Esophageal Adenocarcinoma Tissues Research Articles

MultiOmic Analysis of Esophageal Adenocarcinoma Uncovers Candidate Therapeutic Targets and Cancer-Selective Posttranscriptional Regulation

Efforts to address the poor prognosis associated with esophageal adenocarcinoma (EAC) have been hampered by a lack of biomarkers to identify early disease and therapeutic targets. Despite extensive efforts to understand the somatic mutations associated with EAC over the past decade, a gap remains in understanding how the atlas of genomic aberrations in this cancer impacts the proteome and which somatic variants are of importance for the disease phenotype. We performed a quantitative proteomic analysis of 23 EACs and matched adjacent normal esophageal and gastric tissues. We explored the correlation of transcript and protein abundance using tissue-matched RNA-seq and proteomic data from seven patients and further integrated these data with a cohort of EAC RNA-seq data (n = 264 patients), EAC whole-genome sequencing (n = 454 patients), and external published datasets. We quantified protein expression from 5879 genes in EAC and patient-matched normal tissues. Several biomarker candidates with EAC-selective expression were identified, including the transmembrane protein GPA33. We further verified the EAC-enriched expression of GPA33 in an external cohort of 115 patients and confirm this as an attractive diagnostic and therapeutic target. To further extend the insights gained from our proteomic data, an integrated analysis of protein and RNA expression in EAC and normal tissues revealed several genes with poorly correlated protein and RNA abundance, suggesting posttranscriptional regulation of protein expression. These outlier genes, including SLC25A30, TAOK2, and AGMAT, only rarely demonstrated somatic mutation, suggesting posttranscriptional drivers for this EAC-specific phenotype. AGMAT was demonstrated to be overexpressed at the protein level in EAC compared to adjacent normal tissues with an EAC-selective, posttranscriptional mechanism of regulation of protein abundance proposed. By quantitative proteomic analysis, we have identified GPA33 as an EAC-selective biomarker. Integrated analysis of proteome, transcriptome, and genome in EAC has revealed several genes with tumor-selective, posttranscriptional regulation of protein expression, which may be an exploitable vulnerability.

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.

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.

MiRNA profiling of esophageal adenocarcinoma using transcriptome analysis.

Esophageal adenocarcinoma (EAC) occurs following a series of histological changes through epithelial-mesenchymal transition (EMT). A variable expression of normal and aberrant genes in the tissue can contribute to the development of EAC through the activation or inhibition of critical molecular signaling pathways. Gene expression is regulated by various regulatory factors, including transcription factors and microRNAs (miRs). The exact profile of miRs associated with the pathogenesis of EAC is largely unknown, though some candidate miRNAs have been reported in the literature. To identify the unique miR profile associated with EAC, we compared normal esophageal tissue to EAC tissue using bulk RNA sequencing. RNA sequence data was verified using qPCR of 18 selected genes. Fourteen were confirmed as being upregulated, which include CDH11, PCOLCE, SULF1, GJA4, LUM, CDH6, GNA12, F2RL2, CTSZ, TYROBP, and KDELR3 as well as the downregulation of UGT1A1. We then conducted Ingenuity Pathway Analysis (IPA) to analyze for novel miR-gene relationships through Causal Network Analysis and Upstream Regulator Analysis. We identified 46 miRs that were aberrantly expressed in EAC compared to control tissues. In EAC tissues, seven miRs were associated with activated networks, while 39 miRs were associated with inhibited networks. The miR-gene relationships identified provide novel insights into potentially oncogenic molecular pathways and genes associated with carcinogenesis in esophageal tissue. Our results revealed a distinct miR profile associated with dysregulated genes. The miRs and genes identified in this study may be used in the future as biomarkers and serve as potential therapeutic targets in EAC.

A four-gene signature predicts overall survival of patients with esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) is an aggressive cancer with poor prognosis. Thus, this study aimed to identify a prognostic molecular signature to predict the overall survival (OS) of patients with EAC. The mRNA microarray data sets GSE13898 and GSE26886 were downloaded from the Gene Expression Omnibus (GEO) database. RNA sequencing profile and clinical data of EAC patients were downloaded from The Cancer Genome Atlas (TCGA) database. Differentially expressed genes (DEGs) between EAC tissues and adjacent non-cancerous tissues were obtained using R software. DEGs associated with prognosis of OS were assessed by univariate Cox analysis, and a prognostic signature was built using stepwise multivariate Cox analysis. Time-dependent receiver operating characteristic (ROC) analysis and stratification analysis were conducted to evaluate its predictive performance. Functional enrichment analysis was performed for genes co-expressed with the signature to explore its biological functions in EAC. A total of 336 genes were identified to be differentially expressed between EAC tissues and adjacent non-cancerous tissues. After univariate and multivariate Cox regression analysis, four genes (ALAD, ABLIM3, IL17RB and IFI6) were screened out to construct a prognostic signature. According to this signature, patients could be assigned into high-risk and low-risk group with significantly different OS (P=4.92e-05<0.0001). Multivariate Cox regression analysis suggested that the four-gene signature served as an independent factor in OS prediction. In the time-dependent ROC analysis, the areas under the curves (AUCs) were 0.804, 0.792 and 0.695 for 1-, 3- and 5-year survival prediction, respectively, suggesting a good performance. Functional enrichment analysis showed that the signature was mainly clustered in cell proliferation related biological processes or pathways. The four-gene signature identified in the current study may be a potential prognostic factor for predicting OS of EAC patients.

A Comprehensive Analysis of Tn and STn Antigen Expression in Esophageal Adenocarcinoma.

Differential glycosylation, marked by the presence of truncated O-glycans, is a distinctive feature of epithelial-derived cancers. However, there is a notable gap in research regarding the expression of Tn and STn antigens in esophageal adenocarcinoma (EAC). To address this, we employed commercially available antibodies, previously validated for Tn and STn antigens, to analyze two cohorts of EAC tissues. Initially, large-area tissue sections from formalin-fixed paraffin-embedded (FFPE) EAC and corresponding healthy tissues were subjected to immunohistochemistry (IHC) staining and scoring. Subsequently, we evaluated the RNA expression levels of crucial O-glycosylation related genes-C1GALT1 and C1GALT1C1-using a quantitative real-time polymerase chain reaction (qRT-PCR). In a comprehensive analysis, a substantial cohort of EAC tissues (n = 311 for Tn antigen, n = 351 for STn antigen) was investigated and correlated with clinicopathological data. Our findings revealed that Tn and STn antigens are highly expressed (approximately 71% for both) in EAC, with this expression being tumor-specific. Notably, Tn antigen expression correlates significantly with the depth of tumor cell infiltration (p = 0.026). These antigens emerge as valuable markers and potential therapeutic targets for esophageal adenocarcinoma.

The Expression Patterns and Implications of MALAT1, MANCR, PSMA3-AS1 and miR-101 in Esophageal Adenocarcinoma.

Esophageal adenocarcinoma (EAC) is a malignant tumor with poorly understood molecular mechanisms. This study endeavors to elucidate how the long non-coding RNAs (lncRNAs) MALAT1, MANCR and PSMA3-AS1, as well as the microRNA miR-101, exhibit specific expression patterns in the pathogenesis and prognosis of EAC. A total of 50 EAC tissue samples (tumors and lymph nodes) and a control group comprising 26 healthy individuals were recruited. The samples underwent quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. The relative expression levels of MALAT1, MANCR, PSMA3-AS1, and miR-101 were ascertained and correlated with various clinicopathological parameters including TNM staging, tumor characteristics (size and grade of the tumor) lymphatic invasion, disease-free (DFS) and overall survival (OS) of EAC patients. Quantitative analyses revealed that MALAT1 and MANCR were significantly upregulated in EAC tumors and positive lymph nodes when compared to control tissues (p < 0.05). Such dysregulations correlated positively with advanced lymphatic metastases and a higher N stage. DFS in the subgroup of patients with negative lymph nodes was higher in the setting of low-MANCR-expression patients compared to patients with high MANCR expression (p = 0.02). Conversely, miR-101 displayed a significant downregulation in EAC tumors and positive lymph nodes (p < 0.05), and correlated negatively with advanced tumor stage, lymphatic invasion and the grade of the tumor (p = 0.006). Also, patients with low miR-101 expression showed a tendency towards inferior overall survival. PSMA3-AS1 did not demonstrate statistically significant alterations (p > 0.05). This study reveals MALAT1, MANCR, and miR-101 as putative molecular markers for prognostic evaluation in EAC and suggests their involvement in EAC progression.

The function and immune role of cuproptosis associated hub gene in Barrett's esophagus and esophageal adenocarcinoma.

Barrett's esophagus (BE) is a precancerous lesion of esophageal adenocarcinoma (EAC), with approximately 3-5% of patients developing EAC. Cuproptosis is a kind of programmed cell death phenomenon discovered in recent years, which is related to the occurrence and development of many diseases. However, its role in BE and EAC is not fully understood. We used single sample Gene Set Enrichment Analysis (ssGSEA) for differential analysis of BE in the database, followed by enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO) and GSEA, Protein-Protein Interaction (PPI), Weighted Gene Co-expression Network Analysis (WGCNA), Receiver Operating Characteristic Curve (ROC) and finally Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and immunohistochemistry (IHC) of clinical tissues. Two hub genes can be obtained by intersection of the results obtained from the cuproptosis signal analysis based on BE. The ROC curves of these two genes predicted EAC, and the Area Under the Curve (AUC) values could reach 0.950 and 0.946, respectively. The mRNA and protein levels of Centrosome associated protein E (CENPE) and Shc SH2 domain binding protein 1 (SHCBP1) were significantly increased in clinical EAC tissues. When they were grouped by protein expression levels, high expression of CENPE or SHCBP1 had a poor prognosis. The CENPE and SHCBP1 associated with cuproptosis may be a factor promoting the development of BE into EAC which associated with the regulation of NK cells and T cells.

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.

Smoking induces WEE1 expression to promote docetaxel resistance in esophageal adenocarcinoma

Esophageal adenocarcinoma (EAC) patients have poor clinical outcomes, with an overall 5-year survival rate of 20%. Smoking is a significant risk factor for EAC. The role of WEE1, a nuclear kinase that negatively regulates the cell cycle in normal conditions, in EAC tumorigenesis and drug resistance is not fully understood. Immunohistochemistry staining shows significant WEE1 overexpression in human EAC tissues. Nicotine, nicotine-derived nitrosamine ketone, or 2% cigarette smoke extract treatment induces WEE1 protein expression in EAC, detected by western blot and immunofluorescence staining. qRT-PCR and reporter assay indicates that smoking induces WEE1 expression through miR-195-5p downregulation in EAC. ATP-Glo cell viability and clonogenic assay confirmed that WEE1 inhibition sensitizes EAC cells to docetaxel treatment invitro. A TE-10 smoking machine with EAC patient-derived xenograft mouse model demonstrated that smoking induces WEE1 protein expression and resistance to docetaxel invivo. MK-1775 and docetaxel combined treatment improves EAC patient-derived xenograft mouse survival invivo. Our findings demonstrate, for the first time, that smoking-induced WEE1 overexpression through miRNA dysregulation in EAC plays an essential role in EAC drug resistance. WEE1 inhibition is a promising therapeutic method to overcome drug resistance and target treatment refractory cancer cells.

Attenuated Total Reflection Fourier-Transform Infrared Spectral Discrimination in Human Tissue of Oesophageal Transformation to Adenocarcinoma.

This study presents ATR-FTIR (attenuated total reflectance Fourier-transform infrared) spectral analysis of ex vivo oesophageal tissue including all classifications to oesophageal adenocarcinoma (OAC). The article adds further validation to previous human tissue studies identifying the potential for ATR-FTIR spectroscopy in differentiating among all classes of oesophageal transformation to OAC. Tissue spectral analysis used principal component analysis quadratic discriminant analysis (PCA-QDA), successive projection algorithm quadratic discriminant analysis (SPA-QDA), and genetic algorithm quadratic discriminant analysis (GA-QDA) algorithms for variable selection and classification. The variables selected by SPA-QDA and GA-QDA discriminated tissue samples from Barrett's oesophagus (BO) to OAC with 100% accuracy on the basis of unique spectral "fingerprints" of their biochemical composition. Accuracy test results including sensitivity and specificity were determined. The best results were obtained with PCA-QDA, where tissues ranging from normal to OAC were correctly classified with 90.9% overall accuracy (71.4-100% sensitivity and 89.5-100% specificity), including the discrimination between normal and inflammatory tissue, which failed in SPA-QDA and GA-QDA. All the models revealed excellent results for distinguishing among BO, low-grade dysplasia (LGD), high-grade dysplasia (HGD), and OAC tissues (100% sensitivities and specificities). This study highlights the need for further work identifying potential biochemical markers using ATR-FTIR in tissue that could be utilised as an adjunct to histopathological diagnosis for early detection of neoplastic changes in susceptible epithelium.

Telomere Shortening of Barrett’s Esophagus and Esophageal Adenocarcinoma in Japanese Patients

Telomere shortening is deeply involved in many types of cancer. Telomere length of esophageal adenocarcinoma (EAC) and Barrett’s esophagus (BE) was examined in Japanese patients. Among BE from cancer free patients (Cancer free), BE from patients with EAC (Adjacent) and EAC tissue (Cancer), Cancer free group presented the longest telomeres, while Cancer group presented the shortest telomeres and Adjacent group presented intermediate telomeres. Direction of endoscopic biopsy, 2 o‘clock direction was also significantly associated with shorter telomere length in non-neoplastic BE (p = 0.027). Shortened telomere highlighted the impact of this molecular change in early carcinogenesis in EAC.

Microarchitectures of Barrett's esophagus associated with DNA methylation status.

Aim: DNA methylation is involved in esophageal adenocarcinoma (EAC) and Barrett's esophagus (BE). Microarchitectures of on-neoplastic BE associated with DNA methylation status were examined using magnifying narrow-band imaging (NBI) endoscopy. Patients and methods: Using biopsies from non-neoplastic BE without cancer (n=66; N group), with EAC (n=27; ADJ group) and EAC tissue (n=22; T group), methylation of N33, DPYS, SLC16A12, miR124a3 andmiR34bc genes were quantified. MagnifyingNBI features of non-neoplastic BE were classified according to their morphologies. Results: TheADJ and T groups presented higher DNA methylation compared with theN group. Magnifying NBI endoscopic features of non-neoplastic BE also correlated with DNA methylation as an independent factor. Conclusion: Microarchitectures of BE visualized by magnifying NBI endoscopy correlated with DNA methylation.

Abstract 3130: Extrachromosomal DNA in the cancerous transformation of Barrett’s esophagus

Abstract Oncogene amplification on extrachromosomal DNA (ecDNA) drives rapid tumor evolution, treatment resistance, and poor outcomes for patients. Computational tools can detect ecDNA in whole-genome sequencing (WGS) data from biopsies. However, the lack of longitudinal studies tracking patients from pre-cancer to cancer have made it difficult to determine how ecDNAs contribute to the malignant transformation. Two independent surveillance studies of Barrett’s esophagus (BE) patients enabled us to study this transition, as BE is the precursor lesion of esophageal adenocarcinoma (EAC). The studies included a longitudinal case-control study and an independent, cross-sectional surveillance cohort, both with histological correlatives, providing an unprecedented opportunity to study the role of ecDNA in the transition of BE to EAC. We analyzed WGS data from 206 patients in a cross-sectional surveillance cohort from Cambridge University UK, with biopsy-validated BE, including 42 patients who never developed high-grade dysplasia (HGD) or EAC during multi-year follow-ups, 25 patients with HGD, 51 patients with early-stage EAC, and 88 patients with late-stage EAC. ecDNA frequency increased between early-stage EAC (24%) and late-stage EAC (43%), suggesting continual ecDNA formation during cancer progression. We additionally analyzed WGS and histology data collected from multi-regional sampling of BE and EAC tissue at two time points from 80 BE patients in an observational case-control study at the Fred Hutchinson Cancer Center (FHCC), where 40 of the patients developed BE-associated EAC during the study and 40 did not. Among FHCC patients, 33% who developed EAC had at least one esophageal biopsy with ecDNA prior to, or at EAC diagnosis. In biopsies collected before cancer diagnosis, ecDNA presence in the biopsy was enriched among patients who later developed EAC compared to patients who did not. When linked to histology data, ecDNA-positive biopsies associated with worsened histological states before cancer diagnosis (p=0.015), and also at cancer diagnosis (p=0.037), with the pre-cancer timepoint being on average 2.1 years prior to cancer diagnosis. The ecDNAs had diverse collections of oncogenes and immunomodulatory genes, and showed higher copy number states than other focal amplification types. Furthermore, ecDNAs showed increasing copy number and structural complexity in more advanced disease stages highlighting their positive selection and ongoing structural evolution. In both FHCC and Cambridge cohorts, we found an association of TP53 loss with ecDNA formation demonstrating the role of prior genomic instability to form ecDNA. In total, our findings illustrate that oncogenic ecDNA can develop early in the malignant transformation, and that they are tied to worsening patient disease status. Citation Format: Jens Luebeck, Alvin W. Ng, Patricia C. Galipeau, Xiaohong Li, Annalise Katz-Summercorn, Hoon Kim, Sriganesh Jammula, Yudou He, Roel Verhaak, Carlo C. Maley, Ludmil B. Alexandrov, Rebecca C. Fitzgerald, Thomas G. Paulson, Howard Y. Chang, Sihan Wu, Vineet Bafna, Paul S. Mischel. Extrachromosomal DNA in the cancerous transformation of Barrett’s esophagus [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 3130.

Abstract 1232: Gastroesophageal reflux disease promotes E-cadherin cleavage and activates EMT via APE1-redox function in esophageal adenocarcinoma

Abstract The incidence of esophageal adenocarcinoma (EAC) has increased rapidly over the past four decades. Chronic gastroesophageal reflux disease (GERD), where acidic bile salts (ABS) abnormally refluxate into the esophagus, is the leading risk factor for the development of a metaplastic condition known as Barrett’s esophagus (BE) and its progression to EAC. We used 3D organotypic culture, mouse and human tissue samples to assess the role of ABS in Epithelial-to-Mesenchymal Transition (EMT) in EAC. Analysis of public databases revealed significant enrichment of EMT signaling in EAC progression. RNA-seq analysis of EAC cells showed activation of EMT pathway in response to ABS exposure. ABS induced multiple characteristics of the EMT process, such as downregulation of E-cadherin, upregulation of Vimentin, activation of ß-catenin and EMT-transcription factors (EMT-TFs), cell morphological changes, and enhancement of cell migration and invasion capabilities. Mechanistically, we discovered that ABS induced E-cadherin cleavage via MMP14 proteolytic cascade. APE1 silencing, or APE1-redox-specific inhibitor (E3330), abrogated the ABS-induced EMT process and signaling by downregulating MMP14. Furthermore, APE1 and MMP14 co-expression levels were inversely correlated with E-cadherin expression in gastroesophageal junctions of human EAC tissues, and the L2-IL1ß transgenic mouse model of BE/EAC. EAC patients with high APE1 and EMT signatures had worse relapse free survival than those with low signature. In a summary, this study demonstrates the role of ABS in promoting EMT via the redox-sensitive signaling axis of APE1/MMP14/E-cadherin/ß-catenin. Pharmacological inhibition of APE1-redox function could be a potential therapeutic approach to effectively reduce the risk of Barrett’s carcinogenesis. Citation Format: Heng Lu, Longlong Cao, Farah Ballout, Abbes Belkhiri, Dunfa Peng, Lei Chen, Mohammed Soutto, Oliver McDonald, Alexander Zaika, Jianwen Que, Wael El-Rifai. Gastroesophageal reflux disease promotes E-cadherin cleavage and activates EMT via APE1-redox function in 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 1232.

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.

Construction of the Interaction Network of Hub Genes in the Progression of Barrett's Esophagus to Esophageal Adenocarcinoma.

Esophageal adenocarcinoma (EAC) is one of the histologic types of esophageal cancer with a poor prognosis. The majority of EAC originate from Barrett's esophagus (BE). There are few studies focusing on the dynamic progression of BE to EAC. R software was used to analyze differentially expressed genes (DEGs) based on RNA-seq data of 94 normal esophageal squamous epithelial (NE) tissues, 113 BE tissues and 147 EAC tissues. The overlapping genes of DEGs between BE and EAC were analyzed by Venn diagram tool. The hub genes were selected by Cytoscape software based on the protein-protein interaction network of the overlapping genes using STRING database. The functional analysis of hub genes was performed by R software and the protein expression was identified by immunohistochemistry. In the present study, we found a large degree of genetic similarity between BE and EAC, and further identified seven hub genes (including COL1A1, TGFBI, MMP1, COL4A1, NID2, MMP12, CXCL1) which were all progressively upregulated in the progression of NE-BE-EAC. We have preliminarily uncovered the probable molecular mechanisms of these hub genes in disease development and constructed the ceRNA regulatory network of hub genes. More importantly, we explored the possibility of hub genes as biomarkers in the disease progression of NE-BE-EAC. For example, TGFBI can be used as biomarkers to predict the prognosis of EAC patients. COL1A1, NID2 and COL4A1 can be used as biomarkers to predict the response to immune checkpoint blockade (ICB) therapy. We also constructed a disease progression risk model for NE-BE-EAC based on CXCL1, MMP1 and TGFBI. Finally, the results of drug sensitivity analysis based on hub genes showed that drugs such as PI3K inhibitor TGX221, bleomycin, PKC inhibitor Midostaurin, Bcr-Abl inhibitor Dasatinib, HSP90 inhibitor 17-AAG, and Docetaxel may be potential candidates to inhibit the progression of BE to EAC. This study is based on a large number of clinical samples with high credibility, which is useful for revealing the probable carcinogenic mechanism of BE to EAC and developing new clinical treatment strategies.

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 O09 The clinical utilisation of tissue imaging Mass Spectrometry as a diagnostic model for Oesophageal Adenocarcinoma

Abstract Background The prevalence of Oesophageal adenocarcinoma (OA) in the United Kingdom has steadily risen since the 1970's. The majority of patients present with advanced disease. Early diagnosis utilising point of care testing may enable an increase in the number of patients managed on a potentially curative pathway. Tissue Imaging Mass Spectrometry by Desorption Electrospray (DESI-MS) is one such tool to differentiate the abundance of lipids in OA. This work presents the novel use of Mass Spectrometry (MS) imaging to identify variations in lipid abundance between OA and normal oesophagus (NO). Methods Tissue was sampled from specimens dissected immediately post-resection from the tumour and at 5-centimetre intervals in the proximal NO. Forty-one unique patient OA and NO tissue samples were analysed in triplicate by DESI-MS tissue imaging to differentiate Glycerophospholipids. A total of 246 DESI-MS images were correlated with paired formalin fixed Haematoxylin and Eosin stained tissue slides, the current gold standard cancer diagnostic test. Principle component analysis utilising a supervised model classified tissue types with a significance of p=0.01. Receiver operator curves (ROC) were used to determine the diagnostic sensitivity and specificity of DESI-MS for the identification of adenocarcinoma. Results Tissue analysis confirmed unsaturated Phosphatidylglycerols and Phosphatidic acids were significantly more abundant in OA compared to NO (p&amp;lt;0.001). Tissue analysis by a Partial Least Square (PLS) supervised linear regression model classified cancer and non-cancer tissue with a confusion matrix classification of 93.3% and 70.8% for OA and NO (Figure 1a-c). A diagnostic sensitivity of 85% and specificity of 88% was determined by ROC curve. The abundance of Phosphatidylglycerol and Phosphatidylserine decreased proportionally in analysed tissue sections at 5cm and 10cm from the primary tumour, as did the degree of lipid unsaturation. Conclusions This work presents a novel tissue imaging MS model to differentiate lipid abundance in OA. An abundance ratio of 1.17:1 (OA:NO) of unsaturated Phosphatidylglycerol was diagnostic of cancer by DESI-MS with a high sensitivity and specificity. This MS tissue analysis model has the potential to enable accurate point of care diagnostics for OA. In addition, we suggest oesophageal cancer induced perturbation of Glycerophospholipid metabolism may be a potential target for future chemotherapeutics.