Critical Regulators of Epithelial Homeostasis and Intestinal Crypt Regeneration: Identification of Novel BVES/BCAR3 Signaling Complex in EMT and Tumorigenesis and Biological Implications for MTG16 in Stem Cell Function, Proliferation, Apoptosis and DNA Repair.

Abstract

CANCER BIOLOGY Critical Regulators of Epithelial Homeostasis and Intestinal Crypt Regeneration: Identification of Novel BVES/BCAR3 Signaling Complex in EMT and Tumorigenesis and Biological Implications for MTG16 in Stem Cell Function, Proliferation, Apoptosis and DNA Repair

Shenika Vonsha Poindexter Dissertation under the direction of Dr. Christopher S. Williams Tight junctions regulate cell permeability, cell-cell adhesion and suppress cancer development. Blood Vessel Epicardial Substance (BVES), a tight junction associated protein, is down regulated in colon cancer, and BVES expression in mesenchymal colon cancer cell lines promotes mesenchymal to epithelial transition (MET). However, the mechanism by which BVES exerts its effects is not clearly understood. We have identified Breast Cancer Antiestrogen Resistance 3 (BCAR3), a protein whose overexpression promotes EMT, as a BVES interacting protein. To understand the functional role of BVES and BCAR3 interaction in regulating EMT/MET processes, we applied complementary knockdown and overexpression approaches. We demonstrated loss of BCAR3 rescues BVES dependent effects on cell morphology and migration. The BVES/BCAR3 interaction potentially reveals a unique tight junction to focal adhesion signaling pathway important in regulating EMT potentially through WNT and Rho signaling pathways. Clarifying the role of BVES and BCAR3 in EMT may lead to therapeutic strategies targeting this interaction in colon cancer. Transcriptional regulation is also critical for maintenance of intestinal epithelial integrity. MTG16 is a transcriptional co-repressor implicated in malignancy, hematopoietic stem cell and colonic epithelial functions. Given its’ role in the colon, we hypothesized that MTG16 is important in regulating intestinal responses to injury. Baseline characterization of Mtg16-/- small intestine increased enterocyte proliferation and goblet cell deficiency. After irradiation, we observed increased crypt viability and decreased apoptosis in Mtg16-/- small intestine. In comparison to WT, flow cytometric and immunofluorescence analysis of phospho-Histone H2A.X in Mtg16-/- intestinal epithelial cells indicated decreased DNA damage. After small intestinal crypts were isolated from Mtg16-/- and WT mice, we observed Mtg16-/- crypts had higher enterosphere forming efficiencies and delayed progression to mature enteroids when cultured in the presence of WNT when compared to culturing in EGF, Noggin, and R-spondin; compared with WT, Mtg16-/- intestinal crypts obtained from irradiated mice exhibited increased survival. Interestingly, Mtg16 expression is reduced in a stem cell enriched population at peak time of crypt regeneration. In sum, this study demonstrates that MTG16 contributes to multiple aspects of small intestinal homeostasis and could potentially serve as a biomarker for monitoring response to radiotherapy.