Aspirin and Low-Dose Nitric Oxide–Donating Aspirin Increase Life Span in a Lynch Syndrome Mouse Model

Nonsteroidal anti–inflammatory drugs (NSAID) are a structurally diverse family of compounds that are effective in the prevention of colorectal cancer (1, 2). Acetylsalicylic acid (ASA), commonly known as aspirin, is the archetype of the NSAID family. Epidemiologic studies have reported an inverse relationship between ASA use and the incidence of colorectal cancers (3, 4). Animal models have confirmed that administration of various NSAIDs results in fewer tumors (5, 6).

Nitric oxide–donating NSAIDs (NO-NSAIDs) are novel compounds in which a NO-donating group is attached to the NSAID through a linker molecule. The rationale is that the NO moiety may diminish or alleviate undesirable NSAID-induced side effects such as gastropathy (7, 8). NO-NSAIDs also share many pharmacologic properties with their parental molecules and may possess greater efficacy as chemopreventive agents (7, 8). NO-ASA is the most potent NO-NSAID reported to date, showing at least 100-fold more activity than other NO-NSAIDs in diverse experimental systems (9–12). It is also more effective than aspirin at reducing tumorigenesis in rodent models of cancer (13–16).

The DNA mismatch repair (MMR) pathway recognizes and repairs nucleotide mispairs generated by postreplication misincorporation and genetic recombination between heteroallelic DNAs, as well as several DNA damage–specific lesions (17, 18). There is compelling evidence that links mutations in the MSH2, MSH6, MLH1, and PMS2 genes with Lynch syndrome/hereditary nonpolyposis colorectal cancer (LS/HNPCC; ref. 19). Tumor tissues from most LS/HNPCC cases associated with MMR defects display microsatellite instability (MSI; ref. 20). The mechanism of tumorigenesis has been linked to the mutator phenotype detected by MSI, which provides the environment for the accumulation of multiple secondary mutations that drive tumorigenesis (21).

ASA suppresses the MSI mutator phenotype of MMR-deficient human colon tumor cell lines (22, 23) via a genetic selection that appears to enhance apoptosis in critically unstable cells. The long term outcome is a cell population that has a persistent deficiency in MMR but paradoxically has acquired a largely microsatellite stable (MSS) phenotype (22, 23). NO-ASA also suppresses MSI in MMR-deficient cell lines at concentrations 300- to 3,000-fold less than ASA (23). It was anticipated that treatment with ASA and NO-ASA would delay and/or prevent tumorigenesis in LS/HNPCC animal models of intestinal cancer.

Here, we examined the effect of ASA and NO-ASA in a mouse model of LS/HNPCC (Msh2^flox/floxVpC^+/+) that recapitulates the intestinal-specific disruption of Msh2 function (24). ASA (400 mg/kg) and low-dose NO-ASA (72 mg/kg) increased the life span of the LS/HNPCC mice. Increased survival by NO-ASA occurred at a 10-fold lower equimolar dose than that by ASA. However, dietary exposure of mice to 720 mg/kg NO-ASA, an equimolar dose to 400 mg/kg ASA, as well as 1,500 mg/kg NO-ASA accelerated intestinal tumorigenesis uniquely in MMR-deficient LS/HNPCC mice. This study suggests the long-term use of ASA in LS/HNPCC and should also enable reconsideration of NO-ASA structure such that higher doses, greater efficacy, and reduced toxicity may be accomplished.

The development and subsequent characterization of the LS/HNPCC mouse model used in this study, herein designated Msh2^flox/floxVpC^+/+, has been reported elsewhere (24). The targeting strategy has been outlined in Supplementary Figure S1. Mice were maintained on a mixed background of C57Bl/6J (∼93%) and 129X1/SvJ (∼7%). The following primers were used for genotyping at the floxed Msh2 locus: 184F (TAC TGA TGC GGG TTG AAG G); 130F (TGT GCT GGC TCA CTT AGA CG); 165R (GGC AAA CTC CTC AAA TCA CG). Tissues containing the floxed Msh2 allele yielded a PCR product of 984 bp, amplified by primers 130F and 165R. Cre-mediated loxP deletion of Msh2 exon 12 was detected at the molecular level by PCR with primers 184F and 165R, which yielded a 341-bp product (Supplementary Fig. S2). The Cre transgene was genotyped with the following primers: IMR0015 (CAA ATG TTG CTT GTC TGG TG); IMR0016 (GTC AGT CGA GTG CAC AGT TT); IMR1878 (GTG TGG GAC AGA GAA CAA ACC); IMR1879 (ACA TCT TCA GGT TCT GCG GG). Primers IMR0015/IMR0016 amplified a 200-bp product, indicative of the endogenous wild-type allele. Primers IMR1878/IMR1879 yielded an 1,100-bp product, indicative of the villin promoter-Cre (VpC) transgene. The VpC control mice were obtained from The Jackson Laboratories [strain B6.SJL-Tg(Vil-cre)997Gum/J] and were genotyped as above.

Animals were bred in a barrier one facility and were maintained according to the NIH animal care and use guidelines. All experiments involving animals received prior approval from the OSU Institutional Animal Care and Use Committee.

ASA was obtained from Sigma. The ortho isomer of NO-ASA [2-(acetyloxy)benzoic acid 2-[(nitrooxy)methyl]phenyl ester; NCX-4060] was synthesized by the Indofine Chemical Company, Inc. Animals were fed a powdered diet from Harlan Laboratories, Teklad LM-485 (7912). Aspirin and NO-ASA were mixed with powdered food and administered ad libitum. Treatment was started after weaning when mice were around 25 days of age. Food was formulated on a weekly basis and any food remaining from the prior week was discarded.

The Msh2^flox/floxVpC^+/+ mice used in this study developed intestinal tumors spontaneously over their lifetime. Long term, this resulted in moribund animals that presented with a variety of tumor-related signs including weight loss; marked abdominal distension, possibly accompanied by a hunched posture and poor coat quality; dyspnea; possible enlarged swelling of the lymph nodes around the front and hind legs; and anemia. Animals were inspected daily, in accordance with the guidelines required by the OSU Institutional Animal Care and Use Committee. On observation of these conditions, in addition to lethargy for more than 24 hours, unresponsiveness to stimuli, or anorexia for more than 24 hours, such mice were sacrificed and a complete pathologic examination conducted. Starting at 6 months, all animals were weighed weekly. Weight loss of greater than 20% resulted in removal of the animals from the study for subsequent pathology. If a consensus opinion could not be reached about a particular animal's health, the attending veterinarian was consulted.

All animals in the study were subject to criteria for early removal and were euthanized by CO₂ asphyxiation, followed by cervical dislocation, for pathologic examination before they died from tumor-associated complications. Mice were examined for the presence of tumors. Intestinal tumors were examined macroscopically and with a dissecting microscope. Tumor number and location were recorded. Tissues were fixed in 10% formalin, embedded in paraffin, and 4 μm sections were cut for slides. Sections were stained with hematoxylin and eosin according to standard protocols. A trained pathologist evaluated the slides.

The mouse primers used for MSI analysis have been reported previously as TG₂₇, TA₂₇, GA₂₉, CT₂₅/CA₂₇, and A₃₃ (25). They have been redesignated herein as MSM01 to MSM05, respectively. One oligomer of each primer set was 5′-labeled with FAM, VIC, or NED. Details for all primer pairs are presented in Supplementary Table S3. Genomic DNA was prepared from mouse tissues using a DNeasy tissue kit (QIAGEN). DNA was amplified with Platinum Pfx DNA polymerase. The final components of the reaction mix were as follows: DNA (2–10 ng); amplification buffer (2× concentration); 1 mmol/L MgSO₄; 300 μmol/L deoxynucleotide triphosphates; 300 nmol/L of each primer; 1 unit of Pfx; and H₂O to a final volume of 15 μL. The following amplification conditions were used as a standard: 95°C, 2 minutes (94°C, 20 seconds; 58°C, 30 seconds; 68°C, 30 seconds) for 30 to 35 cycles; 68°C, 2 minutes. Samples were diluted and analyzed on an Applied Biosystems 3730 DNA analyzer. At least 2 independent amplification reactions of each microsatellite sequence were examined for each DNA sample. If there was any ambiguity about the outcome, the amplification was repeated a third time.

Small-pool PCR analysis of MSI was conducted in a similar manner, except that approximately 100 pg of DNA was used as a starting template. Forty-eight reactions were carried out on each DNA sample from matched sets (n = 8) of ear (E), normal intestinal (N), and tumor intestinal (T) tissues from each of the treatment groups. The microsatellite profiles were transformed into distribution patterns similar to the examples presented in Figure 4.

Comparative MSI was calculated by assessing changes at microsatellite loci between matched sets of normal intestinal and tumor tissues from mice in each treatment group. The relative degree of MSI in tumors was subsequently scored as follows: MSS, no comparative changes in microsatellite status; low MSI (MSI-L), MSI displayed in 1 of 5 (≤25%) microsatellite markers; high MSI (MSI-H), MSI displayed in at least 2 of 5 (>25%) markers (See Supplementary Table S2). We often found that one marker was largely uninformative. However, this criteria is identical to the National Cancer Institute definition of MSI (20). Numbers are expressed as a percentage of the total number of tumors analyzed for each group. Fisher's exact test (2 tailed) was used to calculate the significance of comparative changes in MSI between MSI-L and MSI-H tumors from NSAID-treated Msh2^flox/floxVpC^+/+ mice.

All statistical analyses were generated with Graphpad Prism 5.0 software. Survival data from the Kaplan–Meier plots were compared with the log-rank test. Differences in tumor burdens were compared with the Mann–Whitney U test. Comparative MSI was evaluated with a 2-tailed Fisher's exact test. In all cases, a value of P ≤ 0.05 was considered statistically significant.

Only cells of the intestinal epithelia were specifically targeted for Msh2 knockout in our LS/HNPCC (Msh2^flox/flox VpC^+/+) mouse model (Supplementary Figs. S1–S3). Msh2-dependent tumorigenesis was confined to the intestines and had all of the pathologic hallmarks of HNPCC tumors in humans (24). Groups of Msh2^flox/floxVpC^+/+ mice were treated as follows: untreated, 400 mg/kg ASA, 72 mg/kg NO-ASA, 720 mg/kg NO-ASA, and 1,500 mg/kg NO-ASA. The 720-mg/kg NO-ASA dose is equimolar to that of 400 mg/kg ASA. Drugs were mixed with powdered diet and administered ad libitum. Administration of compounds by this method precluded an accurate estimation of the amount of food consumed per animal. Both ASA and NO-ASA, even at the higher doses appeared equally palatable to mice. Thus, there were no apparent issues associated with consumption of drug-laced feeds. We did not examine Msh2^flox/flox mice because in the absence of villin-Cre, these animals would be similar to wild-type mice where tumors do not develop because of functional Msh2 in the intestinal tissues (24). Groups of (Msh2^+/+)VpC^+/+ mice were included to control for confounding effects of constitutive Cre expression in the intestine which could potentially cause tumorigenesis. One group was untreated and the other received 720 mg/kg NO-ASA. Kaplan–Meier survival plots were generated for mice in each group (Fig. 1).

Median survival times for LS/HNPCC mice were as follows: untreated (333 days), 400 mg/kg ASA (393 days), 72 mg/kg NO-ASA (403 days), 720 mg/kg NO-ASA (229 days), 1,500 mg/kg NO-ASA (175 days). Pairwise comparisons between groups were significant, with P < 0.0001 (log-rank test), except untreated mice versus 400 mg/kg ASA (P = 0.0007) and untreated mice versus 72 mg/kg NO-ASA (P = 0.0003). Unexpectedly, dietary exposure to 720 and 1,500 mg/kg of NO-ASA significantly decreased survival of Msh2^flox/floxVpC^+/+ mice in a dose-dependent manner (Fig. 1). In contrast, the untreated and 720 mg/kg VpC^+/+ control groups survived in excess of 800 days (Fig. 1) and their median survival times could not be calculated because they were eventually removed from the study to determine tissue MSI and tumor status, if any. On the basis of previous work (24), it is evident that the effects of high-dose NO-ASA observed in our studies are genotype specific; only manifesting in the LS/HNPCC mouse model but not wild-type mice. These effects appear to be associated with intrinsic pharmacokinetic features of the drug (discussed later). ASA and low-dose NO-ASA regimes increased the survival of LS/HNPCC mouse model by 18% to 21%.

Tumors were confined to the intestines of the LS/HNPCC mice predominantly in the duodenum and jejunum, and rarely in the ileum (Table 1). Although mice treated with ASA and low-dose NO-ASA lived longer and presented with tumors significantly later than untreated mice, they developed statistically equivalent numbers of tumors at the time of death (1.68 ± 0.72 and 1.78 ± 1.25 vs. 1.90 ± 1.02, respectively). Because the endpoint of this study was survival, we cannot determine whether ASA and low-dose NO-ASA affect initiation and/or progression of the tumorigenic process in this mouse model.

In contrast, Msh2^flox/floxVpC^+/+ LS/HNPCC mice treated with 720 and 1,500 mg/kg NO-ASA displayed a 3- to 4-fold increase in tumor number (5.10 ± 2.81 and 5.00 ± 1.73 tumors, respectively; Fig. 2A and Table 1). These values are significant when compared with the untreated, ASA-treated, or 72 mg/kg NO-ASA–treated mice (P ≤ 0.0002; Mann–Whitney U test). Mice receiving 720 mg/kg NO-ASA developed as many tumors as those on 1,500 mg/kg, but the mean survival time of the latter group was only 76% that of the former (175 vs. 229 days). Although more intestinal lesions and tumors developed in mice treated with high-dose NO-ASA, similar types of lesions, including adenomas and adenocarcinomas, were identified in all Msh2^flox/floxVpC^+/+ LS/HNPCC mouse model groups (Fig. 2B and Supplementary Table S1). Taken together with the survival analysis, these results suggest that high-dose NO-ASA accelerates genotype-specific tumorigenesis in the LS/HNPCC mouse model.

To determine whether tissues from the drug-treated LS/HNPCC mouse model displayed MSI characteristic of MMR deficiency, DNA was isolated from (E), (T), and (N) tissues. Analysis of MSS ear tissues (MMR proficient) provided constitutional microsatellite profiles from which subsequent evaluations of corresponding normal and tumor intestinal instability were made. Five microsatellite markers, MSM01 to MSM05, were investigated for each matched set of 3 tissues. Representative examples of the MSI profiles are presented in Figure 3. MSM02 has not been included, as it was only informative for 1 of 158 intestinal samples from Msh2^flox/floxVpC^+/+ LS/HNPCC mice (Supplementary Table S2). The microsatellite profiles of both normal and tumor intestinal tissues remained unstable relative to the MSS patterns of the ear in ASA- and NO-ASA–treated mice (Figs. 3 and 4 and Supplementary Table S2). Intestinal tissues, either normal or tumor, from Msh2^flox/floxVpC^+/+ LS/HNPCC mice displayed MSI, with 147 of 158 tissues showing differences at 2 or more microsatellite markers when compared with the ear samples. However, intestinal tissues and the occasional intestinal tumor which developed in untreated VpC^+/+ mice or VpC^+/+ mice treated with ASA were always MSS (Figs. 3A and 4A and Supplementary Table S2).

Comparative MSI analysis provided a measure of the relative differences in instability between adjacent normal and tumor samples derived from the same tissues. We used the Bethesda Criteria to evaluate MSI status (20): MSS, no changes in microsatellite marker status; low MSI (MSI-L), MSI displayed in 40% or less microsatellite markers; high MSI (MSI-H), MSI displayed in more than 40% of the markers. Fisher's exact test was used to calculate the significance of comparative changes between MSI-L and MSI-H for tumors from NSAID-treated Msh2^flox/floxVpC^+/+ mice. MSI-L and MSI-H tumor numbers from untreated Msh2^flox/floxVpC^+/+ mice were used as the baseline. On the basis of our previous cellular data, we expected only a partial suppression of the MSI phenotype, which might cause a shift in the relative levels of MSI-H to MSI-L (22).

High-dose NO-ASA induced relative increases in MSI where 94% of intestinal tissues from the 720 mg/kg and 86% of tissues from the 1,500 mg/kg treated mice displayed MSI (Table 1). Moreover, 75% of tissues from mice receiving 720 mg/kg NO-ASA were characterized as MSI-H compared with 33% of untreated. When the results of 1,500 mg/kg MSI are included, these data suggest a trend where high-dose NO-ASA may exacerbate MSI in LS/HNPCC mice that correlates with the accelerated tumorigenesis. Treatment with low-dose NO-ASA does not appear to aggravate or attenuate MSI in intestinal tissues of LS/HNPCC mice. In contrast, treatment with ASA appears to provoke partial stabilization of MSI in the intestinal epithelia. Untreated animals had a comparative MSI-L of 39% and MSI-H of 33%, whereas ASA-treated LS/HNPCC mice displayed an MSI-L of 56% and MSI-H of 22% (Table 2 and Supplementary Table S2). These differences also did not attain statistical significance. However, there was a trend toward MSI stabilization by ASA that is consistent with the cellular studies (22, 23).

Additional high-resolution MSI analyses were conducted by small-pool PCR with marker MSM01 (TG₂₇) on matched (E), (N), and (T) DNAs for a subset of mice in each treatment group. This enabled a more extensive assessment of the distribution of novel allelic variants at this locus in the microsatellite unstable normal and tumor intestinal tissues. The resulting profiles comprised a representational distribution of the intrinsic allelic diversity within each tissue (Fig. 4). Ear tissues from these animals were consistently MSS, with the major 137-bp allele always flanked by a relatively unvarying distribution of subsidiary alleles. Normal and intestinal tissues from LS/HNPCC mouse model in all 5 experimental groups showed variation in both major allele sizes and allele distributions. Nevertheless, small-pool PCR analysis suggests that long-term treatment with ASA decreased both the distribution and number of allelic variants compared with untreated mice (Fig. 4). Taken as a whole, these observations appear to suggest that the underlying mechanism(s) through which ASA increases animal survival in our LS/HNPCC mouse model is by suppression of MSI. However, additional studies will be required to fully support this conclusion.

The role of NSAIDs in the prevention of colorectal cancer is mediated through various mechanisms including the inhibition of COX-2 and the induction of apoptosis through COX-2 independent pathways (26). Previous studies from our laboratory suggested that ASA partially suppresses the MSI-H phenotype of human colon cancer cell lines through an apoptotic mechanism that appeared to be COX independent (22). In vitro ASA has been shown to arrest colon cancer cells at the G₁/S checkpoint and induce apoptosis through activation of ATM, p21, and BAX (27). Among NSAIDS, ASA is still a preferred choice for chemoprevention in average-risk individuals. This is not only because of its shown chemopreventive efficacy but also because of its unique potential in cardiovascular protection (3, 4).

In this study, we investigated the chemopreventive potential of ASA and NO-ASA using a newly developed mouse model of LS/HNPCC. LS/HNPCC mice treated with ASA at 400 mg/kg had an increased median survival time compared with untreated controls, with about 18% of the mice living to more than 500 days (Fig. 1). Although ASA reduces tumor numbers in Apc^Min/+ mouse models of colorectal cancer (28), and ASA may have a weak effect on Apc^Min/+Msh2^−/− mice treated in utero (29), this report is the first clear evidence that dietary aspirin increases survival in an LS/HNPCC mouse model (30).

An increase in survival of 18% to 21% could be described as modest. However, this is still an important finding as it suggests that ASA should be considered as a chemopreventive alternative that may delay the rapid progression of tumors between clinical screenings of LS/HNPCC patients (31). Low-dose NO-ASA (72 mg/kg) showed similar efficacy toward increased life span at 10-fold less equimolar dose compared with ASA (Fig. 1). It is possible that this lowered dose could significantly reduce the gastropathies associated with long-term ASA administration.

ASA and low-dose NO-ASA do not completely suppress tumorigenesis. This may be partially a result of our dosage structure, which for ASA was at the lower limit compared with previous colorectal carcinogenesis models (28, 29). Although LS/HNPCC mice treated with both drugs survive for longer than untreated controls, there was little difference in the eventual tumor numbers (Table 1). This indicates that prolonged NSAID treatment is unable to fully inhibit the inherent genomic instability of the MMR-deficient intestinal cells in vivo. It has been shown in a model of chronic colitis that inflammatory stimuli are sufficient to predispose Msh2^−/− mice to intestinal tumors (32). It is possible that inflammation increases in the intestinal compartment of LS/HNPCC over time, despite continual exposure to NSAIDs, or that the intrinsic MSI of the epithelial tissues eventually provides selective escape from aspirin-induced apoptosis in a subset of Msh2-deficient cells.

The available literature supports the chemopreventive superiority of NO-ASA over that of parental ASA (7, 8). It has consistently been shown that animal models treated with NO-ASA can tolerate higher doses than ASA and survive longer with less tumor burden (13–16). The decision to administer ASA at 400 mg/kg was based on an earlier study which showed that similarly treated Msh2^−/− mice remained viable for up to a year without any obvious side effects (29). A dose of 720 mg/kg NO-ASA is equimolar to 400 mg/kg ASA and enables a direct comparison of both compounds at the same effective concentration. These levels of NO-ASA enhanced tumorigenesis in our LS/HNPCC mouse model in a dose-dependent manner. Even though there were significant differences in survival and tumor burden between ASA-treated and 720 or 1,500 mg/kg NO-ASA–treated Msh2^flox/floxVpC^+/+ LS/HNPCC mice, there were no notable disparities in the histologic classifications of intestinal lesions (Fig. 2B and Supplementary Table S2). These results suggest that treatment with high-dose NO-ASA did not cause a shift in tumor origin or type but merely accelerated incipient tumorigenesis. Moreover, the decreased survival caused by high-dose NO-ASA treatment is genotype specific for mice with the intestinal Msh2 deletion.

Why does NO-ASA induce genotype-specific tumorigenesis in vivo? NO-ASA survives passage through the stomach intact and is subject to metabolic transformation only after absorption by the gut (33, 34). The regional increase in tumor formation is likely related to the uptake of NO-ASA and the subsequent appearance of quinone methide: an extremely reactive transient metabolite generated from the benzene linker (35–37). Quinone methides are highly reactive with nucleotides in DNA, among other biologically relevant molecules (38). The remarkably short lifetime of quinone methide intermediates complicates direct experimental assessment (35, 36).

It has been reported that quinone methides react with cellular pools of glutathione and thioredoxin, depleting their levels and contributing to oxidative stress (39, 40). Treatment of TK6 cells with NO-ASA resulted in H2AX phosphorylation and activation of ATM, indicative of double-strand breaks (41–43). NO-ASA also induced 8-oxoguanine lesions, indicative of increased levels of oxidative stress, in both LoVo and LRWZ cells (42, 44). On the basis of these observations, we regard it likely that treatment with high-dose NO-ASA induced sustained levels of DNA damage, which may not be adequately repaired in an Msh2-deficient background. The absence of tumors in NO-ASA–treated wild-type mice, as well as the lack of tumors originating at extraintestinal Msh2-proficient tissues of our LS/HNPCC mouse model support this hypothesis.

Treatment with 72 mg/kg NO-ASA was initiated after it became apparent that 720 or 1,500 mg/kg treatments induced tumorigenesis and following published reports of potential toxicity (16). These subsequent studies were conducted to estimate a lower limit of toxicity in our LS/HNPCC mouse model. A regime of 72 mg/kg NO-ASA is equimolar to that of 40 mg/kg for ASA. To our knowledge, ASA has not shown comparable efficacy when administered at such low doses on MSI or tumorigenesis in any animal model. It was completely unexpected that such a low-dose NO-ASA would improve survival (Fig. 1). Like ASA, NO-ASA exhibits pleiotropic effects on cellular signaling pathways, which include induction of oxidative stress, inhibition of Wnt signaling, activation of the mitogen-activated protein kinase (MAPK) pathway, and inhibition of both inducible nitric oxide synthase and NF-κB (45). This effect appears unique to the LS/HNPCC genotype and has led to studies that explore the specific effect(s) of the NO linker on MSI, which should ultimately result in better NO-ASA agents.

A key prediction from earlier data was that both ASA and NO-ASA might attenuate MSI in the intestinal tissues of Msh2-deficient mice (22, 23). In fact, high-dose NO-ASA treatment appeared to contribute to further instability as persistent MSI was detected in both normal and tumor intestinal tissues (Fig. 3E and F and Table 2). Small-pool PCR analysis of the same tissues supported this observation (Fig. 4E and F). Furthermore, despite promoting animal survival, low-dose NO-ASA did not appear to have a significant effect on MSI in the intestinal compartment. In contrast, ASA appeared to skew the total comparative MSI-H toward MSI-L when assessed in the context of the untreated control group (Fig. 4D and Table 2). These results are consistent with previous cellular data (22, 23) and support the conclusion that long-term dietary exposure to ASA may attenuate MSI in the intestinal tissues of Msh2^flox/floxVpC^+/+ LS/HNPCC mouse model. It is also possible that in utero treatment with ASA or NO-ASA may further reduce the onset of tumorigenesis and attenuate MSI in this mouse model.

One limitation of our current preclinical LS/HNPCC mouse model that is shared by essentially all mouse models of colorectal cancer is that tumors do not spontaneously develop in the colon (Table 1 and ref. 24), which contrasts the pathology observed for LS/HNPCC patients. However, none of the genetically defined MMR-deficient mouse lines reported to date develop colon tumors (30), unless combined with other defective alleles such as Apc^Min/+. The strength of the Msh2^flox/floxVpC^+/+ model is that it restricts Msh2 deletion to a biologically relevant tissue compartment, the intestinal epithelial crypt cells (24). The normal life span of both control groups also indicates that intestinal-specific expression of Cre alone has no discernible phenotype in this mouse background, unlike other systems (46). The Msh2^flox/floxVpC^+/+ mice probably present the best available rodent system for preclinical modeling of LS/HNPCC. This model is amenable to further refinements which may align its resultant tumor phenotype more exactly with that arising in LS/HNPCC patients. Treatment with azoxymethane/dextran sodium sulfate (AOM/DSS) can be used to expand the tumor incidence to the colon (47). It is also possible to restrict Msh2 deletion to only the colon using a recently reported surgical procedure to physically limit subsequent infection of a Cre expressing adenovirus (48).

Recent clinical trials report that regular aspirin use is associated with a lower risk of cancer-specific mortality in individuals already diagnosed with colorectal (49) or breast cancer (50). The prevailing consensus is that the chemopreventive benefits of aspirin only manifest after 10 years or longer of continuous administration (3, 4). Our LS/HNPCC mouse data indicate that ASA and low-dose NO-ASA requires continuous long-term treatment, with a subsequent 18% to 21% increase in life span. Considering that these animals are homozygous nulls for Msh2 (in the intestine), whereas LS/HNPCC patients are heterozygous for MMR defects and require a “second-hit” to promote tumorigenesis, this increase could translate into a dramatic difference in age of onset. Indeed, recently disclosed data suggest that long-term ASA treatment of LS/HNPCC patients appears to result in a 50% decrease in tumor incidence (J. Burn, personal communication).

No potential conflicts of interest were disclosed.

The authors thank Dan Pignatelli for technical assistance.

This work was supported by NIH grant CA67007 and contract CN43302.

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.