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[Cancer Research 64, 6394-6401, September 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Genetic signatures of High- and Low-Risk Aberrant Crypt Foci in a Mouse Model of Sporadic Colon Cancer

Prashant R. Nambiar1,2, Masako Nakanishi1, Rishi Gupta3, Evelyn Cheung4, Ali Firouzi4, Xiao-Jun Ma4, Christopher Flynn1, Mei Dong1, Kishore Guda1, Joel Levine5, Rajiv Raja4, Luke Achenie3 and Daniel W. Rosenberg1

1 Center for Molecular Medicine and Program in Colorectal Cancer, University of Connecticut Health Center, Farmington, Connecticut; Departments of 2 Pathobiology and Veterinary Sciences and 3 Chemical Engineering, University of Connecticut, Storrs, Connecticut; 4 Arcturus Bioscience, Inc., Mountain View, California; and 5 Department of Medicine and Program in Colorectal Cancer, University of Connecticut Health Center, Farmington, Connecticut


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To determine whether cancer risk is related to histopathological features of preneoplastic aberrant crypt foci (ACF), gene expression analysis was performed on ACF from two mouse strains with differing tumor sensitivity to the colonotropic carcinogen, azoxymethane. ACF from sensitive A/J mice were considered at high risk, whereas ACF from resistant AKR/J mice were considered at low risk for tumorigenesis. A/J and AKR/J mice received weekly injections of azoxymethane (10 mg/kg body weight), and frozen colon sections were prepared 6 weeks later. Immunohistochemistry was performed using biomarkers associated with colon cancer, including adenomatous polyposis coli, ß-catenin, p53, c-myc, cyclin D1, and proliferating cell nuclear antigen. Hyperplastic ACF, dysplastic ACF, microadenomas, adjacent normal-appearing epithelium, and vehicle-treated colons were laser captured, and RNA was linearly amplified (LCM-LA) and subjected to cDNA microarray-based expression analysis. Patterns of gene expression were identified using adaptive centroid algorithm. ACF from low- and high-risk colons were not discriminated by immunohistochemistry, with the exception of membrane staining of ß-catenin. To develop genetic signatures that predict cancer risk, LCM-LA RNA from ACF was hybridized to cDNA arrays. Of 4896 interrogated genes, 220 clustered into six broad clusters. A total of 226 and 202 genes was consistently altered in lesions from A/J and AKR/J mice, respectively. Although many alterations were common to both strains, expression profiles stratified high- and low- risk lesions. These data demonstrate that ACF with distinct tumorigenic potential have distinguishing molecular features. In addition to providing insight into colon cancer promotion, our data identify potential biomarkers for determining colon cancer risk in humans.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The majority of colorectal cancer cases are sporadic, characterized by pathologically defined stages, including formation of preneoplastic aberrant crypt foci (ACF), preinvasive adenomas, and malignant carcinomas (1, 2, 3) . ACF, although not yet fully committed to neoplasia, are stratified into two broad classes, hyperplastic and dysplastic, based on morphology and predicted biological behavior (4 , 5) . Histomorphologic grading of ACF, in terms of size and dysplasia, represent one of the few biomarkers of colorectal cancer risk (6, 7, 8, 9, 10) . Therefore, establishing molecular criteria that enhance prediction of ACF progression is relevant to clinical prognostication.

The colonotropic carcinogen, azoxymethane, produces tumors and preneoplastic lesions in mice that closely recapitulate key molecular features of human colorectal cancer, including alterations in Ki-ras, adenomatous polyposis coli (APC), transforming growth factor ß, cyclin D1, and cyclin-dependent kinase 4 (11, 12, 13, 14, 15, 16, 17, 18, 19, 20) . The model provides an appropriate experimental system for studying molecular and pathological changes associated with the human disease. Importantly, inbred mice demonstrate differences in azoxymethane sensitivity. For example, A/J mice develop 10 to 20 tumors in distal colon, whereas tumors in AKR/J mice are rare (10 , 11 , 20) . Interestingly, initiation of ACF upon carcinogen exposure is a feature common to both strains (5 , 10 , 18) . However, a higher percentage of dysplastic ACF form in A/J colons compared with AKR/J. These observations provide the basis for additional stratification of ACF in terms of cancer risk, an approach that may eventually lead to identification of genes involved in early stages of colon tumorigenesis.

ACF are comprised of aggregates of abnormal crypts and characterized by hyperproliferation, increased size, expanded pericryptal zones, and elongated or slit-like crypt lumina (5 , 10 , 21 , 22) . A number of studies support the contention that only limited subsets of ACF have the potential to progress, whereas most lesions remain behaviorally benign (10) . Sequential analyses demonstrate that hyperplastic ACF that are prevalent within the AKR/J colons fail to progress, thereby representing a subpopulation of colon lesions that may be considered low risk (10 , 20 , 23, 24, 25) . Alternatively, ACF that form in sensitive A/J colons are likely to progress to tumors and are therefore considered high risk. This stratification of risk thus provides the basis to our experimental approach.

Although a recent report describes gene expression profiles in 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 differential cancer sensitivity is associated with histopathological features of preneoplastic ACF, we performed a molecular analysis of low- and high-risk ACF with a goal toward developing a molecular signature that enables a reasonable discrimination of their risk potential. Our data demonstrate that ACF with distinct tumorigenic potential have distinguishing genetic signatures, thus providing a potential resource for identifying biomarkers that may be used to establish colon cancer risk in human populations.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Treatment and Laser Capture Microdissected (LCM).
Five A/J and AKR/J male mice (The Jackson Laboratory, Bar Harbor, ME) of 5 weeks of age received injections of azoxymethane (10 mg/kg body weight) weekly for 6 weeks (10 , 20) . A separate group received 0.9% NaCl (vehicle controls). Animals were sacrificed 6 weeks after the last injection and colons were flushed with PBS, embedded in cryoprotectant OCT, and frozen at –80°C. Eight serial cryosections (7-µm thick) with appropriate representations of preneoplastic lesions and abutting normal tissue were prepared. One section was stained with H&E for histologic classification of lesions, whereas the remaining six sections were stained using the HistoGene LCM staining kit (Arcturus, Mountain View, CA). Pure populations of epithelial cells comprising lesions were microdissected using the PixCell II LCM system (Arcturus). A summary of lesions is provided (Table 1)Citation . Because of the rarity of AKR/J dysplastic ACF, step-sections were prepared to obtain five lesions. Laser-captured saline-treated colonic epithelia were pooled and used as reference control, eliminating potential field effects that may exist within azoxymethane-exposed normal-appearing epithelium. The validity of this approach is demonstrated by similar expression profiles from laser-captured, saline-treated colonic epithelium from both strains (correlation coefficient, r = 0.86), validating their use as reference controls and avoiding potentially confounding effects of potentially heterogeneous expression profiles associated with independently captured adjacent normal epithelium from azoxymethane-treated mice. Approximately 50 to 1000 cells were laser-captured from each sample.


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  		Table 1 Summary of tissues harvested from saline and azoxymethane-treated A/J and AKR/J mice

 
ACF Classification.
Histologic evaluation was carried out on H&E-stained colon sections. The criteria used for histologic grading of ACF have been adapted from previous studies (10 , 27, 28, 29) as follows: (1) hyperplastic; small (1 to 5 crypts/focus), moderately hypercellular, normal cellular differentiation with <30% loss of goblet cell differentiation, slight variation in crypt diameter relative to normal, basally arranged and slightly hyperchromatic nucleus; (2) dysplastic; moderate size (5 to 10 crypts/focus), markedly hypercellular, >50% loss of goblet cell differentiation; enlarged, often tortuous crypts with slit-shaped lumina, loss of polarity, dark, elongate to oval, hyperchromatic nuclei with pseudostratification and mitotic figures of 0 to 1/high power field; (3) microadenoma; large size (>10 crypts/focus) but not visible by the naked eye, severely hypercellular, complete loss of goblet cell differentiation, enlarged tortuous crypts; loss of cellular polarity, moderate nuclear anisokaryosis and mitotic figures of 1 to 2/high power field. ACF are defined as high- or low-risk based on established strain sensitivity to azoxymethane (5 , 20) .

RNA Extraction, Linear Amplification, and Array Hybridization.
Total RNA from 50 to 1000 laser-captured tumor cells and vehicle-treated control colons was extracted using the Picopure RNA isolation kit (Arcturus). Because ACF are generally comprised of 20 to 100 cells (400 to 800 pg of total RNA), we used a high-yield T7-based RNA amplification method (RiboAmp HS; Arcturus) to generate adequate amounts of aRNA for hybridization. A 5 to 8 million-fold amplification was achieved with two rounds, yielding ~30 to 70 µg of aRNA. cDNA was transcribed from 8 to 10 µg aRNA in the presence of Cy5-dUTP (Amersham Pharmacia, Piscataway, NJ). Fluorescently labeled cDNA was hybridized to a 14,976-element mouse cDNA array consisting of 4,896 mouse genes, 64 bacterial controls, and 32 targets with only spotting buffer, printed in triplicate across 8 subarrays. Control targets incorporated across all subarrays provide a means of measuring nonspecific target:probe hybridization and potential variation across different regions of the array (11 , 30) .

Data Analysis.
Raw images were analyzed using Imagene 4.2 (BioDiscovery, Los Angeles, CA) and subjected to spot filtering and normalization. Spot filtering was performed by considering only genes above a predetermined level over background threshold. Coefficient of variation was calculated for each replicate spot. Spots that did not satisfy strict coefficient of variation criteria (10%) were not included in the analysis. Data were normalized using a robust 75-percentile method (31 , 32) . In this method, the 75th percentile intensity value for each chip is calculated, and the mean of all these values is used as the reference value to which each chip is scaled. After normalization, genes with expression values close to background were brought to scale by performing floor thresholding using total background of each slide. Normalized ratios were used to represent relative gene expression levels in experimental samples. Because genes were spotted in triplicate, the average ratio was calculated for each gene and used for additional clustering analysis. A novel method for clustering microarray data sets, adaptive centroid algorithm, was used to find gene clusters displaying similar and distinct expression patterns between lesions of similar morphology across strains and also between ACF from the same strain (30 , 33) . The dendrogram of obtained clusters were viewed using Genesite (BioDiscovery).

Quantitative Real-Time PCR Analysis.
Linearly amplified aRNA (4 µg/µL) was converted into cDNA by reverse transcription as described earlier (31) . Quantitative real-time PCR was performed on 100 ng of cDNA using standard conditions. Reactions were run in duplicate for 2 minutes at 50°C, 10 minutes at 95°C, followed by 40 cycles of 10 seconds at 95°C and 1 minute at 60°C. The averages of the threshold cycle (CT) values were analyzed for relative quantitation using the comparative CT method. CT values for lesions were normalized to a reference gene (glyceraldehyde-3-phosphate dehydrogenase) and calibrator (saline control), which were used to determine relative expression.

Immunohistochemistry (IHC).
IHC of formalin-fixed, paraffin-embedded sections for APC, p53, ß-catenin, cyclin D1, and c-myc was performed using a peroxidase-conjugated avidin-biotin method as described previously (11 , 16) . Primary antibodies were used as follows: rabbit polyclonal p53 CM5 antibody (Novacastra, Newcastle-upon-Tyne, United Kingdom) at 1:500 dilution; mouse monoclonal ß-catenin (Sigma-Aldrich, Inc., St. Louis, MO) at 1:1000; cyclin D1 (Novacastra) at 1:40; c-Myc (Upstate, Waltham, MA) at 1:100; APC (N-15 and C-20; Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200 and 1:50, respectively; and goat polyclonal IEX-1 (Santa Cruz Biotechnology) at 1:1000 dilution. Heat-antigen retrieval was used only for APC, cyclin D1, and c-myc antibodies. Lymphoid nodules within colon sections served as internal positive controls for cyclin D1 and p53. Duplicate sections were immunostained with isotype-matched IgG in place of primary antibody (negative control). Immunostained sections were graded by a board-certified veterinary pathologist (P. Nambiar) for cellular localization as follows: nuclear (N), cytoplasmic (C), or membrane (M) and percentage of positively stained cells. For nuclear staining, criteria used were as follows: grade 1, 0 to 20% positive cells; grade 2, 20 to 50% positive cells; and grade 3, >50% positive cells. For cytoplasmic and membrane staining, the criteria were as follows: grade 1, + mild staining; grade 2, ++ moderate staining; and grade 3, +++ marked staining. Samples were considered negative if staining was not greater than negative controls. For immunohistochemistry of human IEX-1, paraffin-embedded tumors and adjacent normal colon sections from colorectal cancer patients were incubated with goat antihuman IEX-1 (Santa Cruz Biotechnology) at 1:1000 dilution. Sections were washed with PBS and incubated with biotinylated antigoat IgG (Vector Laboratories, Burlingame, CA) at a 1:100 dilution for 30 minutes at room temperature. After washing, the sections were incubated with avidin-biotin peroxidase complex provided by Vectastain Elite ABC kit (Vector Laboratories) for 30 minutes at room temperature. Color was developed with 3,3'-diaminobenzidine as the substrate. Sections were then counterstained with hematoxylin.

Statistical Analysis.
Analysis was carried out using Kruskal-Wallis one-way ANOVA by ranks for nonparametric data followed by Dunn’s post hoc analysis for multigroup comparisons (Graphpad Software, San Diego, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Histopathological Examination.
Colon sections were examined microscopically to confirm the histology of each lesion (Fig. 1)Citation . Significant numbers of ACF were observed in colons of both strains, although in A/J, a higher percentage of ACF was dysplastic (5 , 10 , 20) . Hyperplastic ACF from both strains showed increased crypt size, moderate loss of goblet cell differentiation, low crypt multiplicity, and mild to moderate anisokaryosis (Fig. 1, A and C)Citation . Dysplastic ACF had increased crypt size, marked loss of differentiated goblet cells, increased crypt multiplicity, moderate to marked anisokaryosis, and nuclear pseudostratification. In addition, paneth cell metaplasia was noted in multifocal dysplastic A/J ACF (Fig. 1B)Citation .



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  		Fig. 1. Histology of azoxymethane-induced ACF and immunohistochemical analyses of tumor-related proteins. Colon lesions were induced in A/J and AKR/J mice as described under Materials and methods. Representative sections of hyperplastic ACF from A/J (A) and AKR/J (C) mice. Dysplastic ACF from A/J (B) and AKR/J (D) mice. Note the similar histology. The dysplastic ACF from both strains exhibit greater nuclear atypia and increased loss of goblet cell differentiation. In addition, the dysplastic A/J ACF (B) show Paneth cell metaplasia (arrowhead). IHC analyses were done on the colon lesions with antibodies against proliferating cell nuclear antigen (PCNA; E and F); ß-catenin (G and H); APC (I and J); p53 (K and L); cyclin D1 (M and N); and c-myc (O and P). ACF were graded for immunohistochemical staining (percentage of epithelial cells), and the scores are represented in scatterplots adjacent to their respective panels with a median score represented by a horizontal line. For ß-Catenin, the scoring included membrane (M), cytoplasmic (C), and nuclear staining (N).

 
Immunohistochemical Profiling of ACF.
An initial attempt to discriminate populations of ACF from A/J and AKR/J colons was performed by IHC. Expression of several key tumor-related markers, including proliferating cell nuclear antigen, p53, ß-catenin, APC, cyclin D1, and c-Myc, was evaluated (2 , 11 , 13 , 20 , 34, 35, 36) . Deregulation of the Wnt signaling pathway, including mutations in the ß-cateningene and/or APCloss, has been implicated in colon tumorigenesis. Such alterations result in translocation of ß-catenin from membrane to nucleus, with subsequent transcriptional activation of key target genes, including cyclin D1and c-myc. ß-Catenin was detected at comparably high levels in the nucleus and cytoplasm of ACF from either strain, whereas adjacent normal-appearing and vehicle-treated control colons showed only faint membrane staining (Fig. 1)Citation . However, there were differences in immunohistochemical scores for membrane staining of ACF. A/J ACF had a lower score relative to AKR/J (Fig. 1, G and H)Citation . Despite elevated nuclear staining of ß-catenin, there were no mutations identified in either strain within the mutation prone glycogen synthase kinase-3ß phosphorylation sites (data not shown). Because nuclear accumulation of ß-catenin is often associated with loss of APC, we evaluated APC status using antibodies specific to the NH2 terminus. As shown in Fig. 1Citation , we found moderate elevation of APC, eliminating a causal relationship between APC loss and ß-catenin accumulation. To rule out the possibility that accumulated APC protein has undergone truncation, tissues were probed with APC antibody specific for the COOH terminus. APC levels were increased in lesions, primarily localized to the nucleus, an observation that is consistent with the presence of full-length functional protein (37) . Finally, no mutations were detected within the APCmutation cluster region in 10 A/J adenocarcinomas and ACF. These results suggest that ß-catenin accumulation within ACF occurs independently of APC status. To assess the potential for differential activation of the Wnt pathway in A/J and AKR/J colons, we evaluated the status of c-myc and cyclin D1, two important ß-catenin targets. Although elevated nuclear c-Myc and cyclin D1 were detected in ACF from both strains, staining intensity was comparable (Fig. 1)Citation .

While alterations in p53 nuclear staining are typically associated with later stages of colorectal cancer, the status of this tumor suppressor in ACF has not been extensively evaluated (11 , 34 , 35) . Although p53 nuclear staining was consistently observed in ACF, the staining intensity was comparable between the two strains (Fig. 1, K and L)Citation . To extend our previous observations of accumulated wild-type p53 in A/J tumors (11) , we carried out PCR-based sequence analyses (exons 5 to 8) of the p53gene, using 10 laser-captured ACF from both strains and 5 A/J microadenomas. No mutations within this highly mutable region were identified. Thus, immunohistochemical analysis of key tumor biomarkers provided only minimal discrimination of high- and low-risk ACF.

LCM-Based Gene Expression Profiling.
We next carried out a genome-wide, array-based gene expression analysis to identify unique and discriminatory molecular profiles. LCM was used to procure pure cell populations. Hyperplastic and dysplastic ACF, microadenomas, and adjacent normal epithelium were laser-captured (Fig. 2)Citation . Because ACF are comprised of only limited numbers of cells (20 to 500), the RNA yield is minimal ({approx}50 to 500 fg/cell). Therefore, to circumvent the issues of contaminating cells and limiting RNA yield associated with these microscopic lesions, we combined the use of LCM with linear amplification (LCM-LA), using T7-based linear RNA amplification. This method routinely generated 30 to 70 µg of aRNA from laser-captured colonocytes (50 to 1000 cells). However, the size of ACF precluded verification of amplification linearity with a conventional comparison of nonamplified RNA versus amplified aRNA. Therefore, we analyzed expression profiles between nonamplified and amplified RNA from three adenomas and observed high correlation (r = 95). To additionally evaluate reproducibility of our labeling and hybridization strategies, eight random samples were collected in duplicate and hybridized to the array. The correlation coefficient of these duplicate experiments was r = 0.92. These results indicate that the T7-RNA amplification method preserves differential gene expression patterns. This methodology has been independently validated (31) .



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  		Fig. 2. Representative laser-captured sections of ACF from azoxymethane-treated mice and raw fluorescence intensities. Seven-micrograms thick OCT-embedded frozen sections were subjected to LCM as described under Materials and methods. ACF before laser-capture (A–D); same section after laser capture (E–H); captured cells from ACF (I–L).

 
Gene Expression Profiling of ACF.
Using LCM-LA, 50 colon specimens (Table 1Citation ; including random duplicates) resulted in 928,000 data points from 58 separate arrays. Single color hybridization with Cy5 enabled elimination of dye bias, a frequent problem in dual-color hybridization (31) . This strategy, combined with the 75-percentile normalization method, enabled direct comparison between multiple arrays (32) . The similarities in global expression profiles of saline-treated reference controls (r = 0.86) indicate that differences are an outcome of azoxymethane-induced alterations and not from inherent differences in basal gene expression between mouse lines (Fig. 3)Citation . To determine whether ACF could be segregated independent of histologic staging, we carried out cluster analysis on the entire data set (44 samples; refs. 30 , 33 ). As shown in Fig. 4ACitation , of 4896 genes interrogated by adaptive centroid algorithm, there was a clear separation between strains. Six general clusters (a-f, Fig. 4ACitation ) were identified based on unique expression, allowing complete segregation of A/J and AKR/J lesions. The six gene clusters (Fig. 4A)Citation contained a total of 226 genes in A/J ACF and 202 genes in AKR/J ACF that were significantly up- or down-regulated (2-fold) in at least one histologically defined subgroup. Although lesions did not show complete segregation according to histology using unsupervised clustering, subsequent analysis of specific data subsets derived from histologically similar lesions showed clear separation. Although most of the microadenomas clustered together, it was difficult to segregate hyperplastic and dysplastic ACF; additional evidence that subtle differences in gene expression profiles characterize these lesions. There was obvious segregation of adjacent normal-appearing mucosa from ACF (Fig. 4A)Citation .



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  		Fig. 3. Comparison of raw fluorescence intensities of saline-treated colons. Expression levels (in raw fluorescence intensity) of 4896 genes from laser-dissected cells from the colons of saline-treated A/J (n = 3) and AKR/J (n = 3) mice.

 


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  		Fig. 4. Cluster analysis of ACF from azoxymethane-treated mice using the adaptive centroid algorithm. A, dendrogram and heat map of the gene expression levels from hyperplastic ACF (H), dysplastic ACF (D), microadenoma (M), and adjacent normal-appearing (N) from azoxymethane-treated A/J and AKR/J colons, visualized using Genesite software (BioDiscovery). Of the 4896 genes represented on the array, 220 genes (rows) clustered into six broad clusters (A–F) showing similar and distinct expression patterns at different stages (columns) of colon tumor progression in A/J and AKR/J mice. Note the complete segregation of the lesions from sensitive and resistant strains. B, Venn diagram of the total number of significantly altered (2-fold) genes that were identified by adaptive centroid algorithm from A/J and AKR/J colons. Note that 126 of the genes identified were common to A/J and AKR/J ACF. C, expression profiles of a subset of genes from four clusters representing hyperplastic ACF (HACF), dysplastic ACF (DACF), microadenoma (MA), and adjacent normal cells from both strains. Although the histologic phenotype of the corresponding lesions are similar, gene clustering of hyperplastic (D) and dysplastic (E) ACF showed that the observed transcriptional profiles are unique and therefore useful in discriminating low- and high-risk ACF. The expression level of each gene is represented by the ratio of gene intensity in the lesion relative to expression in the saline-treated control colons. The color-bar at the bottom correlates color intensity to gene expression.

 
As shown in the Venn diagram (Fig. 4B)Citation , there was overlap of 126 genes between strains, demonstrating the likelihood of shared pathways in high- and low-risk lesions. Fig. 4CCitation shows four representative gene subclusters with distinct expression profiles. These subclusters were selected on the basis of divergent expression patterns and include genes consistently up- or down-regulated across lesions of variable histology within mouse strains.

Gene Profiles of Low- and High-Risk ACF.
To test the principal that ACF of similar morphology, but different risk potential, have unique molecular signatures, the following analyses were undertaken. Hyperplastic ACF were separated between high (A/J)- and low (AKR/J)-risk colons. Genes that are significantly and differentially regulated (up or down) at least 2-fold between A/J and AKR/J are represented in Fig. 4DCitation . The high-risk-A/J hyperplastic ACF showed increased expression levels of several genes belonging to the GTPase family, including Rac3 and Rab5.6 Genes involved in cell-cell adhesion (Mfge8), immune response (Psmd1), antiangiogenesis (Rnh1), as well as the transcriptional regulator Usf2, were all significantly up-regulated in the low-risk AKR/J hyperplastic ACF.5

Dysplastic ACF were examined using a similar set of criteria. Nine of 11 dysplastic ACF were segregated between the strains. We focused our analysis on clusters representing genes that afforded maximal segregation across strains (Fig. 4E)Citation . Genes that were up-regulated in high-risk A/J ACF and down-regulated in low-risk AKR/J ACF include acid sphingomyelinase-like protein (ASML3a), membrane-associated protein-17 (MAP-17), Rab24, secretory leukocyte serine protease inhibitor (SLPI1), thrombospondin 4, and Grim-19. To validate the array data, eight targets were selected from the various lesions for confirmation by real-time PCR. These results are shown in Table 2Citation and confirm the expression changes observed on the arrays.


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  		Table 2 Target genes validated by quantitative reverse transcription-PCR and their primer sequences

 
One of the genes that was elevated in the low-risk AKR/J ACF was IEX-1(Fig. 4Citation ; Table 2Citation ). IEX-1 plays a role in cell death and is regulated in part by nuclear factor-{kappa}B, p53, and c-Myc (38) . Given its robust increase in the resistant ACF, we examined IEX-1 status in human colon tumors by IHC. Cytoplasmic staining of IEX-1 was present in normal-appearing colonic crypts (Fig. 5)Citation , consistent with previous studies (39) . However, protein staining was minimal in a subset (6 of 10) of matched tumors, providing preliminary evidence for its potential role in colorectal cancer.



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  		Fig. 5. Immunohistochemical analysis of IEX1 expression in human normal and colorectal tumor tissue. A, strong cytoplasmic staining of IEX1 was present in adjacent normal-appearing colonic crypts. B, a markedly reduced level of staining for IEX1 was observed in the tumor crypts from the same patient. C, a second patient showing positive staining within normal colon crypts adjacent to tumor cells that are mostly negative (arrows). D, negative control section in which IEX-1 antibody was replaced with normal serum.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
ACF can be stratified into two broad classes, dysplastic and hyperplastic, based in part on morphologic characteristics and predicted biological behavior (4 , 5) . Histomorphologic evaluation and grading of ACF represent one of the few available biomarkers of colon cancer risk (6, 7, 8, 9, 10) . The present study was undertaken as an initial attempt to develop a molecular signature to discriminate ACF risk potential. IHC analysis of proliferating cell nuclear antigen, p53, and several components of the Wnt signaling pathway, including ß-catenin, APC, cyclin D1, and c-Myc (2 , 11 , 13 , 20 , 34, 35, 36) , afforded minimal discrimination of ACF. Surprisingly, intense ß-catenin nuclear staining was observed in ACF from both strains. This accumulation occurred in the absence of mutations within the glycogen synthase kinase-3 phosphorylation site and was independent of APC loss. Increased nuclear staining was associated with elevated levels of several key ß-catenin target genes, including cyclin D1 and c-myc, an effect that was not strain specific. The significance of the subtle difference in membrane staining of ACF between sensitive and resistant strains is presently under study.

Limited ACF discrimination by conventional IHC prompted a genome-wide, array-based expression analysis to identify discriminatory molecular profiles. Optimization of LCM-LA-MA afforded the opportunity to study gene expression patterns within individual colonic crypts. Although unsupervised clustering provided separation in the A/J and AKR/J colons, segregation of lesions based on histology was incomplete (Fig. 4A)Citation . However, within the complete data set, there were subsets of genes in both strains that were consistently altered by azoxymethane treatment. For example, in A/J colons, elevated levels of Pole2, Hes6, and sf3b1were observed in hyperplastic and dysplastic ACF, microadenomas, and adjacent normal-appearing epithelium. Consistent alterations in these genes throughout the carcinogenic process imply a fundamental role in tumor progression. On the other hand, genes involved in immune response (Ubc-rs2, Psmd13, and Li), cell-cell adhesion (Mfge8), and antiangiogenesis (Rnh1)6 were elevated in ACF from the resistant AKR/J colons. Effective immune surveillance, contact inhibition, and controlled angiogenesis are likely to play a fundamental role in limiting ACF progression, and the additional interrogation of these pathways in human colorectal cancer may prove informative.

To test the principal that ACF of comparable morphology, but distinctly divergent risk potential, are characterized by unique molecular signatures, analysis of hyperplastic and dysplastic ACF was undertaken using adaptive centroid algorithm. These analyses enabled a correlation of transcriptional profile(s) within lesion subtypes with respect to morphologic stage, an approach that will enable our understanding of transcriptional programs that influence tumor progression or growth arrest of putative precancerous lesions. Because dysplastic ACF are associated with tumor progression, we focused our analysis on clusters representing genes that afforded maximal segregation across strains (Fig. 4E)Citation . Genes that are elevated in the A/J dysplastic ACF and down-regulated in the AKR/J dysplastic ACF include ASML3a, MAP-17, Rab24, SPI1, thrombospondin 4, and Grim-19.ASML3a is a recently identified protein that binds with the DBCCR1(deleted in bladder cancer chromosome region 1) tumor suppressor gene (40) . In fact, elevated expression of both genes was reported in human urinary bladder tumors (40) , and it is possible that the high levels of ASML3aobserved in dysplastic A/J ACF contribute to tumor progression. Several other discriminatory targets were also identified. MAP-17was overexpressed in A/J dysplastic ACF, with only moderate levels present in morphologically matched AKR/J lesions. Although the precise role of MAP-17 has not been clarified, previous studies conducted in renal tubular epithelial cells have identified its association with the cytoplasmic aspect of the plasma membrane, implicating its role in cell-cell communication (41) . In addition, abundant levels of MAP-17 have been detected within carcinomas arising from kidney, colon, lung, and breast (41) . Although beyond the scope of the present investigation, additional studies to define the role of MAP-17 in human colon cancer is warranted. Rab24 (part of the Ras superfamily) is involved in vesicle transport, and we found that it was elevated in dysplastic A/J ACF. The potential significance of this observation with respect to colorectal cancer is based in part on earlier reports demonstrating high levels of this protein in hepatocellular carcinomas, prompting speculation that Rab24 may represent a proto-oncogene (42) . Protease inhibitors have been associated with attenuation of tumor progression and metastasis. However, recent studies indicate that expression of secretory leukocyte serine protease inhibitors (SLPI1) may elicit an opposite effect, facilitating tumor progression, metastasis, and even a poor prognosis in humans (43) . Interestingly, high levels of SLPI1 transcripts were observed in A/J dysplastic ACF, representing the first association of this serine protease inhibitor in colon tumorigenesis.

Cluster analysis identified a gene panel that was significantly induced in resistant AKR/J ACF. These genes include IEX-1, Mfge8, Rnh1, Psmd13, and Usfs2 (Fig. 4E)Citation . The increased expression of a subset of genes involved in protein biosynthesis, DNA repair, transcription regulation, members of the nuclear factor-{kappa}B family, ion transport, and cell metabolism raise the possibility for establishing a gene signature for low-risk ACF. In fact, each of the genes represented within this cluster have putative functions that could potentially play a role in limiting ACF progression. For example, IEX-1is induced by various cell stressors, including ionizing radiation, UV radiation, and growth factors (45) . IEX-1, an nuclear factor-{kappa}B target gene, inhibits the activation of nuclear factor-{kappa}B, thereby counteracting its antiapoptotic potential (45) . Whether IEX-1 elicits a comparable protective effect in low-risk ACF is certainly a possibility. To assess the potential relevance of IEX-1 in human colorectal cancer, we evaluated a total of 10 matched tumors with adjacent normal epithelium by IHC. As shown in Fig. 5Citation , there was a marked reduction in IEX-1 staining within the tumor cells, suggesting the potential involvement of this immediate early gene in colon tumorigenesis. Additional analysis of this protein and its functional significance in human colorectal cancer is under way.

Although the importance of ACF as biomarkers of colon cancer risk is well established (5 , 10 , 36 , 46 , 47) , few studies have attempted to additionally stratify these lesions at the molecular/genetic level. The incipient and dynamic nature of ACF is likely to complicate histology-based methods of prognostication. Additionally, it is known that early adenomas that are similar in appearance may be biologically distinct with varying rates of malignant conversion (46 , 47) . Thus, our primary objective was to identify unique transcriptional profiles of ACF at similar histologically defined stages before the onset of adenomas. Although one cannot rule out the possibility that the luminal environment may affect ACF outcome, it is clear from the identified gene clusters that ACF do, in fact, harbor distinct genetic profiles that may result in a divergence of phenotypes and subsequent tumorigenic potential. Interestingly, the genes that were differentially expressed in A/J and AKR/J ACF represent only a small percentage (<5%) of the total number of genes interrogated, underscoring the fundamental similarity of lesions at the earliest stages of tumorigenesis. Despite significant overlap, however, our data have identified a number of discriminatory targets that may affect the biological outcome of ACF. These data thus provide a proof-in-principle of our ability to stratify risk potential of ACF and provide the rationale for assessing the expression characteristics of a unique set of genes in human ACF.

Increased knowledge of gene expression patterns and risks inherent in ACF may additionally inform the relationship between human ACF and tumorigenic potential. Patients with colorectal cancer demonstrate increased numbers of ACF (with greater percentages of large and dysplastic ACF) relative to patients with benign adenomas or those without evidence of pathology. These ACF are not uniformly distributed and cluster, both in cancer and polyp patients, within the distal bowel (48, 49, 50) . Takayama et al. (51) have reported that in patients with adenomas and ACF, 96% of polyps were located in the left colon. The gene expression profiles from high- and low-risk mouse strains suggest that identification of molecular signatures from clinically accessible distal ACF may give new insight into cumulative risk for an individual or population at large. Thus, the use of predictive gene profiling as an adjunct to traditional histologic analysis permits the recognition of differences between histologies that may otherwise be difficult to stratify. By extrapolation, a similar approach to discriminate human ACF on the basis of expression profiles may provide enormous prognostic benefit in individuals at varying risk of colorectal cancer.


   FOOTNOTES
 
Grant support: NIH Grant CA 81248 and a grant from the Robert Leet and Clara Guthrie Patterson Trust.

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 18 U.S.C. Section 1734 solely to indicate this fact.

Note:P.R. Nambiar and M. Nakanishi contributed equally to this publication.

Requests for reprints: Daniel W. Rosenberg, Center for Molecular Medicine, University of Connecticut Health Center, 263 Farmington Avenue, CT 06030-3101. Phone: (860) 679-8704; Fax: (860) 679-1140; E-mail: rosenberg{at}nso2.uchc.edu

6 Internet address: http://www.celera.com. Back

Received 3/18/04. Revised 6/17/04. Accepted 7/15/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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