Tumorigenesis Of Esophageal Squamous Cell Carcinoma Research Articles

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.

Hypoxia inhibits ferritinophagy-mediated ferroptosis in esophageal squamous cell carcinoma via the USP2-NCOA4 axis.

Esophageal squamous cell carcinoma (ESCC) is a prevalent malignancy of the digestive system. Hypoxia is a crucial player in tumor ferroptosis resistance. However, the molecular mechanism of hypoxia-mediated ferroptosis resistance in ESCC remains unclear. Here, USP2 expression was decreased in ESCC cell lines subjected to hypoxia treatment and was lowly expressed in clinical ESCC specimens. Ubiquitin-specific protease 2 (USP2) depletion facilitated cell growth, which was blocked in USP2-overexpressing cells. Moreover, USP2 silencing enhanced the iron ion concentration and lipid peroxidation accumulation as well as suppressed ferroptosis, while upregulating USP2 promoted ferroptotic cell death in ESCC cells. Furthermore, knockout of USP2 in ESCC models discloses the essential role of USP2 in promoting ESCC tumorigenesis and inhibiting ferroptosis. In contrast, overexpression of USP2 contributes to antitumor effect and ferroptosis events in vivo. Specifically, USP2 stably bound to and suppressed the degradation of nuclear receptor coactivator 4 (NCOA4) by eliminating the Lys48-linked chain, which in turn triggered ferritinophagy and ferroptosis in ESCC cells. Our findings suggest that USP2 plays a crucial role in iron metabolism and ferroptosis and that the USP2/NCOA4 axis is a promising therapeutic target for the management of ESCC.

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

BackgroundCD276 (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...

Screening and verification of hub genes in esophageal squamous cell carcinoma by integrated analysis

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors. However, the mechanisms underlying ESCC tumorigenesis have not been fully elucidated. Thus, we aimed to determine the key genes involved in ESCC tumorigenesis. The following bioinformatics analyses were performed: identification of differentially expressed genes (DEGs); gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis; integrated analysis of the protein–protein interaction network and Gene Expression Profiling Interactive Analysis database for validation of hub genes. Finally, western blotting and qPCR were used to explore the expression of cell division cycle 6 (CDC6) in ESCC cell lines. Immunohistochemistry analysis of ESCC samples from patients and matched clinical characteristics was used to determine the effects of CDC6. A total of 494 DEGs were identified, and functional enrichment was mainly focused on cell cycle and DNA replication. Biological pathway analysis of the hub genes was closely related to the cell cycle. We found that CDC6 was upregulated in ESCC cell lines and patient tissues and was related to the clinicopathological characteristics of ESCC. In conclusion, this study identified hub genes and crucial biological pathways related to ESCC tumorigenesis and integrated analyses indicated that CDC6 may be a novel diagnostic and therapeutic target for ESCC.

STK3 kinase activation inhibits tumor proliferation through FOXO1-TP53INP1/P21 pathway in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is an aggressive disease with a poor prognosis, caused by the inactivation of critical cell growth regulators that lead to uncontrolled proliferation and increased malignancy. Although Serine/Threonine Kinase 3 (STK3), also known as Mammalian STE20-like protein kinase 2 (MST2), is a highly conserved kinase of the Hippo pathway, plays a critical role in immunomodulation, organ development, cellular differentiation, and cancer suppression, its phenotype and function in ESCC require further investigation. In this study, we report for the first time on the role of STK3 kinase and its activation condition in ESCC, as well as the mechanism and mediators of kinase activation. In this study, we investigated the expression and clinical significance of STK3 in ESCC. We first used bioinformatics databases and immunohistochemistry to analyze STK3 expression in the ESCC patient cohort and conducted survival analysis. In vivo, we conducted a tumorigenicity assay using nude mouse models to demonstrate the phenotypes of STK3 kinase. In vitro, we conducted Western blot analysis, qPCR analysis, CO-IP, and immunofluorescence (IF) staining analysis to detect molecule expression, interaction, and distribution. We measured proliferation, migration, and apoptosis abilities in ESCC cells in the experimental groups using CCK-8 and transwell assays, flow cytometry, and EdU staining. We used RNA-seq to identify genes that were differentially expressed in ESCC cells with silenced STK3 or FOXO1. We demonstrated the regulatory relationship of the TP53INP1/P21 gene medicated by the STK3-FOXO1 axis using Western blotting and ChIP in vitro. We demonstrate high STK3 expression in ESCC tissue and cell lines compared to esophageal epithelium. Cellular ROS induces STK3 autophosphorylation in ESCC cells, resulting in upregulated p-STK3/4. STK3 activation inhibits ESCC cell proliferation and migration by triggering apoptosis and suppressing the cell cycle. STK3 kinase activation phosphorylates FOXO1Ser212, promoting nuclear translocation, enhancing transcriptional activity, and upregulating TP53INP1 and P21. We also investigated TP53INP1 and P21's phenotypic effects in ESCC, finding that their knockdown significantly increases tumor proliferation, highlighting their crucial role in ESCC tumorigenesis. STK3 kinase has a high expression level in ESCC and can be activated by cellular ROS, inhibiting cell proliferation and migration. Additionally, STK3 activation-mediated FOXO1 regulates ESCC cell apoptosis and cell cycle arrest by targeting TP53INP1/P21. Our research underscores the anti-tumor function of STK3 in ESCC and elucidates the mechanism underlying its anti-tumor effect on ESCC.

Piperlongumine inhibits esophageal squamous cell carcinoma in vitro and in vivo by triggering NRF2/ROS/TXNIP/NLRP3-dependent pyroptosis

Pyroptosis, a type of programmed cell death, is characterized by cell swelling with bubbles, and the release of inflammatory cell cytokines. Piperlongumine (PL) is a natural bioactive product extracted from Piper longum L, which can effectively exert anti-tumor activities in cancer. However, the effects and the exact molecular mechanisms of PL in esophageal squamous cell carcinoma (ESCC) remain unclear. This research aimed to investigate the role and mechanism of PL on ESCC in vitro and in vivo. In vitro, the MTT results showed that the IC50 of PL in ESCC cells was 28.55 μM. Moreover, PL significantly suppressed malignant behavior by promoting pyroptosis of ESCC cells by inhibiting proliferation, migration, invasion, and colony formation of KYSE-30 cells, up-regulating expressions of ASC, Cleaved-caspase-1, NLRP3, and GSDMD, while inducing the generation of ROS. Further, NRF2 knockdown promoted TXNIP expression, while overexpression of NRF2 inhibited TXNIP expression. However, after PL treatment, this effect was reversed. In addition, PL significantly inhibited the malignant behavior of ESCC cells while the inhibitory effects were reversed by DMF (NRF2 activator) or NAC (ROS eliminator) treatment. Finally, PL markedly increased expressions of ASC, Cleaved-caspase-1, NLRP3, GSDMD, and the generation of ROS while the effects were reversed by TXNIP knockdown or RUS (TXNIP inhibitor) treatment. In vivo, the KYSE-30 xenograft model confirmed that PL inhibited the growth of ESCC transplanted tumors by promoting cell pyroptosis. In conclusion, the results suggested that PL inhibited the malignant behavior of ESCC cells in vitro and tumorigenesis of ESCC in vivo by inhibiting NRF2 and promoting ROS-TXNIP-NLRP3-mediated pyroptosis.

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.

The Hippo-YAP signaling pathway drives CD24-mediated immune evasion in esophageal squamous cell carcinoma via macrophage phagocytosis.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies in the world with poor prognosis. Despite the promising applications of immunotherapy, the objective response rate is still unsatisfactory. We have previously shown that Hippo/YAP signaling acts as a powerful tumor promoter in ESCC. However, whether Hippo/YAP signaling is involved in tumor immune escape in ESCC remains largely unknown. Here, we show that YAP directly activates transcription of the "don't eat me" signal CD24, and plays a crucial role in driving tumor cells to avoid phagocytosis by macrophages. Mechanistically, YAP regulates CD24 expression by interacting with TEAD and binding the CD24 promoter to initiate transcription, which facilitates tumor cell escape from macrophage-mediated immune attack. Our animal model data and clinical data show that YAP combined with CD24 in tumor microenvironment redefines the impact of TAMs on the prognosis of ESCC patients which will provide a valuable basis for precision medicine. Moreover, treatment with YAP inhibitor altered the distribution of macrophages and suppressed tumorigenesis and progression of ESCC in vivo. Together, our study provides a novel link between Hippo/YAP signaling and macrophage-mediated immune escape, which suggests that the Hippo-YAP-CD24 axis may act as a promising target to improve the prognosis of ESCC patients. A proposed model for the regulatory mechanism of Hippo-YAP-CD24-signaling axis in the tumor-associated macrophages mediated immune escape.

The RNA m6A writer RBM15 contributes to the progression of esophageal squamous cell carcinoma by regulating miR-3605-5p/KRT4 pathway

Cancer progression can be modulated by N6-methyladenosine (m6A) modification. RNA binding motif protein 15 (RBM15) is an essential RNA m6A writer that influences carcinogenesis, however its significance in esophageal squamous cell carcinoma (ESCC) is uncertain. This research is intended to examine how RBM15 regulates the development of ESCC. We performed qRT-PCR analysis to evaluate the expression of RBM15, microRNA (miR-3605-5p) as well as keratin 4 (KRT4) in ESCC. Target relationship between miR-3605-5p and KRT4 was validated by dual luciferase reporter assay. Western blotting analyzed the protein levels of KRT4, p53, and p21. To demonstrate that RBM15 is responsible for the m6A alteration of miR-3605-5p, RIP and Me-RIP experiments were carried out concurrently. m6A content was measured by m6A quantification assay. Cell growth and migration were assessed using the CCK-8 and transwell assays. In addition, the role of RBM15 in vivo was examined using a mouse tumor xenograft model. RBM15 and miR-3605-5p were both substantially expressed in ESCC, however KRT4 was not expressed highly. Overexpressed RBM15 triggered cell proliferation and migration in ESCC. Besides, RBM15/m6A could mediate pri-3605-5p to form the mature miR-3605-5p, and miR-3605-5p further targeted KRT4. Further investigations showed that upregulation of KRT4 overturned the promoting impact of RBM15 overexpression on cell proliferation as well as on cell migration in ESCC by activating p53 signaling pathway. This work implied the carcinogenic activity of RBM15/m6A in ESCC via miR-3605-5p/KRT4 pathway, providing a novel m6A modification pattern in the tumorigenesis of ESCC.

Phospholipase PLCE1 Promotes Transcription and Phosphorylation of MCM7 to Drive Tumor Progression in Esophageal Cancer.

PLCE1 promotes tumor progression in ESCC by activating PKCα-mediated phosphorylation of E2F1 to upregulate MCM7 and miR-106b-5p expression and by potentiating MCM7 phosphorylation by RIOK2.

Aberrant epithelial cell interaction promotes esophageal squamous-cell carcinoma development and progression

Epithelial-mesenchymal transition (EMT) and proliferation play important roles in epithelial cancer formation and progression, but what molecules and how they trigger EMT is largely unknown. Here we performed spatial transcriptomic and functional analyses on samples of multistage esophageal squamous-cell carcinoma (ESCC) from mice and humans to decipher these critical issues. By investigating spatiotemporal gene expression patterns and cell–cell interactions, we demonstrated that the aberrant epithelial cell interaction via EFNB1-EPHB4 triggers EMT and cell cycle mediated by downstream SRC/ERK/AKT signaling. The aberrant epithelial cell interaction occurs within the basal layer at early precancerous lesions, which expands to the whole epithelial layer and strengthens along the cancer development and progression. Functional analysis revealed that the aberrant EFNB1-EPHB4 interaction is caused by overexpressed ΔNP63 due to TP53 mutation, the culprit in human ESCC tumorigenesis. Our results shed new light on the role of TP53-TP63/ΔNP63-EFNB1-EPHB4 axis in EMT and cell proliferation in epithelial cancer formation.

NRG1 promotes tumorigenesis and metastasis and afatinib treatment efficiency is enhanced by NRG1 inhibition in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is a highly aggressive tumor with significant heterogeneity in incidence and outcomes. The role of Neuregulin 1 (NRG1) in ESCC and its contribution to aggressiveness remain unknown. This study aims to investigate the functions and molecular mechanisms of NRG1 in ESCC as well as the treatment strategy for ESCC with overexpression of NRG1. We firstly demonstrated the upregulation of NRG1 and a negative correlation trend between patients' overall survival (OS) and the expression level of NRG1 in esophageal cancer. And then we found NRG1 promoted cell proliferation, migration, inhibited apoptosis, and accelerated tumorigenesis and metastasis in ESCC using cell lines and xenograft models. Furthermore, we discovered that NRG1 activated the NF-κB/MMP9 signaling pathway, contributing to the metastatic phenotype in ESCC. Finally, we show that afatinib (FDA approved cancer growth blocker) could inhibit ESCC with overexpressed NRG1 and down-regulation of NRG1 along with afatinib treatment provides higher efficient strategy. This study uncovers the critical role and molecular mechanism of NRG1 in ESCC tumorigenesis and metastasis, suggesting its potential as a novel biomarker for ESCC treatment.

A2ML1 Inhibits Esophageal Squamous Cell Carcinoma Progression and Serves as a Novel Prognostic Biomarker.

Studies have established a correlation between α2-macroglobulin-like 1 (A2ML1) and the prognosis of lung, pancreatic, and breast cancers; however, research on its involvement in the pathogenesis of esophageal carcinoma remains limited. Therefore, in this study, we aimed to investigate the role of A2ML1 in the progression of esophageal squamous cell carcinoma (ESCC). Immunohistochemical staining was employed to assess the expression level of A2ML1 protein in both tumor and adjacent normal tissues of patients with ESCC. The Kaplan-Meier method, along with univariate and multivariate Cox risk ratio analyses, was used to determine survival rates and prognostic factors. Furthermore, two human ESCC cell lines, KYSE30 and KYSE150, were used to assess the effect of A2ML1 overexpression on cell proliferation and apoptosis. A human apoptosis antibody kit was also used to analyze the downstream action proteins of A2ML1, and a nude mouse xenotransplantation model was used to evaluate the effect of A2ML1 on ESCC tumorigenesis in vivo. The protein level of A2ML1 in ESCC tissues was significantly lower than that in normal esophageal tissues, and higher A2ML1 protein levels were associated with smaller ESCC tumor sizes and improved tumor-specific survival rates. Multivariate analysis established A2ML1 as a novel independent prognostic factor for ESCC. Moreover, A2ML1 overexpression significantly inhibited ESCC cell proliferation and promoted apoptosis. A2ML1 consistently inhibited tumor growth in mouse models. Furthermore, the human apoptotic antibody kit results showed increased expression of the proliferation-inhibiting protein p21 downstream of KYSE150 cells overexpressing A2ML1. Our findings demonstrate that a correlation exists between A2ML1 and ESCC prognosis and that A2ML1 plays an antitumor role in ESCC progression. This study underscores the potential of A2ML1 as a novel biomarker for predicting the prognosis of ESCC.

Comprehensive analysis of cuproptosis genes and cuproptosis-related genes as prognosis factors in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is a common invasive and pernicious cancer with a low five-year survival rate. To identify potential therapeutic targets, we first investigated the characteristics of cuproptosis genes (CUGs) in ESCC. The expression patterns of 10 CUGs (FDX1, LIPT1, LIAS, DLAT, DLD, PDHA1, PDHB, GLS, MTF1, and CDKN2A) were analyzed to identify ESCC-relevant targets. Weighted correlation network analysis (WGCNA) was performed to obtain CUG-related genes (CRGs). A total of seven differentially expressed genes were identified (FDX1, DLAT, LIAS, PDHB, MTF1, GLS, and CDKN2A). DLAT was upregulated in stage III, and LIPT1 was upregulated in N0 + N1 cancers. The high expression of CDKN2A, and PDHA1, was related to better overall survival, whereas the low expression of LIAS was related to better clinical outcomes. WGCNA was performed to get CUG-related genes (CRGs) and showed three key modules that related to FDX1, DLAT, and LIPT1. Moreover, CRGs (BTLA, CT47A1, and PRRX1) were selected to construct a risk score model in order to predict the survival and prognosis of patients with ESCC. Additionally, the cuproptosis score based on CUGs and a nomogram constructed based on it helped accurately predict the prognosis of patients with ESCC; thus, maybe it can be used for the clinical diagnosis of ESCC. The results also showed that milciclib might inhibit the proliferation and migration of KYSE150 and KYSE510 cells by targeting CDKN2A. In conclusion, the abovementioned CUGs and CRGs play a crucial role in tumorigenesis and cancer progression in ESCC, indicating their potential as therapeutic targets.

PLCD3 promotes malignant cell behaviors in esophageal squamous cell carcinoma via the PI3K/AKT/P21 signaling

BackgroundPhospholipase C Delta 3 (PLCD3) is a member of phospholipase C(PLC) Protein and PLCD3 protein plays a prominent role in many cancers. However, little is known about the role of PLCD3 in esophageal squamous cell carcinoma (ESCC).Material and MethodsWe analyzed PLCD3 mRNA and protein expression in ESCC tissues and cell lines by immunohistochemistry, quantitative real-time PCR, and western blot. The correlation between PLCD3 expression and clinicopathological characteristics was also analyzed. CCK8, colony formation, wound-healing, and transwell assays were conducted to measure cell functional alternations. Flow cytometry was performed to assess the apoptosis rate and cell cycle caused by PLCD3 knockdown. Xenograft models in nude mice to clarify the role of PLCD3 in ESCC. Key proteins in the PI3K / AKT signaling pathway after treatment of ECA109 and KYSE150 cells with the AKT inhibitor MK2206 were analyzed by western blot.ResultsPLCD3 was highly expressed in ESCC tissues and cell lines. PLCD3 expression levels correlated with pathologic stage and lymphatic metastasis. PLCD3 knockdown inhibited cell proliferation, migration, invasion, promoted apoptosis, and caused the cell cycle arrest in the G1 phase. PLCD3 overexpression promoted cell proliferation, migration, and invasion. In vivo experiments with xenografts demonstrated that PLCD3 promoted ESCC tumorigenesis. Finally, Overexpression of PLCD3 activated the PI3K / AKT / P21 signaling.ConclusionPLCD3 promotes malignant cell behaviors in esophageal squamous cell carcinoma via the PI3K/AKT/P21 signaling and could serve as a potential target for ESCC treatment.

MUC1 promotes lymph node metastasis in esophageal squamous cell carcinoma by downregulating DNAJB6 expression.

Aberrant expression of MUC1 correlates with the progression of esophageal squamous cell carcinoma (ESCC), this study aimed to explore the effect of targeting MUC1 by Go-203 on malignant behavior of ESCC and the underlying mechanism. IHC was used to examine the expression of MUC1 and DNAJB6 in ESCC samples. qRT-PCR and western blotting were used to examine the expression of MUC1 and DNAJB6 in ESCC cell lines. CCK8, wound healing, and transwell assays were used to determine the effect of regulating MUC1/DNAJB6 on the proliferation, migration, and invasion of ESCC cells. The effect of overexpressing/targeting MUC1 on the activation of the AKT/HSF-1 pathway was determined by western blotting. A negative correlation was confirmed between the expression of DNAJB6 and MUC1 in ESCC tissue samples by IHC, and high expression of MUC1 and low expression of DNAJB6 correlated with lymph node metastasis in ESCC patients. Overexpressing MUC1 downregulated the expression of DNAJB6, promoted ESCC proliferation, invasion, migration and activated the AKT pathway, while targeting MUC1 suppressed proliferation, invasion, migration, and the AKT pathway and up-regulated DNAJB6 expression in vitro. Moreover, MUC1 increased the phosphorylation of HSF-1 via the AKT pathway, and inhibiting AKT-HSF-1 increased the expression of DNAJB6 in vitro. This study indicated that MUC1 could promote tumorigenesis and metastasis in ESCC by downregulating DNAJB6 expression through AKT-HSF-1 pathway.

Preoperative serum bilirubin is an independent prognostic factor for curatively resected esophageal squamous cell carcinoma

PurposeThis study examines prognostic value of preoperative serum bilirubin, including unconjugated bilirubin (UCB), conjugated bilirubin (CB), and total bilirubin (TB), in esophageal squamous cell carcinoma (ESCC) patients who underwent curative resection.MethodsBetween May 2010 and December 2012, a total of 351 ESCC patients were retrospectively reviewed. All the patients underwent curative resection as their primary treatment. Clinicopathological features and overall survival (OS) rate were investigated. Kaplan-Meier curves were used to calculate the OS rate, and the prognostic factors were identified by Cox regression model. Besides, the potential inhibition effect of UCB on ESCC was investigated with both in vitro and in vivo models.ResultsThe higher-level groups of UCB, CB, and TB demonstrated longer OS than their low counterparts, with hazard ratio (HR) values of 0.567 (95% CI: 0.424–0.759), 0.698 (95% CI: 0.522–0.933), and 0.602 (95% CI: 0.449–0.807), respectively. All three forms of bilirubin were identified as independent prognostic factors for patients with ESCC, and they were found to effectively stratify the survival risk of patients at TNM stage III. In vivo and in vitro models further confirmed the inhibitory effect of unconjugated bilirubin (UCB) on the proliferation of ESCC.ConclusionThe findings of our study have shed new light on the prognostic value and biological functions of bilirubin in relation to ESCC. These results may contribute to a better understanding of the underlying mechanisms involved in ESCC tumorigenesis and provide potential therapeutic pathways for treating ESCC.

GSK3β-driven SOX2 overexpression is a targetable vulnerability in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest forms of human malignancy that currently lacks approved targeted therapeutics. Accumulating evidence suggests that SOX2 overexpression is a key driving factor for ESCC and various squamous cell carcinoma. Here, through screening a small-molecule kinase inhibitor library, we identified GSK3β as a kinase that is critically required for robust SOX2 expression in ESCC cells. GSK3β did not promote SOX2 transcriptionally but was required for SOX2 protein stability. We demonstrated that GSK3β interacts with and phosphorylates SOX2 at residue S251, which blocks SOX2 from ubiquitination and proteasome-dependent degradation instigated by ubiquitin E3 ligase CUL4ADET1-COP1. Pharmacological inhibition or knockdown of GSK3β by RNA interference selectively impaired SOX2-positive ESCC cell proliferation, cancer stemness, and tumor growth in mouse xenograft model, suggesting that GSK3β promotes ESCC tumorigenesis primarily by driving SOX2 overexpression. GSK3β was found to be frequently overexpressed in clinical esophageal tumors, and there was a positive correlation between GSK3β and SOX2 protein levels. Notably, we found that SOX2 enhanced GSK3β expression transcriptionally, suggesting the existence of a vicious cycle that drives a coordinated GSK3β and SOX2 overexpression in ESCC cells. Finally, we demonstrated in tumor xenograft model that GSK3β inhibitor AR-A014418 was effective in suppressing SOX2-positive ESCC tumor progression and inhibited tumor progression cooperatively with chemotherapeutic agent carboplatin. In conclusion, we uncovered a novel role for GSK3β in driving SOX2 overexpression and tumorigenesis and provided evidence that targeting GSK3β may hold promise for the treatment of recalcitrant ESCCs.

A variant in CYP26B1 associated with esophageal squamous cell carcinoma risk by affecting retinoic acid metabolism.

All-trans retinoic acid (ATRA) is the natural and synthetic analogue of vitamin A, playing an essential tumor suppressive role in multiple cancers including the esophageal squamous cell carcinoma (ESCC). Cytochrome P450 family 26 subfamily B member 1 (CYP26B1) exerts a critical regulator of ATRA levels through specific inactivation of ATRA to hydroxylated forms. Our previous exome-wide analyses revealed a rare missense variant in CYP26B1 significantly associated with ESCC risk in the Chinese population. However, it is still unclear whether there are common variants in CYP26B1 affect the susceptibility of ESCC and the tumor promotion role of CYP26B1 in vivo. In this research, we conducted a two-stage case-control study comprised of 5057 ESCC cases and 5397 controls, followed by a series of biochemical experiments to explore the function of CYP26B1 and its common variants in the tumorigenesis of ESCC. Intriguingly, we identified a missense variant rs2241057[A>G] in the fourth exon of CYP26B1 significantly associated with the ESCC risk (combined odds ratio = 1.28; 95% confidence interval = 1.15-1.42; p = 2.96 × 10-6 ). Through further functional analysis, we demonstrated that ESCC cells with the overexpression of rs2241057[G] had a significant lower level of retinoic acid, compared with the overexpression of rs2241057[A] or the control vector. In addition, the CYP26B1 overexpression and knock-out ESCC cells affected cell proliferation rate both in vitro and in vivo. These results highlighted the carcinogenicity of CYP26B1 related to the ATRA metabolism in ESCC risk.

MiR-132-3p promotes heat stimulation-induced esophageal squamous cell carcinoma tumorigenesis by targeting KCNK2.

Epidemiological evidence supports that consumption of high-temperature food and beverages is an important risk factor for esophageal squamous cell carcinoma (ESCC);however, the underlying mechanism still remains unclear. Here, we established a series of animal models and found that drinking 65°C water can promote esophageal tumor progression from preneoplastic lesions to ESCC. RNA sequencing data showed that miR-132-3p was highly expressed in the heat stimulation group compared with controls. Further study verified that miR-132-3p were upregulated in human premalignant lesion tissues of the esophagus, ESCC tissues, and cells. Overexpression of miR-132-3p could promote ESCC cell proliferation and colony formation, whereas knockdown of miR-132-3p could inhibit ESCC progression in vitro and in vivo. Importantly, dual-luciferase reporter assays showed that miR-132-3p could bind with the 3'-untranslated regionof KCNK2 and inhibit KCNK2 gene expression. Knockdown or overexpression of KCNK2 could promote or suppress ESCC progression in vitro. These data suggest that heat stimulation can promote ESCC progression and miR-132-3p mediated this process by directly targeting KCNK2.