Expression In Esophageal Squamous Cell Carcinoma Research Articles

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 acidsmight 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.

Regulation of TMEM100 expression by epigenetic modification, effects on proliferation and invasion of esophageal squamous carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a prevalent malignancy with a high morbidity and mortality rate. TMEM100 has been shown to be suppressor gene in a variety of tumors, but there are no reports on the role of TMEM100 in esophageal cancer (EC). To investigate epigenetic regulation of TMEM100 expression in ESCC and the effect of TMEM100 on ESCC proliferation and invasion. Firstly, we found the expression of TMEM100 in EC through The Cancer Genome Atlas database. The correlation between TMEM100 gene expression and the survival of patients with EC was further confirmed through Kaplan-Meier analysis. We then added the demethylating agent 5-AZA to ESCC cell lines to explore the regulation of TMEM100 expression by epigenetic modification. To observe the effect of TMEM100 expression on tumor proliferation and invasion by overexpressing TMEM100. Finally, we performed gene set enrichment analysis using the Kyoto Encyclopaedia of Genes and Genomes Orthology-Based Annotation System database to look for pathways that might be affected by TMEM100 and verified the effect of TMEM100 expression on the mitogen-activated protein kinases (MAPK) pathway. In the present study, by bioinformatic analysis we found that TMEM100 was lowly expressed in EC patients compared to normal subjects. Kaplan-meier survival analysis showed that low expression of TMEM100 was associated with poor prognosis in patients with EC. Then, we found that the demethylating agent 5-AZA resulted in increased expression of TMEM100 in ESCC cells [quantitative real-time PCR (qRT-PCR) and western blotting]. Subsequently, we confirmed that overexpression of TMEM100 leads to its increased expression in ESCC cells (qRT-PCR and western blotting). Overexpression of TMEM100 also inhibited proliferation, invasion and migration of ESCC cells (cell counting kit-8 and clone formation assays). Next, by enrichment analysis, we found that the gene set was significantly enriched in the MAPK signaling pathway. The involvement of TMEM100 in the regulation of MAPK signaling pathway in ESCC cell was subsequently verified by western blotting. TMEM100 is a suppressor gene in ESCC, and its low expression may lead to aberrant activation of the MAPK pathway. Promoter methylation may play a key role in regulating TMEM100 expression.

Revealing the oncogenic role of elevated GNL3L expression in esophageal squamous cell carcinoma: insights into the STAT3 pathway.

Esophageal squamous cell carcinoma (ESCC) patients carries a poor prognosis, with limited effective therapeutic targets. This study aimed to clarify the clinical significance of guanine nucleotide-binding protein like 3-like (GNL3L) protein expression in ESCC and its role in malignant progression. GNL3L expression and associated cancer-promoting pathways in ESCC were interrogated via bioinformatics analysis through use of The Cancer Genome Atlas (TCGA) database. Subsequent verification of GNL3L protein expression in ESCC, coupled with clinical data, was conducted through immunohistochemistry and followed by a comprehensive prognostic analysis. We further investigated potential signaling pathways facilitating ESCC progression, employing a combination of bioinformatics analysis and immunohistochemical (IHC) experiments. Bioinformatics analysis unveiled a significant elevation in GNL3L expression, particularly in gastrointestinal tumors and ESCC. Immunohistochemistry confirmed elevated GNL3L expression in ESCC tissues. Regression analysis established a correlation between elevated GNL3L expression and advanced tumor node metastasis (TNM) stage, with high expression associated with poor prognosis in patients with ESCC. Our integrated approach of bioinformatics and IHC analysis indicated a potential role of the signal transducers and activators of transcription 3 (STAT3) signaling pathway in ESCC progression. High GNL3L expression significantly contributes to the malignant progression of ESCC. This study further elucidates the mechanisms driving ESCC progression and offers possible insights for more effective diagnosis and treatment strategies.

Abstract 5173: TRAIL expression in tumor cells predicts and drives poor response to neoadjuvant immunochemotherapy in esophageal squamous cell carcinoma

Abstract Background: Neoadjuvant PD-1 blockade combined with chemotherapy has shown promising antitumor efficacy in the therapy of esophageal squamous cell carcinoma (ESCC). However, effective biomarkers and potential mechanisms of resistance to this treatment are still unclear. Methods: We prospectively collected pre- and post-neoadjuvant treatment ESCC tissues from 40 patients who received PD-1blockade (camrelizumab, 200mg) and chemotherapy (albumin-paclitaxel, 260mg/m2 and carboplatin, area under the curve = 5). Based on their pathological responses to neoadjuvant immunochemotherapy (NICT), patients were classified into “responder” and “non-responder” groups. All samples were subjected to bulk RNA-sequencing and whole-exome sequencing. Of note, samples from 14 patients in both groups were subjected to single cell RNA sequencing and TCR sequencing. We characterized and validated the changes in the immunological landscape of “responder” and “non-responder” groups using multi-omics sequencing data, multiplex immunofluorescence, in vitro and in vivo experiments. External validation cohorts of ESCC treated with NICT were analyzed to validate the predictive biomarkers for NICT. Results: We found that TNF-related apoptosis-inducing ligand (TRAIL) signaling was significantly higher in pre-treatment non-responders than in responders, and was inversely correlated with T cell proliferation and TCR expansion. TRAIL was mainly secreted by tumor cells and bound to its specific receptor DR5 in T cells, thereby inhibiting the activation of T cells. Functional co-culture experiments and T cell cytotoxicity assays revealed that TRAIL+ tumor cells mediated T cell suppression and caused CD8+ T cell dysfunction. Furthermore, TRAIL inhibition could improve the efficacy of anti-PD-1 in a xenografted mouse model. In bulk RNA-seq (n=40), TRAIL expression was associated with worse disease-free survival and overall survival, however, these results were not observed in the ESCC cohort treated with upfront surgery from the TCGA database, suggesting that the predictive value of TRAIL is specific to NICT. In an external validation cohort of ESCC patients treated with NICT (n=81), multivariable Cox regression analysis showed that high TRAIL expression was an independent risk factor for unfavorable outcomes. Importantly, TRAIL expression exhibited a higher accuracy in predicting NICT response compared with PD-L1 expression in ESCC (AUC of ROC curves: 0.886 vs 0.607, respectively P<0.001). Meanwhile, the serum concentrations of TRAIL (measured by ELISA) in pre-NICT ESCC patients were also significantly higher in non-responders (n=58). Conclusions: TRAIL expression in tumor cells is negatively correlated with the response of ESCC to NICT. TRAIL expression may serve as a promising biomarker to predict NICT response and guide NICT selection in ESCC patients. Citation Format: Weixiong Yang, Fang Wang, Jianping Guo, Junchao Cai, Zekang Wang, Yao Liu, Zengli Fang, Wenfang Chen, Lixia Xu, Shuishen Zhang, Bo Zeng, Zhenguo Liu, Sui Peng, Sai-Ching Jim Yeung, Chao Cheng. TRAIL expression in tumor cells predicts and drives poor response to neoadjuvant immunochemotherapy in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5173.

LncRNA LUESCC promotes esophageal squamous cell carcinoma by targeting the miR-6785-5p/NRSN2 axis

Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent gastrointestinal malignancies with high mortality worldwide. Emerging evidence indicates that long noncoding RNAs (lncRNAs) are involved in human cancers, including ESCC. However, the detailed mechanisms of lncRNAs in the regulation of ESCC progression remain incompletely understood. LUESCC was upregulated in ESCC tissues compared with adjacent normal tissues, which was associated with gender, deep invasion, lymph node metastasis, and poor prognosis of ESCC patients. LUESCC was mainly localized in the cytoplasm of ESCC cells. Knockdown of LUESCC inhibited cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth in vivo. Mechanistic investigation indicated that LUESCC functions as a ceRNA by sponging miR-6785-5p to enhance NRSN2 expression, which is critical for the malignant behaviors of ESCC. Furthermore, ASO targeting LUESCC substantially suppressed ESCC both in vitro and in vivo. Collectively, these data demonstrate that LUESCC may exerts its oncogenic role by sponging miR-6785-5p to promote NRSN2 expression in ESCC, providing a potential diagnostic marker and therapeutic target for ESCC patients.

FOXD1 promotes proliferation, migration, and epithelial-mesenchymal transformation in esophageal squamous cell carcinoma by targeting SNAI1

The transcription factor Forkhead box D1 (FOXD1) is a crucial member of the FOX family and is widely expressed in human embryonic cells, where it plays a regulatory role in organogenesis. However, the precise functions and molecular mechanisms of FOXD1 in esophageal squamous cell carcinoma (ESCC) are not fully clarified. This study aimed to analyze the expression of FOXD1 in ESCC, investigate its relationship with clinicopathological features and prognosis, and elucidate its role in ESCC tumorigenesis and progression. We found a significant up-regulation of FOXD1 mRNA and protein expression in ESCC (p < 0.01), which exhibited a negative correlation with prognosis. Notably, the expression level of FOXD1, along with tumor TNM stage and lymph node metastasis, emerged as independent prognostic factors for ESCC patients (p < 0.01). In addition, we observed that FOXD1 promoted ESCC cell proliferation, migration, and invasion in vitro. Furthermore, we identified a positive correlation between FOXD1 and SNAI1 in ESCC. Through direct binding to the SNAI1 promoter, FOXD1 enhanced its transcriptional activity, thereby regulating the epithelial-mesenchymal transition (EMT) process and activating TGF-β signaling. These findings suggested that FOXD1 may function as a tumor activator and represent a potential novel therapeutic target for ESCC.

USP4 promotes the proliferation, migration, and invasion of esophageal squamous cell carcinoma by targeting TAK1

Ubiquitin-specific protease 4 (USP4) represents a potential oncogene involved in various human cancers. Nevertheless, the biological roles and precise mechanism of USP4 in esophageal squamous cell carcinoma (ESCC) progression are not understood. Here, USP4 expression was found to be markedly upregulated in ESCC tumor tissues and cells. Loss- and gain-of-function assays suggested that USP4 silencing inhibited ESCC cell proliferation, migration, and invasion, while USP4 overexpression promoted these behaviors. Consistently, USP4 silencing repressed tumor growth and metastasis in an ESCC nude mouse model in vivo. As a target molecule of USP4, transforming growth factor-β-activated kinase 1 (TAK1) also showed high expression in ESCC. Moreover, we observed that USP4 specifically interacted with TAK1 and stabilized TAK1 protein levels via deubiquitination in ESCC cells. Importantly, USP4 promotes ESCC proliferation, migration, and invasion via the MEK/ERK signaling pathway and can be inhibited by U0126. Neutral red (NR), an inhibitor of USP4 can suppress ESCC progression in vitro and in vivo. Overall, this study revealed that USP4/TAK1 plays crucial roles in ESCC progression by modulating proliferation, migration, and invasion, and USP4 might be a potential therapeutic target in ESCC.

The m6A reader IGF2BP2 promotes the progression of esophageal squamous cell carcinoma cells by increasing the stability of OCT4 mRNA.

Esophageal squamous cell carcinoma (ESCC) is a common malignancy with high morbidity and mortality. Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) serves as a reader of RNA m6A (N6 methyladenosine) modification to regulate gene expression at the post-transcriptional level. Emerging evidence suggests that IGF2BP2 plays critical roles in tumorigenesis and malignant development. However, the biological function and molecular mechanism of IGF2BP2 in ESCC are not well understood. Here, we found that IGF2BP2 expression was upregulated in esophageal cancer tissues and ESCC cells, and IGF2BP2 overexpression enhanced proliferation, migration, invasion, and stem cell-like properties of ESCC cells. Conversely, the knockdown of IGF2BP2 expression inhibited malignant phenotype of ESCC cells. Mechanistically, IGF2BP2 upregulated octomer-binding transcription factor 4 (OCT4) mRNA expression, and RNA immunoprecipitation (RIP) assay proved that IGF2BP2 could interact with OCT4 mRNA. Moreover, OCT4 was modified at m6A confirmed by methylated m6A RNA immunoprecipitation (Me-RIP)-qPCR assay, and IGF2BP2 knockdown reduced OCT4 mRNA stability. These results suggested that IGF2BP2 served as a reader for m6A-modified OCT4, thus increased OCT4 mRNA expression by regulating its stability. Furthermore, the knockdown of OCT4 could reverse the effects of IGF2BP2 on ESCC cells. In conclusion, these data indicate that IGF2BP2, as a reader for m6A, plays an oncogenic role by regulating OCT4 expression in ESCC, which provides new insights into targeting IGF2BP2/OCT4 axis for the therapy of ESCC.

NSG1 promotes glycolytic metabolism to enhance Esophageal squamous cell carcinoma EMT process by upregulating TGF-β

As a highly enriched endosomal protein within neuronal cells, NSG1 has been discovered to facilitate the process of epithelial-mesenchymal transition (EMT) in esophageal squamous cell carcinoma (ESCC). However, the precise mechanisms behind this phenomenon have yet to be elucidated. The pivotal role of transforming growth factor-β (TGF-β) in triggering the EMT and its significant contribution towards tumor metabolic reprogramming—responsible for EMT activation—has been robustly established. Nevertheless, the extent of TGF-β involvement in the NSG1-mediated EMT within ESCC and the processes through which metabolic reprogramming participates remain ambiguous. We accessed an array of extensive public genome databases to analyze NSG1 expression in ESCC. Regulation of TGF-β by NSG1 was analyzed by transcriptome sequencing, quantitative Real-Time PCR (qRT-PCR), co-immunoprecipitation (CO-IP), and immunofluorescence (IF). Additionally, cellular functional assays and western blot analyses were conducted to elucidate the effect of NSG1 on TGF-β/Smad signaling pathway, as well as its role in ESCC cell metastasis and proliferation. We validated the influence of the NSG1/TGF-β axis on metabolic reprogramming in ESCC by measuring extracellular acidification, glucose uptake, and lactate production. Our findings identify an oncogenic role for NSG1 in ESCC and show a correlation between high NSG1 expression and poor prognosis in ESCC patients. Additional research indicated TGF-β’s involvement in the NSG1-induced EMT process. From a mechanistic perspective, NSG1 upregulates TGF-β, activating the TGF-β/Smad signaling pathway and subsequently fostering the EMT process by inducing cell metabolic reprogramming—evident from elevated glycolysis levels. In conclusion, our study highlights the NSG1/TGF-β axis as a promising therapeutic target for ESCC.

Low expression of PINK1 and PARK2 predicts poor prognosis in patients with esophageal squamous cell carcinoma

BackgroundThe Parkinson’s disease (PD) gene family expression is strongly linked to tumor development and progression; PINK1 and PARK2 are essential members of the PD gene family. However, the relationship between PINK1 and PARK2 and esophageal squamous cell carcinoma (ESCC) remains unknown. This research aims to clarify the prognostic value of PINK1 and PARK2 in ESCC.MethodsPINK1 and PARK2 protein levels in 232 ESCC specimens, and 125 matched adjacent normal tissues were detected by immunohistochemistry. The relationship between PINK1 and PARK2 protein expression and clinicopathological features were analyzed. Kaplan–Meier survival analysis was performed to estimate the prognostic value of the PINK1 and PARK2 proteins in patients. Cox univariate and multivariate analyses were used to assess the risk factors affecting the OS for patients with ESCC.ResultsPINK1 and PARK2 had low expression in ESCC. Patients with low PINK1 had worse differentiation and advanced T and TNM stages. Lower PARK2 expression was linked to lymph node metastases and an advanced TNM stage. Furthermore, reduced PINK1 and PARK2 levels were associated with a poor prognosis for ESCC. Cox univariate and multivariate analyses revealed that PINK1, PARK2, and tumor size were closely associated with the prognosis of patients with ESCC, and PARK2 was an independent risk factor for patients with ESCC. Finally, the PINK1 and PARK2 proteins were closely related and shared the same signal pathway.ConclusionsPINK1 and PARK2 could work as tumor suppressors in ESCC and are likely to become new treatment targets for ESCC.

Induction of Kallikrein-Related Peptidase 13 and TET2/3 by Anticancer Drugs and Poor Prognosis of Patients with Esophageal Squamous Cell Carcinoma After Preoperative Treatment.

Preoperative chemotherapy/chemoradiotherapy has been generally considered for the treatment of esophageal squamous cell carcinoma (ESCC) to improve prognosis. We examined the effects of anticancer drugs on the expression of kallikrein-related peptidase 13 (KLK13), a potential ESCC prognostic marker, and its clinical relevance in patients who received chemotherapy/chemoradiotherapy for ESCC. Overall, 105 patients with ESCC who received chemotherapy or chemoradiotherapy before esophagectomy were enrolled. The expression of KLK13 in biopsy samples obtained before chemotherapy/chemoradiotherapy and resected ESCC tumors was assessed by immunohistochemical staining. The effects of 5-fluorouracil (5-FU) and/or cisplatin (CDDP) exposure on the expressions of KLK13 and ten-eleven translocation dioxygenases (TET) in ESCC cells were examined by reverse transcription-polymerase chain reaction. Immunohistochemical staining of paired ESCC specimens before (biopsy samples) and after (resected specimens) chemotherapy/chemoradiotherapy demonstrated a change in KLK13 expression. KLK13 and TET2/3 transcriptions were induced when human ESCC cell lines were treated with 5-FU and/or CDDP. Among patients with KLK13-negative status before chemotherapy/chemoradiotherapy, those with KLK13-positive resected tumors had a significantly poorer prognosis than those with KLK13-negative resected tumors (p = 0.0477). By using tumor cells isolated from ESCC biopsy tissues obtained before chemotherapy/chemoradiotherapy, we established a primary culture system and detected the induction of KLK13 expression by anticancer drugs. Preoperative treatments alter KLK13 expression in ESCC. The conversion of KLK13 expression from a negative status in biopsy samples to a positive status in resected tumor samples is a predictor of poor prognosis. KLK13 status is a potential marker for decision making to avoid harmful chemotherapy/chemoradiotherapy in patients with ESCC.

Network Analysis of Differential Expression of ANLN and Its Interacting Proteins in Esophageal Squamous Cell Carcinoma

Anillin (ANLN) is an actin binding protein, which was originally extracted from Drosophila melanogaster embryos. As a key regulatory factor in cytokinesis, ANLN is highly expressed in various tumors, leading to abnormal cell division and promoting the proliferation, migration, and invasion of cancer cells. At present, the role and regulatory mechanism of ANLN in human Esophageal Squamous Cell Carcinoma (ESCC) are not fully understood. The purpose of this study is to construct a Protein-Protein Interaction Network (PPIN) to reveal the characteristics of ANLN and its interacting proteins, by the integration of their expression in ESCC. The differentially expressed ANLN and its interacting proteins in ESCC were identified from our previous RNA-seq data. By constructing a specific PPI network, it was found that many differentially expressed genes/proteins may interact with ANLN. Multiple enrichment pathways of ANLN and its differentially expressed genes were explained by functional enrichment analysis, Gene Ontology (GO) analysis and KEGG pathway analysis. In addition, it is revealed that ANLN, ECT2, ACADM and PPP1R9A play an important role in the occurrence and development of ESCC, and they are proposed as new prognostic factors for ESCC. These bioinformatics analyses provide a comprehensive perspective for the role of ANLN in ESCC.

SPI1-Mediated Upregulation of the CST1 Gene as an Independent Poor Prognostic Factor Accelerates Metastasis in Esophageal Squamous Cell Carcinoma (ESCC) by Interacting with MMP2.

Esophageal squamous cell carcinoma (ESCC) is a highly lethal tumor type, but studies on the ESCC tumor microenvironment are limited. We found that cystatin SN (CST1) plays an important role in the ESCC tumor microenvironment. CST1 has been reported to act as an oncogene in multiple human cancers, but its clinical significance and underlying mechanism in ESCC remain elusive. We performed ESCC gene expression profiling with data from RNA-sequencing and public databases and found CST1 upregulation in ESCC. Then, we assessed CST1 expression in ESCC by RT‒qPCR and Western blot analysis. In addition, immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) were used to estimate the expression of CST1 in ESCC tissue and serum. Moreover, further functional experiments were conducted to verify that the gain and loss of CST1 in ESCC cell lines significantly influenced the proliferation and metastasis of ESCC. Mass spectrometry, coimmunoprecipitation, and gelatin zymography experiments were used to validate the interaction between CST1 and matrix metalloproteinase 2 (MMP2) and the mechanism of CST1 influence on metastasis in ESCC. Here, we found that CST1 expression was significantly elevated in ESCC tissues and serum. Moreover, compared with patients with low CST1 expression, patients with high CST1 expression had a worse prognosis. Overall survival (OS) and disease-free survival (DFS) were significantly unfavorable in the high CST1 expression subgroup. Likewise, the CST1 level was significantly increased in ESCC serum compared with healthy control serum, indicating that CST1 may be a potential serum biomarker for diagnosis, with an area under the curve (AUC) = 0.9702 and p < 0.0001 by receiver operating curve (ROC) analysis. Furthermore, upregulated CST1 can promote the motility and metastatic capacity of ESCC in vitro and in vivo by influencing epithelial mesenchymal transition (EMT) and interacting with MMP2 in the tumor microenvironment (TME). Collectively, the results of this study indicated that high CST1 expression mediated by SPI1 in ESCC may serve as a potentially prognostic and diagnostic predictor and as an oncogene to promote motility and metastatic capacity of ESCC by influencing EMT and interacting with MMP2 in the TME.

Molecular profiles of different PD-L1 expression in patients with esophageal squamous cell carcinoma

ABSTRACT Background PD-1/PD-L1 inhibitors are approved treatments for patients with esophageal squamous cell carcinoma (ESCC). The present investigation aspired to explore the interrelation between molecular phenotype and PD-L1 expression in ESCC. Methods PD-L1 testing and targeted next-generation sequencing (NGS) were performed on tumoral tissues from 139 ESCC patients. Tumor-infiltrating lymphocytes (TILs) were scrutinized using a tyramide signal amplification system combined with immunohistochemistry. Results Among enrolled patients, 36.7% displayed high PD-L1 expression (combined positive score [CPS] ≥10). BRCA1 and NF1 gene mutations were significantly associated with high PD-L1 expression (p < .05) while TGFβ pathway alterations were linked to low PD-L1 expression (p = .02). High copy number instability (CNI) and copy number alterations (CNA) were correlated with low PD-L1 expression. Patients with CDKN2A deletion exhibited higher PD-L1 expression. Varying types of TILs were observed across different PD-L1 expression groups. The ratio of CD8+PD-L1+ T cells and CD8+PD-1+ T cells to CD8+ T cells remained comparable in both tumoral and stromal regions, but the ratio of CD68+PD-L1+ macrophages to CD68+ macrophages was higher than the ratio of CD68+PD-1+ macrophages to CD68+ macrophages. CPS was significantly correlated with PD-L1+ lymphocytes and CD68+ macrophages in the tumoral region. CD8+ T cell infiltration was positively correlated with PD-1+ cells in both tumoral and stromal regions. Conclusion In this study, we presented the prevalence rates of PD-L1 expression in Chinese ESCC patients. The association of genetic profiles with PD-L1 expression levels also provide the clue that genomic phenotype may interact with the immunologic phenotype in ESCC.

Unveiling the prognostic significance of SOX5 in esophageal squamous cell carcinoma: a comprehensive bioinformatic and experimental analysis.

This study aimed to investigate the expression and prognostic significance of SOX5 in esophageal squamous cell carcinoma (ESCC). Gene Expression Omnibus (GEO) data were analyzed to assess SOX5 expression in ESCC and normal tissues. Survival analysis was performed to evaluate its prognostic significance. Pathway enrichment analysis was conducted to identify pathways associated with low SOX5 expression. Methylation status of CpG sites in ESCC cases was examined, and SOX5 expression was evaluated. Differential expression and ChIP-seq data analyses were used to identify genes significantly correlated with SOX5 and to obtain target genes. A protein-protein interaction (PPI) network was constructed using hub genes, and their association with immune cell infiltration was determined. In vitro ESCC cell experiments validated the findings. SOX5 was significantly downregulated in ESCC samples compared to normal samples. Its downregulation was associated with shorter survival in ESCC patients. Pathway enrichment analysis revealed enrichment in regulated necrosis, NLRP3 inflammasome, formation of the cornified envelope, and PD-1 signaling. Methylation status of two CpG sites negatively correlated with SOX5 expression. Differential expression analysis identified 122 genes significantly correlated with SOX5, and 28 target genes were obtained from ChIP-seq analysis. Target genes were enriched in DNA replication, cell cycle, spindle, and ATPase activity. Five hub genes were identified, and the PPI network showed significant associations with immune cell infiltration. In vitro experiments confirmed SOX5 downregulation, upregulation of hub genes, and their functional effects on ESCC cell apoptosis and proliferation. These findings enhance understanding of SOX5 in ESCC and potential therapeutic strategies.

Intracellular Fusobacterium nucleatum infection increases METTL3-mediated m6A methylation to promote the metastasis of esophageal squamous cell carcinoma

IntroductionThe tumor-associated microbiota plays a vital role in cancer development. Accumulating evidence shows that Fusobacterium nucleatum (Fn) participates in the progression of multiple tumor types. However, the underlying mechanisms remain unclear. ObjectivesThis study examined the expression of methyltransferase-like protein 3 (METTL3) during Fn infection and elucidated the function and pathway of Fn-induced m6A methylation in esophageal squamous cell carcinoma (ESCC). MethodsThe abundance of Fn in patient tissues was determined by qPCR. Western blot, qRT-PCR, and immunohistochemistry were performed to measure METTL3 expression in cells and tissues. METTL3 function was evaluated in vitro by colony formation and cell migration assays. MeRIP-qPCR was performed to determine the relationship between METTL3 and c-Myc. In addition, the half-lives of genes that are downstream of METTL3 were determined with RNA stability assays. ResultsFn was enriched in hepatocellular carcinoma (HCC), breast cancer (BRCA), ESCC, and colorectal cancer (CRC) tumor tissues. METTL3 expression was positively associated with Fn abundance in ESCC tissues. Fn could survive and proliferation as well as increase METTL3 expression in ESCC, HCC, CRC, and BRCA cells. Moreover, METTL3 overexpression promoted ESCC cells proliferation, migration in vivo and in vitro. Mechanistically, Intracellular Fn infection increases METTL3 transcription. METTL3 promoted c-Myc mRNA methylation in the 3′-untranslated Region (3′-UTR) and enhanced its mRNA stability in a YTH N6-Methyladenosine RNA binding protein 1(YTHDF1)-dependent manner, which contributes to Fn induced ESCC proliferation and metastasis. ConclusionsThis study indicates that intracellular Fn infection promotes ESCC development and metastasis, and eradicating Fn infection may be a promising strategy for treating ESCC.

NRF2/HO-1 axis, BIRC5, and TP53 expression in ESCC and its correlation with clinical pathological characteristics and prognosis.

Many types of cancer exhibit high nuclear factor erythroid 2-related factor 2 (NRF2), which is effective in resisting drugs and radiation. However, the role of NRF2 gene expression in predicting the prognosis of esophageal squamous cell carcinoma (ESCC) remains unclear. The association between NRF2, heme oxygenase-1 (HO-1), baculovirus IAP repeat 5 (BIRC5), P53 gene expression levels and their relationship to immune-infiltrating cells were assessed using the Cancer Genome Atlas dataset, the Human Protein Atlas and the TISDB database. The expression of NRF2, HO-1, BIRC5, and TP53 in 118 ESCC patients was detected by immunohistochemistry, and the relationship between their expression level and clinicopathological parameters and prognosis was analyzed. In ESCC, NRF2 overexpression was significantly associated with Han ethnicity, lymph node metastasis, and distant metastasis. HO-1 overexpression was significantly associated with differentiation, advanced clinical staging, lymph node metastasis, nerve invasion, and distant metastasis. BIRC5 overexpression was significantly associated with Han ethnicity and lymph node metastasis. TP53 overexpression was significantly associated with Han ethnicity and T staging. The NRF2/HO-1 axis expression was positively correlated with BIRC5 and TP53. Kaplan-Meier and multivariate Cox regression analysis showed that NRF2, BIRC5, and TP53 genes co-expression was an independent prognostic risk factor. TISIDB dataset analysis showed that immune-infiltrating cells were significantly negatively correlated with NRF2 and BIRC5. NRF2, BIRC5, and TP53 axis gene expressions are predictors of poor prognosis for ESCC. The overexpression of the NRF2/HO-1/BIRC5 axis may not be related to immune-infiltrating cells.

Concordance among the three commercially available PD-L1 assays for esophageal squamous cell carcinoma.

4058 Background: Immune checkpoint inhibitors are now standard-of-care for patients with esophageal squamous cell carcinoma (ESCC). The predictive significance of PD-L1 expression in ESCC has been investigated by different antibodies, scoring algorithms and cutoff values in several phase III trials. However, it remains controversial whether PD-L1 is a predictive biomarker for response to PD-1/PD-L1 blockade. Evaluating the concordance of the commercially available PD-L1 assays is essential to understand the discrepancy in the ESCC clinical trials, and helpful to further investigate the predictive value of PD-L1 expression. Methods: 145 archival tumor samples were obtained from 131 patients with ESCC (stage I-IV) at National Taiwan University Hospital. Formalin-fixed, paraffin-embedded archival tumor samples were assessed by three commercially available PD-L1 assays: VENTANA SP263, Dako 22C3 and Dako 28-8 assays. Assays were performed in a College of American Pathologists accredited central laboratory and scored for PD-L1 staining by using multiple metrics including tumor cell score (TC), immune cell score (IC) and combined positive score (CPS) or tumor area percentage (TAP). Analytical concordance was calculated pairwise between assays using the Spearman (ρ) rank correlation coefficient. Classification concordance, including agreement between clinically relevant scoring algorithms, was investigated using positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) at multiple cutoff values to assess the overlap between populations by using different assays. Results: SP263, 22C3 and 28-8 assays showed good analytical correlation for TC staining (Spearman’s rank correlation coefficient 0.8–0.9). Correlation was lower for IC (Spearman’s rank correlation coefficient 0.59-0.61). There was moderate overlap between populations identified by SP263 and 28-8 or 22C3 based on CPS or TAP algorithm at multiple cutoff values. OPAs for these 3 assays ranged from 68%–88% at various matched algorithms. The SP263 and 22C3 PD-L1 assays appeared relatively more sensitive, assigning a higher proportion of patients as PD-L1 positive or high, compared to 28-8 assay. When using assay-specific clinically relevant algorithm, moderate classification agreement was seen for SP263 versus 22C3 or 28-8. Differences were observed between patient populations with tumor classified as PD-L1 high versus PD-L1 low/negative using CPS≥10, TAP≥10% and TC≥1%. The PPA between 22C3 CPS≥10 and SP263 TAP≥10 or 28-8 CPS≥10 or 28-8 TC≥1% were 75%, 57% and 68% respectively. Conclusions: This study is the first dataset to compare various PD-L1 assays in ESCC. Differences in classification of patients with PD-L1 high versus low/negative using clinically relevant algorithms suggest that caution should be taken when comparing data across the trials.

Clinicopathological relevance of stem cell marker growth and differentiation factor 3 in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the second leading cause of cancer-related deaths in Iran, often diagnosed in advanced stages with a poor prognosis. Growth and differentiation factor 3 (GDF3) is a member of the transforming growth factor-beta (TGF-β) superfamily. It acts as an inhibitor of bone morphogenetic proteins (BMPs) signaling pathway associated with pluripotent embryonic and cancer stem cells (CSCs) characteristics. Since its expression in ESCC has not yet been evaluated, the clinicopathological relevance of GDF3 expression was elucidated in ESCC patients. Expression of GDF3 in tumor tissues from 40 ESCC patients was compared to the related margin normal tissues by relatively comparative real-time polymerase chain reaction (PCR). Glyceraldehydes 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control. Likewise, the function of GDF3 in the differentiation and development of embryonic stem cells (ESCs) was also reviewed. GDF3 was significantly overexpressed in 17.5% of tumors and a significant correlation between GDF3 expression and the depth of tumor invasion was observed (P = 0.032). The results suggest that GDF3 expression is likely to have substantial roles in the progression and invasiveness behavior of ESCC. Having considered the importance of CSC markers identification and their exploitation in targeted cancer therapy, GDF3 may be introduced as a promising therapeutic target to inhibit the invasion of tumor cells in ESCC.

SLC1A5, unrelated to prognosis, was associated with CD8+ T-cell exclusion in the tumor microenvironment of squamous cell carcinoma

SLC1A5, short for solute carrier family 1 member 5, is a neutral amino acid transporter whose expression has been reported to be upregulated in various cancers, including esophageal squamous cell carcinoma (ESCC). Despite this, little has been described regarding the immunological involvement of SLC1A5 expression in the tumor microenvironment of ESCC. Given this, we adopted in silico analyses together with a wet lab strategy to investigate the prognostic and clinicopathological meaning of SLC1A5 expression in ESCC. In silico analyses of SLC1A5 expression data available from The Cancer Genome Atlas (TCGA) database revealed that SLC1A5 expression was unrelated to the prognosis of ESCC, which holds true when extended to other types of squamous cell carcinoma (SCC), including head and neck squamous cell carcinoma (HNSC) and lung squamous cell carcinoma (LUSC). Further analyses revealed that SLC1A5 expression correlated markedly with the infiltration density of effector CD8+ T cells in ESCC, and the same was true for HNSC and LUSC when extrapolated. As experimental confirmation, multiplexed immunofluorescent staining was undertaken to verify the correlation between SLC1A5 expression and infiltration of CD8+ T cells in a tissue microarray prepared from ESCC and matched normal control tissues. Our data confirmed that SLC1A5 expression was not associated with prognosis but was associated with the exclusion of CD8+ T cells. Taken together, all the data we curated strongly support the notion that SLC1A5 expression is associated with CD8+ T-cell exclusion in the tumor microenvironment of SCC.