Esophageal Adenocarcinoma Cell Lines Research Articles

Human plasma can modulate micronucleus frequency in TK6 and OE33 cells in vitro

In this paper, we studied the potential genotoxic effects of human plasma from healthy volunteers, as well as patients with gastro-oesophageal reflux disease, Barrett’s oesophagus (BO) and oesophageal adenocarcinoma (OAC) using the oesophageal adenocarcinoma cell line (OE33) and the lymphoblastoid cell line (TK6). Both TK6 and OE33 cells were treated with plasma (10 % volume, replacing foetal bovine serum (FBS) or horse serum (HS)) at different time points of 4 h (for the micronucleus (Mn) assay and the invasion assay) and 24 h (for the cell cycle studies). Plasma-induced effects on DNA damage levels, cell viability and the cell cycle were studied by the micronucleus assay, cytokinesis block proliferation index (CBPI) and flow cytometry respectively. The expression of IL-8 in supernatants of TK6 cells and IFN-β in OE33 cells was also analysed by enzyme-linked immunosorbent assay (ELISA). Finally, we carried out an assessment of cellular invasion of OE33 cells following plasma treatment.The results of the micronucleus assay confirmed the genotoxicity of direct plasma treatment from some participants through the increase in DNA damage in TK6 cells. Conversely, some individual patient plasma samples reduced background levels of TK6 cell Mn frequency, in an anti-genotoxic fashion. In TK6 cells, (on average) plasma samples from patients with Barrett’s oesophagus induced higher micronucleus levels than healthy volunteers (p= 0.0019). There was little difference in Mn induction when using plasma versus serum to treat the cells in vitro. Cell cycle results showed that direct plasma treatment had a marked impact on OE33 cells at 24 h (p=0.0182 for BO and p=0.0320 for OAC) by decreasing the proportion of cells in the S phase, while plasma exposure was less impactful on the cell cycle of TK6 cells. Invasion of OE33 cells was also seen to be non-significantly affected by plasma treatment of OE33 cells.The addition of N-acetyl cysteine NAC in a dose-dependent matter did not alter the formation of Mn in TK6 cells, suggesting that reactive oxygen species (ROS) are not the root cause of plasma’s genotoxicity. The concentration of IL-8 in TK6 cells and IFN-β in OE33 cells was significantly higher in cells treated with OAC-derived plasma than in the untreated negative control. Collectively, our results demonstrate that plasma-specific effects are detectable which helps us better understand some important aspects of the biology of blood-based biomarkers under development.

Abstract 555: Role of TRIM31 in the pathogenesis of esophageal adenocarcinoma

Abstract Background: Esophageal adenocarcinoma (EAC) is one the deadliest cancers in United States (US) and the only major cancer in the US with increasing incidence. Gastroesophageal reflux disease (GERD) can cause Barrett’s esophagus (BE), a columnar metaplasia of the esophagus strongly associated with the development of EAC. GERD leading to BE is a common pre-occurrence in EAC patients, but the mechanism remains obscure. To explore the mechanism and its components, we compared gene expression in BE and EAC cells with normal esophageal cells and found high expression of tripartite motif-containing protein 31 (TRIM31) in BE and EAC compared to normal esophagus (NE) by transcriptomic profiling of RNA extracted from human esophageal tissues. E3 ubiquitin ligase TRIM 31 is a new member of TRIM family and it has been reported to have oncogenic potentials in some cancers. However, its role in EAC pathogenesis is yet to be understood. Methods: RNA sequencing and transcriptomic profiling were performed on human NE, BE, and EAC epithelial tissue samples. TRIM31 expression in NE cell line (Het-1A) and EAC cell lines (OE19, Flo-1, OE33, SK-GT-2, and OACM5.1C) were identified by Western blot analysis. The Het-1A cell line, after exposure to acidic pH and bile acids, was assessed for variable TRIM31 expression. Cell morphology and viability analyses of NE and EAC cell lines after exposure to acidic pH and bile acids either alone or in combinations were observed by microscopy and WST-1 assay respectively. Results: Transcriptomic analysis showed that BE and EAC are similar in their differential gene expression with upregulation of the TRIM31 compared to that in NE. Concordant with their columnar phenotype, BE and EAC groups displayed a multitude of differentially expressed genes relative to NE samples; however, less differentially expressed genes were found comparing BE and EAC groups. Western blot demonstrated frequent overexpression of TRIM31 in EAC cell lines with very low expression of TRIM31 in NE epithelial cell line. When NE epithelial cell line Het-1A was exposed to acidic pH as well as bile acids in acidic pH, we saw a change in cell morphology that may correlate with the metaplasia of these cells to more resilient forms, as seen in human BE and EAC. Interestingly, bile acids in acidic pH increased TRIM31 expression in NE cell line Het-1A. Bile acids had a greater effect on decreasing cell viability at high concentrations and that effect is enhanced when bile acids were added in acidic pH. WST-1 cell viability assay revealed that EAC cells were more resistant to bile acids as well as bile acids in acidic pH induced decreased in cell viability compared to that in NE cell line Het-1A. The sustained cell viability of EAC cell lines under these harsh conditions correlates with the upregulation of TRIM31. Conclusions: Thus, TRIM31 may be an important oncogenic factor in GERD-induced EAC development and may be an innovative therapeutic target and marker for EAC. Citation Format: Md Sazzad Hassan, Jesse Mast, Annie Ritter, Saisantosh Ponna, Niranjan Awasthi, Urs von Holzen. Role of TRIM31 in the pathogenesis of esophageal adenocarcinoma [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 555.

Abstract 3269: APE1/NF-κB/PRDX2 activated by reflux conditions mediates chemoresistance in esophageal adenocarcinoma by suppressing ferroptosis

Abstract Background: Esophageal adenocarcinoma (EAC) stands out as one of the most lethal malignancies, characterized by a discouragingly low 5-year survival rate of less than 20%. Accumulating lines of evidence indicate that ferroptosis, a novel type of cell death, can regulate drug resistance. Peroxiredoxin 2 (PRDX2), an antioxidant enzyme, plays an antioxidant protective role in the cells under stress by regulating redox homeostasis. We aimed to elucidate the role of PRDX2 in regulating reactive oxygen species (ROS), ferroptosis, and chemoresistance in EAC. Methods: In this investigation, we analyzed RNA sequencing data and public databases to uncover the aberrant overexpression and potential chemoresistance function of PRDX2 in EAC. We employed transient acidic bile salts exposure (ABS) and repeated ABS exposure (rABS) to mimic gastroesophageal reflux disease, the main risk factor for EAC, in in vitro cell models. EAC cells and 3D organotypic culture (OTC) served as platforms to delve into the anti-ferroptosis function and regulatory mechanisms of PRDX2. The findings were validated using patient-derived xenografts (PDX) and human EAC tissue microarrays (TMA). Results: Elevated PRDX2 level was observed in both EAC patients and cell lines exhibiting poor responsiveness to chemotherapy. Intrinsic overexpression of PRDX2 emerged as a crucial factor in the recovery from ABS-exposure-induced lipid peroxidation. Silencing PRDX2 heightened the sensitivity of EAC cells to Oxaliplatin by inhibiting ferroptosis in a GPX4-dependent manner. Mechanistically, we found ABS activate the APE1/redox/NF-kB signal axis, leading to the upregulation of PRDX2 transcription. The co-overexpression of APE1 and PRDX2 was identified through both immunofluorescent staining in cells/OTC and immunohistochemistry staining in TMA. Moreover, the APE1-redox-specific inhibitor, APX2009, significantly sensitized EAC cells to Oxaliplatin by repressing PRDX2 and inducing ferroptosis. Remarkably, the combination therapy of Oxaliplatin and APX2009 achieved a synergistic effect in EAC-PDX tumors. Conclusion: The study unveils a novel signaling axis, APE1/NF-kB/PRDX2, linking disease-associated ROS unbalance and ferroptosis-related chemoresistance. Targeting PRDX2 signaling by APE1-redox-specific inhibition provides a potential novel strategy for combined chemotherapies in refractory EAC. Citation Format: Lei Chen, Heng Lu, Farah Ballout, Dunfa Peng, Zheng Chen, Jianwen Que, Steven Chen, Oliver McDonald, Alexander Zaika, Wael El-Rifai. APE1/NF-κB/PRDX2 activated by reflux conditions mediates chemoresistance in esophageal adenocarcinoma by suppressing ferroptosis [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 3269.

Abstract 2177: Postbiotic metabolites secreted by L. acidophilus reduce DNA damage in an experimental model of Barrett’s tumorigenesis

Abstract The origins of esophageal adenocarcinoma (EAC) are commonly attributed to the progression from gastroesophageal reflux disease (GERD) to Barrett’s Esophagus (BE) and finally to esophageal adenocarcinoma. During GERD, various types of bile induce oxidative stress and generate reactive oxygen species (ROS), resulting in DNA damage and inflammation in cells. Although there has been much research documenting the shift in the esophageal microbiome from gram-positive to gram-negative bacteria during this progression, there has also been an observed increase in the presence of Lactobacillus, a gram-positive commensal bacterium with probiotic qualities, in the esophagus during the progression from GERD to BE and eventually to EAC. Our previous research has demonstrated the ability of Lactobacillus spp. to not only colonize the esophagus under in vitro conditions of GERD, but also their anti-inflammatory and anti-genotoxic properties on esophageal cells exposed to bile. To assess the extent of reflux-induced DNA damage and oxidative stress during the progression of BE, we exposed two BE cell lines, CP-B and BAR-T, and the EAC cell line FLO-1 to ox bile or deoxycholic acid. Both bile types demonstrated significant increases in phosphorylated H2AX, as a marker for pronounced DNA damage repair. Next, we supplemented both live L. acidophilus and its postbiotic metabolites to cells exposed to ox bile and deoxycholic acid. We show a significant reduction in DNA damage as measured by pH2AX with both the live treatment and the treatment with postbiotic metabolites when compared to untreated, bile-exposed cells at various stages of BE and EAC. Ongoing experiments include assessing anti-genotoxic effects of postbiotic metabolites through DNA repair protein RAD51, reductions in oxidative stress though ROS marker 8-oxo-guanine, and anti-inflammatory effects through NFkB-associated inflammation. BE and EAC cell viability and proliferation upon bile exposure and postbiotic metabolite administration will also be assessed. This study aims to determine the supplementation with Lactobacillus postbiotic metabolites in the prevention and intervention of esophageal adenocarcinoma development. Citation Format: Divya Joshi, Claudia Andl. Postbiotic metabolites secreted by L. acidophilus reduce DNA damage in an experimental model of Barrett’s tumorigenesis [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 2177.

Abstract 4559: SMAD3 mediates oxaliplatin resistance in esophageal adenocarcinoma

Abstract Background: Resistance to conventional treatments is a significant barrier to esophageal adenocarcinoma (EAC) therapy. TGF-β signaling appears to be important for EAC tumorigenesis; however, its role in EAC chemoresistance remains understudied. This study investigates the role of SMAD3 in EAC resistance to the frontline chemotherapeutic drug oxaliplatin. Methods: Publicly available datasets were analyzed to determine SMAD3 expression in EAC versus normal samples. Western blot, quantitative real-time polymerase chain reaction, immunofluorescence staining, and flow cytometric analyses were utilized to validate bioinformatic results. Human EAC cell lines (OE33 and OE19) were treated with pharmacologic inhibitors or transfected with small interfering RNA and assessed for DNA damage and apoptotic cell death. Patient-derived organoids and patient-derived xenograft models of EAC were used to confirm our findings in vivo. Results: SMAD3 was significantly upregulated in EAC versus normal tissue samples. Total and phosphorylated SMAD3 protein levels were upregulated in EAC tissues accompanied by increased expression of several genes regulated by SMAD3. Notably, oxaliplatin-resistant EAC cells exhibited overexpression of SMAD3. Chemotherapy non-responding patients showed enrichment of SMAD3 gene expression when compared to responders in the clinical dataset GSE165252. Knockdown or inhibition of SMAD3 hindered DNA repair and sensitized EAC cells to oxaliplatin treatment in vitro and in vivo. Mechanistically, SMAD3 promoted ATM phosphorylation by interacting with protein phosphatase 2A (PP2A) and sequentially inhibiting the binding of PP2A to ATM. Conclusion: Our results reveal a pivotal role for SMAD3 in mediating oxaliplatin resistance in EAC and thus hold a significant potential in guiding future drug development and combination strategies. Citation Format: Farah Ballout, Heng Lu, Nadeem Bhat, Lei Chen, Dunfa Peng, Zheng Chen, Alexander Zaika, Oliver McDonald, Wael El-Rifai. SMAD3 mediates oxaliplatin resistance in esophageal adenocarcinoma [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 4559.

High LINC00626 expression promotes esophagogastric junction adenocarcinoma metastasis: the mediating role of the JAK1/STAT3/KHSRP axis

To investigate the role of JAK1/STAT3/KHSRP axis in mediating the regulatory effect of LINC00626 on progression of esophagogastric junction adenocarcinoma. We collected surgical tumor and adjacent tissue specimens from 64 patients with esophagogastric junction adenocarcinoma and examined the expression levels of LINC00626 and KHSRP. qRT-PCR was used to detect the expressions of LINC00626 and KHSRP in 6 esophageal adenocarcinoma cell lines (OE-19, TE-7, Bic-1, Flo-1, SK-GT-4, and BE-3) and a normal esophageal epithelial cell line (HET-1A). OE-19 and TE-7 cell lines with stable LINC00626 knockdown and FLO-1 and SK-GT-4 cells stably overexpressing LINC00626 were constructed by lentiviral transfection, and the changes in proliferation, migration and invasion of the cells were evaluated using Cell Counting Kit-8 (CCK-8) assay and Transwell migration/invasion assay. The expressions of KHSRP and JAK/STAT pathway proteins in the transfected cells were detected with Western blotting. The effects of LINC006266 knockdown and overexpression on subcutaneous tumor formation and lung metastasis of OE-19 and FLO-1 cell xenografts were tested in nude mice. The expression levels of LINC00626 and KHSRP were significantly increased in esophagogastric junction adenocarcinoma tissues and in esophageal adenocarcinoma cells. LINC00626 knockdown obviously inhibited the proliferation, migration and invasion of esophageal adenocarcinoma cells in vitro and decreased their tumor formation and lung metastasis abilities in nude mice, while overexpression of LINC00626 produced the opposite effects. In esophageal adenocarcinoma cells, LINC0626 knockdown significantly decreased and LINC00626 overexpression strongly enhanced the phosphorylation of JAK1 and STAT3. High LINC00626 expression promotes esophageal-gastric junction adenocarcinoma metastasis by activating the JAK1/STAT3/KHSRP signal axis.

Epigenomic analyses identify FOXM1 as a key regulator of anti-tumor immune response in esophageal adenocarcinoma

Unlike most cancer types, the incidence of esophageal adenocarcinoma (EAC) has rapidly escalated in the western world over recent decades. Using whole genome bisulfite sequencing (WGBS), we identify the transcription factor (TF) FOXM1 as an important epigenetic regulator of EAC. FOXM1 plays a critical role in cellular proliferation and tumor growth in EAC patient-derived organoids and cell line models. We identify ERBB2 as an upstream regulator of the expression and transcriptional activity of FOXM1. Unexpectedly, gene set enrichment analysis (GSEA) unbiased screen reveals a prominent anti-correlation between FOXM1 and immune response pathways. Indeed, syngeneic mouse models show that FOXM1 inhibits the infiltration of CD8+ T cells into the tumor microenvironment. Consistently, FOXM1 suppresses CD8+ T cell chemotaxis in vitro and antigen-dependent CD8+ T cell killing. This study characterizes FOXM1 as a significant EAC-promoting TF and elucidates its novel function in regulating anti-tumor immune response.

On-chip Raman spectroscopy of live single cells for the staging of oesophageal adenocarcinoma progression.

The absence of early diagnosis contributes to oesophageal cancer being the sixth most common cause of global cancer-associated deaths, with a 5-year survival rate of < 20%. Barrett's oesophagus is the main pre-cancerous condition to adenocarcinoma development, characterised by the morphological transition of oesophageal squamous epithelium to metaplastic columnar epithelium. Early tracking and treatment of oesophageal adenocarcinoma could dramatically improve with diagnosis and monitoring of patients with Barrett's Oesophagus. Current diagnostic methods involve invasive techniques such as endoscopies and, with only a few identified biomarkers of disease progression, the detection of oesophageal adenocarcinoma is costly and challenging. In this work, single-cell Raman spectroscopy was combined with microfluidic techniques to characterise the development of oesophageal adenocarcinoma through the progression of healthy epithelial, Barrett's oesophagus and oesophageal adenocarcinoma cell lines. Principal component analysis and linear discriminant analysis were used to classify the different stages of cancer progression. with the ability to differentiate between healthy and cancerous cells with an accuracy of 97%. Whilst the approach could also separate the dysplastic stages from healthy or cancer with high accuracy-the intra-class separation was approximately 68%. Overall, these results highlight the potential for rapid and reliable diagnostic/prognostic screening of Barrett's Oesophagus patients.

TRIM31: A Protein With an Oncogenic Role in Esophageal Adenocarcinoma

Background: Esophageal adenocarcinoma (EAC) is a major cancer in the United States with increasing incidence. It is an aggressive cancer involving columnar-type cells different from the normal esophageal (NE) squamous cells. This metaplasia often involves an intermediary morphology called Barrett’s esophagus (BE), which occurs from repeated acid exposure of the esophagus from gastroesophageal reflux disease (GERD). GERD leading to BE is a common pre-occurrence in EAC patients, but the mechanism remains obscure. To explore the mechanism and its components, we compared gene expression in BE and EAC cells with normal cells and discovered the overexpression of TRIM31 in the pathogenic cells. Although previous studies have shown oncogenic potential of TRIM31 in some cancers, its role in EAC is yet to be understood.&#x0D; Methods: RNA sequencing and transcriptomic profiling were performed on human NE, BE, and EAC epithelial tissue samples. TRIM31 expression in NE cell line (Het-1A) and EAC cell lines (OE19, Flo-1, OE33, SK-GT-2, and OACM5.1C) were identified by Western blot. The Het-1A cell line, after exposure to acidic pH and bile acid, was assessed for variable TRIM31 expression. Cell viability analysis of NE and EAC cell lines after exposure to acidic pH and bile acids was observed by WST-1 assay.&#x0D; Results: RNA sequencing, transcriptomic profiling, and western blot revealed overexpression of TRIM31 in BE and EAC epithelium. Exposure of Het-1A cells to bile acids in acidic pH changed the cell morphology with enhanced expression of TRIM31. WST-1 revealed that EAC cells were more resistant to acidic pH and bile acid exposure.&#x0D; Conclusions and potential impact: Our data suggests that increased TRIM31 expression correlates with esophageal epithelium resistance when exposed to bile acids and acidic pH. Consequently, TRIM31 may be a key player in the metaplasia of GERD-induced EAC development and may be an innovative therapeutic target and marker for EAC.

Differential Expression of NEK Kinase Family Members in Esophageal Adenocarcinoma and Barrett's Esophagus.

The incidence of esophageal adenocarcinoma (EAC) has risen rapidly during the past four decades, making it the most common type of esophageal cancer in the USA and Western countries. The NEK (Never in mitosis A (NIMA) related kinase) gene family is a group of serine/threonine kinases with 11 members. Aberrant expression of NEKs has been recently found in a variety of human cancers and plays important roles in tumorigenesis, progression, and drug-resistance. However, the expression of the NEKs in EAC and its precancerous condition (Barrett's esophagus, BE) has not been investigated. In the present study, we first analyzed the TCGA and 9 GEO databases (a total of 10 databases in which 8 contain EAC and 6 contain BE) using bioinformatic approaches for NEKs expression in EAC and BE. We identified that several NEK members, such as NEK2 (7/8), NEK3 (6/8), and NEK6 (6/8), were significantly upregulated in EAC as compared to normal esophagus samples. Alternatively, NEK1 was downregulated in EAC as compared to the normal esophagus. On the contrary, genomic alterations of these NEKs are not frequent in EAC. We validated the above findings using qRT-PCR and the protein expression of NEKs in EAC cell lines using Western blotting and in primary EAC tissues using immunohistochemistry and immunofluorescence. Our data suggest that frequent upregulation of NEK2, NEK3, and NEK7 may be important in EAC.

Cancer Stem Cells of Esophageal Adenocarcinoma are Suppressed by Inhibitors of TRPV2 and SLC12A2.

The potential of membrane transporters activated in cancer stem cells (CSCs) as new therapeutic targets for cancer is attracting increasing interest. Therefore, the present study examined the expression profiles of ion transport-related molecules in the CSCs of esophageal adenocarcinoma (EAC). Cells that highly expressed aldehyde dehydrogenase 1 family member A1 (ALDH1A1) were separated from OE33 cells, a human Barrett's EAC cell line, by fluorescence-activated cell sorting. CSCs were identified based on the formation of tumorspheres. Gene expression profiles in CSCs were examined by a microarray analysis. Among OE33 cells, ALDH1A1 messenger RNA levels were higher in CSCs than in non-CSCs. Furthermore, CSCs exhibited resistance to cisplatin and had the capacity to redifferentiate. The results of the microarray analysis of CSCs showed the up-regulated expression of several genes related to ion channels/transporters, such as transient receptor potential vanilloid 2 (TRPV2) and solute carrier family 12 member 2 (SLC12A2). The cytotoxicities of the TRPV2 inhibitor tranilast and the SLC12A2 inhibitor furosemide were higher at lower concentrations in CSCs than in non-CSCs, and both markedly reduced the number of tumorspheres. The cell population among OE33 cells that highly expressed ALDH1A1 also was significantly decreased by these inhibitors. Based on the present results, TRPV2 and SLC12A2 are involved in the maintenance of CSCs, and their specific inhibitors, tranilast and furosemide, respectively, have potential as targeted therapeutic agents for EAC.

Elevated APE1 Dysregulates Homologous Recombination and Cell Cycle Driving Genomic Evolution, Tumorigenesis, and Chemoresistance in Esophageal Adenocarcinoma

The purpose of this study was to identify drivers of genomic evolution in esophageal adenocarcinoma (EAC) and other solid tumors. An integrated genomics strategy was used to identify deoxyribonucleases correlating with genomic instability (as assessed from total copy number events in each patient) in 6 cancers. Apurinic/apyrimidinic nuclease 1 (APE1), identified as the top gene in functional screens, was either suppressed in cancer cell lines or overexpressed in normal esophageal cells and the impact on genome stability and growth was monitored invitro and invivo. The impact on DNA and chromosomal instability was monitored using multiple approaches, including investigation of micronuclei, acquisition of single nucleotide polymorphisms, whole genome sequencing, and/or multicolor fluorescence in situ hybridization. Expression of 4 deoxyribonucleases correlated with genomic instability in 6 human cancers. Functional screens of these genes identified APE1 as the top candidate for further evaluation. APE1 suppression in EAC, breast, lung, and prostate cancer cell lines caused cell cycle arrest; impaired growth and increased cytotoxicity of cisplatin in all cell lines and types and in a mouse model of EAC; and inhibition of homologous recombination and spontaneous and chemotherapy-induced genomic instability. APE1 overexpression in normal cells caused a massive chromosomal instability, leading to their oncogenic transformation. Evaluation of these cells by means of whole genome sequencing demonstrated the acquisition of changes throughout the genome and identified homologous recombination as the top mutational process. Elevated APE1 dysregulates homologous recombination and cell cycle, contributing to genomic instability, tumorigenesis, and chemoresistance, and its inhibitors have the potential to target these processes in EAC and possibly other cancers.

Abstract 3806: Investigating the role of miRNA-34a in the resistance of esophageal adenocarcinoma to neoadjuvant chemoradiation therapy

Abstract Background: The current standard of care for locally advanced esophageal adenocarcinoma (EAC) involves neo-adjuvant chemoradiation therapy (neo-CRT) followed by surgery. However, response to neo-CRT is poor and resistance remains a significant barrier to effective treatment. There are currently no clinical biomarkers to predict treatment response in EAC. Evidence supports a role for microRNA-34a (miR-34a) as a tumor suppressor in cancer, however, the role of miR-34a in the tumor response to therapy in EAC is largely unknown. Methods: Irradiation of EAC cell lines was performed using an Xstrahl RS225 X-ray irradiator at a clinically-relevant dose of 2 Gy. Radiosensitivity of EAC cell lines (OE33 P, OE33 R, OE33, OE19, Flo-1Par and Flo-1LM) was assessed by the gold standard clonogenic assay. miR-34a expression was assessed by qPCR. EAC tumor biopsies were obtained from consenting patients undergoing diagnostic endoscopy. Pathological response of the resected tumor was assigned by a pathologist using the Mandard Tumor Regression scale. miRTarBase and KEGG pathway analysis were used to identify predicted target genes and pathways of miR-34a. Results: miR-34a was demonstrated to be expressed in a panel of EAC cell lines (OE33 P, OE33 R, OE19, Flo-1Par and Flo-1LM cells). Interestingly, miR-34a expression was significantly decreased in EAC cell line models of both acquired radioresistance (OE33 R) and inherent radioresistance (OE19). miR-34a was also significantly decreased in a model of radioresistant metastatic EAC (Flo-1LM). Supporting in vitro data, in pre-treatment tumor biopsies from EAC patients (n=18), miR-34a was significantly decreased in patients having a subsequent poor pathological response to neo-CRT, when compared to patients having a good pathological response to neo-CRT. Target and pathway analysis demonstrated miR-34a-mediated regulation of genes and pathways associated with treatment resistance, including the complement system, cellular metabolism and p53 signaling, among others. Conclusion: Decreased miR-34a expression is associated with radioresistance across a panel of in vitro EAC models and in pre-treatment tumor biopsies from EAC patients having a poor pathological response to neo-CRT. This highlights a potential role for miR-34a as a novel biomarker predicting response to neo-CRT in EAC. Analysis of validated and predicted gene targets of miR-34a identified a number of pathways associated with treatment resistance. We are currently investigating the functional role of miR-34a in modulating tumor response to radiation in vitro to determine its potential as a novel therapeutic target to boost treatment response in EAC. Citation Format: Christina Cahill, Stephen G. Maher, Rebecca O'Brien, Wei-Lin Winnie Wang, John V. Reynolds, Jacintha O'Sullivan, Niamh Lynam-Lennon. Investigating the role of miRNA-34a in the resistance of esophageal adenocarcinoma to neoadjuvant chemoradiation therapy. [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 3806.

Abstract 4776: NRF2 is overexpressed in esophageal adenocarcinoma and its targeting sensitizes tumor cells to cisplatin through induction of ferroptosis and apoptosis

Abstract Background: Esophageal adenocarcinoma (EAC) is the predominant type of esophageal cancer in the United States, with the 5-year survival rate is below 20%. EAC develops through Barrett’s esophagus (BE)-dysplasia-carcinoma cascade. Gastroesophageal reflux disease (GERD), where acidic bile salts refluxate into the esophagus, is the main risk factor for the development of BE and its progression to EAC. The NFE2-related factor 2 (NRF2) is the master cellular antioxidant regulator, involving in many cancer hallmarks. Methods and Results: Using western blotting and immunohistochemistry technologies, we detected high NRF2 protein levels in EAC cell lines and primary EAC tissues, as compared with normal esophagus and non-neoplastic Barrett’s esophagus samples. The knockdown of NRF2 using NRF2 specific siRNAs significantly increased oxidative stress in response to cellular stress stimuli by bile salts or cisplatin. This was associated with an increase in DNA damage and inhibition of EAC cell growth. Brusatol, a NRF2 inhibitor, significantly inhibited NRF2 transcriptional activity and downregulated NRF2 target genes. We discovered that in addition to inducing apoptosis, Brusatol alone or in combination with CDDP induced significant lipid peroxidation and ferroptosis as evidenced by reduced xCT and GPX4 expression, two known ferroptosis markers. Moreover, the combination of Brusatol and CDDP significantly inhibited EAC tumor xenografts growth in vivo. We confirmed the in vitro data showing ferroptosis as an important mechanism in the xenografted tumors treated with Brusatol or Brusatol and CDDP combination using IHC staining. Conclusion: Our data support the role of NRF2 in protecting against stress-induced apoptosis and ferroptosis in EACs. Targeting NRF2 in combination with platinum therapy can be an effective strategy for eliminating cancer cells in EAC. Citation Format: Farah Ballout, Heng Lu, Zheng Chen, Tianling Hu, Lei Chen, Wael El-Rifai, Dunfa Peng. NRF2 is overexpressed in esophageal adenocarcinoma and its targeting sensitizes tumor cells to cisplatin through induction of ferroptosis and apoptosis. [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 4776.

Abstract 3964: APE1 protects SMAD3 against ROC1 ubiquitin mediated degradation in esophageal adenocarcinoma cells

Abstract Background: The incidence of esophageal adenocarcinoma (EAC) has increased more than six-fold over the past three decades and continues to rise in the Western world. The 5-year survival rate for EAC patients is less than 20% which underscores the need to better understand the underlying mechanisms to identify new therapeutic approaches. This study aimed at investigating the potential role of APE1 in regulating SMAD3 and promoting EAC progression. Methods and Results: Western blot data showed that APE1 and SMAD3 were highly expressed in EAC cell lines. APE1 silencing reduced SMAD3 nuclear expression and downregulated its downstream targets SERPINE1 and c-myc. These results were confirmed by immunofluorescence staining showing loss of nuclear accumulation of SMAD3 after APE1 knockdown. Mechanistically, immunoprecipitation and proximity ligation assays revealed a direct binding between APE1 and SMAD3 in the nucleus. Further investigation showed that APE1 binds to the C-terminal MH2 domain of SMAD3, and this binding protects SMAD3 from ubiquitin mediated proteasomal degradation by blocking its interaction with the RING finger protein, ROC1. Interestingly, APE1-redox-specific inhibition (APX2009) downregulated SMAD3 expression and the APE1 redox-deficient mutant (C65A) disrupted APE1-SMAD3 binding indicating that this regulation depends on APE1 redox activity. Conclusion: Our findings establish a role of APE1 in regulating SMAD3 in EAC. These findings provide a potential therapeutic approach for the treatment of EAC by the pharmacological inhibition of APE1. Citation Format: Farah Ballout, Heng Lu, Dunfa Peng, Wael El-Rifai. APE1 protects SMAD3 against ROC1 ubiquitin mediated degradation in esophageal adenocarcinoma 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 3964.

Abstract 6700: Characterizing circulating tumor DNA release kinetics and fragment length in a chemotherapy resistant model of esophageal adenocarcinoma

Abstract Background: While the incidence of esophageal adenocarcinoma (EAC) is rapidly increasing, the 5-year survival rate of this disease remains poor at less than 25%, with a substantial number of patients exhibiting inherent or acquired resistance to chemotherapy. Circulating tumor DNA (ctDNA) isolated from liquid biopsies of EAC patients holds clinical biomarker potential in this disease, but our understanding of how chemotherapy affects ctDNA release remains limited. Methods: In order to study the impact of cancer treatment on ctDNA emission in vitro, we first sought to establish a chemo-resistant model of EAC. As such, the EAC cell line OE19 was exposed to discontinuous cisplatin treatments for three months in order to establish drug resistance. Following this, established chemo-resistant OE19 cells and parental (chemo-sensitive) OE19 cells were exposed to five daily cisplatin treatments, and cell culture supernatant was collected every day throughout treatment. ctDNA was subsequently isolated from cell culture supernatant and quantified by Qubit fluorometer dsDNA HS assay. ctDNA quantities were additionally assessed by droplet digital PCR (ddPCR) assay using primers and probes targeting the TP53 N310K mutation in the OE19 cell line. Finally, ctDNA fragment lengths were analyzed using the Agilent Bioanalyzer 2100. Results: A chemo-resistant model of the OE19 cell line was established, as exemplified by marked morphological changes in the cells and significantly increased cell viability determined by CCK8 assay after 48-hour treatments of various doses of cisplatin (p&amp;lt;0.05). ctDNA levels, as measured by both Qubit fluorometer and mutant copies detected by ddPCR assay, increased after chemotherapy treatment for both chemo-sensitive and resistant cells. Notably, ctDNA emission was higher with larger amounts cell death, with chemo-sensitive cells emitting significantly higher levels of ctDNA compared to chemo-resistant cells throughout treatment (p&amp;lt;0.05). Fragmentomics analysis revealed ctDNA peaks corresponding to apoptosis (~167 bp) and necrosis (~10,000 bp). Moreover, chemotherapy treatment caused a shift in average fragment size, with larger fragments being observed during chemotherapy treatment in both chemo-sensitive and resistant cells. Conclusion: This study reveals that in vitro cancer models can be used to successfully study ctDNA emission during pre-clinical drug analyses. ctDNA release was found to be correlated to cytotoxicity and cell death, providing us with valuable insight into how clinical liquid biopsy data should be interpreted. Finally, fragment size analysis of ctDNA reveals a potential novel biomarker for treatment response in cancer patients. Citation Format: Alexandra Bartolomucci, Sarah Tadhg Ferrier, Thupten Tsering, Xin Su, Jonathan Cools-Lartigue, Julia V. Burnier. Characterizing circulating tumor DNA release kinetics and fragment length in a chemotherapy resistant model of esophageal adenocarcinoma [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 6700.

New 4,5-Diarylimidazol-2-ylidene-iodidogold(I) Complexes with High Activity against Esophageal Adenocarcinoma Cells.

Inspired by the vascular-disrupting agent combretastatin A-4 and recently published anticancer active N-heterocyclic carbene (NHC) complexes of Au(I), a series of new iodidogold(I)-NHC complexes was synthesized and characterized. The iodidogold(I) complexes were synthesized by a route involving van Leusen imidazole formation and N-alkylation, followed by complexation with Ag2O, transmetalation with chloro(dimethylsulfide)gold(I) [Au(DMS)Cl], and anion exchange with KI. The target complexes were characterized by IR spectroscopy, 1H and 13C NMR spectroscopy, and mass spectrometry. The structure of 6c was validated via single-crystal X-ray diffraction. A preliminary anticancer screening of the complexes using two esophageal adenocarcinoma cell lines showed promising nanomolar activities for certain iodidogold(I) complexes accompanied with apoptosis induction, as well as c-Myc and cyclin D1 suppression in esophageal adenocarcinoma cells treated with the most promising derivative 6b.

Mir-421 and mir-550a-1 are potential prognostic markers in esophageal adenocarcinoma

ObjectiveTo identify the prognostic indicators of esophageal adenocarcinoma (EAC) for future EAC diagnosis and treatment.MethodsThe EAC dataset from The Cancer Genome Atlas was screened for differentially expressed microRNAs (miRNAs) and mRNAs associated with EAC. Weighted gene coexpression network analysis was performed to cluster miRNAs or mRNA with similar expression patterns to identify the miRNAs or mRNA that are highly associated with EAC. Prognostic miRNAs for overall survival (OS) were identified using Cox proportional-hazards regression analysis and least absolute shrinkage and selection operator based on survival duration and status. Two types of miRNAs were selected to develop a prognostic signature model for EAC using multiple Cox regression analysis. Furthermore, the signature was validated using internal validation sets 1 and 2. The receiver operating characteristic curve and concordance index were used to evaluate the accuracy of the signature and validation sets. The expression of miR-421, miR-550a-3p, and miR-550a-5p was assessed using quantitative polymerase chain reaction (qPCR). The proliferation, invasion, and migration of EAC cells were assessed using CCK8 and transwell assays. The OS of target mRNAs was assessed using Kaplan–Meier analysis. Functional enrichment analysis of the target mRNAs was performed using Metascape.ResultsThe prognostic signature and validation sets comprising mir-421 and mir-550a-1 had favorable predictive power in OS. Compared with the patients with EAC in the high-expression group, those assigned to the low-expression group displayed increased OS according to survival analysis. Differential and qPCR analysis showed that miR-421, miR-550a-3p, and miR-550a-5p were highly expressed in the EAC tissues and cell lines. Moreover, the downregulation of miR-421 and miR-550a-3p with inhibitor markedly suppressed the proliferation, invasion, and migration in OE33 cells compared with the negative control. A total of 20 target mRNAs of three miRNAs were predicted, among which seven target mRNAs—ASAP3, BCL2L2, LMF1, PPM1L, PTPN21, SLC18A2, and NR3C2—had prognostic value; PRKACB, PDCD4, RPS6KA5, and BCL2L2 were enriched in the miRNA cancer pathway.ConclusionPrognostic indicators of EAC may be useful in future EAC diagnosis and treatment.

Comparative transcriptome characterization of esophageal squamous cell carcinoma and adenocarcinoma

BackgroundEsophageal cancers are primarily categorized as esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). While various (epi) genomic alterations associated with tumor development in ESCC and EAC have been documented, a comprehensive comparison of the transcriptomes in these two cancer subtypes remains lacking. MethodsWe collected 551 gene expression profiles from publicly available sources, including normal, ESCC, and EAC tissues or cell lines. Subsequently, we conducted a systematic analysis to compare the transcriptomes of these samples at various levels, including gene expression, promoter activity, alternative splicing (AS), alternative polyadenylation (APA), and gene fusion. ResultsSeven distinct cluster gene expression patterns were identified among the differentially expressed genes in normal, ESCC, and EAC tissues. These patterns were enriched in the PI3K-Akt signaling pathway and the activation of extracellular matrix organization and exhibited repression of epidermal development. Notably, we observed additional genes or unique expression levels enriched in these shared pathways and biological processes related to tumor development and immune activation. In addition to the differentially expressed genes, there was an enrichment of lncRNA co-expression networks and downregulation of promoter activity associated with the repression of epidermal development in both ESCC and EAC. This indicates a common feature between these two cancer subtypes. Furthermore, differential AS and APA patterns in ESCC and EAC appear to partially affect the expression of host genes associated with bacterial or viral infections in these subtypes. No gene fusions were observed between ESCC and EAC, thus highlighting the distinct molecular mechanisms underlying these two cancer subtypes. ConclusionsWe conducted a comprehensive comparison of ESCC and EAC transcriptomes and uncovered shared and distinct transcriptomic signatures at multiple levels. These findings suggest that ESCC and EAC may exhibit common and unique mechanisms involved in tumorigenesis.

OGC P21 EZH2: A novel therapeutic target in oesophageal adenocarcinoma

Abstract Background Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of the histone lysine methyltransferase Polycomb Repressor Complex 2 (PRC2), mediates gene silencing of target genes epigenetically. It is over-expressed and associated with poor outcomes in numerous malignancies. EZH2 inhibitors are currently used in the treatment of follicular lymphomas and epithelioid sarcomas. Oesophageal adenocarcinoma (OAC) has predicted sensitivity to EZH2 inhibition due to the high prevalence of potentially sensitizing mutations. This in vitro study aimed to assess the efficacy of EZH2 inhibitors in the treatment of OAC. Methods Bioinformatical analysis of publicly available bulk RNA transcriptomic data from OAC samples included in the Cancer Genome Atlas (TCGA) database was carried out. OAC cell lines OE33, FLO1 and MFD1 cultured in 2D were treated with a panel of EZH2 inhibitors at a range of clinically relevant concentrations either as monotherapies or in combination with OAC standard of care (SOC) chemotherapies. Cell viability was assessed via colorimetric MTS assay at 72hrs and 6 days post-treatment. Results Median EZH2 mRNA expression levels calculated from OAC TCGA data were significantly greater than EZH2 mRNA expression in normal oesophageal tissue (p&amp;lt;0.01). OAC patients with high EZH2 expression had reduced median overall survival compared to patients with low EZH2 expression (13.1 months vs 32 months, p= 0.03). EZH2 inhibitors demonstrated efficacy as monotherapies in all 3 cell lines. EZH2 inhibitors in combination with 5-fluorouracil (5-FU) showed greater efficacy than 5-FU alone, demonstrated by reductions in 5-FU half-maximal inhibitory concentration (IC50) values. Conclusions These early in vitro results demonstrate the potential of EZH2 inhibition as a novel therapy in OAC, particularly in combination with current chemotherapy agents. Further in vitro work is focused on their efficacy in patient derived tumour organoid models.