Investigating the Expression of Ten-Eleven Translocation-2 and Connexin 43 in Inflammatory Bowel Disease and Colorectal Cancer

Abstract

Inflammatory bowel disease (IBD) is a chronic, relapsing inflammation of the gastrointestinal tract. The pathogenesis of IBD involves an abnormal autoimmune response to gut dysbiosis, triggered by genetic and epigenetic predispositions as well as environmental factors. Due to its chronic inflammatory nature, patients with IBD are at an increased risk of developing colorectal cancer (CRC). Connexins (Cx), a large family of transmembrane proteins that form gap junctions, are found in nearly all tissues, and play a critical role in intercellular communication. The expression of Connexin 43 (Cx43), a protein with tumor-suppressive functions, has been shown to be altered under pathological conditions, including IBD and CRC. Furthermore, epigenetic mechanisms, specifically DNA methylation and demethylation, have been implicated in the onset of inflammatory and malignant diseases. The Ten-Eleven Translocation 2 (TET-2) enzyme catalyzes DNA demethylation and is therefore involved in regulating the activity of both tumor-suppressing and cancer-promoting genes. Therefore, there is interest in whether epigenetic mechanisms, such as DNA demethylation, contributes to the alteration of tumor-suppressing genes like Cx43 and whether this pathway underlies the switch from IBD to CRC. To investigate this, HT-29 cells were engineered to over-express or knockdown TET2 or to over-express or knock-out Cx43. These cells were assayed to assess TET2 and Cx43 expressions in each HT-29 cell model at mRNA and protein level. TET2 and Cx43 were shown to have a parallel pattern of expression. To mimic the inflammatory environment of IBD, conditioned media collected from activated THP-1 cells were added to cells which increased both TET2 and Cx43. Furthermore, functionality of these proteins was evaluated with two assays: immunofluorescence imaging for global 5-hmc to indicate TET2 activity and calcein dye transfer assay to measure gap-junction intercellular communication (GJIC). 5-hmc levels increased under TET2/Cx43 overexpression and with inflammation and decreased with TET2/Cx43 overexpression. GJIC also increased under inflammatory conditions. Finally, these findings were translated to human pathology by using formalin-fixed, paraffin-embedded (FFPE) tissues of normal colon, ulcerative colitis and adenocarcinoma samples. Immunofluorescence imaging showed Cx43 increased in ulcerative colitis specimens but decreased in adenocarcinoma tissue. In addition, TET2 decreased in both ulcerative colitis and adenocarcinoma as compared to normal colon tissue. Findings suggest bidirectional cross-talk and potentially reciprocal regulation between epigenetic enzymes such as TET2 and tumor suppressive genes like Cx43 possibly implicating this axis, at least in part and perhaps indirectly, as molecular bridge between inflammation and cancer.