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Complete Deletion of Apc Results in Severe Polyposis in Mice

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Complete Deletion of Apc Results in Severe Polyposis in Mice Oncogene (2010) 29, 1857–1864 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc SHORT COMMUNICATION Complete deletion of Apc results in severe polyposis in mice AF Cheung1, AM Carter1, KK Kostova1, JF Woodruff1, D Crowley1,2, RT Bronson3, KM Haigis4 and T Jacks1,2 1Koch Institute and Department of Biology, MIT, Cambridge, MA, USA; 2Howard Hughes Medical Institute, MIT, Cambridge, MA, USA; 3Department of Pathology, Tufts University School of Medicine and Veterinary Medicine, Boston, MA, USA and 4Masschusetts General Hospital Cancer Center, Harvard Medical School Department of Pathology, Charlestown, MA, USA The adenomatous polyposis coli (APC) gene product is region of APC termed the mutation cluster region and mutated in the vast majority of human colorectal cancers. result in retained expression of an N-terminal fragment APC negatively regulates the WNT pathway by aiding in of the APC protein (Kinzler and Vogelstein, 1996). the degradation of b-catenin, which is the transcription Genotype–phenotype correlations involving germline factor activated downstream of WNT signaling. APC APC mutations suggest that different lengths and levels mutations result in b-catenin stabilization and constitutive of APC expression can influence the number of polyps WNT pathway activation, leading to aberrant cellular in the gut, the distribution of polyps and extra-colonic proliferation. APC mutations associated with colorectal manifestations of the disease (Soravia et al., 1998; cancer commonly fall in a region of the gene termed the Nieuwenhuis and Vasen, 2007). Specifically, patients mutation cluster region and result in expression of an that present clinically with attenuated FAP have N-terminal fragment of the APC protein. Biochemical and reduced polyp counts and a later age of colon cancer molecular studies have revealed localization of APC/Apc to development. Interestingly, germline APC mutations different sub-cellular compartments and various proteins associated with attenuated FAP fall 50 and 30 of the outside of the WNT pathway that associate with truncated mutation cluster region, presumably resulting in APC APC/Apc. These observations and genotype–phenotype truncations bearing different protein domains than in correlations have led to the suggestion that truncated classical FAP and/or hypomorphic APC expression. APC bears neomorphic and/or dominant-negative function These observations suggest a gain-of-function or domi- that support tumor development. To analyze this possibility, nant-negative role for APC truncated at the mutation we have generated a novel allele of Apc in the mouse that cluster region. Paradoxically, patients with whole-gene yields complete loss of Apc protein. Our studies reveal that APC deletions, although rare, have been found to present whole-gene deletion of Apc results in more rapid tumor with classical FAP (Herrera et al., 1986; Sieber et al., development than the APC multiple intestinal neoplasia 2002). More thorough analysis of the phenotypic con- (ApcMin) truncation. Furthermore, we found that adenomas sequences of different heritable APC alleles is hampered by bearing truncated Apc had increased b-catenin activity when the low frequency of FAP and attenuated FAP as well as compared with tumors lacking Apc protein, which could the existence of numerous genetic and environmental lead to context-dependent inhibition of tumorigenesis. modifiers of the disease (de la Chapelle, 2004). Thus, we Oncogene (2010) 29, 1857–1864; doi:10.1038/onc.2009.457; and others have turned to mouse models that faithfully published online 14 December 2009 recapitulate human disease as a tool for examining the consequences of various Apc mutations. Keywords: Apc truncation; Apc null; colon cancer; The APC multiple intestinal neoplasia (ApcMin) was mouse model the first heritable mutant allele of Apc developed in the mouse (Su et al., 1992). ApcMin mice develop numerous intestinal lesions resembling human FAP. The ApcMin Colorectal cancer is one of the leading causes of cancer allele, which was discovered in an ethylnitrosourea death in the Western world. More than 80% of mutagenesis screen, comprises a nonsense mutation in colorectal cancers have mutations in the adenomatous codon 850 of Apc and gives rise to a stable, truncated polyposis coli (APC) gene, which is known to occur early Apc protein similar to those found in human colon in tumor development. Most of these APC gene cancers. Since the characterization of the ApcMin mouse, alterations, which are found both in the germline in additional alleles of Apc have been generated, involving individuals with hereditary familial adenomatous poly- truncations both 50 and 30 to the Min mutation (Oshima posis (FAP) and in sporadic colon cancers, fall in a et al., 1995; Smits et al., 1999; Colnot et al., 2004; Gaspar et al., 2009; Pollard et al., 2009). As observed Correspondence: Dr T Jacks, Koch Institute and Department of in human patients bearing mutations in APC, these Biology, MIT, 77 Massachusetts Avenue, E17-517, Cambridge, alleles have yielded different phenotypic consequences MA, USA. relating to intestinal tumorigenesis (Fodde and Smits, E-mail: [email protected] 2001). The basis for these genotype–phenotype correla- Received 21 August 2009; revised 9 November 2009; accepted 12 November 2009; published online 14 December 2009 tions remains largely unknown. Null Apc yields more severe polyposis than ApcMin AF Cheung et al 1858 The APC/Apc gene product is a critical negative ing novel, conditional and constitutive null alleles of Apc regulator of the canonical Wnt signaling pathway. Apc in mice. We performed sequential gene targetings in forms a complex with Axin and glycogen synthase C57BL/6 embryonic stem cells, inserting LoxP sites kinase-3b that phosphorylates b-catenin on N-terminal 90 kb apart in the Apc locus flanking all 15 protein- residues, allowing for subsequent ubiquitination and coding exons of Apc to generate a Cre-regulatable null proteosomal degradation. When the ability of Apc to allele, Apcfle1–15 (Figure 1a). We then tested our allele by interact with this destruction complex is compromised, crossing Apcfle1–15 mice to Villin-Cre transgenic mice, b-catenin accumulates in the cytoplasm and translocates which express Cre in a mosaic manner throughout the into the nucleus in which it binds to Tcf/Lef transcrip- intestinal epithelium (el Marjou et al., 2004). Apc þ /fle1–15; tion factors to regulate target genes, such as c-myc and Villin-Cre mice developed several lesions in their small Axin2 (Clevers, 2006). Moreover, mutations in b-catenin intestines and colons by 4–5 months of age, whereas that lead to protein stabilization are observed in human Apc þ /fle1–15 mice without Cre never did, even when aged colon cancer and comparable mutations can initiate for >1 year (Figures 1b, d and f; data not shown). An intestinal tumorigenesis in mice (Morin et al., 1997; existing conditional Apc allele developed by Colnot et al. Harada et al., 1999). (2004), Apcfle14, encodes a truncated Apc protein after Beyond b-catenin deregulation, mutant APC has been deletion of exon 14 by exposure to Cre recombinase suggested to contribute to colon tumorigenesis through activity. By histological examination of tumor morphol- other mechanisms. First, the vast majority of sporadic ogy, grade and stromal recruitment, we found that colon cancers in humans have mutations in APC, adenomas from Apc þ /fle1–15;Villin-Cre mice closely re- whereas other cancers with deregulated WNT signaling sembled those found in Apc þ /fle14;Villin-Cre littermates have an equal or greater proportion of mutations in (Figures 1b–g). These data show that conditional b-Catenin or other WNT pathway components (Giles, deletion of Apc grossly phenocopies conditional trunca- 2003). This observation points to a special role for APC tion of Apc. mutation specifically in colon cancer. Second, Apc is a Next, we crossed Apc þ /fle1–15 mice to Meox-Cre relatively large protein of approximately 312 kDa and transgenic animals, which express Cre in the embryo, has a number of protein-binding motifs. In fact, Apc has for generating a heritable null allele of Apc, ApcDe1–15 been shown to interact with a variety of proteins outside (Tallquist and Soriano, 2000; Figure 1a). To confirm the of the Wnt pathway and has been found in various sub- loss of Apc expression upon genomic deletion of the cellular locations (Fodde et al., 2001; Aoki and Taketo, locus, we performed quantitative PCR to detect Apc 2007; Mccartney and Nathke, 2008). Furthermore, the message with Taqman probes spanning exons 9 and 10 majority of APC mutations result in a stable, truncated and 13 and 14. Analysis of complementary DNA polypeptide, lacking most b-catenin and Axin-binding generated from whole colons of 1-month-old animals domains that lie in the middle portion of the protein. In revealed the expected 50% reduction of Apc mRNA these mutant proteins, the microtubule-binding domain in Apc þ /De1–15 mice when compared with a wild-type and EB1-binding site at the C-terminus of APC are also littermate (Figure 1h). Apc þ /De1–15 mice were aged to lost, but the N-terminal armadillo repeats and oligo- allow for spontaneous tumor formation. Histological merization domains are retained (Nathke, 2004). comparison revealed no significant phenotypic differ- We took a genetic approach to dissect the role of ence between tumors derived from Apc þ /De1–15 and truncated Apc in intestinal tumorigenesis by construct- Apc þ /Min mice on a C57BL/6 background (data not Figure 1 Generation of Apcfle1–15 and ApcDe1–15 alleles. (a) Schematic of the wild-type Apc genomic locus showing non-coding exon 0 and coding exons 1, 14 and 15. A floxed Puromycin resistance (PuroR) gene was targeted to intron 1 of Apc in C57BL/6 embryonic stem (ES) cells. ES cells were selected on Puromycin and screened by Southern blot for homologous recombination. Targeted clones were then transfected with a Cre expression plasmid to excise the PuroR cassette leaving behind a single loxP site.
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