Novel diet-related mouse model of colon cancer parallels human colon cancer.

AIM: To investigate the close parallels between our novel diet-related mouse model of colon cancer and human colon cancer. METHODS: Twenty-two wild-type female mice (ages 6-8 wk) were fed the standard control diet (AIN-93G) and an additional 22 female mice (ages 6-8 wk) were fed the control diet supplemented with 0.2% deoxycholic acid [diet + deoxycholic acid (DOC)] for 10 mo. Tumors occurred in the colons of mice fed diet + DOC and showed progression to colon cancer [adenocarcinoma (AC)]. This progression is through the stages of tubular adenoma (TA), TA with high grade dysplasia or adenoma with sessile serrated morphology, intramucosal AC, AC stage T1, and AC stage T2. The mouse tumors were compared to human tumors at the same stages by histopathological analysis. Sections of the small and large intestines of mice and humans were evaluated for glandular architecture, cellular and nuclear morphology including cellular orientation, cellular and nuclear atypia, pleomorphism, mitotic activity, frequency of goblet cells, crypt architecture, ulceration, penetration of crypts through the muscularis mucosa and presence of malignant crypts in the muscularis propria. In addition, preserved colonic tissues from genetically similar male mice, obtained from a prior experiment, were analyzed by immunohistochemistry. The male mice had been fed the control diet or diet + DOC. Four molecular markers were evaluated: 8-OH-dG, DNA repair protein ERCC1, autophagy protein beclin-1 and the nuclear location of beta-catenin in the stem cell region of crypts. Also, male mice fed diet + DOC plus 0.007% chlorogenic acid (diet + DOC + CGA) were evaluated for ERCC1, beclin-1 and nuclear location of beta-catenin. RESULTS: Humans with high levels of diet-related DOC in their colons are at a substantially increased risk of developing colon cancer. The mice fed diet + DOC had levels of DOC in their colons comparable to that of humans on a high fat diet. The 22 mice without added DOC in their diet had no colonic tumors while 20 of the 22 mice (91%) fed diet + DOC developed colonic tumors. Furthermore, the tumors in 10 of these mice (45% of mice) included an adenocarcinoma. All mice were free of cancers of the small intestine. Histopathologically, the colonic tumor types in the mice were virtually identical to those in humans. In humans, characteristic aberrant changes in molecular markers can be detected both in field defects surrounding cancers (from which cancers arise) and within cancers. In the colonic tissues of mice fed diet + DOC similar changes in biomarkers appeared to occur. Thus, 8-OH-dG was increased, DNA repair protein ERCC1 was decreased, autophagy protein beclin-1 was increased and, in the stem cell region at the base of crypts there was substantial nuclear localization of beta-catenin as well as increased cytoplasmic beta-catenin. However, in mice fed diet + DOC + CGA (with reduced frequency of cancer) and evaluated for ERCC1, beclin-1, and beta-catenin in the stem cell region of crypts, mouse tissue showed amelioration of the aberrancies, suggesting that chlorogenic acid is protective at the molecular level against colon cancer. This is the first diet-related model of colon cancer that closely parallels human progression to colon cancer, both at the histomorphological level as well as in its molecular profile. CONCLUSION: The diet-related mouse model of colon cancer parallels progression to colon cancer in humans, and should be uniquely useful in model studies of prevention and therapeutics.

Deficient expression of DNA repair enzymes in early progression to sporadic colon cancer.

BACKGROUND: Cancers often arise within an area of cells (e.g. an epithelial patch) that is predisposed to the development of cancer, i.e. a "field of cancerization" or "field defect." Sporadic colon cancer is characterized by an elevated mutation rate and genomic instability. If a field defect were deficient in DNA repair, DNA damages would tend to escape repair and give rise to carcinogenic mutations. PURPOSE: To determine whether reduced expression of DNA repair proteins Pms2, Ercc1 and Xpf (pairing partner of Ercc1) are early steps in progression to colon cancer. RESULTS: Tissue biopsies were taken during colonoscopies of 77 patients at 4 different risk levels for colon cancer, including 19 patients who had never had colonic neoplasia (who served as controls). In addition, 158 tissue samples were taken from tissues near or within colon cancers removed by resection and 16 tissue samples were taken near tubulovillous adenomas (TVAs) removed by resection. 568 triplicate tissue sections (a total of 1,704 tissue sections) from these tissue samples were evaluated by immunohistochemistry for 4 DNA repair proteins. Substantially reduced protein expression of Pms2, Ercc1 and Xpf occurred in field defects of up to 10 cm longitudinally distant from colon cancers or TVAs and within colon cancers. Expression of another DNA repair protein, Ku86, was infrequently reduced in these areas. When Pms2, Ercc1 or Xpf were reduced in protein expression, then either one or both of the other two proteins most often had reduced protein expression as well. The mean inner colon circumferences, from 32 resections, of the ascending, transverse and descending/sigmoid areas were measured as 6.6 cm, 5.8 cm and 6.3 cm, respectively. When combined with other measurements in the literature, this indicates the approximate mean number of colonic crypts in humans is 10 million. CONCLUSIONS: The substantial deficiencies in protein expression of DNA repair proteins Pms2, Ercc1 and Xpf in about 1 million crypts near cancers and TVAs suggests that the tumors arose in field defects that were deficient in DNA repair and that deficiencies in Pms2, Ercc1 and Xpf are early steps, often occurring together, in progression to colon cancer.

Cancer and age related colonic crypt deficiencies in cytochrome c oxidase I.

AIM: To investigate whether deficiency of expression of cytochrome c oxidase I (CcOI) in colonic crypts is associated with colon cancer. METHODS: The pattern and level of expression of CcOI in non-neoplastic colonic crypts, and in dysplastic tissues, was assessed using standard immunohistochemical methods. Biopsies were obtained from individuals undergoing colonoscopies for screening purposes or for a medically indicated reason. Tissue samples were also obtained from surgical colonic resections. Samples from resections were taken from colonic mucosa 1 and 10 cm from tumors and from the tumors themselves. Samples were evaluated for frequency of crypts with reduced or absent expression of CcOI. In most crypts the loss was apparent throughout the entire crypt, while in a small minority the loss was segmental. The strong immunoreactivity using this monoclonal antibody makes the scoring unambiguous. The percent of crypts with reduced or absent expression of CcOI or (infrequent) segmented loss of expression was then calculated. Data analyses were performed using SPSS statistical package 17.0. RESULTS: The average frequency of CcOI deficient crypts (CcOI-DC) is low in individuals between 20 and 39 years of age, with 0.48% ± 0.40% CcOI-DC for women and 1.80% ± 0.35% for men. CcOI-DC increases after age 40 years, so that between the ages of 40 and 44 years the average frequency of CcOI-DC goes up to 5.89% ± 0.84% in women and 2.15% ± 1.27% in men. By 80-84 years of age, the average frequency of CcOI-DC goes up in women to 15.77% ± 0.97% and in men to 22.6% ± 0.65%. The increases in CcOI-DC from ages 40-44 years compared to 80-84 years in women and men are significantly different with P < 0.01. For women over age 60 years, deficiency of CcOI expression is greater in those women who have had a cancer in their colon. The frequency of CcOI-DC, measured in men, increased in tissues adjacent to colon cancer, being 4.03% ± 0.27% in individuals free of neoplasia in the age range 55-64 years and 14.13% ± 0.35% in resected histologically normal tissue of men with cancer in the same age range, P < 0.001. Similar significant differences were noted in older age ranges. The frequency of CcOI-DC crypts in the cecum and sigmoid colon of an individual are significantly correlated, with an R(2) = 0.414 for women and R(2) = 0.528 for men, P < 0.001. This suggests that the factors determining the level of CcOI deficiency act throughout the colon. Most defective crypts are in clusters of two or more, a likely consequence of crypt fission. In the non-neoplastic margins of cancers, crypts are frequently deficient for CcOI, and such crypts may appear in large clusters, some containing more than 100 deficient crypts. CcOI deficiency is also apparent in colon cancers and sometimes involves a large section of the tumor. Overall, CcOI deficient cells can be visualized in segments of crypts, in whole crypts that increase in frequency with age, in crypts undergoing fission, in clusters of crypts where the clusters increase in size with age, in increased frequency near tumors, in large clusters in the intimate margins of tumors, and in the tumors themselves. There is no clear dividing line between early stages that can be considered aspects of aging and later stages that can be considered aspects of the progression to cancer. This ambiguity may reflect a rather general situation leading to adult cancer where the early stages of cellular change appear to be relatively innocuous features of the aging process but over decades may evolve into malignancy. CONCLUSION: CcOI deficient crypts increase in frequency with age, and clusters of deficient crypts are associated with, and may give rise to, colon cancer.

Deficient Pms2, ERCC1, Ku86, CcOI in field defects during progression to colon cancer.

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia. Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer. DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia. We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.