Developmental abnormalities in multiple proliferative tissues of Apc(Min/+) mice

Germ-line mutation of the Apc gene has been linked to familial adenomatous polyposis (FAP) that predisposes to colon cancer. Apc(Min/+) mice, heterozygous for the Apc gene mutation, progressively develop small intestinal tumours in a manner that is analogous to that observed in the colon of patients with FAP (Su et al. 1992; Fodde et al. 1994; Moser et al. 1995). We have studied the effects of Apc gene mutation on murine intestinal and extra-intestinal, proliferatively active tissues. We have contrasted the histology to that of the age- and sex-matched wild-type C57BL/6 mice. Histological assessment of the normal appearing intestinal mucosa demonstrates minimal change in size of crypts. In contrast, villi are longer in the ileum of Apc(Min/+) mice relative to C57BL/6 mice at 12 and 15 weeks of age. Vigorous splenic haematopoiesis in Apc(Min/+) mice was seen at 12 and 15 weeks of age, as reflected by marked splenomegaly, increased splenic haematopoietic cells and megakaryocytes. Peripheral blood counts, however, did not differ between C57BL/6 and Apc(Min/+) mice at 15 weeks of age. Lymphoid depletion in Apc(Min/+) mice was characterized by diminished numbers of splenic lymphoid follicles and small intestinal Peyer's patches. The ovaries of 12- and 15-week-old Apc(Min/+) mice exhibited increased numbers of atretic follicles, and estrous cycling by serial vaginal smears showed tendency of elongation in the mutant mice during these age ranges. The testicles of 10-week-old Apc(Min/+) mice showed increased numbers of underdeveloped seminiferous tubules. Collectively, these data suggest that, in addition to its obvious effects upon intestinal adenoma formation, Apc gene mutation causes impairment of developmental and apparent differentiation blockade in proliferative tissues, including those of the haematopoietic system, lymphoid and reproductive tract.     

Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers

Murine models of familial adenomatous polyposis harbor a germinal heterozygous mutation on Apc tumor suppressor gene. They are valuable tools for studying intestinal carcinogenesis, as most human sporadic cancers contain inactivating mutations of APC. However, Apc(+/-) mice, such as the well-characterized Apc(Min/+) model, develop cancers principally in the small intestine, while humans develop mainly colorectal cancers. We used a Cre-loxP strategy to achieve a new model of germline Apc invalidation in which exon 14 is deleted. We compared the phenotype of these Apc(Delta14/+) mice to that of the classical Apc(Min/+). The main phenotypic difference is the shift of the tumors in the distal colon and rectum, often associated with a rectal prolapse. Thus, the severity of the colorectal phenotype is partly due to the particular mutation Delta14, but also to environmental parameters, as mice raised in conventional conditions developed more colon cancers than those raised in pathogen-free conditions. All lesions, including early lesions, revealed Apc LOH and loss of Apc gene expression. They accumulated beta-catenin, overexpressed the beta-catenin target genes cyclin D1 and c-Myc, and the distribution pattern of glutamine synthetase, a beta-catenin target gene recently identified in the liver, was mosaic in intestinal adenomas. The Apc(Delta14/+) model is thus a useful new tool for studies on the molecular mechanisms of colorectal tumorigenesis.     

Matrix metalloproteinase‐9 contributes to intestinal tumourigenesis in the adenomatous polyposis coli multiple intestinal neoplasia mouse

Matrix metalloproteinases (MMPs) are a family of 23 extracellular proteases that are best known for their collective ability to degrade all components of the extracellular matrix. We previously demonstrated that genetic ablation of MMP‐7 reduced tumour multiplicity in multiple intestinal neoplasia (Min) mice possessing a genetic alteration in the adenomatous polyposis coli gene (APC). These mice, commonly referred to as APC‐Min mice, are a frequently used model of early intestinal tumourigenesis. To examine further the role of MMPs in intestinal tumour development, we generated APC‐Min mice genetically deficient in MMP‐2, ‐9, ‐12 or ‐19. Genetic ablation of MMP‐2, ‐12 or ‐19 did not affect multiplicity or size of intestinal tumours when crossed into the APC‐Min system. However, MMP‐9 deficient animals developed 40% fewer tumours than littermate controls, although tumour size distribution remained unaffected. Intestinal adenomas from MMP‐9 deficient mice demonstrated a 50% decrease in proliferating cells compared with control tissues, with no difference in apoptosis. To determine the cellular origin of MMP‐9 in these tumours, immunofluorescent co‐staining with markers for different leucocyte lineages was used to demonstrate that intratumoural MMP‐9 is largely a product of neutrophils. These studies extend the potential targets for chemoprevention of intestinal adenomas to MMP‐9 in addition to MMP‐7 and exclude MMP‐2,‐12,‐19 as attractive targets for intervention.     

Apc mice: models, modifiers and mutants

The mouse provides an excellent in vivo system with which to model human diseases and to test therapies. Mutations in the Adenomatous polyposis coli (APC) gene are required to initiate familial adenomatous polyposis (FAP) and are also important in sporadic colorectal cancer tumorigenesis. The (multiple intestinal neoplasia Min) mouse contains a point mutation in the Apc gene, develops numerous adenomas and was the first model used to study the involvement of the Apc gene in intestinal tumorigenesis. The model has provided examples of modifying loci (called Modifiers of Min: Mom) in mice, demonstrating the principle of genetic modulation of disease severity. A spectrum of Apc mutant mice has since been developed, each with defining characteristics, some more able to accurately model human polyposis and colon cancer. We will focus our review on Apc mutant mouse models, the advent of models with concurrent or compound mutations and the importance of genetic background when modeling polyposis and cancer. Brief consideration will be given to the use of these models in drug testing.     

Intestinal epithelial cell-specific CD98 expression regulates tumorigenesis in Apc(Min/+) mice

The transmembrane glycoprotein CD98 regulates integrin signaling that in turn controls cell proliferation and survival. CD98 expression is upregulated in various carcinomas, including colorectal cancer. Recently, by generating gain- and loss-of-function mouse models featuring genetic manipulation of CD98 expression specifically in intestinal epithelial cells (IECs), we have explored the crucial role of CD98 in the regulation of intestinal homeostasis and inflammation-associated tumorigenesis. In the present study, we investigated the contribution of CD98 to intestinal tumorigenesis in Apc(Min/+) mice and the underlying mechanism of action. Mice featuring IEC-specific CD98 overexpression (Tg animals) were crossed with Apc(Min/+) mice, and the characteristics of intestinal adenoma formation were assessed. Compared with Apc(Min/+) mice, Tg/Apc(Min/+) animals exhibited increases in both intestinal tumor incidence and tumor size; these parameters correlated with enhanced proliferation and decreased apoptosis of IECs. IEC-specific CD98 overexpression resulted in increased synthesis of the oncogenic proteins c-myc and cyclin-D1 in Apc(Min/+) mice, independently of the Wnt-APC-β-catenin pathway, suggesting the implication of CD98 overexpression-mediated Erk activation. IEC-specific CD98 overexpression enhanced the production of proinflammatory cytokines and chemokines that are crucial for tumorigenesis. We validated our results in mice exhibiting IEC-specific CD98 downregulation (CD98(flox/+)VillinCre animals). IEC-specific CD98 downregulation efficiently attenuated tumor incidence and growth in Apc(Min/+) mice. The reduction of intestinal tumorigenesis upon IEC-specific CD98 downregulation was caused by the attenuation of IEC proliferation and cytokine/chemokine production. In conclusion, we show that CD98 exerts an oncogenic activity in terms of intestinal tumorigenesis, via an ability to regulate tumor growth and survival.     

Adenomatous polyposis coli is essential for both neuronal differentiation and maintenance of adult neural stem cells in subventricular zone and hippocampus

The tumor suppressor adenomatous polyposis coli (APC) is a multifunctional protein that not only inhibits the Wnt signaling pathway by promoting the degradation of β-catenin but also controls cell polarity, motility, and division. APC is abundantly expressed in the adult central nervous system, but its role in adult neurogenesis remains unknown. Using conditional deletion (or knockout) of APC (APC-CKO) from glial fibrillary acidic protein (GFAP)-expressing cells including adult neural stem cells (NSCs) in the subventricular zone and hippocampal dentate gyrus, we show that APC expression by these cells is a critical component of adult neurogenesis. Loss of APC function resulted in a marked reduction of GFAP-expressing NSC-derived new neurons, leading to the decreased volume of olfactory granule cell layer. Two distinct mechanisms account for impaired neurogenesis in APC-CKO mice. First, APC was highly expressed in migrating neuroblasts and APC deletion disturbed the differentiation from Mash1-expressing transient amplifying cells to neuroblasts with concomitant accumulation of β-catenin. As a result, migrating neuroblasts decreased, whereas Mash1-expressing dividing cells reciprocally increased in the olfactory bulb of APC-CKO mice. Second, APC deletion promoted an exhaustion of the adult germinal zone. Functional NSCs and their progeny progressively depleted with age. These findings demonstrate that APC expression plays a key role in regulating intracellular β-catenin level and neuronal differentiation of newly generated cells, as well as maintaining NSCs in the adult neurogenic niche. STEM CELLS 2010;28:2053-2064.     

Intestinal tumours induced by the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in multiple intestinal neoplasia mice have truncation mutations as well as loss of the wild-type Apc(+) allele.

C57BL/6J-Min/+ (multiple intestinal neoplasia) is a murine model for familial adenomatous polyposis (FAP), where the mice are heterozygous for a nonsense Apc(Min) (adenomatous polyposis coli) mutation, and therefore develop numerous spontaneous adenomas in the small intestine and colon. Neonatal exposure of Min/+ mice to the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (eight subcutaneous injections of 25 or 50 mg/kg PhIP to pups or 50 mg/kg PhIP to lactating dams) markedly increased (2--9-fold) the number of intestinal tumours, especially in the small intestine. We examined whether the Apc gene was affected in small intestinal and colonic tumours induced by PhIP. In spontaneous tumours formed in these mice, the main mechanism for tumour induction is loss of the wild-type Apc(+) allele, i.e. loss of heterozygosity (LOH). Also in the PhIP-induced tumours, this is a major mechanism, since large fractions of PhIP-induced tumours had LOH in APC: However, mechanisms other than LOH must also prevail, since a lower frequency of LOH was found in the small intestinal tumours from male mice exposed to PhIP either via breast milk (65%) or by direct injection (68%), compared with the untreated controls (92%). Tumours that had retained the wild-type Apc(+) allele were further analysed for presence of truncated Apc proteins with in vitro synthesized protein (IVSP) assay. Truncated Apc proteins, indicating truncation mutations in exon 15 of the Apc gene, were detected in 20% (8 of 40) of the tumours not showing LOH from the small intestine after PhIP exposure, all in segment 2 (codons 686--1217). Seventeen percent (2 of 12) of the colonic tumours had a truncated Apc protein in segment 3 (codons 1099--1693). Importantly, no truncated proteins were detected in tumours from unexposed mice with apparently retained wild-type Apc(+) allele. These results show that PhIP induces intestinal tumours in the Min/+ mice both by causing LOH and truncation mutations in the wild-type Apc(+) allele.     

Upregulation of colonic ion channels in APC Min/+ mice

The adenomatous polyposis coli (APC) tumor suppressor gene is mutated in almost all human colonic cancers. Disturbances in Na⁺ absorption have been observed in colonic cancer, and ion channels such as ether a go-go (Eag) or Ca²⁺-sensitive BK channels have been recognized for their oncogenic potential. APC ^Min/+ mice have reduced APC expression and develop multiple intestinal neoplasias (Min). Ion channels in the colonic epithelium were examined using electrophysiology and molecular techniques. APC ^Min/+ mice developed intestinal neoplasia and experienced a significant weight loss. Due to intestinal bleedings, the hematocrit was largely reduced and plasma aldosterone levels were enhanced. Rectal potential measurements in vivo indicated an increase in amiloride-sensitive Na⁺ absorption in APC ^Min/+ mice. Quantitative Ussing chamber studies demonstrated enhanced Na⁺ absorption via epithelial Na⁺ channels (ENaC) and suggested enhanced activity of oncogenic BK and Eag-1 channels. Patch clamp and fluorescence measurements on isolated crypts suggested enhanced K⁺ channel activity in the surface epithelium. ENaC-mRNA and membrane protein expression was enhanced in colonic surface epithelial cells. The data suggest that reduced expression of the APC gene with upregulation of the downstream proteins Akt and mTOR and subsequent hyperaldosteronism is paralleled by upregulation of oncogenic potassium channels and enhanced colonic Na⁺ absorption. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Helicobacter hepaticus infection promotes colon tumorigenesis in the BALB/c-Rag2(-/-) Apc(Min/+) mouse

Adenomatous polyposis coli (APC) mutations are linked to human and mouse colorectal cancers. The Apc multiple intestinal neoplasia (Min) mouse mutation causes adenomas to develop throughout the small and large intestines. The BALB-Min (C.B6-Apc(Min/+)) congenic strain was generated by backcrossing into BALB/c the Apc(Min) allele from C57BL/6J-Apc(Min/+) mice. BALB-Min mice have a low tumor multiplicity (27.4 small intestine tumors/mouse) and a relatively long life span (>1 year) that makes them amenable to long-term studies. To investigate the interplay of the adaptive immune system and intestinal tumorigenesis, the immunodeficient compound mutant strain BALB-RagMin (C.Cg-Rag2(-/-) Apc(Min/+)) was generated. BALB-RagMin mice had a significant increase in tumors in the small, but not large, intestine relative to their BALB-Min counterparts (43.0 versus 24.0 tumors/mouse, respectively). The results suggest that the adaptive immune system plays a role in either the elimination or the equilibrium phase of cancer immunoediting in the small intestine in this model. We investigated the effect of the enterohepatic bacterial pathogen Helicobacter hepaticus on liver and intestine tumorigenesis in BALB-RagMin mice. H. hepaticus-infected BALB-RagMin mice developed moderate hepatitis, moderate typhlitis, and mild colitis. There were no differences in small intestine and cecal tumor multiplicity, regionality, or size relative to that in uninfected mice. However, H. hepaticus-infected BALB-RagMin mice had a significant increase in colon tumor incidence relative to uninfected BALB-RagMin mice (23.5% versus 1.7%, respectively). The data suggest that H. hepaticus, which is present in many research colonies, promotes colon tumorigenesis in the BALB-RagMin mouse and that it has the potential to confound colon tumorigenesis studies.     

Axin, an inhibitor of the Wnt signalling pathway, interacts with β-catenin, GSK-3β and APC and reduces the β-catenin level

Background:

The Wnt/Wingless signalling pathway plays an important role in both embryonic development and tumorigenesis. β-Catenin and Axin are positive and negative effectors of the Wnt signalling pathway, respectively.

Results:

We found that Axin interacts with β-catenin and glycogen synthase kinase-3β (GSK-3β). Furthermore, the regulation of the G-protein signalling (RGS) domain of Axin is associated with the colorectal tumour suppressor adenomatous polyposis coli (APC). Overexpression of Axin in the human colorectal cancer cell line SW480 induced a drastic reduction in the level of β-catenin. Interaction with β-catenin and GSK-3β was required for the Axin-mediated β-catenin reduction.

Conclusion:

Axin interacts with β-catenin, GSK-3β and APC, and negatively regulates the Wnt signalling pathway, presumably by regulating the level of β-catenin.

     

The development of duodenal microadenomas in FAP patients: the human correlate of the Min mouse

The morphological changes associated with the adenoma-carcinoma sequence are well documented in the colorectum. Small intestinal carcinogenesis is thought to progress through a similar adenoma-to-carcinoma pathway, but there is a relative dearth of studies examining the associated morphological changes. The best-known mouse model of intestinal neoplasia, the multiple intestinal neoplasia (Min) mouse, has been criticized as a genetic model of intestinal neoplasia, as the majority of its tumours occur in the small intestine. We examined pancreatico-duodenal resection specimens from seven familial adenomatous polyposis (FAP) patients. Serial sections of these were stained with haematoxylin and eosin for beta-catenin and its downstream target CD44, for BMPR1a, lysozyme, carbonic anhydrase II, and with MIB-1. Individual dysplastic crypts were isolated and mutations in the FAP (APC) gene compared between the top and bottom of the crypt. We found that: (a) duodenal microadenomas are extremely common in FAP patients; (b) these grow in the core of duodenal villi, forming lesions similar to those described in the Min mouse; (c) many lesions arise as monocryptal adenomas and grow by a process of crypt fission and branching; (d) migrating adenomatous cells lose their dysplastic phenotype as they migrate up the crypt villous axis; and (e) Paneth cells lose positional information. In conclusion: (a) the morphological similarity of adenomas in the Min mouse and human suggest the Min mouse is a good model of FAP; (b) duodenal adenomas in FAP originate in monocryptal adenomas and follow the 'bottom-up' rather than the 'top-down' model of morphogenesis; (c) early microadenomas show evidence of cellular differentiation; (d) defects in the positioning of Paneth cells suggests disruption of the EphB2:EphB3 receptor system.     

Expansion of a mutated clone: from stem cell to tumour

Intestinal stem cells are adult, tissue-based stem cells located at the base of the intestinal crypt and are capable of regenerating all intestinal cell types. The progeny of mutated stem cells can expand to fill an entire crypt as a consequence of genetic drift, selective advantage or hitchhiking-eventually forming a clonal crypt population by a process called "niche succession". Cancer is believed to be a disease of stem cells. The digestive tract has a very high cancer prevalence partly due to rapid epithelial cell turnover and exposure to dietary toxins. Work on the hereditary cancer syndromes, including familial adenomatous polyposis (FAP), has led to significant advances, including the adenoma-carcinoma sequence. The initial mutation involved in this stepwise progression is in the "gatekeeper" tumour suppressor gene adenomatous polyposis coli (APC). In FAP somatic, second hits in this gene are non-random events, selected for by the position of the germline mutation. The early growth of adenomas is contentious, with two main theories, the "top-down" and "bottom-up" hypotheses, attempting to explain the spread of dysplastic tissue in the bowel. Initial X chromosome inactivation studies suggested that colorectal tumours were monoclonal; however, work on a rare XO/XY human patient with FAP and chimeric Min mice showed that approximately 76% of adenomas were polyclonal. A reduction in tumour multiplicity in the chimeric mouse model has been achieved by the introduction of a homozygous tumour resistance allele. This model has been used to suggest that short-range interaction between adjacent initiated crypts, not random polyp collision, is responsible for tumour polyclonality.     

Adenomatous polyposis coli (APC) plays multiple roles in the intestinal and colorectal epithelia

The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in most sporadic colorectal tumors. During both embryonic and postnatal periods, APC is widely expressed in a variety of tissues, including the brain and gastrointestinal tract. The APC gene product (APC) is a large multidomain protein consisting of 2843 amino acids. APC downregulates the Wnt signaling pathway through its binding to β-catenin and Axin. Most mutated APC proteins in colorectal tumors lack the β-catenin-binding regions and fail to inhibit Wnt signaling, leading to the overproliferation of tumor cells. Several mouse models (APC ^580D , APC ^Δ716 , APC ¹³⁰⁹ , APC ^Min , APC ^1638T ) have been established to investigate carcinogenesis caused by APC mutations. APC also binds to APC-stimulated guanine nucleotide exchange factor, the kinesin superfamily-associated protein 3, IQGAP1, microtubules, EB1, and discs large (DLG). APC has both nuclear localization signals and nuclear export signals in its molecule, suggesting its occasional nuclear localization and export of β-catenin from the nucleus. APC is highly expressed in the intestinal and colorectal epithelia and may be involved in homeostasis of the enterocyte renewal phenomena, in which proliferation, migration, differentiation, and apoptosis are highly regulated both temporally and spatially. Through the many binding proteins mentioned, APC can exert multiple functions involved in epithelial homeostasis.     

Identification of the modifier of Min 2 (Mom2) locus, a new mutation that influences Apc-induced intestinal neoplasia.

Min (Multiple intestinal neoplasia) mice carry a dominant mutation in the adenomatous polyposis coli (Apc) gene and develop multiple adenomas throughout their intestinal tract (Moser et al. 1990; Su et al 1992). Polyp multiplicity in Min mice is greatly influenced by genetic background. A modifier locus, Mom1 (Modifier of Min 1), was identified and localized to distal mouse chromosome 4 (Moser et al. 1992; Dietrich et al. 1993), and accounts for some of the genetic variance in polyp multiplicity. Mom1 is a semidominant modifier of polyp size and multiplicity in Min mice (Gould and Dove 1997), and encodes the secretory type II nonpancreatic phospholipase A2 (Pla2g2a) gene (MacPhee et al. 1995; Cornier et al. 1997, 2000). We now report the identification of a second Modifier of Min 2 (Mom2) locus that is the result of a spontaneous mutation. One resistant Mom2 allele can suppress 88%-95% of polyps detected in Apc(Min)/+ mice, indicating that Mom2 acts in a dominant fashion. Linkage analysis has localized Mom2 to distal mouse chromosome 18. The effects of the Mom2 locus on reducing polyp multiplicity are stronger than the effects of the Mom1 locus, in both the small and large intestines. Some Apc(Min)/+ mice that carried one resistant Mom2 allele were tumor-free at 21 weeks of age, even in the absence of a resistant Mom1 allele. Thus, the resistant Mom2 allele can, in some cases, completely suppress the penetrance of the Apc(Min) mutation.     

Hypermethylation of APC promoter 1A is associated with moderate activation of Wnt signalling pathway in a subset of colorectal serrated adenomas

Aims:  The role of Wnt signalling pathway in serrated adenomas (SAs) remains to be identified. The aim of this study was to determine whether Wnt signalling plays a role in the pathogenesis of SAs, and to clarify the mechanism of Wnt signalling activation in SAs.
Methods and results:  This study investigated immunoreactivities of adenomatous polyposis coli (APC) and β-catenin, mutations of APC and β-catenin genes, methylation status of APC promoter 1A in 12 SAs, and compared the findings with normal colorectal mucosa, hyperplastic polyps, traditional adenomas (TAs) and colorectal cancers (CRCs). APC expression was moderately decreased in SAs. Cytoplasmic accumulation of β-catenin was demonstrated in 41.7% (5/12) of SAs, but membranous immunoreactivity of β-catenin was lost in only 8.3% (1/12) of SAs. No β-catenin mutation was detected in any of 12 SAs, and only one SA was found to be positive for APC gene mutation. Complete methylation of APC promoter 1A was found in 41.7% (5/12) of SAs, but in no TAs or CRCs.
Conclusions:  Hypermethylation of APC promoter 1A, instead of mutations involving APC and β-catenin , contributes to moderate activation of Wnt signalling in a subset of SAs.     

APC mutation and the crypt cycle in murine and human intestine.

Dysplastic colon adenomas are thought to arise from growth of clones of APC -/- colonic epithelial cells. Isolated clusters of dysplastic crypts are often observed in patients with familial adenomatous polyposis. These patients have genotype APC +/-, and the clusters of dysplastic crypts (called microadenoma or aberrant crypt foci) are thought to represent an early stage in the expansion of a mutant clone of APC -/- cells. It is thought that the growth of these clusters of mutant crypts results from crypt replication through a process similar to what occurs in the normal crypt cycle. We measured the relative replication rate of mutant crypts by analyzing the size of clusters of mutant crypts in APC +/- individuals and found that mutant APC -/- crypts replicate more rapidly than do normal APC +/- (i.e., nonneoplastic) crypts. In contrast, the replication rate of mutant crypts in Apc +/- mice is not significantly different from that of normal crypts, thus supporting previous findings that aberrant crypt foci do not contribute significantly to the colon adenoma population in adult Apc +/- mice. Intriguingly, we found an effect of Apc heterozygosity on the frequency of branching crypts in young mice.