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Aspirin and the Risk of Colorectal Cancer in Relation to the Expression of 15-Hydroxyprostaglandin Dehydrogenase (HPGD)

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Science Translational Medicine  23 Apr 2014:
Vol. 6, Issue 233, pp. 233re2
DOI: 10.1126/scitranslmed.3008481

Abstract

Aspirin use reduces the risk of colorectal neoplasia, at least in part, through inhibition of prostaglandin-endoperoxide synthase 2 (PTGS2, cyclooxygenase 2)–related pathways. Hydroxyprostaglandin dehydrogenase 15-(nicotinamide adenine dinucleotide) (15-PGDH, HPGD) is down-regulated in colorectal cancers and functions as a metabolic antagonist of PTGS2. We hypothesized that the effect of aspirin may be antagonized by low 15-PGDH expression in the normal colon. In the Nurses’ Health Study and the Health Professionals Follow-Up Study, we collected data on aspirin use every 2 years and followed up participants for diagnoses of colorectal cancer. Duplication-method Cox proportional, multivariable-adjusted, cause-specific hazards regression for competing risks data was used to compute hazard ratios (HRs) for incident colorectal cancer according to 15-PGDH mRNA expression level measured in normal mucosa from colorectal cancer resections. Among 127,865 participants, we documented 270 colorectal cancer cases from which we could assess 15-PGDH expression. Compared with nonuse, regular aspirin use was associated with lower risk of colorectal cancer that developed within a background of colonic mucosa with high 15-PGDH expression [multivariable HR, 0.49; 95% confidence interval (CI), 0.34 to 0.71], but not with low 15-PGDH expression (multivariable HR, 0.90; 95% CI, 0.63 to 1.27) (P for heterogeneity = 0.018). Regular aspirin use was associated with lower incidence of colorectal cancers arising in association with high 15-PGDH expression, but not with low 15-PGDH expression in normal colon mucosa. This suggests that 15-PGDH expression level in normal colon mucosa may serve as a biomarker that may predict stronger benefit from aspirin chemoprevention.

INTRODUCTION

Although substantial evidence from randomized controlled trials and observational studies demonstrates that aspirin reduces the risk of colorectal cancer, susceptibility to aspirin likely varies between individuals (110). Understanding of mechanisms underlying the anticancer effects of aspirin is important to establish effective chemopreventive strategies that target those individuals most likely to benefit from long-term use of the drug.

Aspirin, at least at high doses, may exert its effect through inhibition of prostaglandin-endoperoxide synthase 2 (PTGS2, cyclooxygenase 2), which catalyzes metabolic conversion of arachidonic acid to prostaglandins (11, 12). Constitutive up-regulation of PTGS2 activity is a key event in colorectal tumorigenesis (1113). We and others have shown that hydroxyprostaglandin dehydrogenase 15-(nicotinamide adenine dinucleotide) (15-PGDH; Enzyme Commission number 1.1.1.141) (encoded by the HPGD gene) plays an important role as an antagonist of PTGS2 during oncogenesis (1420). 15-PGDH catalyzes prostaglandin degradation and is ubiquitously down-regulated in colorectal cancer (1419). In a mouse model, knockout of HPGD increased colonic prostaglandin E2 (PGE2), markedly increased colon tumor numbers, and conferred resistance to the anti-colon tumor effect of the PTGS2 inhibitor celecoxib (17). Similarly, in a pilot analysis of a clinical trial, low 15-PGDH mRNA expression in normal colon mucosa was associated with lack of response to celecoxib for the prevention of recurrent adenomatous colon polyps (17).

On the basis of these findings, we hypothesized that susceptibility to aspirin might differ according to 15-PGDH expression level in the colon. Within two nationwide cohorts, we examined whether regular aspirin use was associated with a lower risk of colorectal cancer arising in settings of high 15-PGDH mRNA expression in normal colonic mucosa, but not in cancers arising with low normal colon 15-PGDH expression.

RESULTS

Characteristics of the participants

Among 127,865 participants (82,095 women and 45,770 men), we documented 270 colorectal cancer cases [165 cases in the Nurses’ Health Study (NHS) and 105 cases in the Health Professionals Follow-Up Study (HPFS)] with available data on 15-PGDH expression level in normal colonic mucosa and aspirin use status, which developed during 3,166,880 person-years of follow-up (2,272,266 person-years in the NHS and 894,613 person-years in the HPFS). We classified aspirin use status based on the cumulative mean for the number of aspirin tablets consumed per week up to each biennial follow-up cycle. Aspirin use status and other covariates were modeled as time-varying variables to take into account potential changes in exposures over follow-up time. At baseline, compared with nonusers, regular aspirin users were more likely to be older, have a higher body mass index (BMI), smoke, undergo endoscopy, use postmenopausal hormone (women only), consume higher amounts of alcohol and folate, use multivitamins, and engage in less physical activity (Table 1).

Table 1 Age-adjusted demographic characteristics according to regular aspirin use status at baseline.

Values are standardized to the age distribution of the study population. In the NHS, regular aspirin use was defined as the consumption of at least two 325-mg tablets per week, and nonuse was defined as consumption of fewer than two tablets per week. In the HPFS, regular aspirin use was defined as the consumption of aspirin at least two times per week, and nonuse was defined as the consumption of aspirin fewer than two times per week. MET, metabolic equivalent task; SD, standard deviation.

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Correlation of regular aspirin use and incident colorectal cancer with colon 15-PGDH expression

15-PGDH mRNA expression was quantified in matched normal mucosa from 270 colorectal cancer cases, enabling these cases to be divided into those arising in colons with high (greater than median) 15-PGDH expression versus those arising in colons with low (lower than median) 15-PGDH expression. These two groups were respectively designated normal colon high 15-PGDH–type versus normal colon low 15-PGDH–type colon cancers. Both types of cancer were then modeled simultaneously in a competing risk model. Demographic, clinical, and pathological characteristics at diagnosis, including status of BRAF mutation, PIK3CA mutation, and PTGS2 expression, which have been previously noted to differ by aspirin use, were not significantly different according to 15-PGDH expression level (table S1). In the combined cohort, the benefit of regular aspirin use appeared to be confined to decreasing the risk only of normal colon high 15-PGDH colorectal cancers (P for heterogeneity = 0.018) (Table 2). Compared with nonuse, the age-adjusted hazard ratio (HR) of regular aspirin use for developing normal colorectal high 15-PGDH–type cancers was 0.51 [95% confidence interval (CI), 0.35 to 0.73]. This inverse association remained significant after adjustment for life-style and other risk factors (multivariable HR, 0.49; 95% CI, 0.34 to 0.71). In contrast, regular aspirin use was not associated with a lower risk of developing normal colon low 15-PGDH–type cancers, with a multivariable HR of 0.90 (95% CI, 0.63 to 1.27). This finding was independently observed within both study cohorts, with regular aspirin use associated with reduced multivariable HRs for normal colon high 15-PGDH–type colorectal cancers to 0.58 (95% CI, 0.35 to 0.94) in the NHS and to 0.44 (95% CI, 0.25 to 0.76) in the HPFS. In contrast, regular aspirin use was not associated with reduced HR for developing normal colon low 15-PGDH–type cancers in either the NHS or the HPFS. Consistent results were observed in the analyses of aspirin according to dosage and duration (tables S2 and S3).

Table 2 Regular aspirin use and incident colorectal cancer grouped by HPGD (15-PGDH) mRNA expression in normal mucosa.

In the NHS, regular aspirin use was defined as the consumption of at least two 325-mg tablets per week, and nonuse was defined as consumption of fewer than two tablets per week. In the HPFS, regular aspirin use was defined as the consumption of aspirin at least two times per week, and nonuse was defined as the consumption of aspirin fewer than two times per week.

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We further conducted a secondary analysis that additionally adjusted for use of nonsteroidal anti-inflammatory drugs (NSAIDs) (table S4). Inclusion of data on NSAID use did not materially alter the differential association we observed, in which regular aspirin use was associated with reduced risk of only normal colon high 15-PGDH–type cancers. After adjustment for NSAIDs, regular aspirin use was again associated with a lower risk of normal colon high 15-PGDH–type colorectal cancers (multivariable and NSAID-adjusted HR, 0.47; 95% CI, 0.32 to 0.68) but not of normal colon low 15-PGDH–type cancers (multivariable and NSAID-adjusted HR, 0.85; 95% CI, 0.60 to 1.21).

DISCUSSION

In two prospective cohorts, we found that regular aspirin use was associated with a lower incidence of colorectal cancers arising in association with high 15-PGDH expression in the normal colon mucosa, whereas regular aspirin use showed no association with reduced incidence of colorectal cancers that arose in colon mucosa with low normal 15-PGDH expression. Consistent results were observed in the analyses of aspirin according to dosage and duration. These findings support the hypothesis that the anticancer activity of aspirin in colonic mucosa is, at least in part, dependent on high 15-PGDH expression and that low expression of 15-PGDH may impart potential resistance to aspirin’s tumor-preventive effects.

15-PGDH is a physiologic antagonist to the PTGS2 oncogene that mediates oncogenesis through inflammatory responses (1417). In a mouse model, we have previously shown that knockout of the HPGD gene confers resistance to antitumor effect of the PTGS2-selective inhibitor celecoxib, resulting in continued colon tumor development in celecoxib-treated knockout but not in celecoxib-treated wild-type mice (17). In parallel, the ability of celecoxib to lower colonic PGE2 was also attenuated in knockout mice (17). Moreover, in a pilot analysis of patients enrolled in a clinical trial of celecoxib for chemoprevention of recurrent adenoma, low 15-PGDH expression in normal colon mucosa was associated with celecoxib resistance and with continued adenoma development during celecoxib treatment (17). Taken together with previous findings that 15-PGDH expression is consistently abolished within colorectal cancers (1416), our results further confirm a model in which 15-PGDH functions as a key tumor suppressor of colon neoplasms, and suggest that a critical level of 15-PGDH activity is required for aspirin to exert its antineoplastic effects. Conversely, reduction of 15-PGDH expression and its attendant tumor-suppressive activity within normal mucosa may abrogate the chemopreventive benefits of aspirin.

Despite extensive experimental studies on both the anticancer properties of aspirin and the central role of 15-PGDH in colorectal neoplasia (2127), there are no data available regarding whether the effectiveness of aspirin varies among individuals of differing 15-PGDH expression levels (1420). We have previously shown that certain molecular features are associated with a subclass of colon cancers that are resistant to aspirin prevention, including low expression of PTGS2 and mutation of BRAF, which is a key regulator of PTGS2 activity in RAF-MAPK signaling (11, 13). Our present results substantially extend upon these findings by demonstrating that constitutional differences in individuals’ normal colons, namely, in 15-PGDH expression, may also be associated with resistance to chemoprevention with aspirin. Both aspirin and 15-PGDH act to lower prostaglandin levels: aspirin through inhibition of PTGS, and 15-PGDH through catalyzing prostaglandin degradation. Our data suggest that both effective PTGS inhibition and robust prostaglandin degradation are required to lower risk of colorectal cancer. Although aspirin is a highly effective chemopreventive agent, widespread use of the drug is not currently recommended because of uncertainty about its risk-benefit profile (28). Our findings suggest the possibility that 15-PGDH levels in normal colonic mucosa could be exploited to identify individuals who will have greatest likelihood of benefit from regular use of aspirin for chemoprevention.

Our study had several strengths. First, we used prospectively collected data on aspirin use in relation to colorectal cancer, minimizing potential recall bias. Second, our follow-up of more than 28 years allowed us to evaluate the long-term association between regular aspirin use and colorectal cancer incidence. Third, repeated measures and updated assessments of aspirin use in each 2-year questionnaire enabled us to consider potential changes over time. Finally, comprehensive collection of data on other life-style risk factors for colorectal cancer allowed us to adjust for potential confounding variables.

Our study also had limitations. First, we did not have information on expression of 15-PGDH in colon mucosa before the diagnosis of cancer. Second, although normal colon blocks are in most cases prepared from the surgical margins at the edge of a colon resection, the location of the normal colon samples tested would have differed among the cases studied, and we lacked precise information on this point. Mitigating these two limitations, we have recently shown that 15-PGDH expression is stable along the length of the colon and is also stable over time (29). Additionally, we have also shown that aspirin administration does not significantly alter colonic 15-PGDH expression (29). Moreover, in our previous study, we found that levels of 15-PGDH in biopsies taken before treatment with celecoxib predicted likelihood of the development of new adenomas (17). Nonetheless, future studies will be needed in which 15-PGDH levels are assessed prospectively, both before and during aspirin treatment, in individuals monitored for development of colonic neoplasia. A third limitation is that we relied on self-reported aspirin use, but this has previously been shown to be highly reproducible within our cohorts because the participants were all health professionals (11). Fourth, the potential for confounding by unmeasured or unknown factors could not be fully eliminated, although we adjusted for several major life-style and risk factors in the analysis. Fifth, we did not have measurements of 15-PGDH expression in normal mucosa for all cases of colorectal cancer that developed within our cohort. Last, our study population was predominantly non-Hispanic white, and our results might not be generalizable to other ethnic groups.

In summary, we show that regular aspirin use is associated with a lower risk of colorectal cancer that arises in association with high, but not low, expression of 15-PGDH in the normal colon mucosa. Our data provide evidence that the anticancer effect of aspirin requires cooperation with an active 15-PGDH tumor suppressor pathway. Further studies are needed to determine the potential utility of using 15-PGDH expression in colonic mucosa as a biomarker to risk-stratify individuals for aspirin-based chemoprevention.

MATERIALS AND METHODS

Study design

This study aimed to examine whether susceptibility to aspirin might differ according to 15-PGDH expression in the colon. The hypothesis was developed on the basis of our previously reported observations in a mouse model and pilot analysis of a clinical trial (17). Analysis of 15-PGDH mRNA expression was performed by validated real-time polymerase chain reaction (PCR) assay in a blinded fashion. The 15-PGDH mRNA expression was determined as the average levels obtained from three independent real-time PCRs. Information on aspirin use was collected every 2 years prospectively. To assess our hypothesis, duplication-method, multivariable-adjusted, cause-specific Cox proportional hazards regression for competing risks data was used to compute HRs for incident colorectal cancer according to 15-PGDH mRNA expression.

Study population

The NHS was established in 1976, enrolling 121,701 U.S. registered female nurses aged 30 to 55 years. The HPFS was initiated in 1986, including 51,529 U.S. male health professionals aged 40 to 75 years. Every 2 years, questionnaires were mailed to participants, and information on life-style factors and medical history has been collected. We obtained informed consent from all participants. This study was approved by Human Subjects Committees at Harvard School of Public Health and Brigham and Women’s Hospital.

Identification of colorectal cancer cases

In both cohorts, cancer and other disease outcomes were reported by participants on the biennial questionnaires. Deaths were reported by family members or the postal system, or identified through a search of the National Death Index. After obtaining consent from the participants or their next of kin, medical records and pathological reports were obtained and reviewed by study physicians to abstract information on tumor location, stage, and histologic type of the cancer. We retrieved formalin-fixed paraffin-embedded tissue blocks from hospitals throughout the United States where participants with colorectal cancer had undergone surgical resection (30). We collected diagnostic biopsy specimens for rectal cancer patients who received preoperative treatment to minimize biases associated with preoperative radiation treatment. Hematoxylin and eosin–stained tissue sections, including normal and tumor sections from all colorectal cancer cases, were reviewed by a pathologist (S.O.).

Assessment of aspirin use

Detailed description of aspirin assessment in the cohorts has been reported previously (11, 13). In the NHS, we collected the information on aspirin usage and the number of tablets taken per week, beginning in 1980 and every 2 years thereafter except in 1986. In the HPFS, we inquired about aspirin usage, beginning in 1986 and every 2 years thereafter. The number of tablets taken per week was first collected in 1992 for the HPFS. In both cohorts, participants were asked about standard-dose (325-mg) tablets. Participants were also requested to convert intake of four baby aspirin tablets (81 mg) to one standard aspirin tablet in the questionnaires, beginning in 1992. To minimize within-individual variation and to estimate long-term influence, we calculated the cumulative mean for the number of aspirin tablets consumed per week, which was the mean of all available data on tablets per week up to each biennial follow-up cycle (11, 13).

In the NHS, we defined regular aspirin users as women who consumed two or more standard-dose (325-mg) aspirin tablets per week, and nonusers as those who reported intake of fewer aspirin per week. In the HPFS, men who reported consumption of standard-dose (325-mg) aspirin at least two times per week were defined as regular users, and men who reported less frequent aspirin consumption were defined as nonusers. To better reflect potential changes of aspirin use over follow-up time, status of regular aspirin use was updated every 2 years in our analysis.

Analysis of 15-PGDH (HPGD) expression

In most cases, normal colon was less than 20 cm away from the tumor. Four unstained sections of 5-μm thickness were cut from the block of normal colon tissue, and the mucosal layer was retrieved by scraping. RNA was extracted with a High Pure FFPE RNA Micro Kit (Roche). Real-time PCR was performed as described previously (17) to quantitate the expression level of 15-PGDH mRNA, following the MIQE guidelines (31) and the software programs (3234), as fully detailed in Supplementary Methods.

Statistical analysis

We included participants who provided baseline aspirin data in 1980 for the NHS and in 1986 for the HPFS. We excluded participants with a history of cancer (except for nonmelanoma skin cancer), inflammatory bowel disease, or familial polyposis at baseline. Participants were followed from the date of baseline questionnaire return through 1 July 2008, date of colorectal cancer diagnosis, or date of death, whichever came first.

We used a Cox proportional cause-specific hazards regression model with a duplication method for competing risks data and computed HR and 95% CI to examine whether the association between aspirin use and colorectal cancer incidence differed according to 15-PGDH expression in normal colonic mucosa. A heterogeneity test was conducted using a likelihood ratio test, comparing a model that allowed for different associations of aspirin use by 15-PGDH expression with a model that assumed a common association. We used SAS software version 9.3 (SAS Institute Inc.) to stratify all analyses by age (in months), sex (in the analysis using combined cohorts), and year of questionnaire return. We further conducted multivariable analyses, including BMI (<25, 25 to 29.9, or ≥30 kg/m2), smoking status (never, former, or current), family history of colorectal cancer in any first-degree relative (yes or no), previous lower gastrointestinal endoscopy (no endoscopy, confirmed history of adenomatous polyps, or endoscopy without detection of neoplasia), postmenopausal hormone use (for women only, never or ever), physical activity (quintiles of MET hours per week), total red meat intake (quintiles of servings per day), alcohol consumption (0 or quartile of grams per day), total caloric intake (quintiles of kilocalories per day), folate intake (quintiles of micrograms per day), calcium intake (quintiles of milligrams per day), and current multivitamin use (yes or no). MET scores were calculated as previously described (35, 36), and values for individual activities were summed to give a total MET hours per week. A secondary analysis, which adjusted for the regular use of NSAIDs, was conducted using the data from 1990 for the NHS and from 1986 for the HPFS, the time points at which information on NSAIDs use began to be collected routinely in these cohorts.

For all analyses, we used the most updated data before each follow-up cycle and modeled them as time-varying variables to take into account potential changes over time. Cumulative mean was used for the continuous variables, including BMI, physical activity, total red meat intake, alcohol consumption, and intake of calories, folate, and calcium. Before pooling the two cohorts, we examined the possible heterogeneity between cohorts using the Q statistics for the association between aspirin use and colorectal cancer incidence. We did not observe significant heterogeneity between cohorts (P = 0.52 for cancer with low 15-PGDH; P = 0.46 for cancer with high 15-PGDH). We tested the proportional hazards assumption based on the interaction terms between aspirin use and follow-up time and observed no evidence of violation (P = 0.56 for cancer with low 15-PGDH; P = 0.88 for cancer with high 15-PGDH). All analyses were two-sided, and P < 0.05 was considered statistically significant.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/6/233/233re2/DC1

Methods

Table S1. Characteristics at diagnosis according to HPGD (15-PGDH) mRNA expression.

Table S2. Number of aspirin tablets and incident colorectal cancer grouped by HPGD (15-PGDH) mRNA expression.

Table S3. Duration of regular aspirin use and incident colorectal cancer grouped by HPGD (15-PGDH) mRNA expression.

Table S4. Regular aspirin use and incident colorectal cancer grouped by HPGD (15-PGDH) mRNA expression.

Table S5. Comparison of the top four most stable transcripts by program.

REFERENCES AND NOTES

  1. Acknowledgments: We thank the participants and staff of the NHS and the HPFS for their valuable contributions as well as the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, and WY. In addition, this study was approved by the Connecticut Department of Public Health (DPH) Human Investigations Committee. Certain data used in this publication were obtained from the DPH. The authors assume full responsibility for the analyses and interpretation of these data. Funding: Supported by U.S. NIH grants (P01 CA87969 to S. E. Hankinson, P01 CA55075 to W. C. Willett, 1UM1 CA167552 to W. C. Willett, R01 CA136950 to E.C., P50 CA127003 to C.S.F., R01 CA151993 to S.O., P50 CA150964 and U01 CA152756 to S.D.M., and R01 CA137178 and K24 DK 098311 to A.T.C.), the Marguerite Wilson Foundation (to S.D.M.), gifts from the Leonard and Joan Horvitz Foundation (to S.D.M.), the Richard Horvitz and Erica Hartman-Horvitz Foundation (to S.D.M.), the Bennett Family Fund for Targeted Therapies Research (to A.T.C.), and the National Colorectal Cancer Research Alliance (to W. C. Willett and S.D.M.). A.T.C. is a Damon Runyon Clinical Investigator. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or other research foundations. Author contributions: C.S.F., S.O., S.D.M., and A.T.C. contributed to the study concept and design. S.P.F., M.Y., R.N., K.W., E.C., E.G., C.S.F., S.O., S.D.M., and A.T.C. were involved in acquisition of data. All of the authors contributed to the interpretation of the data. S.P.F., M.Y., R.N., S.J., and A.K. performed the statistical analysis. S.P.F., M.Y., R.N., S.O., S.D.M., and A.T.C. drafted the manuscript. All of the authors revised the article critically for important intellectual content and approved the final version of the manuscript submitted for publication. Competing interests: A.T.C. previously served as a consultant for Bayer Healthcare, Millennium Pharmaceuticals, Pfizer Inc., and Pozen Inc. This study was not funded by Bayer Healthcare, Millennium Pharmaceuticals, Pfizer Inc., or Pozen Inc. S.D.M. is an inventor on a patent relating to the use of 15-PGDH levels for selection of individuals for chemoprevention with NSAID-type agents (U.S. Patent 8497084, assigned to Case Western Reserve University). All other authors declare that they have no competing interests.
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