Exploring Novel Properties of Adenomatous Polyposis Coli: Msi1-Knockout Mice Display Alterations in APC and Intestinal Cell Homeostasis; Topoisomerase IIa Binds to Truncated APC in Human Colon Cancer Cells

Abstract

Abstract Colorectal cancer is the second leading cause of cancer-related death in the United States. Adenomatous polyposis coli (APC) is the initiating mutation in 80% of all colorectal cancers. Our lab is focused on understanding functions of APC that promote tumor suppression in the normal intestinal epithelia. As a protein with many functions in the cell, APC is both nuclear and cytoplasmic, with different roles in maintaining cellular homeostasis. In the cytoplasm, APC has been well characterized as a repressor of Wnt signaling, a pathway important in activating proliferation in the intestine. Mutation of APC results in uninhibited Wnt signaling and therefore unrestricted proliferation and expansion of stem cell populations. Though APC has been studied extensively, little is known about how levels of APC are regulated. Recently, our lab showed a double-negative feedback regulatory mechanism between APC and Musashi-1 (MSI1) in cell culture. MSI1 represses translation of APC, while APC, via its role as a negative regulator of Wnt signaling, inhibits transcription of MSI1, a Wnt target gene. To further understand the role of the APC/MSI1 double-negative feedback loop in intestinal homeostasis, I isolated small and large intestines from mice with germ-line knockout of Msi1 and examined differences in proliferation, differentiation, stem cell populations, and apoptosis in the small intestine. In the large intestine, I examined differences in proliferation, stem cell populations, and apoptosis. I hypothesized that loss of Msi1 in the intestine would cause an increase in APC protein, with a subsequent decrease in Wnt signaling. This decrease in Wnt signaling would reduce the rate of proliferation, increase differentiation, decrease stem cell populations, and have an effect on apoptosis. I show here that in the small intestines of MSI1 mutant mice, APC protein levels were higher and Wnt target gene levels were lower with loss of Msi1. However, proliferation and differentiation of goblet cells remained unchanged. Stem cell populations were reduced and there was a decrease in apoptosis. In the large intestines of MSI1 mutant mice, APC levels and Wnt target genes were unaffected, while proliferation and stem cell populations were decreased. Based on this evidence, the APC/Msi1 feedback loop may play a role in homeostasis in the small intestine and loss of Msi1 may affect a different signaling pathway in the large intestine, such as Notch. In the nucleus, APC has been shown to play a role in cell cycle regulation. Our lab showed an interaction between the central portion of the APC protein and topoisomerase IIα, which led to G2 cell cycle arrest. However, whether truncated APC and topo IIα interact in colon cancer cells remained unknown. Here I present data that show that topo IIα interacts with endogenous truncated APC containing previously defined topo IIα binding domains, but not with shorter APC truncations lacking these binding regions. The studies presented here show that disruption of Msi1 regulation of APC has an effect in the small intestine while Msi1 disruption in the colon has a different effect. Also, longer truncated APC proteins endogenous to colon cancer cell lines interact with topo IIα.