Basic Study
Copyright ©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Oct 28, 2022; 28(40): 5845-5864
Published online Oct 28, 2022. doi: 10.3748/wjg.v28.i40.5845
Expression of the methylcytosine dioxygenase ten-eleven translocation-2 and connexin 43 in inflammatory bowel disease and colorectal cancer
Mohammad El-Harakeh, Jessica Saliba, Kawthar Sharaf Aldeen, May Haidar, Layal El Hajjar, Mireille Kallassy Awad, Jana G Hashash, Margret Shirinian, Marwan El-Sabban
Mohammad El-Harakeh, Kawthar Sharaf Aldeen, May Haidar, Layal El Hajjar, Marwan El-Sabban, Department of Anatomy, Cell Biology and Physiological Sciences, Faculty of Medicine, American University of Beirut, Beirut 1107, Lebanon
Mohammad El-Harakeh, Mireille Kallassy Awad, UR GPF Laboratory of Biodiversity and Functional Genomics, Faculty of Science, Université Saint-Joseph de Beyrouth, Beirut 1107, Lebanon
Jessica Saliba, Department of Biology, Faculty of Sciences, Lebanese University, Beirut 1533, Lebanon
Jessica Saliba, Department of Public Health, Faculty of Health Sciences, University of Balamand, Dekwaneh, Sin el Fil 1552, Lebanon
Jana G Hashash, Department of Internal Medicine, Division of Gastroenterology and Hepatology, Faculty of Medicine, American University of Beirut, Beirut 1107, Lebanon
Margret Shirinian, Department of Experimental Pathology, Immunology and Microbiology, Faculty of Medicine, American University of Beirut, Beirut 1107, Lebanon
Author contributions: El-Harakeh M, Saliba J, Haidar M, and Sharaf Aldeen K equally contributed to the study; El-Sabban M conceived and designed the study; Hashash JG, Awad MK, Shirinian M, and El-Sabban M provided the material and resources for the experiments; El-Harakeh M, Haidar M, Sharaf Aldeen K, and El Hajjar L performed the experiments; Saliba J and El-Harakeh M analyzed the data and wrote the manuscript; Shirinian M and Hashash JG critically reviewed the manuscript; Saliba J and El-Sabban M revised the manuscript; All authors read and approved the final version of the manuscript.
Institutional review board statement: Archived formaldehyde-fixed paraffin-embedded (FFPE) colon blocks (ulcerative colitis, sporadic colon adenocarcinoma and normal) were obtained from the American University of Beirut Medical Center (AUBMC). All patients’ identifiers were kept confidential and all samples were anonymous, hence exempted from ethics committee approval.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the American University of Beirut, No. 18-03-476.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: Raw data are available upon reasonable request.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Marwan El-Sabban, PhD, Professor, Department of Anatomy, Cell Biology and Physiological Sciences, Faculty of Medicine, American University of Beirut, Bliss Street, Beirut 1107, Lebanon.
Received: February 6, 2022
Peer-review started: February 6, 2022
First decision: April 10, 2022
Revised: May 6, 2022
Accepted: June 13, 2022
Article in press: June 13, 2022
Published online: October 28, 2022
Research background

Patients with inflammatory bowel disease (IBD) have a higher propensity to acquire colorectal cancer. The link between inflammation and cancer has long been established; however, the molecular players in the switch to cancer are still poorly defined.

Research motivation

In previous work, we have demonstrated a potential role for connexin 43 (Cx43) in inflamed intestinal cells. Recognizing the tumor-suppressor role of Cx43 in several cancers, we set out to explore whether the loss of Cx43 is associated with the switch to carcinogenesis. One potential mechanism for downregulating Cx43 expression is through methylation. We hypothesized that ten-eleven translocation-2 (TET-2), a demethylating enzyme, previously described to have a role in inflammation, may be involved in this process.

Research objectives

Using cell culture, a colitis animal model, and archived human tissues, we assessed the expression of both Cx43 and TET-2 in intestinal inflammation. Specific objectives include: (1) Assessment of the expression levels of Cx43 and TET-2 under inflammatory conditions; (2) Assessment of the expression and activity of TET-2 in HT-29 cell lines with up- or down-regulated for Cx43 expression; (3) Reproduction of a colitis mouse model and assessment of Cx43 and TET-2 levels in colons of mice; and (4) Explore Cx43 and TET-2 expression levels in archived biopsies obtained from patients with ulcerative colitis and colon adenocarcinoma.

Research methods

This study employed several modalities to verify the hypothesis, which include intestinal epithelial cell (IEC) line modified for Cx43 expression grown under inflammatory conditions. A dextran sulfate sodium-induced colitis mouse model was reproduced and tissues from different experimental conditions were analyzed. Gene expression profile, protein expression levels, morphology and cellular localization were described. In addition, archived formalin-fixed paraffin-embedded tissues were sectioned and evaluated for Cx43 and TET-2 expression.

Research results

In vitro, TET-2 expression was elevated under inflammatory conditions and even more so in HT-29 cells devoid of Cx43. The barrier function of IECs was breached when Cx43 levels were down-regulated. These results were corroborated in the murine colitis model. In archived biopsies from ulcerative colitis patients, Cx43 expression was upregulated compared to non-inflamed counterparts. In sporadic colon adenocarcinoma sections, both TET-2 and Cx43 expression levels were decreased.

Research conclusions

Under inflammatory conditions, levels of Cx43 and of the demethylating enzyme TET-2 are upregulated. Through demethylation, TET-2 would turn on the expression of several factors involved in inflammation (presumably Cx43 included). When TET-2 levels were diminished in sporadic colon adenocarcinoma, we also observed that Cx43 was downregulated, which may indicate a role of TET-2 in shutting down Cx43 and its tumor-suppressing potential.

Research perspectives

In vitro, manipulation of TET-2 levels in intestinal cells may yield further insight into the mechanism of action. Methylation studies will also be undertaken. The animal model will be expanded to allow for the development of colon carcinoma, and timed evaluation of molecular players will be performed. More stringent criteria will be implemented for prospective tissue collection from non-inflamed subjects, patients with IBD, and with IBD-associated colorectal cancer.