[CANCER RESEARCH 64, 6394–6401, September 15, 2004] Genetic signatures of High- and Low-Risk Aberrant Crypt Foci in a Mouse Model of Sporadic Colon Cancer Prashant R. Nambiar,1,2 Masako Nakanishi,1 Rishi Gupta,3 Evelyn Cheung,4 Ali Firouzi,4 Xiao-Jun Ma,4 Christopher Flynn,1 Mei Dong,1 Kishore Guda,1 Joel Levine,5 Rajiv Raja,4 Luke Achenie,3 and Daniel W. Rosenberg1 1Center for Molecular Medicine and Program in Colorectal Cancer, University of Connecticut Health Center, Farmington, Connecticut; Departments of 2Pathobiology and Veterinary Sciences and 3Chemical Engineering, University of Connecticut, Storrs, Connecticut; 4Arcturus Bioscience, Inc., Mountain View, California; and 5Department of Medicine and Program in Colorectal Cancer, University of Connecticut Health Center, Farmington, Connecticut ABSTRACT cyclin D1, and cyclin-dependent kinase 4 (11–20). The model pro- vides an appropriate experimental system for studying molecular and To determine whether cancer risk is related to histopathological fea- pathological changes associated with the human disease. Importantly, tures of preneoplastic aberrant crypt foci (ACF), gene expression analysis inbred mice demonstrate differences in azoxymethane sensitivity. For was performed on ACF from two mouse strains with differing tumor sensitivity to the colonotropic carcinogen, azoxymethane. ACF from sen- example, A/J mice develop 10 to 20 tumors in distal colon, whereas sitive A/J mice were considered at high risk, whereas ACF from resistant tumors in AKR/J mice are rare (10, 11, 20). Interestingly, initiation of AKR/J mice were considered at low risk for tumorigenesis. A/J and ACF upon carcinogen exposure is a feature common to both strains (5, AKR/J mice received weekly injections of azoxymethane (10 mg/kg body 10, 18). However, a higher percentage of dysplastic ACF form in A/J weight), and frozen colon sections were prepared 6 weeks later. Immuno- colons compared with AKR/J. These observations provide the basis histochemistry was performed using biomarkers associated with colon for additional stratification of ACF in terms of cancer risk, an ap- cancer, including adenomatous polyposis coli, ␤-catenin, p53, c-myc, cy- proach that may eventually lead to identification of genes involved in clin D1, and proliferating cell nuclear antigen. Hyperplastic ACF, dys- early stages of colon tumorigenesis. plastic ACF, microadenomas, adjacent normal-appearing epithelium, and ACF are comprised of aggregates of abnormal crypts and charac- vehicle-treated colons were laser captured, and RNA was linearly ampli- terized by hyperproliferation, increased size, expanded pericryptal fied (LCM-LA) and subjected to cDNA microarray-based expression analysis. Patterns of gene expression were identified using adaptive cen- zones, and elongated or slit-like crypt lumina (5, 10, 21, 22). A troid algorithm. ACF from low- and high-risk colons were not discrimi- number of studies support the contention that only limited subsets of nated by immunohistochemistry, with the exception of membrane staining ACF have the potential to progress, whereas most lesions remain of ␤-catenin. To develop genetic signatures that predict cancer risk, behaviorally benign (10). Sequential analyses demonstrate that hyper- LCM-LA RNA from ACF was hybridized to cDNA arrays. Of 4896 plastic ACF that are prevalent within the AKR/J colons fail to pro- interrogated genes, 220 clustered into six broad clusters. A total of 226 and gress, thereby representing a subpopulation of colon lesions that may 202 genes was consistently altered in lesions from A/J and AKR/J mice, be considered low risk (10, 20, 23–25). Alternatively, ACF that form respectively. Although many alterations were common to both strains, in sensitive A/J colons are likely to progress to tumors and are expression profiles stratified high- and low- risk lesions. These data dem- therefore considered high risk. This stratification of risk thus provides onstrate that ACF with distinct tumorigenic potential have distinguishing the basis to our experimental approach. molecular features. In addition to providing insight into colon cancer promotion, our data identify potential biomarkers for determining colon Although a recent report describes gene expression profiles in cancer risk in humans. human dysplastic colonic adenomas (26), there have been no attempts to stratify high- and low-risk ACF on the basis of gene expression profiles as an adjunct to histologic staging. To determine whether INTRODUCTION differential cancer sensitivity is associated with histopathological fea- tures of preneoplastic ACF, we performed a molecular analysis of The majority of colorectal cancer cases are sporadic, characterized low- and high-risk ACF with a goal toward developing a molecular by pathologically defined stages, including formation of preneoplastic signature that enables a reasonable discrimination of their risk poten- aberrant crypt foci (ACF), preinvasive adenomas, and malignant tial. Our data demonstrate that ACF with distinct tumorigenic poten- carcinomas (1–3). ACF, although not yet fully committed to neopla- tial have distinguishing genetic signatures, thus providing a potential sia, are stratified into two broad classes, hyperplastic and dysplastic, resource for identifying biomarkers that may be used to establish based on morphology and predicted biological behavior (4, 5). His- colon cancer risk in human populations. tomorphologic grading of ACF, in terms of size and dysplasia, rep- resent one of the few biomarkers of colorectal cancer risk (6–10). Therefore, establishing molecular criteria that enhance prediction of MATERIALS AND METHODS ACF progression is relevant to clinical prognostication. The colonotropic carcinogen, azoxymethane, produces tumors and Animal Treatment and Laser Capture Microdissected (LCM). Five A/J and AKR/J male mice (The Jackson Laboratory, Bar Harbor, ME) of 5 weeks preneoplastic lesions in mice that closely recapitulate key molecular of age received injections of azoxymethane (10 mg/kg body weight) weekly features of human colorectal cancer, including alterations in Ki-ras, for 6 weeks (10, 20). A separate group received 0.9% NaCl (vehicle controls). ␤ adenomatous polyposis coli (APC), transforming growth factor , Animals were sacrificed 6 weeks after the last injection and colons were flushed with PBS, embedded in cryoprotectant OCT, and frozen at Ϫ80°C. Received 3/18/04; revised 6/17/04; accepted 7/15/04. Eight serial cryosections (7-␮m thick) with appropriate representations of Grant support: NIH Grant CA 81248 and a grant from the Robert Leet and Clara preneoplastic lesions and abutting normal tissue were prepared. One section Guthrie Patterson Trust. was stained with H&E for histologic classification of lesions, whereas the 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 remaining six sections were stained using the HistoGene LCM staining kit 18 U.S.C. Section 1734 solely to indicate this fact. (Arcturus, Mountain View, CA). Pure populations of epithelial cells compris- Note: P.R. Nambiar and M. Nakanishi contributed equally to this publication. ing lesions were microdissected using the PixCell II LCM system (Arcturus). Requests for reprints: Daniel W. Rosenberg, Center for Molecular Medicine, Uni- A summary of lesions is provided (Table 1). Because of the rarity of AKR/J versity of Connecticut Health Center, 263 Farmington Avenue, CT 06030-3101. Phone: (860) 679-8704; Fax: (860) 679-1140; E-mail: [email protected]. dysplastic ACF, step-sections were prepared to obtain five lesions. Laser- ©2004 American Association for Cancer Research. captured saline-treated colonic epithelia were pooled and used as reference 6394 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. GENE SIGNATURE OF ACF RISK POTENTIAL Table 1 Summary of tissues harvested from saline and azoxymethane-treated A/J and minutes at 95°C, followed by 40 cycles of 10 seconds at 95°C and 1 minute at AKR/J mice 60°C. The averages of the threshold cycle (CT) values were analyzed for Mouse Saline Normal-appearing Hyperplastic Dysplastic relative quantitation using the comparative CT method. CT values for lesions strain controls epithelium ACF ACF Microadenomas were normalized to a reference gene (glyceraldehyde-3-phosphate dehydro- A/J 3 5 6 6 10 genase) and calibrator (saline control), which were used to determine relative AKR/J 3 6 6 5 expression. Immunohistochemistry (IHC). IHC of formalin-fixed, paraffin-embedded sections for APC, p53, ␤-catenin, cyclin D1, and c-myc was performed using control, eliminating potential field effects that may exist within azoxymethane- a peroxidase-conjugated avidin-biotin method as described previously (11, 16). exposed normal-appearing epithelium. The validity of this approach is dem- Primary antibodies were used as follows: rabbit polyclonal p53 CM5 antibody onstrated by similar expression profiles from laser-captured, saline-treated (Novacastra, Newcastle-upon-Tyne, United Kingdom) at 1:500 dilution; colonic epithelium from both strains (correlation coefficient, r ϭ 0.86), vali- mouse monoclonal ␤-catenin (Sigma-Aldrich, Inc., St. Louis, MO) at 1:1000; dating their use as reference controls and avoiding potentially confounding cyclin D1 (Novacastra) at 1:40; c-Myc (Upstate, Waltham, MA) at 1:100; APC effects of potentially heterogeneous expression profiles