Adenomatous Polyposis Coli
“You are old, Father William,” the young surgeon said,
“And your colon from polyps is free.
Yet most of your siblings are known to be dead –
A really bad family tree”
“In my youth,” Father William replied with a grin,
“I was told that a gene had mutated,
That all who carried this dominant gene
To polyps and cancer were fated.”
Cuthbert Dukes, from a speech entitled “
Familial Intestinal Polyps” delivered to the Royal College of Surgeons of England March 25th, 1952
Familial adenomatous polyposis (FAP) was first thought to be identified by H. Luschka in 1861, who described a colon containing thousands of polyps. The hereditary nature of this disease came into focus with a second case report in 1882 by Cripps who identified polyposis within two members of the same family. Although his findings made allusions to the familial nature of the disease, it wasn’t until 1925 when Lockhart-Mummery was able to demonstrate true Mendelian inheritance in the propagation of FAP. These results were further refined by Gardner in 1951, stating that familial polyposis was characteristic of a dominant gene mutation that affected both sexes equally.
It wasn’t until 1987, however, that the genetic locus for FAP was discovered in three separate studies by Solomon, Leppert, and Bodmer. They were able to show that the long arm of chromosome 5 contained allelic losses that strongly correlated to the presence of the disease. In 1988, Aldred identified adenomatous polyposis coli (APC) as the mutated gene underlying FAP. APC is most well known as the tumor suppressor gene mutated in sporadic and familial colorectal tumors, although it has numerous other functions in other cell types.
The N-terminus oligomerization domain allows APC to form homodimers which may be necessary to form an effective β-catenin destruction complex (see below); although mutations in this region are relatively rare, it has been suggested that dimerization of a mutated, truncated APC protein to a wild-type APC may act in a dominant negative fashion, sequestering the wild-type APC from normal function (Fearnhead et al. 2001).
IQ motif containing GTPase-activating protein 1 (IQGAP1), which regulates the local actin cytoskeleton and recruits key signaling molecules to neuronal synapses.
The regulatory B56 subunit of protein phosphatase 2A (PP2A), which regulates the activation/phosphorylation state of multiple targets, such as Glycogen synthase kinase 3β (GSK3β), and β-catenin. Both proteins function in key signaling pathways, Wnt responsive gene expression and Fragile-X mental retardation protein (FMRP) regulated local protein synthesis at active synapses.
The kinesin-associated protein 3 (KAP3), that interacts with the kinesin 3A-3B microtubule plus-end directed motor proteins. This complex is required for APC transport along microtubules and clustering at their plus ends in membrane protrusions (Jimbo et al. 2002).
APC-stimulated guanine nucleotide exchange factor 1 (Asef1), a guanine nucleotide exchange factor (GEF) for the Rac and Rho GTP binding proteins that regulates the actin cytoskeletal network, cell morphology, and cell migration.
The central portion, consisting of three repeats of 15 amino acids, seven repeats of 20 amino acids, and three SAMP binding motifs, mediates the interaction of both β-catenin and Axin to form the core of the “β-catenin destruction complex,” mutations that lead to colorectal cancers occur most commonly in this region (the mutation cluster region). APC association with β-catenin and Axin is necessary for directing casein kinase 1 (CK1)/GSK-3β mediated phosphorylation of β-catenin, and its subsequent ubiquitination and proteosomal degradation. Disruptions in APC/β-catenin association result in β-catenin accumulation within the cytoplasm, translocation to the nucleus, and subsequent activation of canonical Wnt responsive gene expression (Polakis 1995).
The C-terminal region of APC contains an unstructured basic domain, a binding domain for the microtubule associated plus end-binding protein (EB1), and a PDZ binding motif. The large basic domain facilitates APC’s interaction with the microtubule cytoskeleton and also harbors similarities to many regions found in DNA/RNA binding proteins. APC has been recently identified as an mRNA binding protein, but the interaction domain is not yet defined. The EB1 binding domain functions to capture EB1-tagged microtubule plus-ends at specific cell surface sites and thereby regulates the plane of cell division, cell polarity, migration, and the trafficking of selected cargo.
The PDZ-binding domain mediates the interactions of APC with PDZ domain containing proteins such as Discs large 1 (Dlg1), postsynaptic density 95 (PSD-95, Dlg4, SAP 90), and PSD-93 (Dlg2, Chapsyn110). Interactions with these scaffolding proteins near the plasma membrane are required for normal migration and polarity of epithelial cells and for the maturation and organization of synaptic specializations in peripheral and central nerve cells.
In migrating cells, APC is concentrated at the plus ends of microtubules in cellular protrusions. In polarized epithelial cells, APC is concentrated at the apical plasma membrane near adherens junctions. It is also found to spread diffusely along microtubules in the cytoplasm and localizes within the nucleus. In neurons, punctual distribution of APC is seen in membrane protrusions during migration, within growth cones, as well as in the soma and nucleus. In mature neurons, it is enriched at excitatory postsynaptic sites, but not at inhibitory synapses. The diverse subcellular distributions of APC reflect the wide range of its functions.
In the absence of the soluble Wnt ligand (Fig. 2), APC and Axin recruit β-catenin into a complex with Casein Kinase (CK)1 and GSK3β, promoting a series of phosphorylation events on β-catenin that increase its affinity for binding to APC and mark it for ubiquitination by the E3 ligase β-TRCP and subsequent degradation by the 26S proteosome. Upon binding of Wnt ligand to the frizzled receptor and the LRP 5/6 coreceptor, Axin is recruited to the plasma membrane and dishevelled is activated to inhibit GSK3β activity. Inhibition of GSK3β allows dephosphorylated β-catenin to disassociate from APC. The stable β-catenin accumulates in the cytoplasm, then translocates to the nucleus and activates Wnt target gene expression via binding to the transcriptional cofactors TCF and LEF (list of Wnt target genes, http://web.stanford.edu/group/nusselab/cgi-bin/wnt/target_genes).
Cancer cell lines harboring mutations in APC show high levels of Wnt signaling pathway activation and target gene expression. Most of the APC mutations associated with colorectal cancers are truncations that lead to loss of the first three of the seven 20 amino acid repeats, these domains are necessary for retaining APC’s ability to regulate β-catenin degradation (Fearnhead et al. 2001). APC mutations also cause other cancers, including glioblastoma, primary neuroepithelial tumors of the central nervous system, and pancreatic cancer. APC truncations that cause loss of only the C-terminal regions that function in cytoskeletal regulation, while retaining β-catenin/Wnt regulation capabilities, do not result in cancers. Overall, APC mutations highlight the importance of its role as a tumor suppressor in governing β-catenin and Wnt signal transduction. Moreover, the association of APC mutations with neurological disorders is just beginning to be appreciated.
APC’s Role in Cytoskeletal Regulation
Microtubules (MTs) – directly and with the plus-end tracking protein EB1.
Actin – via cytoskeletal regulators Asef1, IQGAP1, and microtubule-actin crosslinking protein (MACF, ACF7), and β-catenin, as part of the cadherin-based intercellular adhesion complex.
Intermediate filaments (IF) – directly via APC’s armadillo repeats, facilitated by IQGAP1.
Proper regulation of the cytoskeleton is essential for normal cellular differentiation and function. The ability of APC to bind cytoskeletal proteins and dynamic regulators of cytoskeletal organization supports the importance of APC’s role in key cellular functions such as polarity, cell division, migration, and cargo trafficking. APC and its interacting proteins are evolutionarily conserved from Drosophila to mammals.
Microtubules. APC binds directly to microtubules along their length and clusters at the plus ends via APC binding to EB1. APC has been shown to activate tubulin polymerization in vitro. Additionally, APC interactions with EB1 stabilize microtubules and direct their plus-ends to cadherin-catenin adhesion complexes at the cell surface. The APC-EB1 interaction is essential for normal chromosome segregation, planar cell division, migration, and microtubule-mediated transport of cargo to precise regions of the cell.
APC and EB1 also localize at the tips of processes in migrating cells and neuronal growth cones (Fig. 3b, d). In particular, APC is essential in highly polarized neurons for polarity, axon outgrowth, branching, and guidance, as well as proper maturation of synaptic specializations at later stages (Fig. 3e). Axon polarization, outgrowth, and guidance require APC interactions with several proteins. GSK3β regulates the binding of APC to microtubule plus ends. APC, GSK-3β, and the kinesin plus-end motor protein, KIF3A, function together to target Par proteins to the growth cone of the extending axon (Fig. 3d). APC C-terminal truncations or heterozygous deletions, affecting the microtubule binding domains, lead to mislocalization of mPAR3 and deficits in axon specification and neuronal polarity (Shi et al. 2004; Goldstein and Macara 2007). APC-EB1 interactions also direct the targeting of selected proteins down axons and to synaptic sites, and are therefore essential for the proper maturation and function of synapses.
Interestingly, APC modulates microtubule (MT) severing, by regulating the stability and activity of the MT-severing protein p60-katanin in interneurons to promote the rapid remodeling of neuronal processes necessary for their tangential migration (Eom et al. 2014).
APC also functions in the microtubule-mediated transport of mitochondria (Mills et al. 2016). APC directly interacts with the mitochondrial kinesin-motor complex and promotes the localization of mitochondria near the plasma membrane. APC depletion causes a redistribution of mitochondria away from the cell periphery to more perinuclear regions. Disrupted mitochondrial localization is seen in colorectal cancers caused by APC deletions and truncations, and likely has adverse effects in other cell types, such as neurons, where perturbations of mitochondrial transport down axons can lead to neurological disorders.
Actin. APC regulates the polymerization and stabilization of the submembranous actin cytoskeleton through its interactions with β-catenin, EB1-MACF, and the Rho-GTPase effector molecules, IQGAP1, and Asef. Β-catenin binds to alpha-catenin and thereby links the cadherin-based intercellular adhesion complex to the actin cytoskeleton. APC, as the major negative regulator of β-catenin levels, governs cell adhesion dynamics from epithelial cell adherens junctions to neuronal synapses. Additionally, APC interaction with β-catenin serves to target APC to these specialized intercellular adhesion sites. APC brings together key regulators of the local F-actin and microtubule cytoskeleton at these sites, including EB1, Asef, MACF, and IQGAP1. MACF directly binds to EB1, microtubules, and F-actin, and is required for tethering EB1-tagged microtubules to the submembranous F-actin network at specialized junctions (Rosenberg et al. 2008). IQGAP1 enhances F-actin polymerization by stabilizing the Rho-GTPases Rac1 and CDC42 for activation by their respective guanine nucleotide exchange factors (GEFs) (Watanabe et al. 2004).
Asef, a direct binding partner of APC and an actin regulator, is a GEF that activates both Rac1 and CDC42, promoting the subsequent nucleation of F-actin (Kawasaki et al. 2000). Thus APC serves as a hub for organizing several proteins that regulate actin and microtubule cytoskeletal dynamics during cell polarization and directional migration. Migratory response to cytokines and growth factors are dependent on the APC/β-catenin complex being able to spatially and temporally regulate the cytoskeleton. For example, transforming growth factor-β (TGFβ), a key determinant of cellular migration, causes local inactivation of GSK3β and promotes the association of APC with the distal ends of microtubules (Ekman et al. 2012).
APC brings together a multimolecular complex that organizes and maintains a unique microtubule-rich and F-actin-rich region; linking to these cytoskeletal elements stabilizes intercellular junctions and restricts the localization of specific components to these sites in diverse cell types. In mature neurons, these APC functions are essential for the proper maturation and function of excitatory synapses (Temburni et al. 2004; Rosenberg et al. 2008; Rosenberg et al. 2010; Mohn et al. 2014).
Intermediate Filaments. APC interacts with the intermediate filament (IF) cytoskeleton, via its armadillo repeats. APC is responsible for localizing the IF vimentin to microtubules and promoting IF polymerization and elongation. Truncations of APC correlate with disorganization of IFs and perturb the migration of astrocytes (Sakamoto et al. 2013).
All together, these studies identify APC as a crucial regulator and integrator of the cytoskeletal architecture involving microtubules, actin, and intermediate filaments and as a critical determinant of cellular division, migration, and polarization, and of intercellular adhesion sites.
APC’s Role in DNA and mRNA Regulation. Recent studies define novel functions for APC as a regulator of DNA methylation and as an mRNA binding protein. APC has been shown to positively regulate retinoic acid production, thereby inhibiting the DNA demethylase system in the intestine. Genomic DNA methylation regulates chromatin stability and temporally controls cell-type-specific gene expression. Loss of APC results in aberrant DNA methylation and may facilitate tumor initiation and/or progression by triggering genomic instability and changes in gene expression (Andersen and Jones 2013). In APC mutant colorectal cells, retinoic acid production is decreased, leading to continued expression of the demethylase system and inhibition of the epigenetic regulation of genes known to function in pluripotency, differentiation, and cell fate specification, thereby maintaining the cells in an undifferentiated state. Dysregulation of the DNA demethylase system may also lead to chromatin instability that causes an additional deletion on the opposing wild-type APC gene locus, a phenomenon known as a “second-hit” and is often seen in colorectal cancers harboring APC mutations.
mRNA localization and local translation plays important roles in the establishment and maintenance of polarity in multiple cell types as well as in modulating synaptic function and plasticity in neurons. APC interacts, indirectly or directly, with the RNA-binding proteins, FMRP and Fus. APC associates with FMRP and its target mRNAs in the distal tips of protrusions at the leading edge of migrating fibroblasts, and shRNA-mediated reductions in APC disrupt this mRNA localization (Mili et al. 2008). Further, APC association with Fus is required for efficient translation of its associated mRNAs (Yasuda et al. 2013). Importantly, APC itself has been shown to bind selected mRNAs in neurons. Its associated transcripts are highly related to APC functions in Wnt signaling, cytoskeletal regulation, intracellular trafficking, and RNA binding and regulation. APC may regulate the stabilization, localization, and translation of its target mRNAs. As an example, morpholino-mediated suppression of APC leads to decreased β2B-tubulin mRNA levels and localization in the axonal growth cone and to impaired neuron migration (Preitner et al. 2014).
APC acts as an elegantly constructed core regulator of protein and mRNA networks that function in a rich array of cellular processes.
APC and Disease: From Humans to Mouse Models
To date, over one thousand different disease causing loss-of-function APC mutations have been identified. The APC gene is well known for its link to colorectal cancers, but APC heterozygous gene deletions and polymorphisms also link to intellectual disabilities, seizures, and autism spectrum disorders as aspects of a broader syndrome. Most mutations in APC are located in the mutation cluster region (MCR), resulting in a truncated protein product and elevated β-catenin/Wnt signaling due to disruption of β-catenin/Axin binding. Rodent models harboring various APC gene mutations have enabled in vivo validation of its multitude of functions in both healthy and pathogenic states. In total, over twenty APC mouse and rat models have been generated since the early 1990s.
Cancer. APC controls many key cellular functions including proliferation, differentiation, migration, and polarity. In all cancers, these functions are severely altered. The APC gene was discovered in FAP as an inherited mutation. Up to 80% of sporadic colorectal cancers are caused by APC disruptive de novo mutations (Fearon 2011). While APC is best known for its role in colorectal cancers, APC mutations also lead to a variety of other cancers, including cancers of the breast, lung, pancreas, stomach, and glioblastomas. APC mutations have also been found in patients with Turcot syndrome, a rare disorder characterized by tumor development in the primary neuroepithelium of the central nervous system, in conjunction with colorectal polyps (Mori et al. 1994). APC mutations also link to glioblastoma multiforme, a highly aggressive primary brain tumor. APC is also critical for development of the retinal epithelium; congenital hypertrophy of the retinal pigment epithelium, a disorder characterized by poor vision, is found exclusively in FAP patients (Pang et al. 2000).
Many different mouse models of APC truncation mutations have been created to study APC functions and its role in cancer in vivo. One of the best studied models is the multiple intestinal neoplasia mouse, or APCMin mouse (Su et al. 1992). APCMin mice harbor a single allelic mutation within the MCR (T2549A) resulting in a premature stop codon within one copy of the APC gene. This mutation results in a protein, truncated at 850 amino acids, that abolishes the Axin/β-catenin binding domains and C-terminal microtubule, EB1, and PDZ binding domains. Almost all APCMin mice develop 50–100 adenomas in the small intestine by about 4 months of age, invariably associated with loss of the remaining wild-type gene. A small number of female APCMin mice also develop primary mammary tumors, suggesting that APC may play a sex-specific role in these particular cell types. Conditional deletion or truncation of APC in intestinal epithelial crypt cells leads to a similar neoplastic phenotype, with hyperproliferation and expansion of undifferentiated intestinal stem cells outside of the crypt, resulting in disrupted tissue structure and function. Interestingly, conditional and reversible APC depletion via doxycycline-regulated shRNA shows tumor regression and reestablishment of normal intestinal crypt homeostasis. This rescue suggests that restoration of APC function may be a useful target for effective therapeutic strategies.
Nervous System Disorders. APC plays a regulatory role in several cellular processes that are fundamental to normal brain development and function and has recently been identified as a risk gene for neurodevelopmental brain disorders. However, its neural specific functions remain poorly defined. Individuals with heterozygous deletion of APC, de novo or inherited, exhibit intellectual disabilities, ranging from severe to mild, autism spectrum disorder and seizures – often overlapping as a broad syndromic phenotype. Hearing abnormalities have also been reported in some individuals with APC disruptive gene mutations. As APC associates, directly or indirectly, with several proteins and mRNAs implicated as risk factors for neurodevelopmental brain disorders, the precise APC mutation (deletion, truncation, or polymorphism) may play a significant role in the particular phenotypes.
Nonhuman models of APC mutations have played an increasingly large role in elucidating APC functions in the nervous system. APC is required for multiple aspects of brain development. It is essential for maintenance of the polarized radial glial scaffold and for the regulation of cell proliferation, cell-cycle regulated nuclear migration, cell polarity, and cell type specification during cerebral cortical development. Targeted deletion of APC in embryonic neural progenitor cells leads to defective generation and migration of cortical neurons, resulting in malformation of axonal tracts and severe cortical lamination defects.
In neurons, APC is enriched at excitatory, but not inhibitory, postsynaptic sites. The first identification of its role in organizing neuronal synapses, in vivo, was shown in avian autonomic neurons. APC was demonstrated to be required for proper assembly of nicotinic synapses, for the postsynaptic localization of the α3-nicotinic acetylcholine receptor and the cellular adhesion molecules neurexin and neuroligin, and for coordinating the maturation of presynaptic and postsynaptic specializations (Temburni et al. 2004; Rosenberg et al. 2008; Rosenberg et al. 2010). Similarly, APC is concentrated at and regulates the maturation of the nicotinic neuromuscular junction in vertebrate skeletal muscle (Wang et al. 2003). In the mammalian brain, APC is enriched at glutamatergic synapses and it is required in cultured hippocampal neurons for the postsynaptic clustering of PSD-95 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (Shimomura et al. 2007). Mice with conditional deletion of APC predominantly in excitatory neurons of the brain display cognitive deficits, autism-like behaviors (reduced social interest, increased repetitive behaviors), altered synaptic density and function, and excessive levels of β-catenin (Mohn et al. 2014). Β-catenin, in turn, has dual roles in canonical Wnt signaling and cadherin-based intercellular adhesion, both of which are critical for normal synaptic maturation and function. Additionally, APC interacts with many proteins and mRNAs that are either known or predicted to link to neurological diseases such as intellectual disabilities, epilepsy, autism, and Alzheimer’s disease.
APC also plays a role in CNS cell types other than excitatory neurons. Targeted deletion of APC in developing interneurons compromises microtubule dynamics affecting their morphology, migration, and integration into the neural circuitry of the brain (Eom et al. 2014). APC is also critical in nonneuronal glial cells, such as astrocytes and oligodendrocytes, as proper polarity and migration underlie major aspects of their cellular functions in providing targeted modulation of synaptic activity and axonal fidelity (action potential propagation) through proper myelination. APC is also necessary for normal hearing because it regulates the maturation of sensory hair cell ribbon synapses that are critical for cochlear function (Hickman et al. 2015). Deletion of APC in the presynaptic efferent olivocochlear neurons in the developing auditory system results in perturbations in intercellular signaling, aberrant presynaptic ribbon heterogeneity in the sensory hair cells, and decreased sensitivity of the dynamic hearing ranges. APC plays a critical role in the development and function of the diverse neural cell types, and in synaptic maturation and axonal myelination.
Recent studies of this ubiquitously expressed scaffold protein have greatly extended our knowledge of its role in the healthy and diseased state. While APC disruptive mutations are known to be the leading cause of colorectal cancer, it is now appreciated that it also leads to other cancers including highly metastatic glioblastoma. Additionally, APC disruptive mutations link to neurodevelopmental brain disorders, including intellectual disabilities, autism spectrum disorders, and seizures. Knowledge of APC functions has also been expanded. In addition to its roles as the major negative regulator of β-catenin levels in the canonical Wnt signaling pathway and as a key regulator of microtubule and actin cytoskeletal organization, APC is now known to function as an mRNA binding protein. This newly discovered APC function raises important questions about the identity of APC target mRNAs in different cell types and the regulatory role of APC binding to the selected transcripts. Future studies will elucidate the detailed relationship between precise APC mutations, the functions that are perturbed, including cell type specific changes, and the resulting phenotypes. This rich knowledge will lay the foundation for developing new effective treatment strategies to amend the disorders caused by APC disruptive mutations.
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