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Albert Einstein College of Medicine and Cancer Center, Montefiore Hospital/Oncology, Bronx, NY 10467
3To whom correspondence should be addressed: E-mail: augen{at}aecom.yu.edu.
EXPANDED ABSTRACT
Intestinal tumor formation results from perturbations in the normal maturation of intestinal epithelial cells as they migrate from the bottom of the crypt and undergo cell cycle arrest and lineage-specific differentiation during their transit towards the lumen. The complex integration of pathways that govern these processes, leading to homeostasis in the mucosa, is under the control of an intrinsic program that is activated by signals elaborated by the cell and its neighboring cells, signals from the extracellular matrix, and signals from the intestinal contents, especially those generated by the diet.
In the mouse we have manipulated gene loci, such as Apc, p21WAF1/cip1, p27Kip1, and Muc2, each with a role in the intrinsic program of normal cell maturation, and combinations of inactivation of these genes to understand how the activity of these loci influences cell maturation pathways and tumor formation. Although targeted inactivation of the cyclin-dependent kinase inhibitor p21WAF1/cip1 does not cause discernible changes in histology or pathology of the intestinal mucosa (1), it greatly increases the formation of tumors when combined with an inherited mutation in the Apc gene. Increased tumor incidence, frequency, and size, and decreased life span were all a function of p21 gene dosage in the Apc±, p21+/+, ± or –/– compound mice (2). Moreover, feeding the mice a Western-style diet that was high in fat and low in calcium and vitamin D (3,4) was additive with each genotype on tumor formation and histopathology, thus demonstrating the importance of diet even in the presence of strong genetic initiation for intestinal tumor formation (2).
In contrast to inactivation of p21, targeted inactivation of p27Kip1, another cyclin-dependent kinase inhibitor, was sufficient to initiate intestinal tumor formation even in the absence of an inherited Apc mutation. Tumors formed in the intestinal tract when the animals were fed the AIN76A-defined diet, and again tumor number increased dramatically when they were fed the Western-style diet (5). However, no tumors formed when the animals were fed standard rodent laboratory diet, likely because of the suppressive affects of phytochemicals in laboratory food (Yang et al., 2005, unpublished results). When the targeted inactivation of p27 was introduced into the Apc± mouse, not only did many more tumors form but on the defined diets the animals exhibited intestinal intussusception, a pathology seen in humans but not previously reported in mouse genetic models. Moreover, in the compound Apc/p27 model, the Western diet further increased the frequency of tumors, their size, and the formation of intestinal intussusception (Yang et al., 2005, unpublished results). These pathological changes severely shortened the life span of the mice. Thus, the data again showed the importance of diet in modulating tumor formation and pathology, even in mouse models in which genetic initiation of tumor formation is very strong.
In the Apc/p21 mice, we also found that goblet cell number in the intestinal mucosa was decreased (2), indicating that tumor formation may be linked to disruption of the development of this differentiated cell type. This is consistent with the fact that goblet cells tend to be depleted in aberrant crypt foci (6) as well as with our report that in a mouse model with a targeted disruption of the Muc2 gene (the gene that encodes the characteristic mucin that is synthesized and secreted by goblet cells) recognizable goblet cells were not present, the architecture of the intestinal mucosa was aberrant, and tumors developed throughout the gastrointestinal tract (7).
We (8) also reported that the inactivation of the p21 gene was sufficient to abrogate the ability of the nonsteroidal anti-inflammatory drug sulindac to inhibit small intestinal tumors in Apc+/-, p21± or –/– mice. Interestingly, this is not true of p27, for which inactivation of one or both alleles did not affect the tumor inhibitory activity of sulindac. We showed that in the Apc±, p21± mice, the wild-type allele is a target for methylation of a CpG cluster upstream of the transcriptional start site, rendering the wild-type allele nonfunctional (9). Thus, when both p21 alleles are inactivated, whether genetically or epigenetically, p21 cannot be induced by sulindac, and the ability of the drug to inhibit tumor formation is eliminated (9). These data firmly establish that the induction of p21 by sulindac is necessary for it to inhibit tumor formation (8,9). We (8) presented evidence that the cellular mechanism is likely cell cycle arrest. The fact that cytostasis rather than apoptosis is the cellular mechanism of tumor inhibition by the drug is consistent with reports that on cessation of sulindac treatment or with extended treatment, tumors often recur in the intestinal tract.
More recently, Kan Yang, Harold Newmark, and Martin Lipkin [(10) and K. Yang, H. Newmark, M. Lipkin, 2005, unpublished results] developed a new dietary model of wild-type mice that develop tumors of the small and large intestine when maintained on a high-risk diet for extended periods of up to 2 y. This mimics characteristics of sporadic colon cancer in the human. In collaboration with these investigators, we used this model to determine the alterations in pathways that govern the physiology, cell biology, and cell maturation of the intestinal mucosa to understand how dietary factors alter mucosal homeostasis and thus modulate the probability of tumor formation in the intestine (K. Yang, H. Newmark, M. Lipkin, L. Augenlicht, 2005, unpublished results). Using our recently published methodology that allows us to profile and track changes in gene expression as cells in the mouse intestine migrate from the crypt toward the intestinal lumen (11) and our gene expression databases that identify the changes that intestinal cells undergo along different lineages and pathways as they mature (12–14), we are investigating how pathways and sequences that are altered in expression during normal cell maturation overlap with those that are linked to tumor formation, or its inhibition, in these mouse models. These databases are available on our Web site (15).
FOOTNOTES
1 Published in a supplement to The Journal of Nutrition. Presented as part of the International Research Conference on Food, Nutrition, and Cancer held in Washington, DC, July 14–15, 2005. This conference was organized by the American Institute for Cancer Research and the World Cancer Research Fund International and sponsored by (in alphabetical order) California Avocado Commission; California Walnut Commission; Campbell Soup Company; The Cranberry Institute; Danisco USA, Inc.; The Hormel Institute; National Fisheries Institute; The Solae Company; and United Soybean Board. Guest editors for this symposium were Vay Liang W. Go, Ritva R. Butrum, and Helen A. Norman. Guest Editor Disclosure: R. R. Butrum and H. Norman are employed by conference sponsor American Institute for Cancer Research; V.L.W. Go, no relationships to disclose. 
2 Author Disclosure: No relationships to disclose.
LITERATURE CITED
1. Deng C, Zhang P, Harper JW, Elledge SJ, Leder P. Mice lacking p21 cip1/waf1 undergo normal development but are defective in G1 checkpoint control. Cell. 1995;82:675-684.[Medline]
2. Yang WC, Mathew J, Velcich A, Edelmann W, Kucherlapati R, Lipkin M, Yang K, Augenlicht LH. Targeted inactivation of the p21 WAF1/cip1 gene enhances Apc initiated tumor formation and the tumor promoting activity of a Western-style high risk diet by altering cell maturation in the intestinal mucosa. Cancer Res. 2001;61:565-569.
3. Newmark HL, Lipkin M, Maheshwari N. Colonic hyperplasia and hyperproliferation induced by a nutritional stress diet with four components of westen-style diet. J Natl Cancer Inst. 1990;82:491-496.
4. Newmark HL, Lipkin M, Maheshwari N. Colonic hyperproliferation induced in rats and mice by nutritional-stress diets containing four components of a human western-style diet (series 2). Am J Clin Nutr. 1991;54:209S-214S.
5. Yang W, BancroftL Nicholas C, Lozonschi I, Augenlicht LH. Targeted inactivation of p27kip1 is sufficient for large and small intestinal tumorigenesis in the mouse which can be augmented by a western-style high-risk diet. Cancer Res. 2003;63:4990-4996.
6. Pretlow TP, Siddiki B, Augenlicht LH, Pretlow TG, Kim YS. Aberrant crypt foci (ACF)—earliest recognized players or innocent bystanders in colon carcinogenesis. Schmiegel W eds. Aberrant crypt foci (ACF)—earliest recognized players or innocent bystanders in colon carcinogenesis. Colorectal cancer: molecular mechanisms premalignant state and its prevention. :67-82 Kluwer Academic Publishers Lancaster, England.
7. Velcich A, Yang W, Heyer J, Fragale A, Nicholas C, Viani S, Kucherlapati R, Lipkin M, Yang K, et al. Colorectal cancer in mice genetically deficient in the mucin Muc2. Science. 2002;295:1726-1729.
8. Yang WC, Velcich A, Mariadason J, Nicholas C, Corner G, Houston M, Edelmann W, Kucherlapati R, Holt P, et al. p21WAF1/cip1 is an important determinant of intestinal cell response to sulindac in vitro and in vivo. Cancer Res. 2001;61:6297-6302.
9. Yang W, Bancroft L, Augenlicht LH. Methylation in the p21WAF1/cip1 promoter of Apc+/- p21+/- mice and lack of response to sulindac. Oncogene. 2005;24:2104-2109.[Medline]
10. Newmark HL, Yang K, Lipkin M, Kopelovich L, Liu Y, Fan K, Shinozaki HA. Western-style diet induces benign and malignant neoplasms in the colon of normal C57Bl/6 mice. Carcinogenesis. 2001;22:1871-1875.
11. Mariadason JM, Nicholas C, L’Italien KE, Zhuang M, Smartt HJ, Heerdt BG, Yang W, Corner GA, Wilson AJ, et al. Gene expression profiling of intestinal epithelial cell maturation along the crypt-villus axis. Gastroenterology. 2005;128:1081-1088.[Medline]
12. Mariadason JM, Corner GA, Augenlicht LH. Genetic reprogramming in pathways of colonic cell maturation induced by short chain fatty acids: comparison with trichostatin A, sulidac, and curcumin and implications for chemoprevention of colon cancer. Cancer Res. 2000;60:4561-4572.
13. Mariadason JM, Arango D, Corner GA, Aranes MJ, Hotchkiss KA, Yang WC, Augenlicht LH. A gene expression profile that defines colon cell maturation in vitro. Cancer Res. 2002;62:4791-4804.
14. Velcich A, Corner G, Paul D, Zhuang M, Mariadason JM, Laboisse C, Augenlicht L. Quantitative rather than qualitative differences in gene expression predominate in intestinal cell maturation along distinct cell lineages. Exp Cell Res. 2005;304:28-39.[Medline]
15. . ; Augenlicht Lab Online [homepage on the Internet]. Available from: http://www.augenlichtlab.com/.
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