Kishore Guda

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

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, β-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 β-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.

Carcinogen-induced colon tumors in mice are chromosomally stable and are characterized by low-level microsatellite instability

The azoxymethane (AOM)-induced mouse colon tumor model recapitulates many of the histopathological features associated with the multistage progression of human sporadic colorectal cancers (CRCs). To better define the genetic events associated with tumorigenesis in this murine model, we analysed tumors from A/J mice for chromosomal (CIN) and microsatellite (MSI) instabilities, two fundamental pathways of genomic instability that play a critical role in the pathogenesis of human CRCs. Male A/J mice, 6-week old, were injected with either AOM (n=5) (10 mg/kg b.w., i.p.) or vehicle (n=5) (0.9% NaCl solution) once a week for 6 weeks. At 32 weeks after the last dose, comparative genomic hybridization (CGH) was performed on 16 tumors harvested from five animals. Although 25% of the tumors displayed either a gain of chromosome 2 or loss of Y, the majority (75%) showed no genomic imbalances. Further analysis of chromosomal aberrations, using CGH and spectral karyotyping (SKY) was performed in our recently established A/J colon tumor-derived cell line, AJ02-NM0. Results showed a pseudotetraploid karyotype with loss of only the Y chromosome in these cultured cells, thereby providing additional evidence for the minimal role of CIN in the primary AOM-induced tumors. Interestingly, the majority (81%) of A/J tumors displayed low-level microsatellite instability (MSI-L) when analysed using mono- and dinucleotide repeat markers, and showed a significant expansion to high-level instability (MSI-H) in the AJ02-NM0 cells. This finding in cultured cells additionally provides evidence that a mild mutator pathway may contribute to the development of behaviorally benign carcinomas in situ in A/J mice. To better understand the tumorigenic process in the A/J colons, we screened for mutational alterations in key regions of the K-ras and Apc genes. Results showed a very low frequency (6%) of K-ras activating mutations, together with the absence of Apc truncation mutations in primary tumors and AJ02-NM0 cells. However, these tumors displayed intense nuclear accumulation of β-catenin protein, indicating activation of the Wnt signaling pathway. Based on our molecular and cytogenetic findings, we propose that carcinogen-induced tumors may develop via mechanisms independent of the ‘classical’ CIN or MSI pathways.

Dietary iron promotes azoxymethane-induced colon tumors in mice.

Abstract: There is accumulating evidence that high levels of dietary iron may play a role in colon carcinogenesis. We used a mouse model to investigate the impact of elevated dietary iron on incidence of aberrant crypt foci (ACF; a preneoplastic lesion) on tumor formation and on induction of oxidative stress. A/J mice were injected intraperitoneally, once a week for 6 weeks, with the colonotropic carcinogen, azoxymethane (AOM) or saline (vehicle controls). Following AOM or saline treatments, mice were placed on diets of high (3,000 ppm) and low (30 ppm) iron. Mice in each treatment group were sacrificed at 6 and 10 weeks following the final injection with AOM or saline. Colons were removed for subsequent histopathological analysis, which revealed average increases of 4.6 ± 1.3 vs. 10.4 ± 2.5 total tumors at 6 weeks and 30.75 ± 2.7 vs. 41.5 ± 4.4 total tumors at 10 weeks per AOM-treated mouse on low- and high-iron diets, respectively. There were no significant differences in incidence of ACF attributable to iron, although there was a trend toward greater crypt multiplicity per focus in mice on high-iron diets. Notably, no tumors were observed in mice receiving vehicle control injections in place of carcinogen, regardless of the level of dietary iron. These data suggest that iron exerts its effect at the stage of tumor promotion, but is not sufficient to initiate tumor formation. To learn more about mechanisms by which iron promotes tumor growth, colons were assayed for several biomarkers of oxidative stress [BOS; total F2-isoprostanes (F2-IsoPs), 15-F2t-isoprostanes (8-IsoPGF2αs), Isofurans (IsoFs), and 8-hydroxyguanosines (8-OH[d]Gs)], as well as iron absorption, programmed cell death, and cellular proliferation. Elevated PCNA and TUNEL staining of the colon epithelium revealed hyperproliferative and apoptotic responses to iron, while no significant differences between iron groups were observed in each of the BOS that were assayed. Our results suggest that, following carcinogen exposure, elevated dietary iron promotes the growth of tumors with altered cellular homeostasis through a mechanism that is independent of oxidative stress.

Aberrant transforming growth factor-β signaling in azoxymethane-induced mouse colon tumors

Alterations in the transforming growth factor‐β (TGF‐β) pathway are implicated in the pathogenesis of colorectal cancer. We hypothesize that alterations in the TGF‐β pathway contribute to differential sensitivity of mice to the colon carcinogen azoxymethane (AOM). A/J (sensitive) and AKR/J (resistant) mice were injected intraperitoneally with AOM (10 mg/kg of body weight once a week for 6 wk). Twenty‐four weeks after AOM exposure, mutational analysis of TGF‐β type II receptor (TβR‐II) from normal colons and from tumors showed no AOM‐induced alterations. A significant decrease (1.5‐fold, P < 0.05) in TβR‐II mRNA levels, however, was found in A/J tumors with the RNase protection assay. Immunofluorescence of TβR‐II showed marked loss of staining in A/J tumors. The RNase protection assay and sequence analysis of the downstream signaling molecule Smad3 revealed no carcinogen‐induced alterations in either strain. To gain further insight into the functionality of the pathway, expression of TGF‐β, TGF‐β type I receptor (TβR‐I), and several downstream targets of TGF‐β signaling, including Smad7, c‐myc, and p15, was examined. Although no alterations in TGF‐β, TβR‐I, or Smad7 were found in tumors, a significant increase in c‐myc expression (2.5‐fold, P < 0.05 ) and a significant decrease in p15 expression (4.5‐fold, P < 0.05 ) were noted. Concomitant repression of TβR‐II and overexpression of c‐myc may render epithelial cells insensitive to TGF‐β‐mediated growth arrest, a possibility that also is suggested by this model. The significant decrease in p15 expression in tumors provides additional evidence that TGF‐β signaling may be markedly attenuated during colon tumorigenesis.

Multistage gene expression profiling in a differentially susceptible mouse colon cancer model

The DNA alkylating agent, azoxymethane (AOM), induces tumor formation in the distal colon of susceptible mice. Differential susceptibility to this colonotropic carcinogen has been well characterized in A/J (sensitive) and AKR/J (resistant) mice. However, the reasons underlying the differential response to AOM and the molecular mechanisms involved in colon tumor progression remain unclear. To address these issues, we used a cDNA microarray approach to determine time-related changes in gene expression patterns in A/J and AKR/J colons following carcinogen treatment. In the present study, mice were injected intraperitoneally with either AOM (10mg/kg body weight once a week for 6 weeks) or 0.9% NaCl solution (vehicle controls). Total RNA was isolated from the distal colons at 1, 4, and 24 weeks post-AOM exposure. RNA was reverse transcribed and cDNA samples labeled with Cy3 and Cy5 were hybridized to a glass chip containing 4608 mouse cDNA duplicate clones. The resulting mRNA expression levels were analyzed using GLEAMS 3.0, a Unix/Linux-based software program. Genes with more than twofold variations in expression levels were considered significant. Further clustering analysis was performed based on gene expression patterns at different time points using a novel adaptive centroid algorithm (ACA). Of the 4608 genes, 118 clustered into 11 significant groups that displayed similar and distinct expression patterns between the strains following carcinogen treatment. Nine clusters were selected for further analysis based on their divergence in response between A/J and AKR/J and their potential role in tumorigenesis. Overall, our data indicate time- and strain-specific genetic alterations during different stages of colon tumorigenesis following AOM treatment.

p53 and its co-activator p300 are inversely regulated in the mouse colon in response to carcinogen.

We examined the p53 response following acute exposure of mice to the colon-specific carcinogen azoxymethane (AOM). No overall induction of p53-regulated genes was observed in the mouse colon, and only a small subpopulation of apoptotic colonocytes showed increased Bax staining. In contrast, the liver showed dramatic increases in p53-regulated gene expression. Subdued p53 gene activation in the colon did not appear to result from a lack of p53 stabilization, but did correspond to a drop in the expression of its transcriptional co-activator, p300. We propose that inefficient gene activation by p53 in the colon contributes to the organotrophic effects of AOM.

Defective processing of the transforming growth factor-β1 in azoxymethane-induced mouse colon tumors

High levels of the cell growth inhibitor transforming growth factor‐β1 (TGF‐β1) are often found in a variety of human cancers. However, the physiological significance of this overexpression depends on the availability of the biologically active form of TGF‐β1 within the extracellular matrix of the tumor microenvironment. To determine the expression and activation status of TGF‐β1 in chemically induced tumors, 6‐wk‐old A/J mice were injected intraperitoneally with either azoxymethane (AOM) (10 mg/kg body weight, once a week for 6 wk) or normal saline solution, and colon tumors were isolated 24 wk following the last injection. An enzyme‐linked immunosorbent assay for TGF‐β1 revealed a significant increase (1.7‐fold, P < 0.05) in total TGF‐β1 protein in tumors. Interestingly, while 80% of the total TGF‐β1 in the control colon tissues was in the active form, only 50% was found to be active in tumors. Together with our earlier observations that TGF‐β1 mRNA levels are unchanged in A/J tumors, these data further support a mechanism whereby elevated TGF‐β1 levels result from a defective activation and turnover of this protein. Because plasmin is known to be a major activator of TGF‐β1 in vivo, we hypothesized that reduced plasmin activity may be responsible for the observed dysregulation of TGF‐β1 processing in these behaviorally benign tumors. With a fluorogenic peptide substrate for serine proteases, a deficiency in plasmin activity was found in the tumors. Furthermore, semiquantitative reverse transcription (RT)‐polymerase chain reaction (PCR) analysis of a panel of genes involved in the plasminogen activation system, including plasminogen activator inhibitor‐1 (PAI‐1), urokinase‐plasminogen activator (u‐PA), and urokinase‐receptor (u‐PAR‐1), demonstrated a significant upregulation (approximately fourfold to sixfold, P < 0.05) in the expression of each of these genes in the tumor tissue. In addition, no significant changes were observed in the expression levels of thrombospondin‐1 (TSP‐1) and insulin‐like growth factor type II receptor (IGF‐IIR), which also mediate the activation of latent TGF‐β1. To gain further insight into the functionality of the TGF‐β1 pathway, cDNA microarrays were performed and the expression levels of a panel of 21 TGF‐β1–specific target genes were determined in AOM‐induced tumors that overexpress the ligand. A significant dysregulation in the expression of each of these targets was observed, providing evidence of aberrant TGF‐β1 signaling in tumors. Overall, the present study demonstrates a very low plasmin activity in A/J colon tumors, possibly as a result of the potent inhibitory effect of PAI‐1 on the plasminogen activation cascade. The observed deficiency in plasmin activity may not be sufficiently compensated for by other mechanisms of latent TGF‐β1 activation, including TSP‐1 and IGF‐IIR, thereby resulting in a decreased fraction of the biologically active form of TGF‐β1 and subsequent aberration in TGF‐β1‐specific gene regulation in A/J tumors.

Vascular endothelial growth factor and enhanced angiogenesis do not promote metastatic conversion of a newly established azoxymethane‐induced colon cancer cell line

The organo‐specific carcinogen, azoxymethane (AOM), produces colon tumors in mice that share many pathological features with sporadic human colorectal cancer (CRC). An important distinction between AOM‐induced CRC and human CRC is lack of mucosal invasion in the murine model. To assess the role of the microenvironment in preventing the invasive phenotype, multiple benign in situ adenocarcinomas were harvested from AOM‐treated mice and cultured in vitro. However, tumor cell growth was extremely limiting under standard culturing conditions. Thus, we injected tumor cells directly into nude mice and performed two serial transplants, and successfully explanted a rapidly growing epithelial tumor cell line (AJ02nm0). When injected subcutaneously (sc) into nude mice, AJ02nm0 cells formed well‐differentiated adenocarcinomas with minimal tumor invasive capacity. To define whether metastatic and invasive potential were related to lack of angiogenic stimuli, the AJ02nm0 cells were transfected to overexpress murine vascular endothelial growth factor‐164 (VEGF164). AJ02nm‐VEGF cells produced rapidly growing tumors in nude mice that exhibited extensive pseudo‐epithelial ductal architecture and supporting vasculature, but without increased invasive potential compared to controls. The established murine colon epithelial cell line provides a useful experimental model to further elaborate genetic and epigenetic factors that may promote or inhibit colon tumorigenesis and metastasis.