Apc tumor suppressor gene is the "zonation-keeper" of mouse liver

The molecular mechanisms by which liver genes are differentially expressed along a portocentral axis, allowing for metabolic zonation, are poorly understood. We provide here compelling evidence that the Wnt/beta-catenin pathway plays a key role in liver zonation. First, we show the complementary localization of activated beta-catenin in the perivenous area and the negative regulator Apc in periportal hepatocytes. We then analyzed the immediate consequences of either a liver-inducible Apc disruption or a blockade of Wnt signaling after infection with an adenovirus encoding Dkk1, and we show that Wnt/beta-catenin signaling inversely controls the perivenous and periportal genetic programs. Finally, we show that genes involved in the periportal urea cycle and the perivenous glutamine synthesis systems are critical targets of beta-catenin signaling, and that perturbations to ammonia metabolism are likely responsible for the death of mice with liver-targeted Apc loss. From our results, we propose that Apc is the liver "zonation-keeper" gene.     

Expression of Apc2 during mouse development

APC2 (previously known as APCL), a molecule closely related to the adenomatous polyposis coli (APC) tumor suppressor, can deplete cytoplasmic beta-catenin, like APC itself. Recently, it has been shown that APC2 may regulate the localization of p53 and the microtubule stability and/or extension. Although it has been reported that APC2 mRNA is expressed in human brain, the anatomical and ontogenic expression patterns remain unclear. The purpose of this study was to investigate the distribution of mouse Apc2 during mouse development. In the adult brain, Apc2 is expressed predominantly in neurons and throughout the brain. Northern blot analysis demonstrated a high level of Apc2 expression in embryonic and early postnatal brain. Ontogenic analysis has indicated that Apc2 is expressed in neural tissue, including the peripheral nervous system. During development of cortex, retina and cerebellum, Apc2 is expressed in post-mitotic cells. These findings suggest that Apc2 may contribute to the development of neuronal cells.     

Requirement for tumor suppressor Apc in the morphogenesis of anterior and ventral mouse embryo

Tumor suppressor Apc (adenomatous polyposis coli) is implicated in the Wnt signaling pathway that is involved in the early embryonic development and tumorigenesis in vertebrates. While the heterozygous null mutant mice develop intestinal polyps, the homozygous embryos die before gastrulation. To investigate the role of Apc in later embryonic development, we constructed a novel hypomorphic Apc allele whose expression was attenuated by approximately 80%. In the hypomorphic Apc homozygous ES cells, reduction in Apc expression caused beta-catenin accumulation and Wnt signaling activation. The homozygous mutant mouse embryos survived 3 days longer than the null mutant embryos. Interestingly, they showed anterior truncation, partial axis duplication, and defective ventral morphogenesis. To determine the tissues where Apc functions for anterior and ventral morphogenesis, we constructed chimeric embryos whose epiblast was derived predominantly from the Apc hypomorphic homozygous cells but the visceral endoderm was from the wild type. Although these chimeric embryos still showed some anterior defects, their ventral morphogenesis was rescued. In addition, marker studies indicated that the axial mesendoderm was also defective in the homozygous embryos. Our results provide genetic evidence that expression of Apc at the normal level is essential for both anterior and ventral development, in the epiblast derivatives and visceral endoderm.     

The Apc5 subunit of the anaphase-promoting complex/cyclosome interacts with poly(A) binding protein and represses internal ribosome entry site-mediated translation

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that mediates the proteolysis of cell cycle proteins in mitosis and G(1). We used a yeast three-hybrid screen to identify proteins that interact with the internal ribosome entry site (IRES) of platelet-derived growth factor 2 mRNA. Surprisingly, this screen identified Apc5, although it does not harbor a classical RNA binding domain. We found that Apc5 binds the poly(A) binding protein (PABP), which directly binds the IRES element. PABP was found to enhance IRES-mediated translation, whereas Apc5 overexpression counteracted this effect. In addition to its association with the APC/C complex, Apc5 binds much heavier complexes and cosediments with the ribosomal fraction. In contrast to Apc3, which is associated only with the APC/C and remains intact during differentiation, Apc5 is degraded upon megakaryocytic differentiation in correlation with IRES activation. Expression of Apc5 in differentiated cells abolished IRES activation. This is the first report implying an additional role for an APC/C subunit, apart from its being part of the APC/C complex.     

Apc mutation enhances PyMT-induced mammary tumorigenesis

The Adenomatous Polyposis Coli (APC) tumor suppressor gene is silenced by hypermethylation or mutated in up to 70% of human breast cancers. In mouse models, Apc mutation disrupts normal mammary development and predisposes to mammary tumor formation; however, the cooperation between APC and other mutations in breast tumorigenesis has not been studied. To test the hypothesis that loss of one copy of APC promotes oncogene-mediated mammary tumorigenesis, Apc(Min/+) mice were crossed with the mouse mammary tumor virus (MMTV)-Polyoma virus middle T antigen (PyMT) or MMTV-c-Neu transgenic mice. In the PyMT tumor model, the Apc(Min/+) mutation significantly decreased survival and tumor latency, promoted a squamous adenocarcinoma phenotype, and enhanced tumor cell proliferation. In tumor-derived cell lines, the proliferative advantage was a result of increased FAK, Src and JNK signaling. These effects were specific to the PyMT model, as no changes were observed in MMTV-c-Neu mice carrying the Apc(Min/+) mutation. Our data indicate that heterozygosity of Apc enhances tumor development in an oncogene-specific manner, providing evidence that APC-dependent pathways may be valuable therapeutic targets in breast cancer. Moreover, these preclinical model systems offer a platform for dissection of the molecular mechanisms by which APC mutation enhances breast carcinogenesis, such as altered FAK/Src/JNK signaling.     

Progressive changes in adherens junction structure during intestinal adenoma formation in Apc mutant mice

The C57BL/6J-Min/+ (Min/+) mouse bears a mutant Apc gene and therefore is an important in vivo model of intestinal tumorigenesis. Min/+ mice develop adenomas that exhibit loss of the wild-type Apc allele (Apc(Min/-)). Previously, we found that histologically normal enterocytes bearing a truncated Apc protein (Apc(Min/+)) migrated more slowly in vivo than enterocytes with either wild-type Apc (Apc(+/+)) or with heterozygous loss of Apc protein (Apc(1638N)). To study this phenotype further, we determined the effect of the Apc(Min) mutation upon cell-cell adhesion by examining the components of the adherens junction (AJ). We observed a reduced association between E-cadherin and beta-catenin in Apc(Min/+) enterocytes. Subcellular fractionation of proteins from Apc(+/+), Apc(Min/+), and Apc(Min/-) intestinal tissues revealed a cytoplasmic localization of intact E-cadherin only in Apc(Min/+), suggesting E-cadherin internalization in these enterocytes. beta-Catenin tyrosine phosphorylation was also increased in Apc(Min/+) enterocytes, consistent with its dissociation from E-cadherin. Furthermore, Apc(Min/+) enterocytes showed a decreased association between beta-catenin and receptor protein-tyrosine phosphatase beta/zeta (RPTPbeta/zeta), and Apc(Min/-) cells demonstrated an association between beta-catenin and receptor protein-tyrosine phosphatase gamma. In contrast to the Apc(Min/+) enterocytes, Apc(Min/-) adenomas displayed increased expression and association of E-cadherin, beta-catenin, and alpha-catenin relative to Apc(+/+) controls. These data show that Apc plays a role in regulating adherens junction structure and function in the intestine. In addition, discovery of these effects in initiated but histologically normal tissue (Apc(Min/+)) defines a pre-adenoma stage of tumorigenesis in the intestinal mucosa.     

Unique mechanisms of growth regulation and tumor suppression upon Apc inactivation in the pancreas

beta-catenin signaling is heavily involved in organogenesis. Here, we investigated how pancreas differentiation, growth and homeostasis are affected following inactivation of an endogenous inhibitor of beta-catenin, adenomatous polyposis coli (Apc). In adult mice, Apc-deficient pancreata were enlarged, solely as a result of hyperplasia of acinar cells, which accumulated beta-catenin, with the sparing of islets. Expression of a target of beta-catenin, the proto-oncogene c-myc (Myc), was increased in acinar cells lacking Apc, suggesting that c-myc expression is essential for hyperplasia. In support of this hypothesis, we found that conditional inactivation of c-myc in pancreata lacking Apc completely reversed the acinar hyperplasia. Apc loss in organs such as the liver, colon and kidney, as well as experimental misexpression of c-myc in pancreatic acinar cells, led to tumor formation with high penetrance. Surprisingly, pancreas tumors failed to develop following conditional pancreas Apc inactivation. In Apc-deficient acini of aged mice, our studies revealed a cessation of their exaggerated proliferation and a reduced expression of c-myc, in spite of the persistent accumulation of beta-catenin. In conclusion, our work shows that beta-catenin modulation of c-myc is an essential regulator of acinar growth control, and unveils an unprecedented example of Apc requirement in the pancreas that is both temporally restricted and cell-specific. This provides new insights into the mechanisms of tumor pathogenesis and tumor suppression in the pancreas.     

Novel interaction between Apc5p and Rsp5p in an intracellular signaling pathway in Saccharomyces cerevisiae

The ubiquitin-targeting pathway is evolutionarily conserved and critical for many cellular functions. Recently, we discovered a role for two ubiquitin-protein ligases (E3s), Rsp5p and the Apc5p subunit of the anaphase-promoting complex (APC), in mitotic chromatin assembly in Saccharomyces cerevisiae. In the present study, we investigated whether Rsp5p and Apc5p interact in an intracellular pathway regulating chromatin remodeling. Our genetic studies strongly suggest that Rsp5p and Apc5p do interact and that Rsp5p acts upstream of Apc5p. Since E3 enzymes typically require the action of a ubiquitin-conjugating enzyme (E2), we screened E2 mutants for chromatin assembly defects, which resulted in the identification of Cdc34p and Ubc7p. Cdc34p is the E2 component of the SCF (Skp1p/Cdc53p/F-box protein). Therefore, we analyzed additional SCF mutants for chromatin assembly defects. Defective chromatin assembly extracts generated from strains harboring a mutation in the Cdc53p SCF subunit or a nondegradable SCF target, Sic1(Deltaphos), confirmed that the SCF was involved in mitotic chromatin assembly. Furthermore, we demonstrated that Ubc7p physically and genetically interacts with Rsp5p, suggesting that Ubc7p acts as an E2 for Rsp5p. However, rsp5CA and Deltaubc7 mutations had opposite genetic effects on apc5CA and cdc34-2 phenotypes. Therefore, the antagonistic interplay between Deltaubc7 and rsp5CA, with respect to cdc34-2 and apc5CA, indicates that the outcome of Rsp5p's interaction with Cdc34p and Apc5p may depend on the E2 interacting with Rsp5p.     

Apc1 is required for maintenance of local brain organizers and dorsal midbrain survival

The tumor suppressor Apc1 is an intracellular antagonist of the Wnt/β-catenin pathway, which is vital for induction and patterning of the early vertebrate brain. However, its role in later brain development is less clear. Here, we examined the mechanisms underlying effects of an Apc1 zygotic-effect mutation on late brain development in zebrafish. Apc1 is required for maintenance of established brain subdivisions and control of local organizers such as the isthmic organizer (IsO). Caudal expansion of Fgf8 from IsO into the cerebellum is accompanied by hyperproliferation and abnormal cerebellar morphogenesis. Loss of apc1 results in reduced proliferation and apoptosis in the dorsal midbrain. Mosaic analysis shows that Apc is required cell-autonomously for maintenance of dorsal midbrain cell fate. The tectal phenotype occurs independently of Fgf8-mediated IsO function and is predominantly caused by stabilization of β-catenin and subsequent hyperactivation of Wnt/β-catenin signalling, which is mainly mediated through LEF1 activity. Chemical activation of the Wnt/β-catenin in wild-type embryos during late brain maintenance stages phenocopies the IsO and tectal phenotypes of the apc mutants. These data demonstrate that Apc1-mediated restriction of Wnt/β-catenin signalling is required for maintenance of local organizers and tectal integrity.     

Min基因突变小鼠模型在肠道肿瘤研究中的应用

迄今为止, 肠道肿瘤相关的基因突变小鼠或敲除小鼠大约有30多种, Min(multiple intestinal neoplasia)小鼠是具有肠道多发性腺瘤特征的Apc基因突变小鼠, 被认为是当前较为理想的家族性腺瘤性息肉病(FAP)的研究模型。APC基因是Wnt途径中重要的抑癌基因, 该途径不仅是动物胚胎发育过程中关键的信号转导途径, 也对结直肠肿瘤的发生发展起到不同寻常的作用。对Min小鼠模型历史、分子遗传学与表型特征、肠道肿瘤与Wnt途径异常、抑癌基因甲基化、TGF-b途径和多药耐药基因等内容进行了介绍, 并分析了该小鼠模型在抗结直肠肿瘤药物研究中的应用和意义。     

Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop

The anaphase‐promoting complex/cyclosome (APC/C), a multi‐subunit ubiquitin ligase essential for cell cycle control, is regulated by reversible phosphorylation. APC/C phosphorylation by cyclin‐dependent kinase 1 (Cdk1) promotes Cdc20 co‐activator loading in mitosis to form active APC/C‐Cdc20. However, detailed phospho‐regulation of APC/C dynamics through other kinases and phosphatases is still poorly understood. Here, we show that an interplay between polo‐like kinase (Plx1) and PP2A‐B56 phosphatase on a flexible loop domain of the subunit Apc1 (Apc1‐loop⁵⁰⁰) controls APC/C activity and mitotic progression. Plx1 directly binds to the Apc1‐loop⁵⁰⁰ in a phosphorylation‐dependent manner and promotes the formation of APC/C‐Cdc20 via Apc3 phosphorylation. Upon phosphorylation of loop residue T532, PP2A‐B56 is recruited to the Apc1‐loop⁵⁰⁰ and differentially promotes dissociation of Plx1 and PP2A‐B56 through dephosphorylation of Plx1‐binding sites. Stable Plx1 binding, which prevents PP2A‐B56 recruitment, prematurely activates the APC/C and delays APC/C dephosphorylation during mitotic exit. Furthermore, the phosphorylation status of the Apc1‐loop⁵⁰⁰ is controlled by distant Apc3‐loop phosphorylation. Our study suggests that phosphorylation‐dependent feedback regulation through flexible loop domains within a macromolecular complex coordinates the activity and dynamics of the APC/C during the cell cycle.     

Cyclin D1 is not an immediate target of beta-catenin following Apc loss in the intestine

Cyclin D1 is postulated to be a target of the canonical Wnt pathway and critical for intestinal adenoma development. We show here that, unlike cyclin D1 reporter assays, endogenous cyclin D1 levels are not affected following antagonism of the Wnt pathway in vitro, nor is cyclin D1 immediately up-regulated following conditional loss of Apc in vivo. Cyclin D1 levels do, however, increase in a delayed manner in a small subset of cells, suggesting such up-regulation occurs as a secondary event. We also analyzed the immediate consequences of Apc loss in a cyclin D1(-/-) background and failed to find any cyclin D1-dependent phenotypes. However, we did observe elevated cyclin D1 expression in lesions developing 20 days after Apc loss. In these circumstances, all adenomas (but not smaller lesions) showed cyclin D1 up-regulation. Finally in a smaller study, we analyzed whether cyclin D1 deficiency affected adenoma formation 20 days following induced loss of Apc. Unlike AhCre(+) Apc(fl/fl) mice (which all developed adenomas), doubly mutant AhCre(+) Apc(fl/fl) cyclin D1(-/-) mice only developed small lesions. Taken together, this argues that cyclin D1 up-regulation in intestinal neoplasia is important for tumor progression rather than initiation.     

Apc+/Min colonic epithelial cells express TNF receptors and ICAM-1 when they are co-cultured with large intestine intra-epithelial lymphocytes

Adenomatous polyposis coli (APC) functions are involved in the heterotypic interactions occurring between intestinal epithelial cells (IECs) and intra-epithelial lymphocytes (IELs). These interactions may be of interest in cancer prevention, since recent data provide evidence for lymphocyte mediated immunosurveillance of epithelial cancers. The present study attempts to determine if APC inactivation induces changes in the cross-talk between IEC and large intestine IEL (LI-IEL) through intercellular adhesion molecule (ICAM-1)/leukocyte function-associated (LFA-1) interactions. Mouse Apc+/+ and Apc+/Min colonocytes were co-cultivated with LI-IEL. When co-cultured with LI-IEL Apc+/Min IEC but not Apc+/+ IEC expressed high levels of ICAM-1. The presence of ICAM-1 was linked to TNFalpha production in both co-cultures and TNFR expression only in co-cultivated Apc+/Min IEC. Finally, butyrate enhanced the expression of ICAM-1 in Apc+/Min IEC co-cultured with LI-IEL, and the secretion of TNFalpha by both types of co-cultures. These events could participate in determining the Apc+/Min IEC immunogenicity under different in vivo conditions.     

Implications for the ubiquitination reaction of the anaphase-promoting complex from the crystal structure of the Doc1/Apc10 subunit

The anaphase-promoting complex (APC) is a multi-subunit E3 protein ubiquitin ligase that is responsible for the metaphase to anaphase transition and the exit from mitosis. One of the subunits of the APC that is required for its ubiquitination activity is Doc1/Apc10, a protein composed of a Doc1 homology domain that has been identified in a number of diverse putative E3 ubiquitin ligases. Here, we present the crystal structure of Saccharomyces cerevisiae Doc1/Apc10 at 2.2A resolution. The Doc1 homology domain forms a beta-sandwich structure that is related in architecture to the galactose-binding domain of galactose oxidase, the coagulation factor C2 domain and a domain of XRCC1. Residues that are invariant amongst Doc1/Apc10 sequences, including a temperature-sensitive mitotic arrest mutant, map to a beta-sheet region of the molecule, whose counterpart in galactose oxidase, the coagulation factor C2 domains and XRCC1, mediate bio-molecular interactions. This finding suggests the identification of the functionally important and conserved region of Doc1/Apc10 and, since invariant residues of Doc1/Apc10 colocalise with conserved residues of other Doc1 homology domains, we propose that the Doc1 homology domains perform common ubiquitination functions in the APC and other E3 ubiquitin ligases.     

Disruption of early proximodistal patterning and AVE formation in Apc mutants

In the postimplantation mouse embryo, axial patterning begins with the restriction of expression of a set of genes to the distal visceral endoderm (DVE). This proximodistal (PD) axis is subsequently transformed into an anteroposterior axis as the VE migrates anteriorly to form the anterior visceral endoderm (AVE). Both Nodal and Wnt signaling pathways are involved in these events. We show here that loss of function in the adenomatous polyposis coli gene (Apc) leads to constitutive beta-catenin activity that induces a proximalization of the epiblast with the activation of a subset of posterior mesendodermal genes, and loss of ability to induce the DVE. The loss of some DVE genes such as Hex and goosecoid is rescued in chimeras where only the epiblast was wild type; however, these DVE markers were no longer restricted distally but covered the entire epiblast. Thus, the Apc gene is needed in both embryonic and extraembryonic lineages for normal PD patterning around implantation, suggesting that early restricted activation of the Wnt pathway may be important for initiating axial asymmetries. In addition, we found that nuclear beta-catenin and other molecular markers are asymmetrically expressed by 4.5 days.     

The tumor suppressor Apc controls planar cell polarities central to gut homeostasis

The stem cells (SCs) at the bottom of intestinal crypts tightly contact niche-supporting cells and fuel the extraordinary tissue renewal of intestinal epithelia. Their fate is regulated stochastically by populational asymmetry, yet whether asymmetrical fate as a mode of SC division is relevant and whether the SC niche contains committed progenitors of the specialized cell types are under debate. We demonstrate spindle alignments and planar cell polarities, which form a novel functional unit that, in SCs, can yield daughter cell anisotropic movement away from niche-supporting cells. We propose that this contributes to SC homeostasis. Importantly, we demonstrate that some SC divisions are asymmetric with respect to cell fate and provide data suggesting that, in some SCs, mNumb displays asymmetric segregation. Some of these processes were altered in apparently normal crypts and microadenomas of mice carrying germline Apc mutations, shedding new light on the first stages of progression toward colorectal cancer.     

More than two decades of Apc modeling in rodents

Mutation of tumor suppressor gene adenomatous polyposis coli (APC) is an initiating step in most colon cancers. This review summarizes Apc models in mice and rats, with particular concentration on those most recently developed, phenotypic variation among different models, and genotype/phenotype correlations.     

Analysis of follicle-cell functions in Drosophila: the Fs(3)Apc mutation and the development of chorionic appendages

Fs(3)Apc is a dominant female-sterile mutation of Drosophila melanogaster which causes an incomplete migration of follicle cells between the oocyte and the nurse cells. This leads to leakage of anterior egg cytoplasm followed by degeneration of the egg primordium or deposition of flaccid eggs with reduced anterior egg coverings including the dorsal appendages. Analysis of ovarian and germ-line chimeras revealed that the focus of the Apc phenotype is located in the ovarian soma. Apc+ clones, induced by mitotic recombination, lead to the formation of "exceptional" eggs with (often partial) rescue of the mutant phenotype. Analysis of Apc+ mosaics shows that the Apc mutant phenotype depends on the genotype of the anterior follicle cells. The patterns of apparent Apc+ clones suggest that there is no lineage restriction between the follicle cells that form the anterior egg coverings and those that form the dorsal appendages at the follicle envelope stage.