Understanding phenotypic variation in rodent models with germline Apc mutations

Adenomatous Polyposis Coli (APC) is best known for its crucial role in colorectal cancer suppression. Rodent models with various Apc mutations have enabled experimental validation of different Apc functions in tumors and normal tissues. Since the development of the first mouse model with a germline Apc mutation in the early 1990s, twenty other Apc mouse and rat models have been generated. This article compares and contrasts currently available Apc rodent models with particular emphasis on providing potential explanations for their reported variation in three areas: 1) intestinal polyp multiplicity, 2) intestinal polyp distribution, and 3) extra intestinal phenotypes

Constitutive Musashi1 expression impairs mouse postnatal development and intestinal homeostasis

Evolutionarily conserved RNA-binding protein Musashi1 (Msi1) can regulate developmentally relevant genes. Here we report the generation and characterization of a mouse model that allows inducible Msi1 overexpression in a temporal and tissue-specific manner. We show that ubiquitous Msi1 induction in ∼5-wk-old mice delays overall growth, alters organ-to-body proportions, and causes premature death. Msi1-overexpressing mice had shortened intestines, diminished intestinal epithelial cell (IEC) proliferation, and decreased growth of small intestine villi and colon crypts. Although Lgr5-positive intestinal stem cell numbers remained constant in Msi1-overexpressing tissue, an observed reduction in Cdc20 expression provided a potential mechanism underlying the intestinal growth defects. We further demonstrated that Msi1 overexpression affects IEC differentiation in a region-specific manner, with ileum tissue being influenced the most. Ilea of mutant mice displayed increased expression of enterocyte markers, but reduced expression of the goblet cell marker Mucin2 and fewer Paneth cells. A higher hairy and enhancer of split 1:mouse atonal homolog 1 ratio in ilea from Msi1-overexpressing mice implicated Notch signaling in inducing enterocyte differentiation. Together, this work implicates Msi1 in mouse postnatal development of multiple organs, with Notch signaling alterations contributing to intestinal defects. This new mouse model will be a useful tool to further elucidate the role of Msi1 in other tissue settings

APC controls Wnt-induced β-catenin destruction complex recruitment in human colonocytes

This work is licensed under a Creative Commons Attribution 4.0 International License.Wnt/β-catenin signaling is essential for intestinal homeostasis and is aberrantly activated in most colorectal cancers (CRC) through mutation of the tumor suppressor Adenomatous Polyposis Coli (APC). APC is an essential component of a cytoplasmic protein complex that targets β-catenin for destruction. Following Wnt ligand presentation, this complex is inhibited. However, a role for APC in this inhibition has not been shown. Here, we utilized Wnt3a-beads to locally activate Wnt co-receptors. In response, the endogenous β-catenin destruction complex reoriented toward the local Wnt cue in CRC cells with full-length APC, but not if APC was truncated or depleted. Non-transformed human colon epithelial cells displayed similar Wnt-induced destruction complex localization which appeared to be dependent on APC and less so on Axin. Our results expand the current model of Wnt/β-catenin signaling such that in response to Wnt, the β-catenin destruction complex: (1) maintains composition and binding to β-catenin, (2) moves toward the plasma membrane, and (3) requires full-length APC for this relocalization

APC controls Wnt-induced β-catenin destruction complex recruitment in human colonocytes

Wnt/β-catenin signaling is essential for intestinal homeostasis and is aberrantly activated in most colorectal cancers (CRC) through mutation of the tumor suppressor Adenomatous Polyposis Coli (APC). APC is an essential component of a cytoplasmic protein complex that targets β-catenin for destruction. Following Wnt ligand presentation, this complex is inhibited. However, a role for APC in this inhibition has not been shown. Here, we utilized Wnt3a-beads to locally activate Wnt co-receptors. In response, the endogenous β-catenin destruction complex reoriented toward the local Wnt cue in CRC cells with full-length APC, but not if APC was truncated or depleted. Non-transformed human colon epithelial cells displayed similar Wnt-induced destruction complex localization which appeared to be dependent on APC and less so on Axin. Our results expand the current model of Wnt/β-catenin signaling such that in response to Wnt, the β-catenin destruction complex: (1) maintains composition and binding to β-catenin, (2) moves toward the plasma membrane, and (3) requires full-length APC for this relocalization

Oncogenic Serine 45-Deleted β-Catenin Remains Susceptible to Wnt Stimulation and APC Regulation in Human Colonocytes

This work is licensed under a Creative Commons Attribution 4.0 International License.The Wnt/β-catenin signaling pathway is deregulated in nearly all colorectal cancers (CRCs), predominantly through mutation of the tumor suppressor Adenomatous Polyposis Coli (APC). APC mutation is thought to allow a “just-right” amount of Wnt pathway activation by fine-tuning β-catenin levels. While at a much lower frequency, mutations that result in a β-catenin that is compromised for degradation occur in a subset of human CRCs. Here, we investigate whether one such “stabilized” β-catenin responds to regulatory stimuli, thus allowing β-catenin levels conducive for tumor formation. We utilize cells harboring a single mutant allele encoding Ser45-deleted β-catenin (β-catΔS45) to test the effects of Wnt3a treatment or APC-depletion on β-catΔS45 regulation and activity. We find that APC and β-catΔS45 retain interaction with Wnt receptors. Unexpectedly, β-catΔS45 accumulates and activates TOPflash reporter upon Wnt treatment or APC-depletion, but only accumulates in the nucleus upon APC loss. Finally, we find that β-catenin phosphorylation at GSK-3β sites and proteasomal degradation continue to occur in the absence of Ser45. Our results expand the current understanding of Wnt/β-catenin signaling and provide an example of a β-catenin mutation that maintains some ability to respond to Wnt, a possible key to establishing β-catenin activity that is “just-right” for tumorigenesis.National Science Foundation (IOS-1456538)NIH P30CA16852

Oncogenic Serine 45-Deleted β-Catenin Remains Susceptible to Wnt Stimulation and APC Regulation in Human Colonocytes

The Wnt/β-catenin signaling pathway is deregulated in nearly all colorectal cancers (CRCs), predominantly through mutation of the tumor suppressor Adenomatous Polyposis Coli (APC). APC mutation is thought to allow a “just-right” amount of Wnt pathway activation by fine-tuning β-catenin levels. While at a much lower frequency, mutations that result in a β-catenin that is compromised for degradation occur in a subset of human CRCs. Here, we investigate whether one such “stabilized” β-catenin responds to regulatory stimuli, thus allowing β-catenin levels conducive for tumor formation. We utilize cells harboring a single mutant allele encoding Ser45-deleted β-catenin (β-catΔS45) to test the effects of Wnt3a treatment or APC-depletion on β-catΔS45 regulation and activity. We find that APC and β-catΔS45 retain interaction with Wnt receptors. Unexpectedly, β-catΔS45 accumulates and activates TOPflash reporter upon Wnt treatment or APC-depletion, but only accumulates in the nucleus upon APC loss. Finally, we find that β-catenin phosphorylation at GSK-3β sites and proteasomal degradation continue to occur in the absence of Ser45. Our results expand the current understanding of Wnt/β-catenin signaling and provide an example of a β-catenin mutation that maintains some ability to respond to Wnt, a possible key to establishing β-catenin activity that is “just-right” for tumorigenesis

Novel association of APC with intermediate filaments identified using a new versatile APC antibody

<p>Abstract</p> <p>Background</p> <p>As a key player in suppression of colon tumorigenesis, Adenomatous Polyposis Coli (APC) has been widely studied to determine its cellular functions. However, inconsistencies of commercially available APC antibodies have limited the exploration of APC function. APC is implicated in spindle formation by direct interactions with tubulin and microtubule-binding protein EB1. APC also interacts with the actin cytoskeleton to regulate cell polarity. Until now, interaction of APC with the third cytoskeletal element, intermediate filaments, has remained unexamined.</p> <p>Results</p> <p>We generated an APC antibody (APC-M2 pAb) raised against the 15 amino acid repeat region, and verified its reliability in applications including immunoprecipitation, immunoblotting, and immunofluorescence in cultured cells and tissue. Utilizing this APC-M2 pAb, we immunoprecipitated endogenous APC and its binding proteins from colon epithelial cells expressing wild-type APC. Using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), we identified 42 proteins in complex with APC, including β-catenin and intermediate filament (IF) proteins lamin B1 and keratin 81. Association of lamin B1 with APC in cultured cells and human colonic tissue was verified by co-immunoprecipitation and colocalization. APC also colocalized with keratins and remained associated with IF proteins throughout a sequential extraction procedure.</p> <p>Conclusion</p> <p>We introduce a versatile APC antibody that is useful for cell/tissue immunostaining, immunoblotting and immunoprecipitation. We also present evidence for interactions between APC and IFs, independent of actin filaments and microtubules. Our results suggest that APC associates with all three major components of the cytoskeleton, thus expanding potential roles for APC in the regulation of cytoskeletal integrity.</p

The 15-aa repeat region of Adenomatous polyposis coli is intrinsically disordered and retains conformational flexibility upon binding β-catenin

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.biochem.0c00479.The tumor suppressor Adenomatous polyposis coli (APC) is a large, multidomain protein with many identified cellular functions. The best characterized role of APC is to scaffold a protein complex that negatively regulates Wnt signaling via β-catenin destruction. This destruction is mediated by β-catenin binding to centrally located 15- and 20-amino acid repeat regions of APC. More than 80% of cancers of the colon and rectum present with an APC mutation. Most carcinomas with mutant APC express a truncated APC protein that retains the ∼200-amino acid long′ 15-amino acid repeat region′. This study demonstrates that the 15-amino acid repeat region of APC is intrinsically disordered. We investigated the backbone dynamics in the presence of β-catenin and predicted residues that may contribute to transient secondary features. This study reveals that the 15-amino acid region of APC retains flexibility upon binding β-catenin and that APC does not have a single, observable “highest-affinity” binding site for β-catenin. This flexibility potentially allows β-catenin to be more readily captured by APC and then remain accessible to other elements of the destruction complex for subsequent processing

Branched actin networks are assembled on microtubules by adenomatous polyposis coli for targeted membrane protrusion

Cell migration is driven by pushing and pulling activities of the actin cytoskeleton, but migration directionality is largely controlled by microtubules. This function of microtubules is especially critical for neuron navigation. However, the underlying mechanisms are poorly understood. Here we show that branched actin filament networks, the main pushing machinery in cells, grow directly from microtubule tips toward the leading edge in growth cones of hippocampal neurons. Adenomatous polyposis coli (APC), a protein with both tumor suppressor and cytoskeletal functions, concentrates at the microtubule-branched network interface, whereas APC knockdown nearly eliminates branched actin in growth cones and prevents growth cone recovery after repellent-induced collapse. Conversely, encounters of dynamic APC-positive microtubule tips with the cell edge induce local actin-rich protrusions. Together, we reveal a novel mechanism of cell navigation involving APC-dependent assembly of branched actin networks on microtubule tips

A knock-in mouse model reveals roles for nuclear Apc in cell proliferation, Wnt signal inhibition and tumor suppression

Mutation of the tumor suppressor adenomatous polyposis coli (APC) is considered an initiating step in the genesis of the vast majority of colorectal cancers. APC inhibits the Wnt signaling pathway by targeting proto-oncogene β-catenin for destruction by cytoplasmic proteasomes. In the presence of a Wnt signal, or in the absence of functional APC, β-catenin can serve as a transcription co-factor for genes required for cell proliferation such as cyclin D1 and c-Myc. In cultured cells, APC shuttles between the nucleus and cytoplasm, with nuclear APC implicated in inhibition of Wnt target gene expression. Taking a genetic approach to evaluate the functions of nuclear APC in the context of a whole organism, we generated a mouse model with mutations that inactivate the nuclear localization signals of Apc (ApcmNLS). ApcmNLS/mNLS mice are viable and fractionation of embryonic fibroblasts (MEFs) isolated from these mice revealed a significant reduction in nuclear Apc compared to Apc+/+ MEFs. The levels of Apc and β-catenin protein were not significantly altered in small intestinal epithelia from ApcmNLS/mNLS mice. Compared to Apc+/+ mice, ApcmNLS/mNLS mice displayed increased proliferation in epithelial cells from the jejunum, ileum, and colon. These same tissues from ApcmNLS/mNLS mice displayed more mRNA from three genes up-regulated in response to canonical Wnt signal, c-Myc, Axin2, and Cyclin D1, and less mRNA from Hath 1 which is down-regulated in response to Wnt. These observations suggest a role for nuclear Apc in inhibition of canonical Wnt signaling and control of epithelial proliferation in intestinal tissue. Furthermore, we found ApcMin/+ mice, which harbor a mutation that truncates Apc, have increased polyp size and multiplicity if they also carry the ApcmNLS allele. Taken together, this analysis of the novel ApcmNLS mouse model supports a role for nuclear Apc in control of Wnt target genes, intestinal epithelial cell proliferation and polyp formation