Several key signal transduction pathways play critical roles in animal development, and also are inappropriately activated in cancer. Among these is the Wnt pathway. Its role in human cancer was first identified through the tumor suppressor Adenomatous polyposis coli (APC), mutated in most colon cancers. We now know that APC is a critical negative regulator of Wnt signaling. APC is part of a multiprotein """"""""destruction complex"""""""" targeting the key Wnt effector _catenin for phosphorylation and eventual proteasomal destruction. Understanding APC function is essential both to understanding how Wnt signaling shapes normal development and how its inappropriate activation contributes to cancer. In addition to its role in Wnt regulation, APC also plays roles in cytoskeletal regulation. Diverse cytoskeletal functions have been ascribed to APC. Among these is a proposed role in chromosome instability in APC mutant tumors, via effects on chromosome segregation. We established a model system to study APC function in Wnt signaling and cytoskeletal regulation during normal development and to model how its inactivation leads to cancer. We use the fruit fly Drosophila, making use of the powerful combination of genetic, cell biological and biochemical tools available in flies. Both mammals and Drosophila have two APC family members, which all share a core set of protein domains but which differ at their N- and C-termini. In the past funding period, we addressed several key questions in the field. First, we discovered that the two fly APC family members act redundantly in Wnt signaling in many tissues, despite their divergent structures and striking differences in intracellular localization. Second, we generated a null allele of APC2. Using existing null alleles of APC1, this allowed us to create, for the first time, both tissues and whole animals null for both APC family members, revealing the null phenotypes of APC function in both Wnt and cytoskeletal regulation. We also generated a series of new APC2 alleles;among other findings these revealed that the truncated APC proteins characteristic of colon tumors are reduced for Wnt regulation but not null, supporting the """"""""just right"""""""" hypothesis. They also revealed that these truncated proteins have dominant negative effects on the cytoskeleton but not in Wnt signaling. Despite the great interest in APC, key questions remain regarding its roles in the destruction complex and cytoskeletal regulation. We propose 3 Specific Aims, each addressing key questions:
Aim 1 : Define the mechanism(s) of action of APC proteins in the destruction complex.
Aim 2 : Determine how APC structure influences the assembly and activity of the destruction complex and regulates _catenin transfer to the E3 ligase Aim 3: Explore mechanisms by which APC family proteins regulate the cytoskeleton

Public Health Relevance

The body's cells communicate with one another during normal development of an embryo, and in adult tissues to regulate tissue maintenance and repair wounds. Altered cell communication underlies several common cancers including colon cancer, while loss of cell communication causes some forms of congenital bone malformation. We have developed a model system to explore how the tumor suppressor APC normally regulates cell communication and cell behavior, to allow better understanding of what goes wrong in human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067236-08
Application #
8122120
Study Section
Special Emphasis Panel (ZRG1-CB-N (02))
Program Officer
Maas, Stefan
Project Start
2002-09-01
Project End
2013-07-31
Budget Start
2011-08-01
Budget End
2013-07-31
Support Year
8
Fiscal Year
2011
Total Cost
$252,018
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Pronobis, Mira I; Deuitch, Natalie; Posham, Vinya et al. (2017) Reconstituting regulation of the canonical Wnt pathway by engineering a minimal ?-catenin destruction machine. Mol Biol Cell 28:41-53
Poulton, John S; Cuningham, John C; Peifer, Mark (2017) Centrosome and spindle assembly checkpoint loss leads to neural apoptosis and reduced brain size. J Cell Biol 216:1255-1265
Pronobis, Mira I; Deuitch, Natalie; Peifer, Mark (2016) The Miraprep: A Protocol that Uses a Miniprep Kit and Provides Maxiprep Yields. PLoS One 11:e0160509
Lerit, Dorothy A; Poulton, John S (2016) Centrosomes are multifunctional regulators of genome stability. Chromosome Res 24:5-17
Lerit, Dorothy A; Jordan, Holly A; Poulton, John S et al. (2015) Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function. J Cell Biol 210:79-97
Terzo, Esteban A; Lyons, Shawn M; Poulton, John S et al. (2015) Distinct self-interaction domains promote Multi Sex Combs accumulation in and formation of the Drosophila histone locus body. Mol Biol Cell 26:1559-74
Pronobis, Mira I; Rusan, Nasser M; Peifer, Mark (2015) A novel GSK3-regulated APC:Axin interaction regulates Wnt signaling by driving a catalytic cycle of efficient ?catenin destruction. Elife 4:e08022
Poulton, John S; Cuningham, John C; Peifer, Mark (2014) Acentrosomal Drosophila epithelial cells exhibit abnormal cell division, leading to cell death and compensatory proliferation. Dev Cell 30:731-45
Poulton, John S; Mu, Frank W; Roberts, David M et al. (2013) APC2 and Axin promote mitotic fidelity by facilitating centrosome separation and cytoskeletal regulation. Development 140:4226-36
Roberts, David M; Pronobis, Mira I; Alexandre, Kelly M et al. (2012) Defining components of the ýý-catenin destruction complex and exploring its regulation and mechanisms of action during development. PLoS One 7:e31284

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