTPN23[Thr] variant reduces susceptibility and tumorigenesis in esophageal squamous cell carcinoma through dephosphorylation of EGFR.

Post-translational modifications (PTMs) have emerged as pivotal regulators of the development of cancers, including esophageal squamous cell carcinoma (ESCC). Here, we conducted a comprehensive analysis of PTM-related genetic variants associated with ESCC risk using large-scale genome-wide and exome-wide association datasets. We observed significant enrichment of PTM-related variants in the ESCC risk loci and identified five variants that were significantly associated with ESCC risk. Among them, rs6780013 in PTPN23 exhibited the highest level of significance in ESCC susceptibility in 9,728 ESCC cases and 10,977 controls (odds ratio [OR] = 0.85, 95 % confidence interval [CI] = 0.81- 0.89, P = 9.77 × 10-14). Further functional investigations revealed that PTPN23[Thr] variant binds to EGFR and modulates its phosphorylation at Thr699. PTPN23[Thr] variant substantially inhibited ESCC cell proliferation both in vitro and in vivo. Our findings underscore the critical role of PTPN23[Thr]-EGFR interaction in ESCC development, providing more insights into the pathogenesis of this cancer.

Molecular Landscape and Clinical Implication of CCNE1-amplified Esophagogastric Cancer.

Cyclin E overexpression as a result of CCNE1 amplification is a critical driver of genomic instability in gastric cancer, but its clinical implication is largely unknown. Thus, we integrated genomic, transcriptomic, and immune profiling analysis of 7,083 esophagogastric tumors and investigated the impact of CCNE1 amplification on molecular features and treatment outcomes. We identified CCNE1 amplification in 6.2% of esophageal adenocarcinoma samples, 7.0% of esophagogastric junction carcinoma, 4.2% of gastric adenocarcinoma samples, and 0.8% of esophageal squamous cell carcinoma. Metastatic sites such as lymph node and liver showed an increased frequency of CCNE1 amplification relative to primary tumors. Consistent with a chromosomal instability phenotype, CCNE1 amplification was associated with decreased CDH1 mutation and increased TP53 mutation and ERBB2 amplification. We observed no differences in immune biomarkers such as PD-L1 expression and tumor mutational burden comparing CCNE1-amplified and nonamplified tumors, although CCNE1 amplification was associated with changes in immune populations such as decreased B cells and increased M1 macrophages from transcriptional analysis. Real-world survival analysis demonstrated that patients with CCNE1-amplified gastric cancer had worse survival after trastuzumab for HER2-positive tumors, but better survival after immunotherapy. These data suggest that CCNE1-amplified gastric cancer has a distinct molecular and immune profile with important therapeutic implications, and therefore further investigation of CCNE1 amplification as a predictive biomarker is warranted. SIGNIFICANCE: Advanced gastric cancer has a relatively dismal outcome with a 5-year overall survival of less than 10%. Furthermore, while comprehensive molecular analyses have established molecular subtypes within gastric cancers, biomarkers of clinical relevance in this cancer type are lacking. Overall, this study demonstrates that CCNE1 amplification is associated with a distinct molecular profile in gastric cancer and may impact response to therapy, including targeted therapy and/or immunotherapy.

Targeting leucine-rich PPR motif-containing protein/LRPPRC by 5,7,4'-trimethoxyflavone suppresses esophageal squamous cell carcinoma progression.

JAK2/STAT3/MYC axis is dysregulated in nearly 70 % of human cancers, but targeting this pathway therapeutically remains a big challenge in cancer therapy. In this study, genes associated with JAK2, STAT3, and MYC were analyzed, and potential target genes were selected. Leucine-rich PPR motif-containing protein (LRPPRC) whose function and regulation are not fully understood, emerged as one of top 3 genes in terms of RNA epigenetic modification. Here, we demonstrate LRPPRC may be an independent prognostic indicator besides JAK2, STAT3, and MYC. Mechanistically, LRPPRC impairs N6-methyladenosine (m6A) modification of JAK2, STAT3, and MYC to facilitate nuclear mRNA export and expression. Meanwhile, excess LRPPRC act as a scaffold protein binding to JAK2 and STAT3 to enhance stability of JAK2-STAT3 complex, thereby facilitating JAK2/STAT3/MYC axis activation to promote esophageal squamous cell carcinoma (ESCC) progression. Furthermore, 5,7,4'-trimethoxyflavone was verified to bind to LRPPRC, STAT3, and CDK1, dissociating LRPPRC-JAK2-STAT3 and JAK2-STAT3-CDK1 interaction, leading to impaired tumorigenesis in 4-Nitroquinoline N-oxide induced ESCC mouse models and suppressed tumor growth in ESCC patient derived xenograft mouse models. In summary, this study suggests regulation of m6A modification by LRPPRC, and identifies a novel triplex target compound, suggesting that targeting LRPPRC-mediated JAK2/STAT3/MYC axis may overcome JAK2/STAT3/MYC dependent tumor therapeutic dilemma.