Investigating The Dynamic Interaction Between Cx43 and TET2 in Inflammatory Bowel Disease and Colorectal Cancer

Abstract

Inflammatory Bowel Disease (IBD) is comprised of two disorders, Ulcerative Colitis and Chron’s disease; both result in chronic inflammation of the gastrointestinal tract accompanied by repetitive acute inflammatory flare ups. Statistically this has shown to significantly increase the risk for the development of colorectal cancer (CRC). Gap junction proteins such as Connexin 43 (Cx43) are thought to be tumor suppressor genes and have been observed to be altered in pathological conditions including IBD and CRC. There is a current interest in studying the role of epigenetic mechanisms such as DNA methylation in the context of disease. In one such instance, the DNA demethylase Ten-Eleven-Translocation-2 (TET2) is implicated in altering the methylation status of various cancer–promoting, as well as tumor-suppressor genes. Our interest lies in interrogating whether TET-2 contributes to the alterations in the tumor suppressor gene Cx43’s expression and function and whether this signaling axis contributes to CRC development in an IBD model. To mirror the inflammatory environment of IBD, activated monocyte supernatants were added to HT-29 human colorectal adenocarcinoma cells. Furthermore, HT-29 cells were stably transfected to either overexpress or knock out Cx43 or TET2. These cells were then assayed to assess Cx43 and TET2 expression in a reciprocal fashion by looking at RNA expression, relative protein expression, and sub-cellular localization by immunofluorescence. The addition of activated monocyte supernatant resulted in an increase in the expression of Cx43. However, TET2 overexpression resulted in Cx43 downregulation, and, somewhat surprisingly, Cx43 overexpression resulted in a downregulation of TET2 expression. This seemingly paradoxical finding opened the possibility of a two-way interaction between TET2 and Cx43 rather than a unilateral effect of TET2 on Cx43 gene methylation. These findings indicate that there is a dynamic relationship between TET2 and Cx43 in the setting of inflammation and opens the doors to investigate how this relationship results in the progression from an inflammatory state to the development and progression of colorectal cancer.