Human Esophageal Epithelial Cells Research Articles

NEK2 Promotes ESCC Malignant Progression by Inhibiting Cellular Senescence via the FOXM1/c-Myc/p27 Signaling Pathway.

Never in mitosis gene A (NIMA)-related kinase 2 (NEK2) is a crucial serine-threonine kinase involved in the process of cell mitosis. However, the precise relationship between NEK2 and esophageal squamous cell carcinoma (ESCC) remains inadequately understood. NEK2 expression in ESCC tissues was assessed through bioinformatics analysis, reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry, revealing a correlation with ESCC patient prognosis. Cultured ESCC cells and human normal esophageal epithelial cells (HEEC) were used to investigate the effects of NEK2 knockdown on the development and progression of ESCC by integrated confluence algorithm, colony formation, wound-healing, transwell, and ESCC xenograft tumor model, in vitro and in vivo. In ESCC tissues, NEK2 was found to be significantly upregulated, and its expression correlated with poor prognosis in ESCC patients. NEK2 may facilitate ESCC development by regulating cell proliferation, migration, and invasion. Additionally, results from in vivo experiments suggested that NEK2 knockdown can inhibit tumor growth. Moreover, forkhead box M1 (FOXM1) was identified as a potential downstream target of NEK2 in the regulation of ESCC, with its overexpression reversing the effects of NEK2 knockdown on ESCC. Mechanistic studies also indicated that NEK2 may promote the malignant progression of ESCC by inhibiting cellular senescence through the activation of the FOXM1/c-Myc/p27 signaling pathways, which may provide a novel perspective for the management of ESCC.

Chronic exposure to hexavalent chromium induces esophageal tumorigenesis via activating the Notch signaling pathway.

Hexavalent chromium Cr(VI), as a well-established carcinogen, contributes to tumorigenesis for many human cancers, especially respiratory and digestive tumors. However, the potential function and relevant mechanism of Cr(VI) on the initiation of esophageal carcinogenesis are largely unknown. Here, immortalized human esophageal epithelial cells (HEECs) were induced to be malignantly transformed cells, termed HEEC-Cr(VI) cells, via chronic exposure to Cr(VI), which simulates the progress of esophageal tumorigenesis. In vitro and in vivo experiments demonstrated that HEEC-Cr(VI) cells obtain the ability of anchorage-independent growth, greater proliferative capacity, cancer stem cell properties, and the capacity to form subcutaneous xenografts in BALB/c nude mice when compared to their parental cells, HEECs. Additionally, HEEC-Cr(VI) cells exhibited weakened cell motility and enhanced cell adhesion. Interestingly, HEECs with acute exposure to Cr(VI) failed to display those malignant phenotypes of HEEC-Cr(VI) cells, suggesting that Cr(VI)‍-induced malignant transformation, but not Cr(VI) itself, is the cause for the tumor characteristics of HEEC-Cr(VI) cells. Mechanistically, chronic exposure to Cr(VI) induced abnormal activation of Notch signaling, which is crucial to maintaining the capacity for malignant proliferation and stemness of HEEC-Cr(VI) cells. As expected, N-‍[N-‍(3,5-difluorophenacetyl)‍-L-alanyl]‍-S-phenylglycine t-butyl ester (DAPT), an inhibitor for the Notch pathway, drastically attenuated cancerous phenotypes of HEEC-Cr(VI) cells. In conclusion, our study clarified the molecular mechanism underlying Cr(VI)‍-induced esophageal tumorigenesis, which provides novel insights for further basic research and clinical therapeutic strategies about Cr(VI)‍-associated esophageal cancer.

Global Transcriptomic Analysis of Topical Sodium Alginate Protection against Peptic Damage in an In Vitro Model of Treatment-Resistant Gastroesophageal Reflux Disease.

Breakthrough symptoms are thought to occur in roughly half of all gastroesophageal reflux disease (GERD) patients despite maximal acid suppression (proton pump inhibitor, PPI) therapy. Topical alginates have recently been shown to enhance mucosal defense against acid-pepsin insult during GERD. We aimed to examine potential alginate protection of transcriptomic changes in a cell culture model of PPI-recalcitrant GERD. Immortalized normal-derived human esophageal epithelial cells underwent pretreatment with commercial alginate-based anti-reflux medications (Gaviscon Advance or Gaviscon Double Action), a matched-viscosity placebo control, or pH 7.4 buffer (sham) alone for 1 min, followed by exposure to pH 6.0 + pepsin or buffer alone for 3 min. RNA sequencing was conducted, and Ingenuity Pathway Analysis was performed with a false discovery rate of ≤0.01 and absolute fold-change of ≥1.3. Pepsin-acid exposure disrupted gene expressions associated with epithelial barrier function, chromatin structure, carcinogenesis, and inflammation. Alginate formulations demonstrated protection by mitigating these changes and promoting extracellular matrix repair, downregulating proto-oncogenes, and enhancing tumor suppressor expression. These data suggest molecular mechanisms by which alginates provide topical protection against injury during weakly acidic reflux and support a potential role for alginates in the prevention of GERD-related carcinogenesis.

Modeling Epithelial Homeostasis and Perturbation in Three-Dimensional Human Esophageal Organoids.

Background: Esophageal organoids from a variety of pathologies including cancer are grown in Advanced Dulbecco's Modified Eagle Medium-Nutrient Mixture F12 (hereafter ADF). However, the currently available ADF-based formulations are suboptimal for normal human esophageal organoids, limiting the ability to compare normal esophageal organoids with those representing a given disease state. Methods: We have utilized immortalized normal human esophageal epithelial cell (keratinocyte) lines EPC1 and EPC2 and endoscopic normal esophageal biopsies to generate three-dimensional (3D) organoids. To optimize the ADF-based medium, we evaluated the requirement of exogenous epidermal growth factor (EGF) and inhibition of transforming growth factor-(TGF)-β receptor-mediated signaling, both key regulators of the proliferation of human esophageal keratinocytes. We have modeled human esophageal epithelial pathology by stimulating esophageal 3D organoids with interleukin (IL)-13, an inflammatory cytokine, or UAB30, a novel pharmacological activator of retinoic acid signaling. Results: The formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGFβ-receptor-mediated signaling. Optimized HOME0 improved normal human esophageal organoid formation. In the HOME0-grown organoids, IL-13 and UAB30 induced epithelial changes reminiscent of basal cell hyperplasia, a common histopathologic feature in broad esophageal disease conditions including eosinophilic esophagitis. Conclusions: HOME0 allows modeling of the homeostatic differentiation gradient and perturbation of the human esophageal epithelium while permitting a comparison of organoids from mice and other organs grown in ADF-based media.

The role of LncRNA TUG1 in DNA damage and malignant transformation induced by Helicobacter pylori and N-methyl-N′-nitro-N-nitrosoguanidine on human esophageal epithelial cells

N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) and Helicobacter pylori might synergistically promote the malignant transformation of human esophageal epithelial cells (HEECs) through inducing DNA double-strand breaks (DSBs) and inhibition of PAXX protein expression. The long noncoding RNA (lncRNA) TUG1 is associated with multiple cancers, and its overexpression can promote cancer by interfering with the functions of oncogenes. LncRNA TUG1 is also associated with DNA methyltransferase 1 (DNMT1) and the epithelial signaling pathway of H. pylori infection. To explore the role of LncRNA TUG1 in the malignant transformation of HEECs induced by H.pylori + MNNG, a stable strain of HEECs with LncRNA TUG1 knockdown (LncRNA TUG1-KD) was constructed using RNA interference technology with lentivirus as a vector. Set up negative controls LncRNA TUG1-NC (null carrier lentivirus was selected to transfect HEECs) and block controls (normal HEECs without exposure). H. pylori + MNNG were added to the LncRNA TUG1-KD and LncRNA TUG1-NC groups for 6 h and then passaged until their malignant transformation. From each group, cells in the early, intermediate, and late stages of malignant transformation were used for the alkaline comet assay and determination of protein expression, including γ-H2AX and PAXX, by western blotting assay to assess DNA damage and repair processes. Clone formation assay in soft agar and nude mouse xenograft model was used to assess malignancy. This study suggests that H. pylori + MNNG promotes the malignant transformation of HEECs by inducing DNA DSBs and inhibiting PAXX expression, and this effect may be alleviated by LncRNA TUG1 knockdown. It elucidates the pathogenesis of EC from the perspective of the combined effect of epigenetic and environmental carcinogens, offering new insights for the comprehensive prevention and treatment of EC.

KLF5 inhibition initiates epithelial-mesenchymal transition in non-transformed human squamous epithelial cells

The transcriptional regulator Krüppel-like factor 5 (KLF5) is highly expressed in squamous epithelial cells of the esophagus. Increased KLF5 activity induces tumorigenesis and promotes metastasis in several cancers, although this function appears to be context-dependent. Here, we demonstrate that acute KLF5 inhibition, both genetically and with the potent KLF5 inhibitor ML264, causes non-transformed human primary esophageal squamous epithelial cells to enter the epithelial to mesenchymal transition (EMT). Moreover, chronic KLF5 inhibition with ML264 leads to the development of cells with a mesenchymal phenotype characterized by the expression of mesenchymal markers and functionally by reduced cell growth and increased migration and cellular invasion. This EMT resulting from chronic KLF5 inhibition is not driven by β-Catenin or TGF-β signaling. Pharmacologically, ML264 inhibits KLF5 by promoting proteasomal-mediated degradation. Taken together, we demonstrate that reduced KLF5 activity reprograms epithelial cells towards a mesenchymal phenotype and enhances their migratory and invasive potential. These findings have potential implications not only for esophageal cancers but also for normal processes such as esophageal tissue repair following injury.

Abstract 6609: NRF2/ACSS2 axis regulates alcohol-induced metabolic reprogramming in esophageal squamous epithelial cells

Abstract Esophageal diseases such as gastroesophageal reflux disease (GERD) and esophageal cancer have a strong association with alcohol drinking. However, the molecular mechanisms of alcohol-induced esophageal pathology remain unclear. In our previous study, we identified acyl-CoA synthetase short-chain family member 2 (ACSS2) as a transcriptional target of nuclear factor erythroid 2-related factor 2 (NRF2) in the esophageal epithelial cells in vitro and in vivo. Ethanol exposure enhanced de novo lipogenesis in NRF2high cells and generated a more energetic and invasive phenotype. In this study, we aimed to investigate the role of the NRF2/ACSS2 axis in regulating alcohol-induced metabolic reprogramming in esophageal squamous epithelial cells. We first demonstrated that ethanol exposure activated NRF2 signaling and upregulated the expression of NRF2 and ACSS2 in human esophageal squamous epithelial cells. 13C-ethanol/13C-acetate metabolic flux assay showed a dramatic NRF2-dependent increase of labeling of the TCA cycle intermediates and de novo lipogenesis in NRF2high cells in comparison with NRF2null or NRF2low cells. Long-term ethanol exposure caused metabolic reprogramming, in particular induced de novo lipogenesis, in the mouse esophagus according to metabolomics profiling of 833 metabolites. PET/CT and autoradiography showed increased 11C-acetate uptake in the NRF2high esophagus of a conditional tissue-specific mouse (Sox2CreER;LSL-Nrf2E79Q) due to upregulation of transporters and metabolic enzymes. In summary, Our data demonstrated the functional role of the NRF2-ACSS2 axis in alcohol-induced metabolic reprogramming in esophageal squamous epithelial cells both in vitro and in vivo, suggesting NRF2/ACSS2 inhibition as a novel mechanism-based prevention for alcohol-associated esophageal pathology. Citation Format: Zhaohui Xiong, Boopathi Subramaniyan, Chorlada Paiboonrungruang, Yahui Li, Wentao He, Hong Yuan, Guofang Zhang, Xiaoxin Chen. NRF2/ACSS2 axis regulates alcohol-induced metabolic reprogramming in esophageal squamous epithelial cells [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 6609.

LINC00963 Promotes Cisplatin Resistance in Esophageal Squamous Cell Carcinoma by Interacting with miR-10a to Upregulate SKA1 Expression.

Long non-coding RNA (lncRNA) is associated with a large number of tumor cellular functions together with chemotherapy resistance in a variety of tumors. LINC00963 was identified to regulate the malignant progression of various cancers. However, whether LINC00963 affects drug resistence in esophageal squamous cell carcinoma (ESCC) and the relevant molecular mechanisms have never been reported. This study aims to investigate the effect of LINC00963 on cisplatin resistance in ESCC. After detecting the level of LINC00963 in human esophageal squamous epithelial cells (HET-1A), ESCC cells (TE-1) and cisplatin resistant cells of ESCC (TE-1/DDP), TE-1/DDP cell line and nude mouse model that interfered with LINC00963 expression were established. Then, the interaction among LINC00963, miR-10a, and SKA1 was clarified by double luciferase and RNA immunoprecipitation (RIP) assays. Meanwhile, the biological behavior changes of TE-1/DDP cells with miR-10a overexpression or SKA1 silencing were observed by CCK-8, flow cytometry, scratch, Transwell, and colony formation tests. Finally, the biological function of the LINC00963/SKA1 axis was elucidated by rescue experiments. LINC00963 was upregulated in TE-1 and TE-1/DDP cell lines. LINC00963 knockdown inhibited SKA1 expression of both cells and impaired tumorigenicity. Moreover, LINC00963 has a target relationship with miR-10a, and SKA1 is a target gene of miR-10a. MiR-10a overexpression or SKA1 silencing decreased the biological activity of TE-1/DDP cells and the expression of SKA1. Furthermore, SKA1 overexpression reverses the promoting effect of LINC00963 on cisplatin resistance of ESCC. LINC00963 regulates TE-1/DDP cells bioactivity and mediates cisplatin resistance through interacting with miR-10a and upregulating SKA1 expression.

In Human Esophageal Epithelial and Muscle Cells Treated With Th2 Cytokines, Upadacitinib Decreases Eotaxin-3 Secretion and Muscle Tension

In Human Esophageal Epithelial and Muscle Cells Treated With Th2 Cytokines, Upadacitinib Decreases Eotaxin-3 Secretion and Muscle Tension

Mononuclear cell composition and activation in blood and mucosal tissue of eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic, inflammatory, antigen-driven disease of the esophagus. Tissue EoE pathology has previously been extensively characterized by novel transcriptomics and proteomic platforms, however the majority of surface marker determination and screening has been performed in blood due to mucosal tissue size limitations. While eosinophils, CD4+ T cells, mast cells and natural killer (NK) T cells were previously investigated in the context of EoE, an accurate picture of the composition of peripheral blood mononuclear cells (PBMC) and their activation is missing. In this study, we aimed to comprehensively analyze the composition of peripheral blood mononuclear cells and their activation using surface marker measurements with multicolor flow cytometry simultaneously in both blood and mucosal tissue of patients with active EoE, inactive EoE, patients with gastroesophageal reflux disease (GERD) and controls. Moreover, we set out to validate our data in co-cultures of PBMC with human primary esophageal epithelial cells and in a novel inducible mouse model of eosinophilic esophagitis, characterized by extensive IL-33 secretion in the esophagus. Our results indicate that specific PBMC populations are enriched, and that they alter their surface expression of activation markers in mucosal tissue of active EoE. In particular, we observed upregulation of the immunomodulatory molecule CD38 on CD4+ T cells and on myeloid cells in biopsies of active EoE. Moreover, we observed significant upregulation of PD-1 on CD4+ and myeloid cells, which was even more prominent after corticosteroid treatment. With co-culture experiments we could demonstrate that direct cell contact is needed for PD-1 upregulation on CD4+ T cells. Finally, we validated our findings of PD-1 and CD38 upregulation in an inducible mouse model of EoE. Herein we show significant alterations in the PBMC activation profile of patients with active EoE in comparison to inactive EoE, GERD and controls, which could have potential implications for treatment. To our knowledge, this study is the first of its kind expanding the multi-color flow cytometry approach in different patient groups using in vitro and in vivo translational models.

Circ_0003855 involvement of esophageal cancer progression through miR-622/FLOT1.

To confirm the relationship between Circ_0003855 and EC, we purchased the Human esophageal carcinoma cell line Eca109 and normal human esophageal epithelial cells HEEC, and the expression levels of Circ_0003855, miR-622, and FLOT1 were detected. The results show that Circ_0003855 and FLOT1 were highly expressed in Eca109 cells, while miR-622 was lowly expressed (p < 0.05). Subsequently, Circ_0003855 small interfering RNA (si-Circ_0003855) and its negative control (si-NC) were used to detect changes in cellular biological behaviors. We found that the activity of Eca109 cells was reduced after interfering with the expression of Circ_0003855, and miR-622 expression was elevated, while FLOT1 was decreased (p < 0.05). Additionally, si-Circ_0003855 and miR-622 inhibitor sequence (miR-622-inhibition) were co-transfected into cells with miR-622-inhibition alone, and untreated Eca109 cells were used as a control to detect the expression of FLOT1. Co-transfection of si-Circ_0003855 and miR-622-inhibition showed no significant difference in FLOT1 expression compared to the control cells (p > 0.05). Synthesizing the results of these experiments above, we believe that interfering with the expression of Circ_0003855 can inhibit the activity of EC cells, and its mechanism is related to miR-622 and FLOT1.

RAE1 promotes nitrosamine-induced malignant transformation of human esophageal epithelial cells through PPARα-mediated lipid metabolism

Esophageal cancer (EC) is the sixth cause of cancer-related deaths and still is a significant public health problem globally. Nitrosamines exposure represents a major health concern increasing EC risks. Exploring the mechanisms induced by nitrosamines may contribute to the prevention and early detection of EC. However, the mechanism of nitrosamine carcinogenesis remains unclear. Ribonucleic acid export 1 (RAE1), has an important role in mediating diverse cancer types, but, to date, there has been no study for any functional role of RAE1 in esophageal carcinogenesis. Here, we successfully verified the nitrosamine-induced malignant transformation cell (MNNG-M) by xenograft tumor model, based on which it was found that RAE1 was upregulation in the early stage of nitrosamine-induced esophageal carcinogenesis and EC tissues. RAE1 knockdown led to severe blockade in G2/M phase and significant inhibition of proliferation of MNNG-M cells, whereas RAE1 overexpression had the opposite effect. In addition, peroxisome proliferator-activated receptor-alpha (PPARα), was demonstrated as a downstream target gene of RAE1, and its down-regulation reduced lipid accumulation, resulting in causing cells accumulation in the G2/M phase. Mechanistically, we found that RAE1 regulates the lipid metabolism by maintaining the stability of PPARα mRNA. Taken together, our study reveals that RAE1 promotes malignant transformation of human esophageal epithelial cells (Het-1A) by regulating PPARα-mediated lipid metabolism to affect cell cycle progression, and offers a new explanation of the mechanisms underlying esophageal carcinogenesis.

MUC13 promotes the development of esophageal cancer by upregulating the expression of o-glycan process-related molecules

BackgroundEsophageal cancer is one of the most common malignant tumors in the world, which is characterized by poor prognosis, aggressiveness, and poor survival. Mucin 13 (MUC13) is a member of the membrane-bound mucin and located on chromosome 3q21.2 and consists of α and β subunits. It has been found that MUC13 is overexpressed in a variety of tumor cells and acts a vital role in the invasiveness and malignant progression of several types of tumors. However, the role and regulatory mechanism of MUC13 in the progression of esophageal cancer remain unclear.MethodsThe expression level of MUC13 was detected in 15 esophageal cancer tissues and 15 pairs of adjacent nontumor tissues by immunohistochemistry (IHC). In addition, the expression of MUC13 mRNA level in human esophageal cancer cell lines (EC9706 and ECA109 and TE-1) was measured by qRT-PCR. In vitro, after silencing MUC13 with lentiviral interference technology, CCK8 assay, clone formation assay, and flow cytometry were applied to investigate the proliferation activity, clone formation ability and anti-apoptosis ability of EC9706 and ECA109 cells. The tumor xenograft growth assay was used to confirm the influence of MUC13 knockdown on the growth of esophageal tumors in vivo. The qRT-PCR assay and western blot experiments were taken to study the mechanism of MUC13 regulating the proproliferation and antiapoptotic of esophageal cancer.ResultsThe results showed that MUC13 was overexpressed in esophageal cancer tissues and cell lines (EC9706 and ECA109 and TE-1), especially in EC9706 and ECA109 cells, but low expressed in human esophageal epithelial cell line (HEEC). Next, silencing MUC13 inhibits proliferation, blocks cell cycle progression, and promotes cell apoptosis in vitro, and restrains the growth of esophageal cancer tissues in vivo. Finally, MUC13 affects the proproliferation and antiapoptotic by regulating the expression of GLANT14, MUC3A, MUC1, MUC12, and MUC4 that closely related to O-glycan process.ConclusionsThis study proved that MUC13 is an important molecule that regulates the O-glycan process and then affects the progress of esophageal cancer. MUC13 may be a novel therapeutic target for patients with esophageal cancer.

Linc01614 Regulates the Proliferation, Apoptosis, and Chemotherapy Resistance in Esophageal Squamous Cell Carcinoma by Targeting Mir-4775.

Esophageal squamous cell carcinoma (ESCC), a widely known esophageal disease, severely affects people's health. Numerous investigations demonstrated that long non-coding RNAs (lncRNAs) performed key jobs inside a wide scope of organic cycles and stand out in malignant growth. Our study planned to investigate the roles and mechanisms of linc01614 in ESCC. A Total of 60 ESCC tissue samples including 30 patients with cisplatin sensitivity and 30 patients with cisplatin resistance, who received DDP-based treatment, were obtained from Zhuhai People's Hospital, Zhuhai City during 2021. These tissues were frozen and saved in a -80 °C ultra-low temperature freezer. We performed CCK-8, clone formation, flow cytometry assays to determine the effect of linc01614 on ESCC progression, and explored the specific mechanism of linc01614 in ESCC cell proliferation, apoptosis, and chemotherapy resistance. linc01614 expression was upregulated in ESCC tissues and cells compared with non-tumor tissues and human normal esophageal epithelial cells (Het-1A). Knockdown of linc01614 repressed cell expansion, chemotherapy opposition, and advanced cell apoptosis in ESCC. Besides, linc01614 regulated the expression of miR-4775 as a competitive endogenous RNA (ceRNA). The linc01614/miR-4775 axis played an important role in ESCC progression and drug resistance, revealing that linc01614 is a promising target in ESCC treatment.

The Regulation of Exosome-Mediated miR-132-3p/miR-132-3p-UUU on Radiation-Induced Esophageal Injury.

Radiation-induced esophageal injury (RIEI) is a major dose-limiting complication of radiotherapy, mainly acute esophagitis. However, understanding of radiation injury and repair mechanisms in esophageal epithelial cells remains limited. MiR-132-3p and its uridylated isoform (miR-132-3p-UUU) are upregulated in radiation esophageal injury, yet their role in radiation-induced esophageal injury progression remains unexplored. We expressed miR-132-3p and its uridine form in irradiated human esophageal epithelial cells (HEEC) and secreted exosomes was examined by real-time polymerase chain reaction (RT-PCR). Cell proliferation, migration, apoptosis and colony formation were used to determine biological effects. Cell cycle assays and dual luciferase reporter assays were used to assess the relationship between miR-132-3p and its uridylated isoforms and MEF2A. The addition of miR-132-3p mimics or overexpression of miR-132-3p significantly inhibited the proliferation and migration of esophageal epithelial cells (HEEC cells as well as primary cells) and increased radiation damage. This was reversed by its uridylated isoform by reducing binding to MEF2A and regulating the cell cycle. Furthermore, miR-132-3p and its triuridylated isomer also regulate apoptosis after irradiation through pathways other than reactive oxygen species (ROS). In conclusion, our data reveal that radiation-induced miR-132-3p uridylation and exosome-mediated intercellular communication and tri-uridylated isoforms are protective against radiation-induced esophageal injury. Furthermore, miR-132-3p offers new opportunities as a promising biomarker widely present in human body fluids for the prediction of radiation esophagitis as a biomarker.

NEK2 promotes esophageal squamous cell carcinoma cell proliferation, migration and invasion through the Wnt/β-catenin signaling pathway

ObjectivesThe NEK2 (never in mitosis gene A-related kinase 2), a serine/threonine kinase involved in chromosome instability and tumorigenesis. Hence, this study aimed to explore the molecular function of NEK2 in esophageal squamous cell carcinoma (ESCC).MethodsBy available transcriptome datasets (GSE53625 cohort, GSE38129 cohort, and GSE21293 cohort), we analyzed the differentially expressed genes in invading and non-invading ESCC. Subsequently, we evaluated the association between NEK2 expression level and clinical outcomes through Kaplan–Meier analysis method. The quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB) analyses were performed to determine the expression levels of NEK2 mRNA and protein, respectively. We knocked down the NEK2 expression in ESCC cells (ECA109 and TE1), and evaluated the NEK2 biology function associated with ESCC cell proliferation, migration, invasion, and colony formation abilities. Finally, the downstream pathway of NEK2 was analyzed through Gene Set Enrichment Analysis (GSEA) and validated the regulatory mechanism of NEK2 on the potential pathway through WB.ResultsWe found that NEK2 was highly expressed in ESCC cells compared with human esophageal epithelial cells (HEEC) (P < 0.0001), and high NEK2 expression was remarkably associated with poor survival (P = 0.019). Knockdown of NEK2 showed the significant inhibitory effect for tumorigenesis, and suppressed the ESCC cells proliferation, migration, invasion, and formation of colonies abilities. Additionally, GSEA revealed that Wnt/β-catenin pathway was a downstream pathway of NEK2. WB results further validated the regulatory mechanism of NEK2 for Wnt/β-catenin signaling.ConclusionsOur results indicated that NEK2 promotes ESCC cell proliferation, migration and invasion by activating the Wnt/β-catenin pathway. NEK2 could be a promising target for ESCC.

Direct and indirect effects of alcohol and its toxic metabolite acetaldehyde on human esophageal myofibroblasts and epithelial cells.

Mechanisms by which alcohol increases the risk of esophageal squamous cell carcinoma remain undefined. Human esophageal myofibroblasts (HEMFs) subjacent to the squamous epithelium are exposed directly to these agents via epithelial barrier defects and indirectly via factors derived from the exposed epithelium. Our aim was to investigate the cellular biology of HEMFs and HEMF-esophageal epithelial cell interactions in response to alcohol and its toxic metabolite acetaldehyde. An immortalized HEMF and a human esophageal epithelial cell line (Epi) were treated with alcohol (0 to 200 mM) or acetaldehyde (0 to 100 μM) in a cyclic fashion or incubated with supernatants collected from treated cells. Healthy cell %, reactive oxygen species (ROS), and proliferation were assessed via flow cytometry, luminescence, scratch wound, and colorimetric assays, respectively. A 15-plex multiplex assay was performed on cell supernatants, followed by IL-6 and IL-8 qRT-PCR and ELISA. Healthy HEMF decreased to less than 80% at 30 mM alcohol and 70 μM acetaldehyde, with microscopic changes at 40 μM acetaldehyde. HEMF ROS was detected at 100 mM alcohol and 80 μM acetaldehyde. Supernatants from 30 mM alcohol- or 40 μM acetaldehyde-treated HEMFs increased Epi proliferation more than two-fold that of lower doses. In the complementary studies, healthy Epi cells decreased to less than 80% at 50 mM and 70 μM acetaldehyde, with microscopic changes at 40 μM. Supernatants from Epi treated with 50 mM alcohol or 40 μM acetaldehyde increased HEMF proliferation more than two-fold that of lower doses. A multiplex assay of supernatants showed the greatest increase in concentrations of IL-6 and IL-8 in HEMFs and in Epi treated with higher doses of alcohol or acetaldehyde. Neutralization of IL-6 and IL-8 in supernatants of HEMFS and esophageal epithelial cells inhibited the proliferation of Epi and HEMFs, respectively. Alcohol and acetaldehyde doses in which the majority of HEMFs and epithelial cells are healthy, elicit the production of paracrine mediators with pro-proliferative effects on neighboring cells. Understanding the effect of alcohol and acetaldehyde on HEMFs and HEMF-epithelial interactions could help to identify the molecular basis by which alcohol increases the risk for esophageal cancer.

Helicobacter pylori plus N-Methyl-N'-nitro-N-nitrosoguanidine: DNA damage and repair: Malignant transformation of human esophageal epithelial cells.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG), found in pickled foods and in chlorinated water, has been used to induce malignant transformation and gastrointestinal cancer in rats. Helicobacter pylori (HP) is implicated in human gastric cancer and possibly also in esophageal cancer. These two agents - one chemical and the other biological - might act together to induce esophageal cancer. In this study, human esophageal epithelial cells (HEECs) were divided into four groups: HP, MNNG, HP +MNNG, and control. The HP-to-HEEC ratio was 100:1. Cells were exposed for 6h and then passaged until malignant transformation. HEEC at early, intermediate, and late stages of malignant transformation were used for proliferation, cell-cycle, and invasion assays. The alkaline comet assay was performed and expression of proteins, including γ-H2AX and PAXX, was studied by western blotting, to explore DNA damage and repair processes. Measurements of cell morphology, soft-agar clone formation, and invasiveness, and a nude mouse xenograft model, were used to examine malignancy. The effect of HP was stronger than that of MNNG. The combination HP +MNNG exerted a stronger malignant transformation effect than either HP or MNNG alone. Mechanisms of this combined carcinogenesis may include promotion of cell proliferation, perturbation of the cell cycle, promotion of invasiveness, DNA double-strand break induction, or PAXX inhibition.

Research on essential performance of oxidized chitosan-crosslinked acellular porcine aorta modified with bioactive SCPP/DOPA for esophageal scaffold with enhanced mechanical strength, biocompatibility and anti-inflammatory

Acellular porcine aorta (APA) is an excellent candidate for an implanted scaffold but needs to be modified with appropriate cross-linking agent to increase its mechanical property and storage time in vitro as well as to give itself some bioactivities and eliminate its antigenicity for acting as a novel esophageal prosthesis. In this paper, a polysaccharide crosslinker (oxidized chitosan, OCS) was prepared by oxidizing chitosan using NaIO4 and further used to fix APA to prepare a novel esophageal prosthesis (scaffold). And then the surface modification with dopamine (DOPA) and strontium-doped calcium polyphosphate (SCPP) were performed one after another to prepare DOPA/OCS-APA and SCPP-DOPA/OCS-APA to improve the biocompatibility and inhibit inflammation of the scaffolds. The results showed that the OCS with a feeding ratio of 1.5:1.0 and a reaction time of 24 h had a suitable molecular weight and oxidation degree, almost no cytotoxicity and good cross-linking effect. Compared with glutaraldehyde (GA) and genipin (GP), OCS-fixed APA could provide a more suitable microenvironment for cell proliferation. The vital cross-linking characteristics and cytocompatibility of SCPP-DOPA/OCS-APA were evaluated. Results suggested that SCPP-DOPA/OCS-APA exhibited suitable mechanical properties, excellent resistance to enzymatic degradation/acid degradation, suitable hydrophilicity, and the ability to promote the proliferation of Human normal esophageal epithelial cells (HEECs) and inhibit inflammation in vitro. In vivo tests also confirmed that SCPP-DOPA/OCS-APA could diminish the immunological response to samples and had a positive impact on bioactivity and anti-inflammatory. In conclusion, SCPP-DOPA/OCS-APA could act as an effective, bioactive artificial esophageal scaffold and be expected to be used for clinical in the future.

Abstract 2594: Alcohol drinking inhibits NOTCH-PAX9 signaling in esophageal squamous epithelial cells

Abstract Alcohol drinking has been established as a major risk factor for esophageal diseases, such as gastroesophageal reflux disease (GERD) and esophageal squamous cell carcinoma (ESCC). Certain mechanisms of alcohol-induced esophageal injury are supported by experimental data, whereas most are speculative or extrapolated from studies on cancers of other organ sites. Our previous study showed that ethanol exposure inhibited PAX9 expression in human esophageal squamous epithelial cells in vitro and in vivo, and promoted ESCC. In this study, we aimed to investigate the molecular pathways through which alcohol drinking suppresses PAX9 in esophageal squamous epithelial cells. Gene microarray, qRT-PCR, ChIP-PCR, Western blotting, and immunostaining were used to identify and validate inhibition of NOTCH-PAX9 signaling in human esophageal squamous epithelial cells and mouse esophagus after ethanol exposure. Human tissue samples were analyzed to further validate NOTCH inhibition by alcohol drinking. We first demonstrated the inhibition of NOTCH by ethanol exposure in vitro. NOTCH regulated PAX9 expression in esophageal squamous epithelial cells in vitro and in vivo. RBPJ and NOTCH intracellular domain (NICD1) ChIP-PCR confirmed Pax9 as a direct downstream target of NOTCH signaling in mouse esophagus. NOTCH inhibition by alcohol drinking was further validated in mouse esophagus and human tissue samples. In conclusion, ethanol exposure inhibited NOTCH signaling and thus suppressed PAX9 expression in esophageal squamous epithelial cells in vitro and in vivo. Our data support a novel mechanism of alcohol-induced esophageal injury through the inhibition of NOTCH-PAX9 signaling. As a result, chemical NOTCH activators may be used to prevent or treat alcohol-associated esophageal diseases such as GERD and ESCC. Citation Format: Zhaohui Xiong. Alcohol drinking inhibits NOTCH-PAX9 signaling in esophageal squamous epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2594.