Kunhua Fan

Mutation in the mismatch repair gene Msh6 causes cancer susceptibility

Mice carrying a null mutation in the mismatch repair gene Msh6 were generated by gene targeting. Cells that were homozygous for the mutation did not produce any detectable MSH6 protein, and extracts prepared from these cells were defective for repair of single nucleotide mismatches. Repair of 1, 2, and 4 nucleotide insertion/deletion mismatches was unaffected. Mice that were homozygous for the mutation had a reduced life span. The mice developed a spectrum of tumors, the most predominant of which were gastrointestinal tumors and B- as well as T-cell lymphomas. The tumors did not show any microsatellite instability. We conclude that MSH6 mutations, like those in some other members of the family of mismatch repair genes, lead to cancer susceptibility, and germline mutations in this gene may be associated with a cancer predisposition syndrome that does not show microsatellite instability.

Haploinsufficiency of Flap endonuclease (Fen1) leads to rapid tumor progression

Flap endonuclease (Fen1) is required for DNA replication and repair, and defects in the gene encoding Fen1 cause increased accumulation of mutations and genome rearrangements. Because mutations in some genes involved in these processes cause cancer predisposition, we investigated the possibility that Fen1 may function in tumorigenesis of the gastrointestinal tract. Using gene knockout approaches, we introduced a null mutation into murine Fen1. Mice homozygous for the Fen1 mutation were not obtained, suggesting absence of Fen1 expression leads to embryonic lethality. Most Fen1 heterozygous animals appear normal. However, when combined with a mutation in the adenomatous polyposis coli (Apc) gene, double heterozygous animals have increased numbers of adenocarcinomas and decreased survival. The tumors from these mice show microsatellite instability. Because one copy of the Fen1 gene remained intact in tumors, Fen1 haploinsufficiency appears to lead to rapid progression of cancer.

Western-style diet-induced colonic tumors and their modulation by calcium and vitamin D in C57Bl/6 mice: a preclinical model for human sporadic colon cancer

We reported previously that a new Western-style diet (NWD) for 18 months, consisting of elevated lipids and decreased calcium, vitamin D and methyl-donor nutrients, induced colonic tumors in normal C57Bl/6 mice [Newmark, H.L. et al. (2001) A Western-style diet induces benign and malignant neoplasms in the colon of normal C57Bl/6 mice. Carcinogenesis, 22, 1871-1875], suggesting a new mouse model for human sporadic colon cancer. Here, we have extended this study during a longer feeding period of 2 years wherein tumor formation, tumor inhibition by addition of dietary calcium and vitamin D and their effects on gene expression were determined. We also similarly tested individual supplements of methyl donor (transfer) nutrients (folic acid, choline, methionine and dietary fiber), but these had no significant effect on colonic tumor incidence or multiplicity, whereas supplementation with combined calcium and vitamin D produced significant decrease in both colon tumor incidence and multiplicity, during 2 years of feeding. No visible colonic tumors were found at 6 months, very few at 12 months, more at 18 months and significantly at 24 months. In a related study of gene changes of the mouse colonic mucosa at 6 months of feeding taken from this study, long before any tumors were visibly detectable, indicated altered profiles of gene expression linked to later risk of dietary initiation of colon tumor formation. This type of early genetic altered profile, an indication of increased risk of later colonic tumor development, may become a useful tool for prediction of colon tumor risk while the colon grossly still appears histologically and physiologically normal.

Mbd4 inactivation increases C→T transition mutations and promotes gastrointestinal tumor formation

Mbd4 (methyl-CpG binding domain 4) is a novel mammalian repair enzyme that has been implicated biochemically in the repair of mismatched G-T residues at methylated CpG sites. In addition, the human protein has been shown to interact with the DNA mismatch repair protein MLH1. To clarify the role of Mbd4 in DNA repair in vivo and to examine the impact of Mbd4 inactivation on gastrointestinal (GI) tumorigenesis, we introduced a null mutation into the murine Mbd4 gene by gene targeting. Heterozygous and homozygous Mbd4 mutant mice develop normally and do not show increased cancer susceptibility or reduced survival. Although Mbd4 inactivation did not increase microsatellite instability (MSI) in the mouse genome, it did result in a 2- to 3-fold increase in C→T transition mutations at CpG sequences in splenocytes and epithelial cells of the small intestinal mucosa. The combination of Mbd4 deficiency with a germ line mutation in the adenomatous polyposis coli (Apc) gene increased the tumor number in the GI tract and accelerated tumor progression. The change in the GI cancer phenotype was associated with an increase in somatic C→T mutations at CpG sites within the coding region of the wild-type Apc allele. These studies indicate that, although inactivation of Mbd4 does not by itself cause cancer predisposition in mice, it can alter the mutation spectrum in cancer cells and modify the cancer predisposition phenotype.

A mouse model of human familial adenomatous polyposis.

In an effort to generate a good mouse model for human colorectal cancer, we generated mice which carry a mutation in the adenomatous polyposis coli (Apc) gene. Mice which are heterozygous for the mutation, designated Apc1638, develop colonic polyps and tumors of the small intestine. Neoplasms were found in 96% of animals studied, and they included adenomas, adenocarcinomas, and polypoid hyperplasias. The mice developed an average of 3.3 tumors, with the highest number in duodenum, followed by jejunum, stomach, ileum, and colon. Focal areas of dysplasias were observed in the colonic mucosa in 50% of mice which were 10 months old or older. These results suggest that mice carrying the Apc1638 mutation can serve as a good model to study the initiation, progression, and inhibition of gastrointestinal tumors.

An Msh2 conditional knockout mouse for studying intestinal cancer and testing anticancer agents.

BACKGROUND & AIMS Mutations in the DNA mismatch repair (MMR) gene MSH2 cause Lynch syndromes I and II and sporadic colorectal cancers. Msh2(null) mice predominantly develop lymphoma and do not accurately recapitulate the colorectal cancer phenotype. METHODS We generated and examined mice with a conditional Msh2 disruption (Msh2(LoxP)), permitting tissue-specific gene inactivation. ECMsh2(LoxP/LoxP) mice carried an EIIa-Cre transgene, and VCMsh2(LoxP/LoxP) mice carried a Villin-Cre transgene. We combined the VCMsh2(LoxP) allele with either Msh2(Delta7null) (VCMsh2(LoxP/null)) or Msh2(G674D) mutations (VCMsh2(LoxP/G674D)) to create allelic phase mutants. These mice were given cisplatin or 5-fluorouracil/leucovorin and oxaliplatin (FOLFOX), and their tumors were measured by magnetic resonance imaging. RESULTS Embryonic fibroblasts from ECMsh2(LoxP/LoxP) mice do not express MSH2 and are MMR deficient. Reverse transcription, polymerase chain reaction, and immunohistochemistry from VCMsh2(LoxP/LoxP) mice demonstrated specific loss of Msh2 messenger RNA and protein from epithelial cells of the intestinal tract. Microsatellite instability was observed in all VCMsh2 strains and limited to the intestinal mucosa. Resulting adenomas and adenocarcinomas had somatic truncation mutations to the adenomatous polyposis coli (Apc) gene. VCMsh2(LoxP/LoxP) mice did not develop lymphoma. Comparison of allelic phase tumors revealed significant differences in multiplicity and size. When treated with cisplatin or FOLFOX, tumor size was reduced in VCMsh2(LoxP/G674D) but not VCMsh2(LoxP/null) tumors. The apoptotic response to FOLFOX was partially sustained in the intestinal mucosa of VCMsh2(LoxP/G674D) animals. CONCLUSIONS Msh2(LoxP/LoxP) mice in combination with appropriate Cre recombinase transgenes have excellent potential for preclinical modeling of Lynch syndrome, MMR-deficient tumors of other tissue types, and use in drug development.

Preclinical mouse models for cancer chemoprevention studies

ABSTRACT To aid in identifying the ability of chemopreventive agents to inhibit tumor development, new preclinical in vivo rodent models have recently been developed. Some of the models contain targeted mutations capable of increasing the incidence and progression of neoplastic lesions, whereas in other models dietary nutrients induce preneoplastic lesions in normal mice. These new preclinical models are assisting the analysis of genetic and environmental factors leading to neoplasia, and clinical studies to evaluate the chemopreventive efficacy of specific nutrients and pharmacological agents.

Modulation of abnormal colonic epithelial cell proliferation and differentiation by low-fat dairy foods

CONTEXT Before the development of human colonic neoplasms, colonic epithelial cells showed altered growth and differentiation. These alterations characterized mucosa at risk for cancer formation and were termed intermediate biomarkers of risk. Modifications of the mucosa toward more normal features by nutrients or drugs are putative approaches to chemoprevention of colon cancer. OBJECTIVE To determine whether increasing calcium intake via dairy products alters colonic biomarkers toward normal. DESIGN Randomized, single-blind, controlled study. SETTING Outpatient clinic. PARTICIPANTS Seventy subjects with a history of polypectomy for colonic adenomatous polyps. INTERVENTION Low-fat dairy products containing up to 1200 mg/d of calcium. Subjects were randomized to 4 strata by diet (control vs higher calcium) and age (<60 vs > or = 60 years). MAIN OUTCOME MEASURES Changes in total colonic epithelial cells and number and position of thymidine-labeled epithelial cells and changes in the ratio of sulfomucins (predominantly secreted by distal colorectal epithelial cells) to sialomucins and expression of cytokeratin AE1, 2 markers of colonic cell differentiation. RESULTS During 6 and 12 months of treatment, reduction of colonic epithelial cell proliferative activity (P<.05), reduction in size of the proliferative compartment (P<.05), and restoration of acidic mucin (P<.02), cytokeratin AE1 distribution (P<.05), and nuclear size (P<.05) toward that of normal cells occurred. Control subjects showed no differences from baseline proliferative values at 6 and 12 months (P>.05). CONCLUSION Increasing the daily intake of calcium by up to 1200 mg via low-fat dairy food in subjects at risk for colonic neoplasia reduces proliferative activity of colonic epithelial cells and restores markers of normal cellular differentiation.

Tumor progression in Apc 1638N mice with Exo1 and Fen1 deficiencies

Flap endonuclease 1 (Fen1) and exonuclease 1 (Exo1) have sequence homology and similar nuclease capabilities. Both function in multiple pathways of DNA metabolism, but appear to have distinct in vivo nucleic acid substrates, and therefore distinct metabolic roles. When combined with Apc1638N, Fen1 promotes tumor progression. Because of functional similarity to Fen1, and because Exo1 is involved in DNA mismatch repair (MMR) by interaction with Msh2 and Mlh1, genes that cause hereditary nonpolyposis colorectal cancer (HNPCC), we investigated the possibility that Exo1 might also act as a modifier to Apc1638N. We present evidence that mice with combined mutations in Apc1638N and Exo1 and Apc1638N, Exo1 and Fen1 genes show moderate increased tumor incidence and multiplicity in comparison to Apc1638N siblings, implying a low penetrance role for Exo1 in early gastrointestinal (GI) tumorigenesis. Despite a decrease in median survival (10 months) in Apc1638N Exo1 mice, their tumors do not progress any more rapidly than those of Apc1638N. Instead these animals die from infections that are the result of impaired immune response. Apc1638N Exo1 Fen1 mice survive longer (18 months), and therefore appear relatively immune competent. They die of invasive GI tumors that display microsatellite instability (MSI). Our results show that Exo1 has a modest tumor suppressor function.

Loss of Rb1 in the gastrointestinal tract of Apc1638N mice promotes tumors of the cecum and proximal colon

To examine the role of Rb1 in gastrointestinal (GI) tumors, we generated mice with an Apc1638N allele, Rbtm2brn floxed alleles, and a villin-cre transgene (RBVCA). These animals had exon 19 deleted from Rb1 throughout the GI tract. We have shown previously that Rb1 deficiency is insufficient for GI tumor initiation, with inactivation of an Apc allele capable of overcoming the insufficiency. In this study we demonstrate that RBVCA mice have reduced median survival because of an increase in tumor incidence and multiplicity in the cecum and the proximal colon. Large intestinal tumors are predominantly adenomas, whereas the tumors of the small intestine are a mixture of adenomas and adenocarcinomas. We find truncation mutations to the second Apc allele in tumors of both the large and small intestine. Expression profiles of duodenal and cecal tumors relative to each other show unique gene subsets up and down regulated. Substantial expression patterns compare to human colorectal cancer, including recapitulation of embryonic genes. Our results indicate that Rb1 has significant influence over tumor location in the GI tract, and that both cecal and duodenal tumors initiate through inactivation of Apc. Expression profile analysis indicates the two tumor types differentially regulate distinct sets of genes that are over-expressed in a majority of human colorectal carcinomas.