Wnt signaling in human colonocytes: roles for the tumor suppressor APC in β-catenin cytoplasmic retention and destruction complex localization

Abstract

The Wnt signaling pathway is remarkably conserved across all metazoans. This pathway coordinates a large variety of processes involved in development, establishment of cell polarity, maintenance of tissue homeostasis, and promotion of cellular proliferation. Intriguingly, components of the Wnt pathway are frequently altered in diseases such as cancer and result in aberrant Wnt activation. In the case of colorectal cancer (CRC), mutation to the Wnt antagonist Adenomatous Polyposis Coli (APC) occurs in over 80% of human CRCs resulting in expression of a truncated form of APC. The APC protein is a component of the cytoplasmic β-catenin destruction complex which functions in the absence of Wnt ligands to phosphorylate β-catenin, marking it for ubiquitination and proteasomal degradation. However, Wnt ligand engagement with cell surface receptors inhibits the β-catenin destruction complex through an incompletely understood mechanism. Functions of APC in normal destruction complex activity and in Wnt signal transduction have remained elusive. To better understand how Wnt-induced destruction complex inhibition occurs and whether APC has a role, I utilized Wnt3a-Dynabeads to study the localization of the destruction complex in response to Wnt. In this work, I found that the β-catenin destruction complex re-orients following Wnt exposure toward a localized Wnt cue in colon epithelial cells of normal and malignant origin, and that this process occurs in an APC-dependent fashion. Mutation of APC has been theorized to act in a “just-right” manner, in which specific regions of the APC protein are retained in order to allow a specific level of β-catenin activity that is optimal for tumor growth. My data also provides support that less frequently occurring β-catenin mutations may work in a “just-right” manner as well. I find that a mutation thought to “stabilize” the downstream Wnt pathway effector, β-catenin, does not interfere with the ability of cells to respond to Wnt and find that APC loss results in further accumulation and nuclear translocation of this stabilized β-catenin. Kinetic analysis of the destruction complex response to Wnt reveals that components of the complex can traffic to the Wnt-receptors immediately, suggesting that this trafficking is an initial Wnt response. I also find that APC loss impairs cells ability to accumulate β-catenin in response to Wnt and that β-catenin degradation is compromised in these cells. The mechanisms by which APC functions in the destruction complex to regulate β-catenin are not completely understood, nor are the reasons why certain regions of APC are commonly lost or retained due to truncation in CRC. In this work, I find that full-length APC is required for Wnt-induced complex localization and propose that regions found within the frequently lost C-terminus of APC mediate destruction complex trafficking and inhibition. These findings provide key mechanistic details to better understand and therapeutically target the Wnt signaling pathway.