Published online Oct 28, 2022. doi: 10.3748/wjg.v28.i40.5845
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
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.
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.
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.
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.
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.
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.
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.