[CANCER RESEARCH 61, 7934 –7942, November 1, 2001] The Distinct Spectra of Tumor-associated Apc Mutations in Mismatch Repair-deficient Apc 1638N Mice Define the Roles of MSH3 and MSH6 in DNA Repair and Intestinal Tumorigenesis 1 Mari Kuraguchi, Kan Yang, Edmund Wong, Elena Avdievich, Kunhua Fan, Richard D. Kolodner, Martin Lipkin, Anthony M. C. Brown, Raju Kucherlapati, and Winfried Edelmann 2 Strang Cancer Research Laboratory at The Rockefeller University, New York, New York 10021 [M. K., K. Y., K. F., M. L., A. M. C. B.]; Department of Cell Biology and Anatomy, Weill Medical College of Cornell University, New York, New York 10021 [A. M. C. B.]; Departments of Cell Biology [E. W., E. A., W. E.] and Molecular Genetics [R. K.], Albert Einstein College of Medicine, Bronx, New York 10461; and Ludwig Institute for Cancer Research, La Jolla, California 92093 [R. D. K.] ABSTRACT In mammalian cells, mismatch recognition has been attributed to two partially redundant heterodimeric protein complexes of MutS homo- logues, MSH2-MSH3 and MSH2-MSH6. We have conducted a compara- tive analysis of Msh3 and Msh6 deficiency in mouse intestinal tumorigen- esis by generating Apc 1638N mice deficient in Msh3, Msh6 or both. We have found that Apc 1638N mice defective in Msh6 show reduced survival and a 6 –7-fold increase in intestinal tumor multiplicity. In contrast, Msh3-deficient Apc 1638N mice showed no difference in survival and intes- tinal tumor multiplicity as compared with Apc 1638N mice. However, when Msh3 deficiency is combined with Msh6 deficiency (Msh3 / Msh6 / Apc 1638N ), the survival rate of the mice was further reduced compared to Msh6 / Apc 1638N mice because of a high multiplicity of intestinal tumors at a younger age. Almost 90% of the intestinal tumors from both Msh6 / Apc 1638N and Msh3 / Msh6 / Apc 1638N mice contained truncation mu- tations in the wild-type Apc allele. Apc mutations in Msh6 / Apc 1638N mice consisted predominantly of base substitutions (93%) creating stop codons, consistent with a major role for Msh6 in the repair of base-base mis- matches. However, in Msh3 / Msh6 / Apc 1638N tumors, we observed a mixture of base substitutions (46%) and frameshifts (54%), indicating that in Msh6 / Apc 1638N mice frameshift mutations in the Apc gene were suppressed by Msh3. Interestingly, all except one of the Apc mutations detected in mismatch repair-deficient intestinal tumors were located up- stream of the third 20-amino acid -catenin binding repeat and before all of the Ser-Ala-Met-Pro repeats, suggesting that there is selection for loss of multiple domains involved in -catenin regulation. Our analysis there- fore has revealed distinct mutational spectra and clarified the roles of Msh3 and Msh6 in DNA repair and intestinal tumorigenesis. INTRODUCTION DNA MMR 3 is critical for maintaining genomic stability and multiple homologues of the bacterial methyl-directed MMR proteins, MutS and MutL, have been shown to be essential for MMR in humans (1–3). Inherited mutations in MMR genes in humans are directly involved in the etiology of HNPCC, an autosomal dominant disorder that confers predisposition to colonic and other tumors, whereas somatic mutations in MMR genes underlie some sporadic cancers (4 – 8). In human cells, mismatch recognition is attributed to two heterodimeric protein complexes of MutS homologues, MSH2-MSH3 and MSH2-MSH6 (9 –12). Both of these complexes interact with a heterodimer of two MutL homologues, MLH1-PMS2. Previous stud- ies in human cells have indicated that base-base mismatches are preferentially targeted by the MSH2-MSH6 complex. In contrast, MSH2-MSH6 and MSH2-MSH3 appear to be redundant in insertion/ deletion loop repair with possibly some specificity of the latter com- plex for larger (4 –5 bases) insertion/deletion loops (10, 11, 13–17). Because loss of MSH2 inactivates the activity of both mismatch recognition heterodimers, it is understandable that MSH2 deficiency causes strong cancer predisposition in both mice (18 –20) and humans (21, 22). Loss of MSH3 or MSH6 function alone causes a partial MMR defect, consistent with their roles in MMR (15, 23). This may explain the rarity of MSH6 and the absence of MSH3 germ-line mutations in typical HNPCC families (24 –26). Germ-line mutations in the tumor suppressor gene APC lead to familial adenomatous polyposis, another autosomal dominant syn- drome that imparts predisposition to colorectal cancer (27, 28). In familial adenomatous polyposis patients, mutation or loss of the wild-type APC allele is considered a rate-limiting step in tumor initiation. APC is also mutated in the majority of sporadic cases of colorectal cancers (4) and in a subset of HNPCC-derived tumors (29, 30). Wild-type APC encodes a 2843-amino acid protein, one of the normal functions of which is to facilitate the destabilization of - catenin, a protein involved in both cell adhesion and signal transduc- tion (31, 32). APC acts as a component of the Axin or Conductin complex, which targets -catenin for degradation by the proteasome pathway (33, 34). The mechanism by which APC functions in this process is poorly understood, but the activity is localized to the central region of APC. This region contains a series of seven 20-amino acid -catenin binding repeats (35) and three SAMP repeats, which are binding sites for Axin (or Conductin; Refs. 33, 34). In addition, recent reports suggest that APC also contains highly conserved NESs in this region, and that it shuttles -catenin from the nucleus and cytoplasm to a junctional compartment where the axin complex may be anchored (36, 37). Nearly all of the tumor-associated mutations in APC occur within the first 1500 codons, and approximately two-thirds of these somatic mutations are confined to a MCR located between codons 1286 and 1513 (38, 39). These tumor-associated mutations give rise to truncated APC proteins that retain one or two of the 20-amino acid repeats in addition to all three of the more NH 2 -terminal 15-amino acid -catenin binding sites. These truncated APC products are unable to down-regulate -catenin (31, 35). The inability to regulate -cate- nin concentrations in the cells results in excessive levels of cytosolic -catenin and entry of this protein into the nucleus, where it acts as a transcriptional coactivator of the DNA binding protein Tcf-4 (40 – 42). To clarify the role of the MMR proteins in vivo, we previously developed a series of mouse lines, each carrying an inactivating mutation in a different MMR gene. Mice carrying an Msh6 null mutation have a cancer predisposition phenotype, associated with a significantly reduced life span (43). Msh3 -/- mice develop tumors Received 6/11/01; accepted 8/27/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by NIH Grants CA76329 (to W. E.), CA84301 (to R. K. and W. E.), CA29502 and CA47207 (to A. M. C. B.), CA67944 and N01-CN-65031 (to M. L. and R. K.), GM50006 (to R. D. K.), and Center Grant CA13330 to Albert Einstein College of Medicine; the AACR-Cancer Research Foundation of America Fellowship in Prevention Research (to M. K.); and an Irma T. Hirschl Career Scientist Award (to A. M. C. B.). 2 To whom requests for reprints should be addressed, at Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: (718) 430-2030; Fax: (718) 430-8574; E-mail: edelmann@aecom.yu.edu. 3 The abbreviations used are: MMR, mismatch repair; APC, adenomatous polyposis coli; HNPCC, hereditary nonpolyposis colorectal cancer; SAMP, Ser-Ala-Met-Pro; NES, nuclear export signal; MCR, mutation cluster region; GI, gastrointestinal; IVTT, in vitro transcription and translation. 7934 Research. on March 7, 2016. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from