Familial Adenomatous Polyposis

FAP is a rare hereditary syndrome characterized by development of hundreds of colonic polyps during late teens or early twenties of the affected individual. Some of the polyps inevitably transform into colonic carcinomas. Almost all FAP is caused by a mutation in the adenomatous polyposis coli ( APC ) gene. Less frequently, mutations in the base excision repair gene, MYH , can cause an attenuated form of FAP. To better mimic common mutations found in FAP patients, several mouse models of Apc mutation have been constructed using gene knockout methods other than Apc ^min . Apc ^▵716 contains a truncating mutation at codon 716, and Apc ^1638N contains a truncating mutation at codon 1638. All three mutations produced different polyp burden in the small intestine on the same background; Apc ^min producing the most polyps of 100 or more and Apc ¹⁶³⁸ the least number at approximately 3. The polyps were phenotypically indistinguishable from one another in all three mutant models. The advantage of the Apc ^1638N model for FAP is that with a lower polyp burden, these animals have an increased life span and develop more advanced tumors useful for studying tumor progression and metastasis. They are also useful for looking at positive synergy with mutations in other candidate tumor suppressors, whereas Apc ^Min are better for testing chemopreventive drugs (eg, assaying for reduced adenoma burden). A confounding finding in the Apc deficient mice is that adenomas form in the small intestine instead of colon as seen more commonly in FAP patients. However, Apc mouse models have shed much light on the role of Wnt pathway in the initiation, development, and progression of CRCs. Furthermore, these mouse models have been useful in studying the effect of modifiers of Apc mutations and activated WNT signaling, as well as studying the impact of diet in the process of CRC development. Several modifiers of min-1 have been identified in mice which have provided insight into their epistatic interaction with APC . Other mutations that affect the formation of intestinal adenomas in Apc ^min mice are genes in archidonic acid pathway. One of these genes is COX-2 Ptgs2, and the double mutant, Apc ⁷¹⁶ ; Ptgs2, has been helpful in interpreting the impact of nonsteroidal anti-inflammatory drugs on mechanisms of colon polyp development. Moreover, the introduction of a cyclooxygenase 1 and 2 (COX-1 an COX-2) mutations in the Apc ^min mice had a pronounced effect on the size and number of intestinal polyps. This mouse model has been useful for providing a model system for chemoprevention studies, using nonsteroidal anti-inflammatory drugs to treat patients suffering from familial and sporadic polyposis.

Lynch Syndrome

Mouse models for DNA mismatch repair (MMR) genes, MLH1/MSH2/MSH6/PMS2 , have been under investigation for many years; MMR mouse models have been highly valuable in revealing the mechanisms of MMR genes in cancer biology. MMR prevents cancer and suppresses tumors through several mechanisms. These include (1) single base substitution repair, (2) insertion/deletion frameshift repair (also called microsatellite instability), (3) anti-apoptosis, and (4) suppression of promiscuous homeologous recombination. MLH1 and MSH2 mutations are responsible for greater than 90% of all cases of Lynch syndrome; other MMR genes mutations are less deleterious. Msh2 is an essential part of the MutS protein complex and several knockout mouse models of Msh2 have been generated. These mice have a reduced life span as a result of aggressive lymphomas, specifically T-cell lymphomas, intestinal tumors, and other tumor types as well as sebaceous gland tumors similar to those in Muir-Torre syndrome patients. Mice with biallelic mutations in Mlh1/Msh2/Msh6/Pms2 exhibits some of the same malignances seen in humans with biallelic inactivation of the same genes, such as hematologic disorders, that results in shorter life span. One of the hallmarks of MMR deficiency in mice and in humans is the microsatellite instability (MSI) phenotype, reflecting the accumulation of repetitive sequences in the genome. There are, however, differences in the development of the disease in GEM that are deficient in MMR genes as compared to human patients; in humans, most of the tumor development occurs in the colon and extracolonic cancers, including endometrium. In order for mice to develop endometrial cancer, MMR-deficient mice must be crossed with Pten or other tumor suppressor gene knockout mice. A new mouse model that addresses the difference in tumor development between mice and humans is the conditional mouse line of Msh2 ^loxp ; Vill -cre, which directs tumor development to the colon instead of the intestine as was seen in eariler mouse models for Lynch syndrome, and avoids development of lymphomas. Msh6 -deficient mice show a much later onset of tumor development as compared with Msh2 ^−/− mice. Since the role of Msh6 in the MMR system is mostly repair of base substitution and repair of single base IDs, its deficiency does not affect the repair of 2 to 4 base insertion-deletion loops; therefore, mice with Msh6 mutation mostly accumulate base substitutions mutations as opposed to frameshift mutations seen in Msh2 ^−/− mice, and the phenotype of MSI is not seen in tumors from these animals. Msh6 ^−/− mouse models show a similar phenotype of cancer onset and progression seen in Lynch syndrome patients with mutations in MSH6 gene, where most cancer development and progression occurs at around 60 years or older with variable MSI phenotypes. MSH6 mutation has also been linked to endometrial cancers in Lynch syndrome patients and Msh6 ^−/− mice are reported to develop endometrial cancers. Mouse models for Msh3 deficiency show a slight predisposition to the development of cancer due to moderate repair defects and display a normal life span. Msh3 -deficient mouse cells are not able to efficiently repair 1 to 4 base insertion-deletion loops, yet due to the presence of Msh6 they are efficiently able to repair single base substitution. Similarly, human patients with MSH3 mutations develop late-onset tumorigenesis. The MutL homologs ( MLH and PMS genes) play important roles in DNA excision during repair, acting as molecular scaffold for additional proteins to coalesce. At the heart of the three Mutl complexes, Mlh1/Mlh3, Mlh1/Pms2 , and Mlh1/Pms1 , lies Mlh1 . MLH1 deficiency in humans is responsible for shortened life span and a strong predisposition to cancer. Mlh1 knockout mice do not have MMR capacity; therefore, due to the high rate of base substitution, as well as small insertions and deletions in mono- and dinucleotide repeats, these mice have high mutator phenotype similar to Msh2 ^−/− mice. Mice deficient in Mlh1 also have a shortened life span and high degree of predisposition for cancer development. Combined mutations of Mlh1 and Msh2 do not change the tumor suppressor phenotype, consistent with the idea that they both participate in the same complex (M. Liskay, personal communication, 2004). The tumor spectrum of Mlh1 ^−/− mice includes T-cell lymphomas, intestinal adenomas, and adenocarcinomas as well as skin tumors. PMS2 deficiency in humans is associated with Lynch syndrome as well as Turcot syndrome, a rare genetic condition that also predisposes patients to both multiple adenomatous colon polyps and brain tumors. Pms2 ^−/− mice exhibit different disease progression; they show a milder mutator phenotype and an increase in mutation frequency at mononucleotide repeat tracts, but they do not develop any intestinal or brain adenomas; rather, they mostly develop lymphomas and sarcomas with a delayed onset. Recently characterized Mlh3 gene deficiency has been shown to have similar effects as Pms2 deficiency in mice and human patients with the same mutation. One major difference between Pms2 ^−/− and Mlh3 ^−/− deficiencies is that Mlh3 ^−/− mice develop small intestinal adenomas and adenocarcinomas as well as extra-gastrointestinal tumors, including lymphomas and basal cell carcinomas of the skin. Similar to Msh3 and Msh6 combined inactivation mimicking Msh2 ^−/− , combined inactivation of Pms2 and Mlh3 increases the level of mutator phenotype to that of Mlh1 ^−/− . Thus, Mlh3 ^−/− Pms2 ^−/− mice display similar disease development and progression as Mlh1 ^−/− mice. From these mouse models, it has become clear that Pms2 and Mlh3 and Msh3 and Msh6 have redundant roles in repair and tumor suppression functions and that is the most likely reason penetrance is lower in patients deficient in any one of these genes. In a recently reported Pms2 -cre mouse model, where a cell division–activated cre-lox system for stochastic recombination of Lox-P-flanked loci was used, this system was able to better mimic the spontaneous mutation that occurs in cancer.

MMR deficiency in mice results in DNA repair and DNA damage response defect, where both of these mechanisms are important in suppression of tumorigenesis. To study the role of each mechanism, knock-in mouse models have been generated where the mice carrying a recurrent mutation in a gene renders DNA repair mechanism null. Msh2 ^G67A ( Msh2 ^GA ) was one of the first knock-in mutant model with separation-of-function mutation similar to MSH2 missense mutation seen in patients. As predicted, this mutant lost its DNA repair capability but retained its DNA damage response, and MEF s from these mice responded to cisplatin, 6-thioguanine (6-TG), and N-methyl-N-nitro-N-nitrosoquanidine (MNNG). Mouse models of Mlh1 ^G67R caused a separation of function in the Mlh1 protein; the DNA damage repair was impaired whereas DNA damage response, including apoptotic response to cisplatin, remained intact. These mice displayed strong cancer predisposition similar to Mlh1 ^−/− ; however, they developed fewer intestinal adenomas. To date, more than 250 different germline mutations have been identified in MLH1 patients. These new mouse models of different missense mutations, which can elicit different phenotype and, more importantly, different response to therapy, are promising in terms of tailored therapy for variances that exist in patients.

Somatic DNA Mismatch Repair Inactivation

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn disease, has been linked to the development of cancer. The immune system has been shown to play an important role in colonic inflammation. Several mouse models with deletion of immune-specific genes, such as those for interleukin (IL)-2, IL-10, T-cell receptor chains, and major histocompatibility complex class II molecules, have been generated. These mice have a strong predisposition for developing colorectal adenocarcinomas. IL-2– and IL-10–defective mice display immune system aberrations as well as IBD similar to humans. Mucin2 (MUC2) is a secretory protein in the intestinal mucosa, and mice made deficient of Muc2 become prone to inflammation and subsequently develop intestinal adenomas and invasive adenocarcinomas. G-protein alpha subunit (Gia2) knockout mouse develops spontaneous colitis and nonpolyposis, right-sided, multifocal CRCs with mucinous histology; and a Crohn-like inflammatory infiltrate. Gi α 2−/− mice is the first mouse model of somatically acquired MMR deficiency due to inflammation through repression of Mlh1 promoter. These mice also show MSI phenotype as the result of Mlh1 repression. Treatment of Gi α 2−/− mice with histone deacetylase inhibitor (HDACi) decreases colitis and relieves epigenetic repression of Mlh1 expression. IBD mouse models are important because gastrointestinal inflammation is considered a strong risk for developing CRC. Suberoylanilide hydroxamic acid is an HDACi that is currently used in the treatment of cutaneous T-cell lymphoma and is in clinical trial for other cancers; the use of this compound could be extended to treatment of IBD and associated diseases.

Peutz-Jeghers Syndrome

PJS is autosomal dominant disease with variable inheritance caused by germline mutation in LKB1/STK11. LKB1/STK11 is a serine/theonine kinase and is considered to be a tumor suppressor. PJS is characterized by hamartomatous polyposis of the gastrointestinal tract, mostly in the small intestine, with a strong predisposition to malignancy, as well as developing mammary tumors and mucocutenous pigmentation. Lkb1/Stk11 null mutation in mice is lethal and Lkb1/Stk11 heterozygous mice exhibit a similar phenotype to PJS patients as they also develop gastrointestinal hamartomatous without wild-type allele inactivation. Furthermore, heterozygous mutation of Lkb1 is sufficient for the development of gastrointestinal hamartomas that exhibited histologic features similar to PJS patients. This mouse model has showed that biallelic inactivation is not necessary for hamartoma development and, therefore, it is plausible that LKB1 loss of heterozygosity (LOH) would result in malignant transformation of hamartomas along with other mutations. Furthermore, the life span of a mouse is too short to allow for the inactivation of wild-type allele and any further genetic hits necessary for progression to malignancy as seen in LKB1 inactivation in humans. A proposed mechanism for LKB1’s tumor suppression capability is that it is involved in the p53 -dependent apoptosis and decreased level of LKB1 protein would suppress growth arrest and apoptosis, which leads to accumulation of somatic mutations. Furthermore, the Wnt pathway is not activated in Lkb1± hamartomas; however, LKB1 LOH adenomatous lesions showed β-catenin mutation, suggesting that mutation in the Wnt pathway is also important for the progression of hamartomas to carcinomas along with LKB1 LOH.