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Herms A, Fernandez-Antoran D, Alcolea MP, Kalogeropoulou A, Banerjee U, Piedrafita G, Abby E, Valverde-Lopez JA, Ferreira IS, Caseda I, Bejar MT, Dentro SC, Vidal-Notari S, Ong SH, Colom B, Murai K, King C, Mahbubani K, Saeb-Parsy K, Lowe AR, Gerstung M, Jones PH. Self-sustaining long-term 3D epithelioid cultures reveal drivers of clonal expansion in esophageal epithelium. Nat Genet 2024; 56:2158-2173. [PMID: 39313617 PMCID: PMC11525200 DOI: 10.1038/s41588-024-01875-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2023] [Accepted: 07/18/2024] [Indexed: 09/25/2024]
Abstract
Aging epithelia are colonized by somatic mutations, which are subjected to selection influenced by intrinsic and extrinsic factors. The lack of suitable culture systems has slowed the study of this and other long-term biological processes. Here, we describe epithelioids, a facile, cost-effective method of culturing multiple mouse and human epithelia. Esophageal epithelioids self-maintain without passaging for at least 1 year, maintaining a three-dimensional structure with proliferative basal cells that differentiate into suprabasal cells, which eventually shed and retain genomic stability. Live imaging over 5 months showed that epithelioids replicate in vivo cell dynamics. Epithelioids support genetic manipulation and enable the study of mutant cell competition and selection in three-dimensional epithelia, and show how anti-cancer treatments modulate competition between transformed and wild-type cells. Finally, a targeted CRISPR-Cas9 screen shows that epithelioids recapitulate mutant gene selection in aging human esophagus and identifies additional drivers of clonal expansion, resolving the genetic networks underpinning competitive fitness.
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Affiliation(s)
- Albert Herms
- Wellcome Sanger Institute, Hinxton, UK
- Department of Biomedical Sciences, Universitat de Barcelona, Barcelona, Spain
- Lipid Trafficking and Disease Group, Institut d'Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), Barcelona, Spain
| | - David Fernandez-Antoran
- Wellcome Sanger Institute, Hinxton, UK
- Wellcome/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK
- ARAID Foundation, Aragón Health Research Institute (IIS Aragón), Zaragoza, Spain
| | - Maria P Alcolea
- Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK
| | | | | | - Gabriel Piedrafita
- Wellcome Sanger Institute, Hinxton, UK
- Department of Biochemistry and Molecular Biology, Complutense University of Madrid, Madrid, Spain
- Spanish National Cancer Research Centre (CNIO), Madrid, Spain
| | | | | | - Inês S Ferreira
- Wellcome/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK
| | - Irene Caseda
- Department of Biomedical Sciences, Universitat de Barcelona, Barcelona, Spain
- Lipid Trafficking and Disease Group, Institut d'Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), Barcelona, Spain
| | - Maria T Bejar
- Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK
| | - Stefan C Dentro
- Wellcome Sanger Institute, Hinxton, UK
- European Molecular Biology Laboratory, European Bioinformatics Institute, Cambridge, UK
- Artificial Intelligence in Oncology (B450), Deutsches Krebsforschungszentrum, Heidelberg, Germany
| | - Sara Vidal-Notari
- Department of Biochemistry and Molecular Biology, Complutense University of Madrid, Madrid, Spain
- Spanish National Cancer Research Centre (CNIO), Madrid, Spain
| | | | - Bartomeu Colom
- Wellcome Sanger Institute, Hinxton, UK
- Cambridge Institute of Science, Altos Labs, Cambridge, UK
| | | | | | - Krishnaa Mahbubani
- Department of Surgery, University of Cambridge, Cambridge, UK
- Collaborative Biorepository for Translational Medicine (CBTM), Cambridge NIHR Biomedical Research Centre, Cambridge, UK
| | - Kourosh Saeb-Parsy
- Department of Surgery, University of Cambridge, Cambridge, UK
- Collaborative Biorepository for Translational Medicine (CBTM), Cambridge NIHR Biomedical Research Centre, Cambridge, UK
| | - Alan R Lowe
- Institute for Structural and Molecular Biology, University College London, London, UK
- Institute for the Physics of Living Systems, University College London, London, UK
- Department of Physics and Astronomy, University College London, London, UK
| | - Moritz Gerstung
- European Molecular Biology Laboratory, European Bioinformatics Institute, Cambridge, UK
- Artificial Intelligence in Oncology (B450), Deutsches Krebsforschungszentrum, Heidelberg, Germany
| | - Philip H Jones
- Wellcome Sanger Institute, Hinxton, UK.
- Department of Oncology, Hutchison Research Centre, University of Cambridge, Cambridge, UK.
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2
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Iijima K. Etiologic factors for Barrett's esophagus: toward countermeasures in Asia. Expert Rev Gastroenterol Hepatol 2024; 18:407-420. [PMID: 39072626 DOI: 10.1080/17474124.2024.2386367] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/14/2024] [Accepted: 07/26/2024] [Indexed: 07/30/2024]
Abstract
INTRODUCTION Over the past several decades, Europe and the United States have experienced a rapid increase in esophageal adenocarcinoma. Research and countermeasures against Barrett's esophagus, its precancerous lesion, are progressing. Because esophageal adenocarcinoma has an extremely poor prognosis when diagnosed in an advanced stage, recommendations for early cancer detection have been made based on the various proven etiological factors of Barrett's esophagus and the actual cancer risk of Barrett's esophagus. In recent years, there have been indications of an increase in esophageal adenocarcinoma in Japan, and a similar trend of cancer will occur shortly in other Asian countries. Consequently, Asian countries must implement similar countermeasures against Barrett's esophagus and esophageal adenocarcinoma, referencing the knowledge gained thus far in Europe and the United States. AREAS COVERED This review summarizes the latest findings on the etiologic factors of Barrett's esophagus and discusses the differences between Westerners and Asians. The current status of Barrett's esophagus in Japan and other Asian countries is also summarized. EXPERT OPINION The etiological factors and cancer incidence of Barrett's esophagus in Asia diverge somewhat from those observed in Europe and America. Therefore, it is imperative to implement measures that are tailored to the actual circumstances of Asian people.
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Affiliation(s)
- Katsunori Iijima
- Department of Gastroenterology, Akita University Graduate School of Medicine, Akita, Japan
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3
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Abdulnour-Nakhoul SM, Kolls JK, Flemington EK, Ungerleider NA, Nakhoul HN, Song K, Nakhoul NL. Alterations in gene expression and microbiome composition upon calcium-sensing receptor deletion in the mouse esophagus. Am J Physiol Gastrointest Liver Physiol 2024; 326:G438-G459. [PMID: 38193195 PMCID: PMC11213479 DOI: 10.1152/ajpgi.00066.2023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/29/2023] [Revised: 12/17/2023] [Accepted: 12/23/2023] [Indexed: 01/10/2024]
Abstract
The calcium-sensing receptor (CaSR), a G protein-coupled receptor, regulates Ca2+ concentration in plasma by regulating parathyroid hormone secretion. In other tissues, it is reported to play roles in cellular differentiation and migration and in secretion and absorption. We reported previously that CaSR can be conditionally deleted in the mouse esophagus. This conditional knockout (KO) (EsoCaSR-/-) model showed a significant reduction in the levels of adherens and tight junction proteins and had a marked buildup of bacteria on the luminal esophageal surface. To further examine the role of CaSR, we used RNA sequencing to determine gene expression profiles in esophageal epithelia of control and EsoCaSR-/-mice RNA Seq data indicated upregulation of gene sets involved in DNA replication and cell cycle in EsoCaSR-/-. This is accompanied by the downregulation of gene sets involved in the innate immune response and protein homeostasis including peptide elongation and protein trafficking. Ingenuity pathway analysis (IPA) demonstrated that these genes are mapped to important biological networks including calcium and Ras homologus A (RhoA) signaling pathways. To further explore the bacterial buildup in EsoCaSR-/- esophageal tissue, 16S sequencing of the mucosal-associated bacterial microbiome was performed. Three bacterial species, g_Rodentibacter, s_Rodentibacter_unclassified, and s_Lactobacillus_hilgardi were significantly increased in EsoCaSR-/-. Furthermore, metagenomic analysis of 16S sequences indicated that pathways related to oxidative phosphorylation and metabolism were downregulated in EsoCaSR-/- tissues. These data demonstrate that CaSR impacts major pathways of cell proliferation, differentiation, cell cycle, and innate immune response in esophageal epithelium. The disruption of these pathways causes inflammation and significant modifications of the microbiome.NEW & NOTEWORTHY Calcium-sensing receptor (CaSR) plays a significant role in maintaining the barrier function of esophageal epithelium. Using RNA sequencing, we show that conditional deletion of CaSR from mouse esophagus causes upregulation of genes involved in DNA replication and cell cycle and downregulation of genes involved in the innate immune response, protein translation, and cellular protein synthesis. Pathway analysis shows disruption of signaling pathways of calcium and actin cytoskeleton. These changes caused inflammation and esophageal dysbiosis.
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Affiliation(s)
- Solange M Abdulnour-Nakhoul
- Deming Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States
- Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana, United States
| | - Jay K Kolls
- Deming Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States
- Center for Translational Research in Infection and Inflammation, Tulane University School of Medicine, New Orleans, Louisiana, United States
| | - Erik K Flemington
- Department of Pathology, Tulane University, New Orleans, Louisiana, United States
| | - Nathan A Ungerleider
- Department of Pathology, Tulane University, New Orleans, Louisiana, United States
| | - Hani N Nakhoul
- Department of Pathology, Tulane University, New Orleans, Louisiana, United States
| | - Kejing Song
- Deming Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States
- Center for Translational Research in Infection and Inflammation, Tulane University School of Medicine, New Orleans, Louisiana, United States
| | - Nazih L Nakhoul
- Deming Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States
- Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana, United States
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Sugano K, Spechler SJ, El-Omar EM, McColl KEL, Takubo K, Gotoda T, Fujishiro M, Iijima K, Inoue H, Kawai T, Kinoshita Y, Miwa H, Mukaisho KI, Murakami K, Seto Y, Tajiri H, Bhatia S, Choi MG, Fitzgerald RC, Fock KM, Goh KL, Ho KY, Mahachai V, O'Donovan M, Odze R, Peek R, Rugge M, Sharma P, Sollano JD, Vieth M, Wu J, Wu MS, Zou D, Kaminishi M, Malfertheiner P. Kyoto international consensus report on anatomy, pathophysiology and clinical significance of the gastro-oesophageal junction. Gut 2022; 71:1488-1514. [PMID: 35725291 PMCID: PMC9279854 DOI: 10.1136/gutjnl-2022-327281] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/01/2022] [Accepted: 05/03/2022] [Indexed: 02/07/2023]
Abstract
OBJECTIVE An international meeting was organised to develop consensus on (1) the landmarks to define the gastro-oesophageal junction (GOJ), (2) the occurrence and pathophysiological significance of the cardiac gland, (3) the definition of the gastro-oesophageal junctional zone (GOJZ) and (4) the causes of inflammation, metaplasia and neoplasia occurring in the GOJZ. DESIGN Clinical questions relevant to the afore-mentioned major issues were drafted for which expert panels formulated relevant statements and textural explanations.A Delphi method using an anonymous system was employed to develop the consensus, the level of which was predefined as ≥80% of agreement. Two rounds of voting and amendments were completed before the meeting at which clinical questions and consensus were finalised. RESULTS Twenty eight clinical questions and statements were finalised after extensive amendments. Critical consensus was achieved: (1) definition for the GOJ, (2) definition of the GOJZ spanning 1 cm proximal and distal to the GOJ as defined by the end of palisade vessels was accepted based on the anatomical distribution of cardiac type gland, (3) chemical and bacterial (Helicobacter pylori) factors as the primary causes of inflammation, metaplasia and neoplasia occurring in the GOJZ, (4) a new definition of Barrett's oesophagus (BO). CONCLUSIONS This international consensus on the new definitions of BO, GOJ and the GOJZ will be instrumental in future studies aiming to resolve many issues on this important anatomic area and hopefully will lead to better classification and management of the diseases surrounding the GOJ.
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Affiliation(s)
- Kentaro Sugano
- Division of Gastroenterology, Department of Medicine, Jichi Medical University, Shimotsuke, Japan
| | - Stuart Jon Spechler
- Division of Gastroenterology, Center for Esophageal Diseases, Baylor University Medical Center, Dallas, Texas, USA
| | - Emad M El-Omar
- Microbiome Research Centre, St George & Sutherland Clinical Campuses, School of Clinical Medicine, Faculty of Medicine & Health, Sydney, New South Wales, Australia
| | - Kenneth E L McColl
- Division of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK
| | - Kaiyo Takubo
- Research Team for Geriatric Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
| | - Takuji Gotoda
- Division of Gastroenterology and Hepatology, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan
| | - Mitsuhiro Fujishiro
- Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Katsunori Iijima
- Department of Gastroenterology, Akita University Graduate School of Medicine, Akita, Japan
| | - Haruhiro Inoue
- Digestive Disease Center, Showa University Koto Toyosu Hospital, Tokyo, Japan
| | - Takashi Kawai
- Department of Gastroenterological Endoscopy, Tokyo Medical University, Tokyo, Japan
| | | | - Hiroto Miwa
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Hyogo College of Medicine, Kobe, Japan
| | - Ken-Ichi Mukaisho
- Education Center for Medicine and Nursing, Shiga University of Medical Science, Otsu, Japan
| | - Kazunari Murakami
- Department of Gastroenterology, Oita University Faculty of Medicine, Yuhu, Japan
| | - Yasuyuki Seto
- Department of Gastrointestinal Surgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Hisao Tajiri
- Jikei University School of Medicine, Minato-ku, Tokyo, Japan
| | | | - Myung-Gyu Choi
- Gastroenterology, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, The Republic of Korea
| | - Rebecca C Fitzgerald
- Medical Research Council Cancer Unit, Hutchison/Medical Research Council Research Centre, University of Cambridge, Cambridge, UK
| | - Kwong Ming Fock
- Department of Gastroenterology and Hepatology, Duke NUS School of Medicine, National University of Singapore, Singapore
| | | | - Khek Yu Ho
- Department of Medicine, National University of Singapore, Singapore
| | - Varocha Mahachai
- Center of Excellence in Digestive Diseases, Thammasat University and Science Resarch and Innovation, Bangkok, Thailand
| | - Maria O'Donovan
- Department of Histopathology, Cambridge University Hospital NHS Trust UK, Cambridge, UK
| | - Robert Odze
- Department of Pathology, Tuft University School of Medicine, Boston, Massachusetts, USA
| | - Richard Peek
- Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | - Massimo Rugge
- Department of Medicine DIMED, Surgical Pathology and Cytopathology Unit, University of Padova, Padova, Italy
| | - Prateek Sharma
- Department of Gastroenterology and Hepatology, University of Kansas School of Medicine, Kansas City, Kansas, USA
| | - Jose D Sollano
- Department of Medicine, University of Santo Tomas, Manila, Philippines
| | - Michael Vieth
- Institute of Pathology, Klinikum Bayreuth, Friedrich-Alexander University Erlangen, Nurenberg, Germany
| | - Justin Wu
- Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China
| | - Ming-Shiang Wu
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Duowu Zou
- Department of Gastroenterology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | | | - Peter Malfertheiner
- Medizinixhe Klinik und Poliklinik II, Ludwig Maximillian University Klinikum, Munich, Germany
- Klinik und Poliklinik für Radiologie, Ludwig Maximillian University Klinikum, Munich, Germany
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Nrf2/Keap1-Pathway Activation and Reduced Susceptibility to Chemotherapy Treatment by Acidification in Esophageal Adenocarcinoma Cells. Cancers (Basel) 2021; 13:cancers13112806. [PMID: 34199909 PMCID: PMC8200109 DOI: 10.3390/cancers13112806] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2021] [Revised: 06/02/2021] [Accepted: 06/03/2021] [Indexed: 12/24/2022] Open
Abstract
Simple Summary Inflammation caused by acidic reflux contributes to disease progression in Barrett’s esophagus. Little is known, whether esophageal cancer cells are influenced by acidic reflux and whether reflux influences cancer cell physiology, targeting the Nrf2/Kepa1- and the NFκB-pathway. The understanding mechanisms of the acidic susceptibility in cells from advanced stages of Barrett’s esophagus will provide further evidence, whether it should be prevented during chemotherapy for EAC treatment. Abstract Chronic acid reflux causes cellular damage and inflammation in the lower esophagus. Due to these irritating insults, the squamous epithelium is replaced by metaplastic epithelium, which is a risk factor for the development of esophageal adenocarcinoma (EAC). In this study, we investigated the acid susceptibility in a Barrett’s cell culture in vitro model, using six cell lines, derived from squamous epithelium (EPC1 and EPC2), metaplasia (CP-A), dysplasia (CP-B), and EAC (OE33 and OE19) cells. Cells exposed to acidic pH showed a decreased viability dependent on time, pH, and progression status in the Barrett’s sequence, with the highest acid susceptibility in the squamous epithelium (EPC1 and EPC2), and the lowest in EAC cells. Acid pulsing was accompanied with an activation of the Nrf2/Keap1- and the NFκB-pathway, resulting in an increased expression of HO1—independent of the cellular context. OE33 showed a decreased responsiveness towards 5-FU, when the cells were grown in acidic conditions (pH 6 and pH 5.5). Our findings suggest a strong damage of squamous epithelium by gastroesophageal reflux, while Barrett’s dysplasia and EAC cells apparently exert acid-protective features, which lead to a cellular resistance against acid reflux.
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Ren Z, Rajani C, Jia W. The Distinctive Serum Metabolomes of Gastric, Esophageal and Colorectal Cancers. Cancers (Basel) 2021; 13:cancers13040720. [PMID: 33578739 PMCID: PMC7916516 DOI: 10.3390/cancers13040720] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2020] [Revised: 01/18/2021] [Accepted: 02/07/2021] [Indexed: 12/14/2022] Open
Abstract
Simple Summary Cancer of the stomach, esophagus and colon are often fatal. Ways are being sought to establish patient-friendly screening tests that would allow these cancers to be detected earlier. Examination of the metabolomics results of cancer patient’s serum for certain metabolites unique for a particular cancer was the goal of this review. From studies conducted within the past five years several metabolites were found to be changed in cancer compared to non-cancer patients for each of the three cancers. Further confirmation of what was discovered in this review coupled with establishment of standard protocols may allow for cancer screening on patient blood samples to become routine clinical tests. Abstract Three of the most lethal cancers in the world are the gastrointestinal cancers—gastric (GC), esophageal (EC) and colorectal cancer (CRC)—which are ranked as third, sixth and fourth in cancer deaths globally. Early detection of these cancers is difficult, and a quest is currently on to find non-invasive screening tests to detect these cancers. The reprogramming of energy metabolism is a hallmark of cancer, notably, an increased dependence on aerobic glycolysis which is often referred to as the Warburg effect. This metabolic change results in a unique metabolic profile that distinguishes cancer cells from normal cells. Serum metabolomics analyses allow one to measure the end products of both host and microbiota metabolism present at the time of sample collection. It is a non-invasive procedure requiring only blood collection which encourages greater patient compliance to have more frequent screenings for cancer. In the following review we will examine some of the most current serum metabolomics studies in order to compare their results and test a hypothesis that different tumors, notably, from EC, GC and CRC, have distinguishing serum metabolite profiles.
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Affiliation(s)
- Zhenxing Ren
- Center for Translational Medicine, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China;
| | - Cynthia Rajani
- Cancer Biology Program, University of Hawaii Cancer Center, Honolulu, HI 96813, USA
- Correspondence: (C.R.); or (W.J.)
| | - Wei Jia
- Center for Translational Medicine, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China;
- Cancer Biology Program, University of Hawaii Cancer Center, Honolulu, HI 96813, USA
- School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China
- Correspondence: (C.R.); or (W.J.)
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Lipid droplet biogenesis and COX-2 pathway activation are triggered by Barrett's esophagus and adenocarcinoma, but not esophageal squamous cell carcinoma risk factors. Sci Rep 2021; 11:981. [PMID: 33441691 PMCID: PMC7807011 DOI: 10.1038/s41598-020-80035-4] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2019] [Accepted: 11/24/2020] [Indexed: 12/09/2022] Open
Abstract
Esophageal cancer (EC) is an aggressive disease, presenting two main histological subtypes: adenocarcinoma (EAC) and squamous cell carcinoma (ESCC). The two EC subtypes widely differ concerning virtually all factors. ESCC development is mainly associated with tobacco and alcohol abuse, whereas obesity and chronic gastroesophageal reflux disease (GERD) are important risk factors not only for EAC, but also for for Barrett’s esophagus (BE), an intestinal metaplasia that precedes EAC. Obesity triggers ectopic lipid droplets (LD) accumulation in non-adipose tissues. LD are organelles involved in cell metabolism, signaling, proliferation and production of inflammatory mediators. Therefore, the aim of this work was to investigate LD occurrence and role in EC. This study shows progressive LD levels increase along EAC development, in esophageal samples from non-obese through obese individuals, as well as BE, and EAC patients, whereas no significant changes were observed in ESCC samples, when compared to non-tumor samples. Additionally, in order to mimic BE and EAC risk factors exposure, a non-tumor esophageal cell line was incubated with oleic acid (OA) and acidified medium and/or deoxycholic acid (DCA), revealing a significant increment in LD amount as well as in COX-2 and CXCL-8 expression, and in IL-8 secretion. Further, COX-2 expression and LD amount presented a significant positive correlation and were detected co-localized in EAC, but not in ESCC, suggesting that LD may be the site for eicosanoid production in EAC. In conclusion, this study shows that obesity, and BE- and EAC-associated inflammatory stimuli result in a gradual increase of LD, that may be responsible for orchestrating inflammatory mediators’ production and/or action, thus contributing to BE and EAC genesis and progression.
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Abdulnour-Nakhoul SM, Nakhoul NL. Ussing Chamber Methods to Study the Esophageal Epithelial Barrier. Methods Mol Biol 2021; 2367:215-233. [PMID: 32946026 DOI: 10.1007/7651_2020_324] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/11/2023]
Abstract
The Ussing chamber was developed in 1949 by Hans Ussing and quickly became a powerful tool to study ion and solute transport in epithelia. The chamber has two compartments strictly separating the apical and basolateral sides of the tissue under study. The two sides of the tissue are connected via electrodes to a modified electrometer/pulse generator that allows measurement of electrical parameters, namely, transepithelial voltage, current, and resistance. Simultaneously, permeability of the tissue to specific solutes or markers can be monitored by using tracers or isotopes to measure transport from one side of the tissue to the other. In this chapter, we will describe the use of the Ussing chamber to study the barrier properties of the mouse esophageal epithelium. We will also briefly describe the use of the modified Ussing chamber to simultaneously study transepithelial and cellular electrophysiology in the rabbit esophageal epithelium. Lastly, we will cover the use of the Ussing chamber to study bicarbonate secretion in the pig esophagus. These examples highlight the versatility of the Ussing chamber technique in investigating the physiology and pathophysiology of epithelia including human biopsies.
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Affiliation(s)
| | - Nazih L Nakhoul
- Departments of Medicine and Physiology, Tulane University School of Medicine, New Orleans, LA, USA
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Xia Y, Fang Y, Zhang H, Shen C, Wang P, Yan W, Li J, Xu Y, Shao S, Zhang Y, Yu X, Peng Z, Peng G, Chen W, Fang D. Role of Kruppel-Like Factor 5 in Deoxycholic Acid-Mediated Intestinal Transdifferentiation of Esophageal Squamous Epithelium. J Cancer 2019; 10:5597-5607. [PMID: 31632504 PMCID: PMC6775683 DOI: 10.7150/jca.30050] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2018] [Accepted: 05/26/2019] [Indexed: 12/15/2022] Open
Abstract
Barrett's esophagus (BE) is an acquired condition in which normal squamous epithelium is replaced with metaplastic columnar epithelium as a consequence of gastroesophageal reflux disease. BE is known as a precursor of esophageal adenocarcinoma. Currently, the molecular mechanism underlying epithelial metaplasia in BE patients remains unknown. Therefore, we investigated the role of Krüppel-like factor 5 (KLF5) signaling in the initiation of BE-associated metaplasia. Sprague-Dawley (SD) rats were used to create a surgical model of bile reflux injury. Immunohistochemistry was performed to analyze human and mouse esophageal specimens. Human esophageal squamous epithelial (HET-1A) cells were treated with bile acid and used in transfection experiments. Quantitative real-time PCR and western blot analysis were performed to detect the expression of KLF5, CDX2, MUC2 and villin. Epithelial tissue from both the rat BE model and human BE patients strongly expressed KLF5, CDX2, MUC2, and villin. Bile acid treatment also increased the expression of KLF5, CDX2, MUC2 and villin in esophageal epithelial cells in a time-dependent manner. Moreover, siRNA-mediated knockdown of KLF5 blocked the expression of CDX2, MUC2 and villin, but transfection of a KLF5 expression vector into esophageal epithelial cells promoted their transdifferentiation into columnar-like cells, as demonstrated by increased expression of the intestinal markers CDX2, MUC2 and villin. Thus, in addition to its function as a transcription factor, KLF5 may be linked to an increased risk of BE development.
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Affiliation(s)
- Yiju Xia
- Department of Gastroenterology, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R. China
| | - Yu Fang
- Department of Thoracic and Cardiovascular Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400042, China
| | - Haoxiang Zhang
- Department of Gastroenterology, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R. China
| | - Caifei Shen
- Department of Gastroenterology, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R. China
| | - Pu Wang
- Department of Gastroenterology, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R. China
| | - Wu Yan
- Department of Gastroenterology, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R. China
| | - Jingwen Li
- Department of Gastroenterology, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R. China
| | - Yin Xu
- Department of Gastroenterology, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R. China
| | - Shunzi Shao
- Department of Gastroenterology, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R. China
| | - Yafei Zhang
- Department of Gastroenterology, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R. China
| | - Xiaona Yu
- Department of Gastroenterology, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R. China
| | - Zhihong Peng
- Department of Gastroenterology, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R. China
| | - Guiyong Peng
- Department of Gastroenterology, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R. China
| | - Wensheng Chen
- Department of Gastroenterology, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R. China
| | - Dianchun Fang
- Department of Gastroenterology, Southwest Hospital, Army Medical University, Chongqing, 400038, P.R. China
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Caspa Gokulan R, Garcia-Buitrago MT, Zaika AI. From genetics to signaling pathways: molecular pathogenesis of esophageal adenocarcinoma. Biochim Biophys Acta Rev Cancer 2019; 1872:37-48. [PMID: 31152823 PMCID: PMC6692203 DOI: 10.1016/j.bbcan.2019.05.003] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2019] [Revised: 05/10/2019] [Accepted: 05/10/2019] [Indexed: 02/07/2023]
Abstract
Esophageal adenocarcinoma (EAC) has one of the fastest rising incidence rates in the U.S. and many other Western countries. One of the unique risk factors for EAC is gastroesophageal reflux disease (GERD), a chronic digestive condition in which acidic contents from the stomach, frequently mixed with duodenal bile, enter the esophagus resulting in esophageal tissue injury. At the cellular level, progression to EAC is underlined by continuous DNA damage caused by reflux and chronic inflammatory factors that increase the mutation rate and promote genomic instability. Despite recent successes in cancer diagnostics and treatment, EAC remains a poorly treatable disease. Recent research has shed new light on molecular alterations underlying progression to EAC and revealed novel treatment options. This review focuses on the genetic and molecular studies of EAC. The molecular changes that occur during the transformation of normal Barrett's esophagus to esophageal adenocarcinoma are also discussed.
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Affiliation(s)
| | | | - Alexander I Zaika
- Department of Surgery, University of Miami, Miami, FL, United States of America; Department of Veterans Affairs, Miami VA Healthcare System, Miami, FL, United States of America.
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11
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Abstract
Chronic injury and inflammation in the esophagus can cause a change in cellular differentiation known as metaplasia. Most commonly, the differentiation changes manifest as Barrett's esophagus (BE), characterized by the normal stratified squamous epithelium converting into a cuboidal-columnar, glandular morphology. BE cells can phenotypically resemble specific normal cell types of the stomach or intestine, or they can have overlapping phenotypes in disorganized admixtures. The stomach can also undergo metaplasia characterized by aberrant gastric or intestinal differentiation patterns. In both organs, it has been argued that metaplasia may represent a recapitulation of the embryonic or juvenile gastrointestinal tract, as cells access a developmental progenitor genetic program that can help repair damaged tissue. Here, we review the normal development of esophagus and stomach, and describe how BE represents an intermixing of cells resembling gastric pseudopyloric (SPEM) and intestinal metaplasia. We discuss a cellular process recently termed "paligenosis" that governs how mature, differentiated cells can revert to a proliferating progenitor state in metaplasia. We discuss the "Cyclical Hit" theory in which paligenosis might be involved in the increased risk of metaplasia for progression to cancer. However, somatic mutations might occur in proliferative phases and then be warehoused upon redifferentiation. Through years of chronic injury and many rounds of paligenosis and dedifferentiation, eventually a cell with a mutation that prevents dedifferentiation may arise and clonally expand fueling stable metaplasia and potentially thereafter acquiring additional mutations and progressing to dysplasia and cancer.
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Affiliation(s)
- Ramon U Jin
- Division of Oncology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA
| | - Jason C Mills
- Division of Gastroenterology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA.
- Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO, USA.
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA.
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12
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Huo X, Zhang X, Yu C, Cheng E, Zhang Q, Dunbar KB, Pham TH, Lynch JP, Wang DH, Bresalier RS, Spechler SJ, Souza RF. Aspirin prevents NF-κB activation and CDX2 expression stimulated by acid and bile salts in oesophageal squamous cells of patients with Barrett's oesophagus. Gut 2018; 67:606-615. [PMID: 28442495 PMCID: PMC5656558 DOI: 10.1136/gutjnl-2016-313584] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/13/2016] [Revised: 03/28/2017] [Accepted: 04/03/2017] [Indexed: 12/11/2022]
Abstract
OBJECTIVE In previous studies using oesophageal squamous cells from patients with Barrett's oesophagus (normal oesophageal squamous (NES)-B cells) and from patients without Barrett's oesophagus (NES-G cells), we showed that acid and bile salts induced caudal-related homeobox transcription factor 2 (CDX2) expression only in NES-B cells. CDX2, a transcription factor required to form intestinal epithelium, is a target of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling, which can be inhibited by aspirin. We explored mechanisms underlying differences between NES-B and NES-G cells in CDX2 expression and effects of aspirin on that CDX2 expression. DESIGN We exposed NES-B and NES-G cells to acid and bile salts, with and without aspirin, and evaluated effects on IκB-NF-κB-PKAc complex activation, p65 NF-κB subunit function, and CDX2 expression. RESULTS In both NES-B and NES-G cells, acid and bile salts activated nicotinamide adenine dinucleotide phosphate oxidase to generate H2O2, which activated the IκB-NF-κB-PKAc complex. NES-B cells exhibited higher levels of phosphorylated IκB and p65 and greater NF-κB transcriptional activity than NES-G cells, indicating greater IκB-NF-κB-PKAc complex activation by acid and bile salts in NES-B cells, and p65 siRNA prevented their increased expression of CDX2. Aspirin blocked IκB phosphorylation, p65 nuclear translocation, CDX2 promoter activation and CDX2 expression induced by acid and bile salts in NES-B cells. CONCLUSIONS Differences between NES-B and NES-G cells in NF-κB activation by acid and bile salts can account for their differences in CDX2 expression, and their CDX2 expression can be blocked by aspirin. These findings might explain why some patients with GORD develop Barrett's oesophagus while others do not, and why aspirin might protect against development of Barrett's oesophagus.
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Affiliation(s)
- Xiaofang Huo
- Esophageal Diseases Center, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Medicine, Center for Esophageal Diseases, Baylor University Medical Center and Center for Esophageal Research, Baylor Scott and White Research Institute, Dallas, Texas
| | - Xi Zhang
- Esophageal Diseases Center, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Medicine, Center for Esophageal Diseases, Baylor University Medical Center and Center for Esophageal Research, Baylor Scott and White Research Institute, Dallas, Texas
| | - Chunhua Yu
- Esophageal Diseases Center, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX
| | - Edaire Cheng
- Esophageal Diseases Center, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Pediatrics, Children's Medical Center and the University of Texas Southwestern Medical Center, Dallas, TX
| | - Qiuyang Zhang
- Esophageal Diseases Center, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Medicine, Center for Esophageal Diseases, Baylor University Medical Center and Center for Esophageal Research, Baylor Scott and White Research Institute, Dallas, Texas
| | - Kerry B. Dunbar
- Esophageal Diseases Center, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX
| | - Thai H. Pham
- Esophageal Diseases Center, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Surgery, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX
| | - John P. Lynch
- Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
| | - David H. Wang
- Esophageal Diseases Center, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX
| | - Robert S. Bresalier
- Department of Medicine, University of Texas MD Anderson Cancer Center, Houston, TX
| | - Stuart J. Spechler
- Esophageal Diseases Center, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Medicine, Center for Esophageal Diseases, Baylor University Medical Center and Center for Esophageal Research, Baylor Scott and White Research Institute, Dallas, Texas,Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX
| | - Rhonda F. Souza
- Esophageal Diseases Center, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical Center, Dallas, TX,Department of Medicine, Center for Esophageal Diseases, Baylor University Medical Center and Center for Esophageal Research, Baylor Scott and White Research Institute, Dallas, Texas,Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX
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13
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Lin B, Xie F, Xiao Z, Hong X, Tian L, Liu K. Basal progenitor cells bridge the development, malignant cancers, and multiple diseases of esophagus. J Cell Physiol 2017; 233:3855-3866. [PMID: 28777465 DOI: 10.1002/jcp.26136] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2017] [Accepted: 08/03/2017] [Indexed: 12/19/2022]
Abstract
The esophagus is a pivotal organ originating from anterior foregut that links the mouth and stomach. Moreover, its development involves precise regulation of multiple signal molecules and signal transduction pathways. After abnormal regulation of these molecules in the basal cells of the esophagus occurs, multiple diseases, including esophageal atresia with or without tracheoesophageal fistula, Barrett esophagus, gastroesophageal reflux, and eosinophilic esophagitis, will take place as a result. Furthermore, expression changes of signal molecules or signal pathways in basal cells and the microenvironment around basal cells both can initiate the switch of malignant transformation. In this review, we highlight the molecular events underlying the transition of normal development to multiple esophageal diseases. Additionally, the animal models of esophageal development and related diseases, challenges, and strategies are extensively discussed.
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Affiliation(s)
- Baoshun Lin
- Institute for Laboratory Medicine, Fuzhou General Hospital, PLA, Fuzhou, Fujian, P. R. China
| | - Fuan Xie
- Institute for Laboratory Medicine, Fuzhou General Hospital, PLA, Fuzhou, Fujian, P. R. China
| | - Zhangwu Xiao
- Emergency Department of the 476 Hospital, Fuzhou General Hospital, PLA, Fuzhou, Fujian, P. R. China
| | - Xiaoqian Hong
- Dong fang Hospital, Xiamen University, Fuzhou, Fujian, P. R. China
| | - Liming Tian
- Dong fang Hospital, Xiamen University, Fuzhou, Fujian, P. R. China
| | - Kuancan Liu
- Institute for Laboratory Medicine, Fuzhou General Hospital, PLA, Fuzhou, Fujian, P. R. China.,Dong fang Hospital, Xiamen University, Fuzhou, Fujian, P. R. China.,Department of Medicine, Columbia University Medical Center, New York, New York
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14
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Souza RF. Reflux esophagitis and its role in the pathogenesis of Barrett's metaplasia. J Gastroenterol 2017; 52:767-776. [PMID: 28451845 PMCID: PMC5488728 DOI: 10.1007/s00535-017-1342-1] [Citation(s) in RCA: 42] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/07/2017] [Accepted: 04/11/2017] [Indexed: 02/04/2023]
Abstract
Reflux esophagitis damages the squamous epithelium that normally lines the esophagus, and promotes replacement of the damaged squamous lining by the intestinal metaplasia of Barrett's esophagus, the precursor of esophageal adenocarcinoma. Therefore, to prevent the development of Barrett's metaplasia and esophageal adenocarcinoma, the pathogenesis of reflux esophagitis must be understood. We have reported that reflux esophagitis, both in a rat model and in humans, develops as a cytokine-mediated inflammatory injury (i.e., cytokine sizzle), not as a caustic chemical injury (i.e., acid burn), as traditionally has been assumed. Moreover, reflux induces activation of hypoxia inducible factor (HIF)-2α, which enhances the transcriptional activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) causing increases in pro-inflammatory cytokines and in migration of T lymphocytes, an underlying molecular mechanism for this cytokine-mediated injury. In some individuals, reflux esophagitis heals with Barrett's metaplasia. A number of possibilities exist for the origin of the progenitor cells that give rise to this intestinal metaplasia including those of the esophagus, the proximal stomach, or the bone marrow. However, intestinal cells are not normally found in the esophagus, the stomach, or the bone marrow. Thus, the development of Barrett's intestinal metaplasia must involve some molecular reprogramming of key developmental transcription factors within the progenitor cell, a process termed transcommitment, which may be initiated by the noxious components of the gastric refluxate. This review will highlight recent studies on the pathogenesis of reflux esophagitis and on reflux-related molecular reprogramming of esophageal squamous epithelial cells in the pathogenesis of Barrett's metaplasia.
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Affiliation(s)
- Rhonda F. Souza
- Center for Esophageal Research, Baylor Scott and White Research Institute, Dallas, TX, USA
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15
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Carr DF, Ayehunie S, Davies A, Duckworth CA, French S, Hall N, Hussain S, Mellor HR, Norris A, Park BK, Penrose A, Pritchard DM, Probert CS, Ramaiah S, Sadler C, Schmitt M, Shaw A, Sidaway JE, Vries RG, Wagoner M, Pirmohamed M. Towards better models and mechanistic biomarkers for drug-induced gastrointestinal injury. Pharmacol Ther 2017; 172:181-194. [DOI: 10.1016/j.pharmthera.2017.01.002] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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16
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Colleypriest BJ, Burke ZD, Griffiths LP, Chen Y, Yu WY, Jover R, Bock M, Biddlestone L, Quinlan JM, Ward SG, Mark Farrant J, Slack JMW, Tosh D. Hnf4α is a key gene that can generate columnar metaplasia in oesophageal epithelium. Differentiation 2016; 93:39-49. [PMID: 27875772 PMCID: PMC5293356 DOI: 10.1016/j.diff.2016.11.001] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2016] [Revised: 11/02/2016] [Accepted: 11/07/2016] [Indexed: 12/20/2022]
Abstract
Barrett's metaplasia is the only known morphological precursor to oesophageal adenocarcinoma and is characterized by replacement of stratified squamous epithelium by columnar epithelium. The cell of origin is uncertain and the molecular mechanisms responsible for the change in cellular phenotype are poorly understood. We therefore explored the role of two transcription factors, Cdx2 and HNF4α in the conversion using primary organ cultures. Biopsy samples from cases of human Barrett's metaplasia were analysed for the presence of CDX2 and HNF4α. A new organ culture system for adult murine oesophagus is described. Using this, Cdx2 and HNF4α were ectopically expressed by adenoviral infection. The phenotype following infection was determined by a combination of PCR, immunohistochemical and morphological analyses. We demonstrate the expression of CDX2 and HNF4α in human biopsy samples. Our oesophageal organ culture system expressed markers characteristic of the normal SSQE: p63, K14, K4 and loricrin. Ectopic expression of HNF4α, but not of Cdx2 induced expression of Tff3, villin, K8 and E-cadherin. HNF4α is sufficient to induce a columnar-like phenotype in adult mouse oesophageal epithelium and is present in the human condition. These data suggest that induction of HNF4α is a key early step in the formation of Barrett's metaplasia and are consistent with an origin of Barrett's metaplasia from the oesophageal epithelium.
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Affiliation(s)
- Benjamin J Colleypriest
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK; Department of Gastroenterology, Royal United Hospital, Combe Park, Bath BA1 3NG, UK
| | - Zoë D Burke
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
| | - Leonard P Griffiths
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK; Department of Gastroenterology, Royal United Hospital, Combe Park, Bath BA1 3NG, UK
| | - Yu Chen
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
| | - Wei-Yuan Yu
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
| | - Ramiro Jover
- Unidad Mixta Hepatologia Experimental & CIBERehd, Departamento de Bioquimica y Biologia Molecular, Universidad de Valencia, Spain
| | - Michael Bock
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
| | - Leigh Biddlestone
- Department of Gastroenterology, Royal United Hospital, Combe Park, Bath BA1 3NG, UK
| | - Jonathan M Quinlan
- Department of Gastroenterology, Royal United Hospital, Combe Park, Bath BA1 3NG, UK
| | - Stephen G Ward
- Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK
| | - J Mark Farrant
- Department of Gastroenterology, Royal United Hospital, Combe Park, Bath BA1 3NG, UK
| | - Jonathan M W Slack
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK; Stem Cell Institute, University of Minnesota, Minneapolis 55455, USA
| | - David Tosh
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.
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17
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Kusaka G, Uno K, Iijima K, Shimosegawa T. Role of nitric oxide in the pathogenesis of Barrett’s-associated carcinogenesis. World J Gastrointest Pathophysiol 2016; 7:131-137. [PMID: 26909236 PMCID: PMC4753179 DOI: 10.4291/wjgp.v7.i1.131] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2015] [Revised: 09/01/2015] [Accepted: 11/11/2015] [Indexed: 02/06/2023] Open
Abstract
Barrett’s esophagus (BE), a premalignant condition to Barrett’s adenocarcinoma (BAC), is closely associated with chronic inflammation due to gastro-esophageal reflux. Caudal type homeobox 2 (CDX2), a representative marker of BE, is increased during the metaplastic and neoplastic transformation of BE. Nitric oxide (NO) has been proposed to be a crucial mediator of Barrett’s carcinogenesis. We previously demonstrated that CDX2 might be induced directly under stimulation of large amounts of NO generated around the gastro-esophageal junction (GEJ) by activating epithelial growth factor receptor in a ligand-independent manner. Thus, we reviewed recent developments on the role of NO in Barrett’s carcinogenesis. Notably, recent studies have reported that microbial communities in the distal esophagus are significantly different among groups with a normal esophagus, reflux esophagitis, BE or BAC, despite there being no difference in the bacterial quantity. Considering that microorganism components can be one of the major sources of large amounts of NO, these studies suggest that the bacterial composition in the distal esophagus might play an important role in regulating NO production during the carcinogenic process. Controlling an inflammatory reaction due to gastro-esophageal reflux or bacterial composition around the GEJ might help prevent the progression of Barrett’s carcinogenesis by inhibiting NO production.
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18
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Wang DH, Souza RF. Transcommitment: Paving the Way to Barrett's Metaplasia. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2016; 908:183-212. [PMID: 27573773 DOI: 10.1007/978-3-319-41388-4_10] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Barrett's esophagus is the condition in which metaplastic columnar epithelium that predisposes to cancer development replaces stratified squamous epithelium in the distal esophagus. Potential sources for the cell or tissue of origin for metaplastic Barrett's epithelium are reviewed including native esophageal differentiated squamous cells, progenitor cells native to the esophagus located within the squamous epithelium or in the submucosal glands or ducts, circulating bone marrow-derived stem cells, and columnar progenitor cells from the squamocolumnar junction or the gastric cardia that proximally shift into the esophagus to fill voids left by damaged squamous epithelium. Wherever its source the original cell must undergo molecular reprogramming (i.e., either transdifferentiation or transcommitment) to give rise to specialized intestinal metaplasia. Transcription factors that specify squamous, columnar, intestinal, and mucus-secreting epithelial differentiation are discussed. An improved understanding of how esophageal columnar metaplasia forms could lead to development of effective treatment or prevention strategies for Barrett's esophagus. It could also more broadly inform upon normal tissue development and differentiation, wound healing, and stem cell biology.
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Affiliation(s)
- David H Wang
- Division of Hematology and Oncology, Department of Internal Medicine, Harold C. Simmons Comprehensive Cancer Center, Esophageal Diseases Center, Medical Service, VA North Texas Health Care System, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390-8852, USA.
| | - Rhonda F Souza
- Division of Digestive and Liver Diseases, Department of Internal Medicine, Harold C. Simmons Comprehensive Cancer Center, Esophageal Diseases Center, Medical Service (111B1), VA North Texas Health Care System, University of Texas Southwestern Medical Center, 4500 S. Lancaster Road, Dallas, TX, 75216, USA
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19
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Abdulnour-Nakhoul S, Brown KL, Rabon EC, Al-Tawil Y, Islam MT, Schmieg JJ, Nakhoul NL. Cytoskeletal changes induced by allosteric modulators of calcium-sensing receptor in esophageal epithelial cells. Physiol Rep 2015; 3:3/11/e12616. [PMID: 26603452 PMCID: PMC4673644 DOI: 10.14814/phy2.12616] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell-cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell-cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett's, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin-catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells.
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Affiliation(s)
- Solange Abdulnour-Nakhoul
- Medicine/Gastroenterology, Tulane Medical School, New Orleans, Louisiana South Louisiana Veterans Health Care System (SLVHCS), New Orleans, Louisiana
| | - Karen L Brown
- South Louisiana Veterans Health Care System (SLVHCS), New Orleans, Louisiana Medicine/Nephrology, Tulane Medical School, New Orleans, Louisiana
| | - Edd C Rabon
- South Louisiana Veterans Health Care System (SLVHCS), New Orleans, Louisiana
| | - Youhanna Al-Tawil
- Pediatric Gastroenterology and Nutrition-GI for Kids, East Tennessee Children's Hospital, Knoxville, Tennessee
| | - Mohammed T Islam
- South Louisiana Veterans Health Care System (SLVHCS), New Orleans, Louisiana Medicine/Nephrology, Tulane Medical School, New Orleans, Louisiana
| | - John J Schmieg
- Pathology, Tulane Medical School, New Orleans, Louisiana
| | - Nazih L Nakhoul
- South Louisiana Veterans Health Care System (SLVHCS), New Orleans, Louisiana Medicine/Nephrology, Tulane Medical School, New Orleans, Louisiana
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20
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Kameoka Y, Kitazawa R, Ariasu K, Tachibana R, Mizuno Y, Haraguchi R, Kitazawa S. Reactivation of CDX2 in Gastric Cancer as Mark for Gene Silencing Memory. Acta Histochem Cytochem 2015; 48:115-24. [PMID: 26379313 PMCID: PMC4564377 DOI: 10.1267/ahc.15014] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2015] [Accepted: 06/30/2015] [Indexed: 01/05/2023] Open
Abstract
To explore the epigenetic mechanism that reactivates CDX2 (a homeobox transcription factor that serves as a tumor-suppressor gene) in intestinal-type gastric cancer during cancer progression, we examined the methylation status of the CDX2 gene promoter and the expression pattern of methyl-CpG binding protein-2 (MeCP2). From archives of the pathology records of surgically excised advanced stomach cancer cases in the Department of Molecular Pathology, Ehime University in a past decate (n=265), 10 cases of intestinal-type tubular adenocarcinoma, well-differentiated type (wel) with minor poorly-differentiated adenocarcinoma (por) components were selected. The expression pattern of CDX2, MUC2 and MeCP2 in these 10 cases was analyzed by immunohistochemistry. The cancerous and non-cancerous areas were selectively obtained by microdissection, and the methylation status of the CDX2 promoter of each area was assessed by methylation-specific polymerase chain reaction (MSP). In all 10 cases, CDX2 expression was clearly observed in the nucleus of the non-cancerous background of the intestinal metaplasic area, where the unmethylation pattern of the CDX2 gene promoter prevailed with reduced MeCP2 expression. In this metaplastic area, CDX2 expression was co-localized with its target gene, MUC2. CDX2 expression then disappeared from the deep invasive wel area. Reflecting the reduced CDX2 expression, microdissected samples from all the wel areas showed hypermethylation of the CDX2 gene promoter by MSP, with prominent MeCP2 expression. Interestingly, while hypermethylation of the CDX2 gene promoter was maintained in the por area in 8 of the 10 cases, CDX2 expression was restored in por areas where MeCP2 expression was markedly and selectively reduced. The other two cases, however, showed a constant MeCP2 expression level comparable to the surrounding deep invasive wel area with negative CDX2 expression. Therefore, gene silencing by hypermethylation may be overcome by the reduction of methyl-CpG binding proteins, resulting in apparent but non-functional reactivation of CDX2 as a mere molecular mark for gene silencing memory.
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Affiliation(s)
- Yuri Kameoka
- Department of Molecular Pathology, Ehime University Graduate School of Medicine
| | - Riko Kitazawa
- Department of Molecular Pathology, Ehime University Graduate School of Medicine
- Department of Diagnostic Pathology, Ehime University Hospital
| | - Kanazu Ariasu
- Department of Molecular Pathology, Ehime University Graduate School of Medicine
| | - Ryosuke Tachibana
- Department of Molecular Pathology, Ehime University Graduate School of Medicine
| | - Yosuke Mizuno
- Department of Diagnostic Pathology, Ehime University Hospital
| | - Ryuma Haraguchi
- Department of Molecular Pathology, Ehime University Graduate School of Medicine
| | - Sohei Kitazawa
- Department of Molecular Pathology, Ehime University Graduate School of Medicine
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21
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Park SY, Lee YJ, Cho EJ, Shin CY, Sohn UD. Intrinsic resistance triggered under acid loading within normal esophageal epithelial cells: NHE1- and ROS-mediated survival. J Cell Physiol 2015; 230:1503-14. [DOI: 10.1002/jcp.24896] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2013] [Accepted: 12/12/2014] [Indexed: 01/27/2023]
Affiliation(s)
- Sun Young Park
- Department of Pharmacology; College of Pharmacy; Chung-Ang University; Seoul South Korea
| | - Yeon Joo Lee
- Department of Pharmacology; College of Pharmacy; Chung-Ang University; Seoul South Korea
| | - Eun Jeong Cho
- Department of Pharmacology; College of Pharmacy; Chung-Ang University; Seoul South Korea
| | - Chang Yell Shin
- Dong-A Pharmaceutical; Research Center; Yongin-si Gyeonggi-do South Korea
| | - Uy Dong Sohn
- Department of Pharmacology; College of Pharmacy; Chung-Ang University; Seoul South Korea
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Nakagawa H, Whelan K, Lynch JP. Mechanisms of Barrett's oesophagus: intestinal differentiation, stem cells, and tissue models. Best Pract Res Clin Gastroenterol 2015; 29:3-16. [PMID: 25743452 PMCID: PMC4352719 DOI: 10.1016/j.bpg.2014.11.001] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/13/2014] [Accepted: 11/02/2014] [Indexed: 01/31/2023]
Abstract
Barrett's oesophagus (BE) is defined as any metaplastic columnar epithelium in the distal oesophagus which replaces normal squamous epithelium and which predisposes to cancer development. It is this second requirement, the predisposition to cancer, which makes this condition both clinically highly relevant and an important area for ongoing research. While BE has been defined pathologically since the 1950's (Allison and Johnstone, Thorax 1955), and identified as a risk factor for esophageal adenocarcinoma since the 1970's (Naef A.P., et al J Thorac Cardiovasc Surg. 1975), our understanding of the molecular events giving rise to this condition remains limited. Herein we will examine what is known about the intestinal features of BE and how well it recapitulates the intestinal epithelium, including stem identity and function. Finally, we will explore laboratory models of this condition presently in use and under development, to identify new insights they may provide into this important clinical condition.
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Affiliation(s)
- Hiroshi Nakagawa
- Research Associate Professor of Medicine, Division of Gastroenterology, 421 Curie Boulevard, 956 Biomedical Research Building, Philadelphia, PA 19104, Office: 215-573-1867, Fax: 215-573-2024
| | - Kelly Whelan
- Division of Gastroenterology, 421 Curie Boulevard, 956 Biomedical Research Building, Philadelphia, PA, 19104, USA.
| | - John P Lynch
- Division of Gastroenterology, 421 Curie Boulevard, 956 Biomedical Research Building, Philadelphia, PA, 19104, USA.
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Hartman KG, Bortner JD, Falk GW, Ginsberg GG, Jhala N, Yu J, Martín MG, Rustgi AK, Lynch JP. Modeling human gastrointestinal inflammatory diseases using microphysiological culture systems. Exp Biol Med (Maywood) 2014; 239:1108-23. [PMID: 24781339 PMCID: PMC4156523 DOI: 10.1177/1535370214529388] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Gastrointestinal illnesses are a significant health burden for the US population, with 40 million office visits each year for gastrointestinal complaints and nearly 250,000 deaths. Acute and chronic inflammations are a common element of many gastrointestinal diseases. Inflammatory processes may be initiated by a chemical injury (acid reflux in the esophagus), an infectious agent (Helicobacter pylori infection in the stomach), autoimmune processes (graft versus host disease after bone marrow transplantation), or idiopathic (as in the case of inflammatory bowel diseases). Inflammation in these settings can contribute to acute complaints (pain, bleeding, obstruction, and diarrhea) as well as chronic sequelae including strictures and cancer. Research into the pathophysiology of these conditions has been limited by the availability of primary human tissues or appropriate animal models that attempt to physiologically model the human disease. With the many recent advances in tissue engineering and primary human cell culture systems, it is conceivable that these approaches can be adapted to develop novel human ex vivo systems that incorporate many human cell types to recapitulate in vivo growth and differentiation in inflammatory microphysiological environments. Such an advance in technology would improve our understanding of human disease progression and enhance our ability to test for disease prevention strategies and novel therapeutics. We will review current models for the inflammatory and immunological aspects of Barrett's esophagus, acute graft versus host disease, and inflammatory bowel disease and explore recent advances in culture methodologies that make these novel microphysiological research systems possible.
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Affiliation(s)
- Kira G Hartman
- Division of Gastroenterology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
| | - James D Bortner
- Division of Gastroenterology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
| | - Gary W Falk
- Division of Gastroenterology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
| | - Gregory G Ginsberg
- Division of Gastroenterology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
| | - Nirag Jhala
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
| | - Jian Yu
- Departments of Pathology and Radiation Oncology, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA
| | - Martín G Martín
- Department of Pediatrics, Division of Gastroenterology and Nutrition, Mattel Children's Hospital and the David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California 90095, USA
| | - Anil K Rustgi
- Division of Gastroenterology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
| | - John P Lynch
- Division of Gastroenterology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
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McAdam E, Haboubi HN, Griffiths AP, Baxter JN, Spencer-Harty S, Davies C, Jenkins GJ. Reflux composition influences the level of NF-κB activation and upstream kinase preference in oesophageal adenocarcinoma cells. Int J Cancer 2014; 136:527-35. [PMID: 24931696 DOI: 10.1002/ijc.29029] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2014] [Revised: 06/03/2014] [Accepted: 06/06/2014] [Indexed: 01/17/2023]
Abstract
Oesophageal adenocarcinoma (OA) incidence is rising and prognosis is poor. Understanding the molecular basis of this malignancy is key to finding new prevention and treatment strategies. Gastroesophageal reflux disease is the primary cause of OA, usually managed with acid suppression therapy. However, this often does little to control carcinogenic bile acid reflux. The transcription factor nuclear factor kappa B (NF-κB) plays a key role in the pathogenesis of OA and its activity is associated with a poor response to chemotherapy, making it an attractive therapeutic target. We sought to decipher the role of different bile acids in NF-κB activation in oesophageal cell lines using short, physiologically relevant exposure times. The effect of an acidic or neutral extracellular pH was investigated concurrently, to mimic in vivo conditions associated with or without acid suppression. We found that some bile acids activated NF-κB to a greater extent when combined with acid, whereas others did so in its absence, at neutral pH. The precise composition of an individual's reflux, coupled with whether they are taking acid suppressants may therefore dictate the extent of NF-κB activation in the oesophagus, and hence the likelihood of histological progression and chemotherapy success. Regardless of pH, the kinase inhibitor of κB kinase was pivotal in mediating reflux induced NF-κB activation. Its importance was confirmed further as its increased activation was associated with histological progression in patient samples. We identified further kinases important in acid or bile induced NF-κB signalling in oesophageal cells, which may provide suitable targets for therapeutic intervention.
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Affiliation(s)
- E McAdam
- Institute of Life Science, School of Medicine, Swansea University, Singleton Park, Swansea, SA2 8PP, United Kingdom
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25
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Clemons NJ, Wang DH, Croagh D, Tikoo A, Fennell CM, Murone C, Scott AM, Watkins DN, Phillips WA. Sox9 drives columnar differentiation of esophageal squamous epithelium: a possible role in the pathogenesis of Barrett's esophagus. Am J Physiol Gastrointest Liver Physiol 2012; 303:G1335-46. [PMID: 23064761 DOI: 10.1152/ajpgi.00291.2012] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The molecular mechanism underlying the development of Barrett's esophagus (BE), the precursor to esophageal adenocarcinoma, remains unknown. Our previous work implicated sonic hedgehog (Shh) signaling as a possible driver of BE and suggested that bone morphogenetic protein 4 (Bmp4) and Sox9 were downstream mediators. We have utilized a novel in vivo tissue reconstitution model to investigate the relative roles of Bmp4 and Sox9 in driving metaplasia. Epithelia reconstituted from squamous epithelial cells or empty vector-transduced cells had a stratified squamous phenotype, reminiscent of normal esophagus. Expression of Bmp4 in the stromal compartment activated signaling in the epithelium but did not alter the squamous phenotype. In contrast, expression of Sox9 in squamous epithelial cells induced formation of columnar-like epithelium with expression of the columnar differentiation marker cytokeratin 8 and the intestinal-specific glycoprotein A33. In patient tissue, A33 protein was expressed specifically in BE, but not in normal esophagus. Expression of Cdx2, another putative driver of BE, alone had no effect on reconstitution of a squamous epithelium. Furthermore, epithelium coexpressing Cdx2 and Sox9 had a phenotype similar to epithelium expressing Sox9 alone. Our results demonstrate that Sox9 is sufficient to drive columnar differentiation of squamous epithelium and expression of an intestinal differentiation marker, reminiscent of BE. These data suggest that Shh-mediated expression of Sox9 may be an important early event in the development of BE and that the potential for inhibitors of the hedgehog pathway to be used in the treatment of BE and/or esophageal adenocarcinoma could be tested in the near future.
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Affiliation(s)
- Nicholas J Clemons
- Surgical Oncology Research Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.
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Chen X, Jiang K, Fan Z, Liu Z, Zhang P, Zheng L, Peng N, Tong J, Ji G. Aberrant expression of Wnt and Notch signal pathways in Barrett's esophagus. Clin Res Hepatol Gastroenterol 2012; 36:473-83. [PMID: 22889748 DOI: 10.1016/j.clinre.2012.06.001] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/19/2012] [Revised: 05/30/2012] [Accepted: 06/06/2012] [Indexed: 02/04/2023]
Abstract
BACKGROUND Specialized intestinal metaplasia (SIM) mucosa observed in Barrett's esophagus (BE) has been a hot research topic due to the close relationship with adenocarcinoma of esophagus (ACE). Earlier studies found that SIM mucosa was similar to normal intestinal epithelium on morphology and histology. The signal pathway involved in conformation of intestinal epithelium may be a critical initiator for BE. METHODS The expressions of key regulators (Tcf4, Cdx2, Hes1 and Math1) in Wnt and Notch signal pathways were measured in 41 paired esophageal biopsies. Cell morphological changes in normal esophagus were compared among groups treated by acid, bile acid and the mixture of both media. RESULTS The expression levels of regulators (Tcf4, Cdx2, Hes1 and Math1) were found significantly increased in SIM, C ≥ 3Mn and high-grade dysplasia (HGD) groups. Distinct ultrastructure changes and highly expressed key regulators were also detected at the seventh day for group treated by 400 μmol bile acid. CONCLUSIONS Aberrant expressed regulators in Wnt and Notch pathways were observed in BE tissue and normal esophageal cells treated by acid, bile acid and the mixture of both media. This study provided preliminary data to understand the mechanism of BE conformation.
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Affiliation(s)
- Xia Chen
- Institute of Digestive Endoscopy and Medical Center for Digestive Diseases, Second Affiliated Hospital of Nanjing Medical University, 121, Jing Jiayuan, Nanjing 210011, China
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Bile Exposure Inhibits Expression of Squamous Differentiation Genes in Human Esophageal Epithelial Cells. Ann Surg 2012; 255:1113-20. [DOI: 10.1097/sla.0b013e3182512af9] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
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Cox2 and β-catenin/T-cell factor signaling intestinalize human esophageal keratinocytes when cultured under organotypic conditions. Neoplasia 2012; 13:792-805. [PMID: 21969813 DOI: 10.1593/neo.11788] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2011] [Revised: 07/14/2011] [Accepted: 07/18/2011] [Indexed: 12/12/2022] Open
Abstract
The incidence of esophageal adenocarcinoma (EAC) is rising in the United States. An important risk factor for EAC is the presence of Barrett esophagus (BE). BE is the replacement of normal squamous esophageal epithelium with a specialized columnar epithelium in response to chronic acid and bile reflux. However, the emergence of BE from squamous keratinocytes has not yet been demonstrated. Our research has focused on this. Wnt and cyclooxygenase 2 (Cox2) are two pathways whose activation has been associated with BE and progression to EAC, but their role has not been tested experimentally. To explore their contribution, we engineered a human esophageal keratinocyte cell line to express either a dominant-active Wnt effector CatCLef or a Cox2 complementary DNA. In a two-dimensional culture environment, Cox2 expression increases cell proliferation and migration, but neither transgene induces known BE markers. In contrast, when these cells were placed into three-dimensional organotypic culture conditions, we observed more profound effects. CatCLef-expressing cells were more proliferative, developed a thicker epithelium, and upregulated Notch signaling and several BE markers including NHE2. Cox2 expression also increased cell proliferation and induced a thicker epithelium. More importantly, we observed cysts form within the epithelium, filled with intestinal mucins including Muc5B and Muc17. This suggests that Cox2 expression in a three-dimensional culture environment induces a lineage of mucin-secreting cells and supports an important causal role for Cox2 in BE pathogenesis. We conclude that in vitro modeling of BE pathogenesis can be improved by enhancing Wnt signaling and Cox2 activity and using three-dimensional organotypic culture conditions.
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Castillo D, Puig S, Iglesias M, Seoane A, de Bolós C, Munitiz V, Parrilla P, Comerma L, Poulsom R, Krishnadath KK, Grande L, Pera M. Activation of the BMP4 pathway and early expression of CDX2 characterize non-specialized columnar metaplasia in a human model of Barrett's esophagus. J Gastrointest Surg 2012; 16:227-37; discussion 237. [PMID: 22076569 DOI: 10.1007/s11605-011-1758-5] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/29/2011] [Accepted: 05/31/2011] [Indexed: 01/31/2023]
Abstract
BACKGROUND A human model of gastroesophageal reflux disease was used to examine the contribution of a non-specialized columnar type of metaplasia (NSCM) and key molecular events (BMP4 and CDX2) in the development of Barrett's esophagus. METHODS Biopsies of the remnant esophagus from 18 patients undergoing esophagectomy with gastric preservation were taken at 6-36-month intervals postoperatively and examined for activation of the BMP pathway (BMP4/P-Smad 1/5/8) and CDX2 and CDX1 expression by imunohistochemistry, quantitative real-time PCR, Western blot, and in situ hybridization. RESULTS A short segment (mean 15.6 mm) of NSCM was detected in 10 (56%) patients, with an increasing prevalence from 17% at 6 months to 62% at 36 months. Nuclear expression of P-Smad 1/5/8 in the squamous epithelium close to the anastomosis with strong expression in all epithelial cells of NSCM areas was found. Forty-eight (63%) biopsies with NSCM showed scattered nuclear expression of CDX2. Two cases showed isolated glands at 18, 24, and 36 months that fully expressed CDX2 and co-expressed CDX1. BMP4 mRNA and CDX2 mRNA levels were significantly greater in NSCM than in squamous epithelium. CONCLUSIONS BMP4 activation in NSCM and early expression of CDX2 are involved in the columnar epithelial differentiation of Barrett's esophagus.
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Affiliation(s)
- Daniel Castillo
- Section of Gastrointestinal Surgery, Parc de Salut Mar and Institut de Recerca Hospital del Mar (IMIM-Hospital del Mar), Universitat Autònoma de Barcelona, Passeig Maritim 25-29, 08003, Barcelona, Spain
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Chen H, Fang Y, Tevebaugh W, Orlando RC, Shaheen NJ, Chen X. Molecular mechanisms of Barrett's esophagus. Dig Dis Sci 2011; 56:3405-20. [PMID: 21984436 PMCID: PMC3750118 DOI: 10.1007/s10620-011-1885-6] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/16/2011] [Accepted: 08/16/2011] [Indexed: 12/11/2022]
Abstract
Barrett's esophagus (BE) is defined as the metaplastic conversion of esophageal squamous epithelium to intestinalized columnar epithelium. As a premalignant lesion of esophageal adenocarcinoma (EAC), BE develops as a result of chronic gastroesophageal reflux disease (GERD). Many studies have been conducted to understand the molecular mechanisms of this disease. This review summarizes recent results involving squamous and intestinal transcription factors, signaling pathways, stromal factors, microRNAs, and other factors in the development of BE. A conceptual framework is proposed to guide future studies. We expect elucidation of the molecular mechanisms of BE to help in the development of improved management of GERD, BE, and EAC.
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Affiliation(s)
- Hao Chen
- Cancer Research Program, JLC-BBRI, North Carolina Central University, Durham, NC 27707, USA
| | - Yu Fang
- Cancer Research Program, JLC-BBRI, North Carolina Central University, Durham, NC 27707, USA
| | - Whitney Tevebaugh
- Cancer Research Program, JLC-BBRI, North Carolina Central University, Durham, NC 27707, USA
| | - Roy C. Orlando
- Center for Esophageal Diseases and Swallowing, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7080, USA
| | - Nicholas J. Shaheen
- Center for Esophageal Diseases and Swallowing, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7080, USA
| | - Xiaoxin Chen
- Cancer Research Program, JLC-BBRI, North Carolina Central University, Durham, NC 27707, USA,Center for Esophageal Diseases and Swallowing, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7080, USA,Corresponding authors: Xiaoxin Luke Chen, MD, PhD, Cancer Research Program, Julius L. Chambers Biomedical Biotechnology Research Institute, North Carolina Central University, 700 George Street, Durham, NC 27707, USA. Tel: 919-530-6425; Fax: 919-530-7780;
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Fujimura T, Oyama K, Sasaki S, Nishijima K, Miyashita T, Ohta T, Koichi M, Takanori H. Inflammation-related carcinogenesis and prevention in esophageal adenocarcinoma using rat duodenoesophageal reflux models. Cancers (Basel) 2011; 3:3206-24. [PMID: 24212953 PMCID: PMC3759194 DOI: 10.3390/cancers3033206] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2011] [Revised: 08/02/2011] [Accepted: 08/04/2011] [Indexed: 12/30/2022] Open
Abstract
Development from chronic inflammation to Barrett's adenocarcinoma is known as one of the inflammation-related carcinogenesis routes. Gastroesophageal reflux disease induces regurgitant esophagitis, and esophageal mucosa is usually regenerated by squamous epithelium, but sometimes and somewhere replaced with metaplastic columnar epithelium. Specialized columnar epithelium, so-called Barrett's epithelium (BE), is a risk factor for dysplasia and adenocarcinoma in esophagus. Several experiments using rodent model inducing duodenogastroesophageal reflux or duodenoesophageal reflux revealed that columnar epithelium, first emerging at the proliferative zone, progresses to dysplasia and finally adenocarcinoma, and exogenous carcinogen is not necessary for cancer development. It is demonstrated that duodenal juice rather than gastric juice is essential to develop esophageal adenocarcinoma in not only rodent experiments, but also clinical studies. Antireflux surgery and chemoprevention by proton pump inhibitors, nonsteroidal anti-inflammatory drugs, selective cyclooxygenase-2 inhibitors, green tea, retinoic acid and thioproline showed preventive effects on the development of Barrett's adenocarcinoma in rodent models, but it remains controversial whether antireflux surgery could regress BE and prevent esophageal cancer in clinical observation. The Chemoprevention for Barrett's Esophagus Trial (CBET), a phase IIb, multicenter, randomized, double-masked study using celecoxib in patients with Barrett's dysplasia failed to prove to prevent progression of dysplasia to cancer. The AspECT (Aspirin Esomeprazole Chemoprevention Trial), a large multicenter phase III randomized trial to evaluate the effects of esomeprazole and/or aspirin on the rate of progression to high-grade dysplasia or adenocarcinoma in patients with BE is now ongoing.
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Affiliation(s)
- Takashi Fujimura
- Gastroenterologic Surgery, Kanazawa University Hospital, Kanazawa, Japan, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan; E-Mails: (K.O.); (S.S.); (K.N.); (T.M.); (T.O.)
- Author to whom correspondence should be addressed; E-Mail: ; Tel.: +81-76-265-2362; Fax: +81-76-234-4260
| | - Katsunobu Oyama
- Gastroenterologic Surgery, Kanazawa University Hospital, Kanazawa, Japan, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan; E-Mails: (K.O.); (S.S.); (K.N.); (T.M.); (T.O.)
| | - Shozo Sasaki
- Gastroenterologic Surgery, Kanazawa University Hospital, Kanazawa, Japan, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan; E-Mails: (K.O.); (S.S.); (K.N.); (T.M.); (T.O.)
| | - Koji Nishijima
- Gastroenterologic Surgery, Kanazawa University Hospital, Kanazawa, Japan, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan; E-Mails: (K.O.); (S.S.); (K.N.); (T.M.); (T.O.)
| | - Tomoharu Miyashita
- Gastroenterologic Surgery, Kanazawa University Hospital, Kanazawa, Japan, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan; E-Mails: (K.O.); (S.S.); (K.N.); (T.M.); (T.O.)
| | - Tetsuo Ohta
- Gastroenterologic Surgery, Kanazawa University Hospital, Kanazawa, Japan, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan; E-Mails: (K.O.); (S.S.); (K.N.); (T.M.); (T.O.)
| | - Miwa Koichi
- Houju Memorial Hospital, Nomi, Japan, 11-71 Midorigaoka, Nomi, Ishikawa 923-1226, Japan; E-Mail:
| | - Hattori Takanori
- Division of Molecular and Diagnostic Pathology, Shiga University of Medical Science, Otsu, Japan, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan; E-Mail:
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Kong J, Crissey MA, Funakoshi S, Kreindler JL, Lynch JP. Ectopic Cdx2 expression in murine esophagus models an intermediate stage in the emergence of Barrett's esophagus. PLoS One 2011; 6:e18280. [PMID: 21494671 PMCID: PMC3071814 DOI: 10.1371/journal.pone.0018280] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2010] [Accepted: 02/24/2011] [Indexed: 01/27/2023] Open
Abstract
Barrett's esophagus (BE) is an intestinal metaplasia that occurs in the setting of chronic acid and bile reflux and is associated with a risk for adenocarcinoma. Expression of intestine-specific transcription factors in the esophagus likely contributes to metaplasia development. Our objective was to explore the effects of an intestine-specific transcription factor when expressed in the mouse esophageal epithelium. Transgenic mice were derived in which the transcription factor Cdx2 is expressed in squamous epithelium using the murine Keratin-14 gene promoter. Effects of the transgene upon cell proliferation and differentiation, gene expression, and barrier integrity were explored. K14-Cdx2 mice express the Cdx2 transgene in esophageal squamous tissues. Cdx2 expression was associated with reduced basal epithelial cell proliferation and altered cell morphology. Ultrastructurally two changes were noted. Cdx2 expression was associated with dilated space between the basal cells and diminished cell-cell adhesion caused by reduced Desmocollin-3 mRNA and protein expression. This compromised epithelial barrier function, as the measured trans-epithelial electrical resistance (TEER) of the K14-Cdx2 epithelium was significantly reduced compared to controls (1189 Ohm*cm(2) ±343.5 to 508 Ohm*cm(2)±92.48, p = 0.0532). Secondly, basal cells with features of a transitional cell type, intermediate between keratinocytes and columnar Barrett's epithelial cells, were observed. These cells had reduced keratin bundles and increased endoplasmic reticulum levels, suggesting the adoption of secretory-cell features. Moreover, at the ultrastructural level they resembled "Distinctive" cells associated with multilayered epithelium. Treatment of the K14-Cdx2 mice with 5'-Azacytidine elicited expression of BE-associated genes including Cdx1, Krt18, and Slc26a3/Dra, suggesting the phenotype could be advanced under certain conditions. We conclude that ectopic Cdx2 expression in keratinocytes alters cell proliferation, barrier function, and differentiation. These altered cells represent a transitional cell type between normal squamous and columnar BE cells. The K14-Cdx2 mice represent a useful model to study progression from squamous epithelium to BE.
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Affiliation(s)
- Jianping Kong
- Division of Gastroenterology, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
| | - Mary Ann Crissey
- Division of Gastroenterology, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
| | - Shinsuke Funakoshi
- Division of Gastroenterology, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
| | - James L. Kreindler
- Division of Pulmonary Medicine, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America
| | - John P. Lynch
- Division of Gastroenterology, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
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Abstract
Barrett's esophagus is an acquired metaplastic abnormality in which the normal stratified squamous epithelium lining of the esophagus is replaced by an intestinal-like columnar epithelium. While in itself a benign and asymptomatic disorder, the clinical importance of this relatively common condition relates to its role as a precursor lesion to esophageal adenocarcinoma, the incidence of which has dramatically increased in Western populations in recent years. Although known to arise as a consequence of chronic gastroesophageal reflux, the cellular and molecular mechanisms underlying development Barrett's esophagus and its progression to cancer remain unclear.
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Affiliation(s)
- Wayne A Phillips
- Division of Cancer Surgery, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.
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Abstract
The past few years have brought new advances in our understanding of the molecular mechanisms underlying the development of Barrett's esophagus and esophageal adenocarcinoma. Although knowledge of the genetic basis for these conditions has not yet translated into clinically useful biomarkers, the current pace of biomedical discovery holds endless possibilities for molecular medicine to improve the diagnosis and management of patients with these conditions. This article provides a useful conceptual basis for understanding the molecular events involved in the making of Barrett metaplasia and in its neoplastic progression, and provides a rationale for evaluating studies on the application of molecular medicine to the diagnosis and management of patients with Barrett's esophagus and esophageal adenocarcinoma.
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Affiliation(s)
- David H. Wang
- Assistant Professor, Departments of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical School, and the Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas
| | - Rhonda F. Souza
- Associate Professor, Departments of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical School, and the Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas
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35
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Abstract
Gastroesophageal reflux disease (GERD) affects an estimated 20% of the population in the United States. About 10%-15% of patients with GERD develop Barrett’s esophagus, which can progress to adenocarcinoma, currently the most prevalent type of esophageal cancer. The esophagus is normally lined by squamous mucosa, therefore, it is clear that for adenocarcinoma to develop, there must be a sequence of events that result in transformation of the normal squamous mucosa into columnar epithelium. This sequence begins with gastroesophageal reflux, and with continued injury metaplastic columnar epithelium develops. This article reviews the pathophysiology of Barrett’s esophagus and implications for its treatment. The effect of medical and surgical therapy of Barrett’s esophagus is compared.
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36
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Huo X, Zhang HY, Zhang X, Lynch JP, Strauch ED, Wang JY, Melton SD, Genta RM, Wang DH, Spechler SJ, Souza RF. Acid and bile salt-induced CDX2 expression differs in esophageal squamous cells from patients with and without Barrett's esophagus. Gastroenterology 2010; 139:194-203.e1. [PMID: 20303354 PMCID: PMC2902607 DOI: 10.1053/j.gastro.2010.03.035] [Citation(s) in RCA: 88] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/27/2009] [Revised: 02/18/2010] [Accepted: 03/11/2010] [Indexed: 12/22/2022]
Abstract
BACKGROUND & AIMS It is not clear why only a minority of patients with gastroesophageal reflux disease (GERD) develop Barrett's esophagus. We hypothesized that differences among individuals in molecular pathways activated when esophageal squamous epithelium is exposed to reflux underlie the development of Barrett's metaplasia. METHODS We used esophageal squamous cell lines from patients who had GERD with Barrett's esophagus (normal esophageal squamous [NES]-B3T and NES-B10T) and without Barrett's esophagus (NES-G2T and NES-G4T) to study effects of acid and bile salts on expression of the CDX2 gene. Bay 11-705, Ad5 inhibitor kappaB(IkappaB)alpha-SR, and site-directed mutagenesis were used to explore effects of nuclear factor-kappaB (NF-kappaB) inhibition on CDX2 promoter activity; DNA binding of the NF-kappaB subunits p50 and p65 was assessed by chromatin immune-precipitation. RESULTS Acid and bile salts increased CDX2 messenger RNA (mRNA), protein, and promoter activity in NES-B3T and NES-B10T cells, but not in NES-G2T or NES-G4T cells. Inhibition of NF-kappaB abolished the increase in CDX2 promoter activity. Increased CDX2 promoter activity was associated with nuclear translocation of p50, which bound to the promoter. We found CDX2 mRNA in 7 of 10 esophageal squamous biopsy specimens from patients with Barrett's esophagus, but in only 1 of 10 such specimens from patients who had GERD without Barrett's esophagus. CONCLUSIONS Acid and bile salts induce CDX2 mRNA and protein expression in esophageal squamous cells from patients with Barrett's esophagus, but not from GERD patients without Barrett's esophagus. We speculate that these differences in acid- and bile salt-induced activation of molecular pathways may underlie the development of Barrett's metaplasia.
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Affiliation(s)
- Xiaofang Huo
- Department of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical School, Dallas, TX
| | - Hui Ying Zhang
- Department of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical School, Dallas, TX
| | - Xi Zhang
- Department of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical School, Dallas, TX
| | - John P. Lynch
- Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
| | - Eric D. Strauch
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD
| | - Jian-Ying Wang
- Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD,Department of Pathology, University of Maryland School of Medicine, Baltimore, MD,Baltimore Veterans Affairs Medical Center, Baltimore, MD
| | - Shelby D. Melton
- Department of Pathology, VA North Texas Health Care System and the University of Texas Southwestern Medical School, Dallas, TX,Caris Life Sciences, Inc., Irving, TX
| | - Robert M. Genta
- Department of Pathology, VA North Texas Health Care System and the University of Texas Southwestern Medical School, Dallas, TX,Caris Life Sciences, Inc., Irving, TX
| | - David H. Wang
- Department of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical School, Dallas, TX,Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX
| | - Stuart J. Spechler
- Department of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical School, Dallas, TX
| | - Rhonda F. Souza
- Department of Medicine, VA North Texas Health Care System and the University of Texas Southwestern Medical School, Dallas, TX,Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX
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Abstract
Barrett's metaplasia is discussed in the context of a general theory for the formation of metaplasias based on developmental biology. The phenotype of a particular tissue type becomes established during embryonic development by the expression of a specific set of transcription factors. If this combination becomes altered, then the tissue type can be altered. Such events may occur by mutation or by environmental effects on gene expression, normally within the stem cell population of the tissue. A macroscopic patch of metaplastic tissue will arise only if the new gene activity state is self-sustaining in the absence of its original causes, and if the new tissue type can outgrow the parent tissue type. An important candidate gene for the causation of Barrett's metaplasia is Cdx2 (Caudal-type homeobox 2). In normal development, this is expressed in the future intestine, but not the future foregut. Mouse knockout studies have shown that it is needed for intestinal development, and that its loss from adult intestine can lead to squamous transformations. It is also expressed in Barrett's metaplasia and can be activated in oesophageal cell cultures by treatment with bile acids. We have investigated the ability of Cdx2 to bring about intestinal transformations in oesophageal epithelium. Our results show that Cdx2 can activate a programme of intestinal gene expression when overexpressed in HET-1A cells, or in fetal epithelium, but not in the adult epithelium. This suggests that Cdx2, although necessary for formation of intestinal tissue, is not sufficient to provoke Barrett's metaplasia in adult life and that overexpression of additional transcription factors is necessary. In terms of diet and nutrition, there is a known association of Barrett's metaplasia with obesity. This may work through an increased risk of gastro-oesophageal reflux. Acid and bile are known to activate Cdx2 expression in oesophageal cells. It may also increase circulating levels of TNFalpha (tumour necrosis factor alpha), which activates Cdx2. In addition, there may be effects of diet on the composition of the bile.
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Abstract
The precise mechanisms whereby gastro-oesophageal reflux disease causes reflux oesophagitis and Barrett's oesophagus are not clear, even though these diseases have been known to be linked for many years. Recent studies indicate a role for the reflux-induced inflammatory response of oesophageal squamous epithelial cells and the immune cells in the pathogenesis of reflux oesophagitis. Although reflux oesophagitis commonly heals with oesophageal squamous cell regeneration, in some individuals the oesophagus heals through the process of metaplasia, a condition termed Barrett's oesophagus. Recent studies indicate that individual differences in the reflux-mediated response of oesophageal squamous epithelial cells in the type of immune response and/or in signalling pathways that regulate cell proliferation or cell phenotype may determine whether the oesophagus heals with the regeneration of squamous cells or through Barrett's metaplasia.
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Abstract
Barrett's oesophagus is the replacement of normal squamous oesophageal epithelium with an intestinalized columnar epithelium. Although some insight has been gained as to what Barrett's oesophagus is, how this columnar epithelium emerges from within a stratified squamous epithelium remains an unanswered question. We have sought to determine whether oesophageal keratinocytes can be trans-differentiated into Barrett's oesophagus cells. Using an Affymetrix microarray, we found unexpectedly that gene-expression patterns in the Barrett's oesophagus were only slightly more similar to the normal small intestine than they were to the normal oesophagus. Thus gene-expression patterns suggest significant molecular similarities remain between Barrett's oesophagus cells and normal squamous oesophageal epithelium, despite their histological resemblance with intestine. We next determined whether directed expression of intestine-specific transcription factors could induce intestinalization of keratinocytes. Retroviral-mediated Cdx2 (Caudal-type homeobox 2) expression in immortalized human oesophageal keratinocytes engineered with human telomerase reverse transcriptase (EPC2-hTERT cells) could be established transiently, but not maintained, and was associated with a reduction in cell proliferation. Co-expression of cyclin D1 rescued proliferation in the Cdx2-expressing cells, but co-expression of dominant-negative p53 did not. Cdx2 expression in the EPC2-hTERT.D1 cells did not induce intestinalization. However, when combined with treatments that induce chromatin remodelling, there was a significant induction of Barrett's oesophagus-associated genes. Studies are ongoing to determine whether other intestinal transcription factors, either alone or in combination, can provoke greater intestinalization of oesophageal keratinocytes. We conclude that, on the basis of gene-expression patterns, Barrett's oesophagus epithelial cells may represent an intermediate between oesophageal keratinocytes and intestinal epithelial cells. Moreover, our findings suggest that it may be possible to induce Barrett's oesophagus epithelial cells from oesophageal keratinocytes by altering the expression of certain critical genes.
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Stairs DB, Kong J, Lynch JP. Cdx genes, inflammation, and the pathogenesis of intestinal metaplasia. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2010; 96:231-70. [PMID: 21075347 PMCID: PMC6005371 DOI: 10.1016/b978-0-12-381280-3.00010-5] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Intestinal metaplasia (IM) is a biologically interesting and clinically relevant condition in which one differentiated type of epithelium is replaced by another that is morphologically similar to normal intestinal epithelium. Two classic examples of this are gastric IM and Barrett's esophagus (BE). In both, a chronic inflammatory microenvironment, provoked either by Helicobacter pylori infection of the stomach or acid and bile reflux into the esophagus, precedes the metaplasia. The Caudal-related homeodomain transcription factors Cdx1 and Cdx2 are critical regulators of the normal intestinal epithelial cell phenotype. Ectopic expression of Cdx1 and Cdx2 occurs in both gastric IM as well as in BE. This expression precedes the onset of the metaplasia and implies a causal role for these factors in this process. We review the observations regarding the role of chronic inflammation and the Cdx transcription factors in the pathogenesis of gastric IM and BE.
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Affiliation(s)
- Douglas B Stairs
- Department of Medicine, Division of Gastroenterology, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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Retracted article: Interleukin-1beta and tumor necrosis factor-alpha regulation of CDX2 homeobox gene through nuclear factor kappa B-dependent pathway in the intestinal-type gastric cancer. Med Oncol 2009; 27:1155. [PMID: 19908169 DOI: 10.1007/s12032-009-9352-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2009] [Accepted: 10/26/2009] [Indexed: 10/20/2022]
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Vaninetti N, Williams L, Geldenhuys L, Porter GA, Guernsey DL, Casson AG. Regulation of CDX2 expression in esophageal adenocarcinoma. Mol Carcinog 2009; 48:965-74. [PMID: 19415720 DOI: 10.1002/mc.20549] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Reflux of acidic gastric contents and bile acids into the lower esophagus has been identified to have a central role in esophageal malignancy and is reported to upregulate caudal-related homologue 2 (CDX2), a regulatory gene involved in embryonic development and axial patterning of the alimentary tract. The aim of this study was to characterize the expression of CDX2 in a well-defined series of human esophageal tissues, comprising reflux-induced esophagitis, premalignant Barrett esophagus (BE), and primary esophageal adenocarcinoma (EADC). To explore potential molecular regulatory mechanisms, we also studied the expression of beta-catenin, SOX9, and CDX2 promoter methylation in esophageal tissues, in addition to the effect of bile acids and nitric oxide (NO) on CDX2 expression in the normal human esophageal cell line Het1A. Relative to matched normal esophageal epithelia, CDX2 was overexpressed in esophagitis (37% for RNA; cytoplasmic immunoreactivity in 48% of tissues), a high proportion (91%) of BE tissues, and in EADC (57% for RNA; cell nuclear immunopositivity in 80%). An association with beta-catenin expression was seen, but not with SOX9 or CDX2 promoter methylation. In Het1A cells, CDX2 was upregulated following exposure to bile acids and NO, alone and in combination. These results further implicate CDX2 and beta-catenin in the molecular pathogenesis of human EADC. The observed synergistic effect of NO on the efficacy of bile acid-induction of CDX2 suggests a novel role for NO in modulating the development of the Barrett phenotype and esophageal adenocarcinogenesis.
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Affiliation(s)
- Nadine Vaninetti
- Department of Pathology, Dalhousie University, Halifax, NS, Canada
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Ingravallo G, Dall'Olmo L, Segat D, Fassan M, Mescoli C, Dazzo E, Castoro C, Polimeno L, Rizzetto C, Baroni MD, Zaninotto G, Ancona E, Rugge M. CDX2 hox gene product in a rat model of esophageal cancer. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2009; 28:108. [PMID: 19664209 PMCID: PMC3225830 DOI: 10.1186/1756-9966-28-108] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/12/2009] [Accepted: 08/07/2009] [Indexed: 12/19/2022]
Abstract
Background Barrett's mucosa is the precursor of esophageal adenocarcinoma. The molecular mechanisms behind Barrett's carcinogenesis are largely unknown. Experimental models of longstanding esophageal reflux of duodenal-gastric contents may provide important information on the biological sequence of the Barrett's oncogenesis. Methods The expression of CDX2 hox-gene product was assessed in a rat model of Barrett's carcinogenesis. Seventy-four rats underwent esophago-jejunostomy with gastric preservation. Excluding perisurgical deaths, the animals were sacrificed at various times after the surgical treatment (Group A: <10 weeks; Group B: 10–30 weeks; Group C: >30 weeks). Results No Cdx2 expression was detected in either squamous epithelia of the proximal esophagus or squamous cell carcinomas. De novo Cdx2 expression was consistently documented in the proliferative zone of the squamous epithelium close to reflux ulcers (Group A: 68%; Group B: 64%; Group C: 80%), multilayered epithelium and intestinal metaplasia (Group A: 9%; Group B: 41%; Group C: 60%), and esophageal adenocarcinomas (Group B: 36%; Group C: 35%). A trend for increasing overall Cdx2 expression was documented during the course of the experiment (p = 0.001). Conclusion De novo expression of Cdx2 is an early event in the spectrum of the lesions induced by experimental gastro-esophageal reflux and should be considered as a key step in the morphogenesis of esophageal adenocarcinoma.
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Affiliation(s)
- Giuseppe Ingravallo
- Department of Medical Diagnostic Sciences & Special Therapies, Pathology Unit, University of Padova, Padova, Italy.
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Sun YG, Wang XW, Yang SM, Zhou G, Wang WQ, Wang HB, Wang RQ, Fang DC. Inhibition of nucleostemin upregulates CDX2 expression in HT29 cells in response to bile acid exposure: implications in the pathogenesis of Barrett's esophagus. J Gastrointest Surg 2009; 13:1430-9. [PMID: 19449081 DOI: 10.1007/s11605-009-0899-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/10/2009] [Accepted: 04/15/2009] [Indexed: 01/31/2023]
Abstract
BACKGROUND Barrett's esophagus (BE), a squamous-to-columnar metaplasia, may originate from growth-promoting mutations in metaplastic stem cells. Nucleostemin is a protein highly expressed in undifferentiated embryonic stem cells. The objectives of this study were to explore the potential role of nucleostemin in the pathogenesis of BE METHODS: The expression profiles of 30,968 genes were compared between BE and normal esophageal tissues (n = 6 in each group) by using oligo microarray. Three siRNA plasmid expression vectors against nucleostemin, pRNAi-1, pRNAi-2, and pRNAi-3, were constructed and transfected into HT29 cells. In addition, HT29 cells were exposed to 100-1,000 microM chenodeoxycholic acid (CDC), a bile acid, for 2, 12, and 24 h, and then messenger RNA and protein expressions of nucleostemin and CDX2 were determined by reverse-transcriptase polymerase chain reaction and Western blotting. RESULTS Four hundred and twenty-six differentially expressed genes were detected in BE; 142 were upregulated and 284 downregulated. Nucleostemin was downregulated while CDX2 was upregulated. In vitro, all the recombinant plasmids inhibited the nucleostemin expression in transfected HT29 cells, with pRNAi-1 being the most effective. CDX2 expression was significantly increased in pRNAi-1-transfected HT29 cells, compared with that in the empty plasmid (pRNAT-U6.1/Neo) transfected or untransfected HT29 cells. In addition, CDX2 expression was increased whereas nucleostemin expression was decreased in a dose- and time-dependent manner in HT29 cells treated with CDC. CONCLUSION These findings suggest that the inhibition of nucleostemin expression in "esophageal stem cells" in response to bile acid exposure may be involved in the pathogenesis of BE through upregulating CDX2 expression.
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Affiliation(s)
- Yong-Gang Sun
- Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China
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Colleypriest BJ, Palmer RM, Ward SG, Tosh D. Cdx genes, inflammation and the pathogenesis of Barrett's metaplasia. Trends Mol Med 2009; 15:313-22. [PMID: 19564133 DOI: 10.1016/j.molmed.2009.05.001] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2009] [Revised: 05/12/2009] [Accepted: 05/12/2009] [Indexed: 01/07/2023]
Abstract
Metaplasia is the conversion of one cell or tissue type to another and can predispose patients to neoplasia. Perhaps one of the best-known examples of metaplasia is Barrett's metaplasia (BM), a pathological condition in which the distal oesophageal epithelium switches from stratified squamous to intestinal-type columnar epithelium. BM predisposes to oesophageal adenocarcinoma and is the consequence of long-term acid bile reflux. The incidence of BM and oesophageal adenocarcinoma has risen dramatically in recent years. A key event in the pathogenesis of BM is the induction of oesophageal CDX2 expression. Importantly, recent data reveal the molecular mechanisms that link inflammation in the development of Barrett's metaplasia, CDX2 and the progression to cancer. This review highlights the relationship between inflammation, metaplasia and carcinogenesis.
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Affiliation(s)
- Benjamin J Colleypriest
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.
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di Pietro M, Fitzgerald RC. Barrett’s oesophagus: an ideal model to study cancer genetics. Hum Genet 2009; 126:233-46. [DOI: 10.1007/s00439-009-0665-2] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2009] [Accepted: 04/01/2009] [Indexed: 12/16/2022]
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Avissar NE, Toia L, Hu Y, Watson TJ, Jones C, Raymond DP, Matousek A, Peters JH. Bile acid alone, or in combination with acid, induces CDX2 expression through activation of the epidermal growth factor receptor (EGFR). J Gastrointest Surg 2009; 13:212-22. [PMID: 18854960 DOI: 10.1007/s11605-008-0720-7] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/24/2008] [Accepted: 09/24/2008] [Indexed: 01/31/2023]
Abstract
OBJECTIVES Bile acids and acid are implicated in the development of Barrett's esophagus. Evidence suggests that Barrett's esophagus intestinal metaplasia may occur via induction of caudal homeobox gene 2 (CDX2). We hypothesized that induction of CDX2 by bile acids may be due to ligand-dependent transactivation of epidermal growth factor receptor (EGFR). METHODS Human mucosal epithelial cells (SEG-1) were treated for 0 to 24 h with up to 300 microM deoxycholic acid (DCA) at pH 7 or 5 with or without (w/wo) antibodies against EGFR ligand-binding site (Mab528, 3-5 mug/ml). Treatment with 100 ng/ml EGF served as control. CDX2 mRNA expression was determined by real-time polymerase chain reaction. EGFR activation was analyzed by Westerns of phosphorylated EGFR tyrosines. RESULTS Acid (pH 5) increased the induction of CDX2 mRNA expression caused by DCA. CDX2 mRNA induction was markedly reduced by EGFR blockade with Mab528. Each treatment (pH 5, DCA or pH 5 plus DCA) activated the EGFR on all tyrosines tested but in different time courses. Phosphorylation by DCA was inhibited by Mab528. Activation of EGFR by DCA at pH 5 resulted in EGFR degradation, while that by DCA alone did not. CONCLUSION Thus, CDX2 induction by DCA w/wo acid occurs through ligand-dependent transactivation of the EGFR. The variations in EGFR degradation pattern with DCA or DCA at pH 5 indicate differential transactivation pathways. The molecular pathogenesis of Barrett's esophagus may occur via bile-stimulated cell signaling through the EGFR.
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Affiliation(s)
- Nelly E Avissar
- Department of Surgery, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA.
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Zheng LD, Tong QS, Weng MX, He J, Lv Q, Pu JR, Jiang GS, Cai JB, Liu Y, Hou XH. Enhanced expression of resistin-like molecule beta in human colon cancer and its clinical significance. Dig Dis Sci 2009; 54:274-81. [PMID: 18594973 DOI: 10.1007/s10620-008-0355-2] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/02/2007] [Accepted: 06/03/2008] [Indexed: 01/08/2023]
Abstract
Previous studies have indicated that resistin-like molecule beta (RELM beta), an intestinal goblet cell-specific protein, is markedly increased in the intestinal tumors of min mice and over-expressed in a human colon cancer cell line. We hypothesized that RELM beta might be enhanced in human colon cancer. The aim of this study was to examine the clinical importance of RELM beta expression in colon cancer patients and to correlate its expression with various clinicopathological parameters, upstream regulatory molecule expression, tumor proliferative capacity, and patients' survival. Of the 80 colon cancer patients studied, 65 (81.25%) tested positive for RELM beta, mainly in the cytoplasm of colon mucosa. Contrasting sharply with the strongly RELM beta-positive tumors, normal colon mucous membrane was negative or weakly positive. RELM beta positivity in colon cancer was correlated with histological grade of differentiation and lymph node metastasis, but not with age, gender, tumor location and size, tumor infiltration, Dukes' stage, liver metastasis, and venous invasion. RELM beta expression was significantly correlated with the expression of transcription factor CDX-2 (P < 0.01) but not with that of proliferative index Ki-67 (P > 0.05). The mean postoperative survival time (2.76 years) of RELM beta-positive patients was significantly longer than that (1.26 years) of RELM beta-negative patients (P = 0.032). These findings support evidence of the enhanced RELM beta expression in colon cancer patients and suggest that further investigation is warranted to explore the role of RELM beta in colon cancer.
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Affiliation(s)
- Li-Duan Zheng
- Department of Pathology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, Hubei Province, China
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Wang XG, Zhang Z, Sun LP, Yuan Y. Relationship between the expression of CDX2 and different types of gastric intestinal metaplasia. Shijie Huaren Xiaohua Zazhi 2009; 17:86-89. [DOI: 10.11569/wcjd.v17.i1.86] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To inquire the relationship between the expression of CDX2 and different types of gastric intestinal metaplasia.
METHODS: Expression of CDX2 in 58 normal gastric mucosa, 184 IM and 36 gastric cancer were detected using immunohistochemical method. One hundred and eighty four IM cases were classified into three subtypes, including 81 cases of IM Ⅰ, 62 cases of IM Ⅱ and 41 cases of IM Ⅲ using HID-ABpH2.5-PAS methods.
RESULTS: The positive rates of CDX2 expression in IM and gastric cancer were significantly higher than normal gastric mucosa in which negative expression was detected (P < 0.01), but lower than IM (27.8% vs 59.8%, P < 0.01). The positive rates of CDX2 expression in IM Ⅰ and IM Ⅱ were significantly higher than either gastric cancer group or IM Ⅲ. However, there was no significant difference between IM Ⅲ and gastric cancer group. CDX2 was located in IM cellular nucleus.
CONCLUSION: The IM without expression of CDX2 has close relationship with gastric cancer, and CDX2 is a valuable biomarker to judge the differentiation of IM.
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Kong J, Nakagawa H, Isariyawongse BK, Funakoshi S, Silberg DG, Rustgi AK, Lynch JP. Induction of intestinalization in human esophageal keratinocytes is a multistep process. Carcinogenesis 2009; 30:122-30. [PMID: 18845559 PMCID: PMC2722140 DOI: 10.1093/carcin/bgn227] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2008] [Revised: 08/28/2008] [Accepted: 09/24/2008] [Indexed: 12/22/2022] Open
Abstract
Barrett's esophagus (BE) is the replacement of normal squamous esophageal mucosa with an intestinalized columnar epithelium. The molecular mechanisms underlying its development are not understood. Cdx2 is an intestine-specific transcription factor that is ectopically expressed in BE, but its role in this process is unclear. Herein, we describe a novel cell culture model for BE. Retroviral-mediated Cdx2 expression in immortalized human esophageal keratinocytes [EPC-human telomerase reverse transcriptase (hTERT)] could transiently be established but not maintained and was associated with a reduction in cell proliferation. Coexpression of cyclin D1, but not a dominant-negative p53, rescued proliferation in the Cdx2-expressing cells. Cdx2 expression in the EPC-hTERT.D1 cells decreased cell proliferation but did not induce intestinalization. We investigated for other treatments to enhance intestinalization and found that acidic culture conditions uniformly killed EPC-hTERT.D1.Cdx2 cells. However, treatment with 5-aza-2-deoxycytidine (5-AzaC) to demethylate epigenetically silenced genes did appear to be tolerated. Multiple Cdx2 target genes, markers of intestinal differentiation and markers of BE, were induced by this 5-AzaC treatment. More interestingly, the expression level of several of these genes was enhanced only in the EPC-hTERT.D1-Cdx2 cells treated with 5-AzaC. Two of these, SLC26a3/DRA (downregulated in adenoma) and Na+/H+ exchanger 2 (NHE2), were not previously known to be elevated in BE; however, we confirmed their elevation in BE tissue samples. 5-AzaC treatment also induced cell senescence, even at low doses. We conclude that ectopic proliferation signals, alterations in epigenetic gene regulation and the inhibition of tumor suppressor mechanisms are required for Cdx2-mediated intestinalization of human esophageal keratinocytes in BE.
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Affiliation(s)
- Jianping Kong
- Department of Medicine, Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Hiroshi Nakagawa
- Department of Medicine, Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Brandon K. Isariyawongse
- Department of Medicine, Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Shinsuke Funakoshi
- Department of Medicine, Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Debra G. Silberg
- Department of Medicine, Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, USA
- AstraZeneca LP, Wilmington, DE 19850-5437, USA
| | - Anil K. Rustgi
- Department of Medicine, Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - John P. Lynch
- Department of Medicine, Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, USA
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