TGFß1 polymorphism (L10P) and risk of colorectal adenomatous and hyperplastic polyps

Rachel Sparks1,2, Jeannette Bigler1, Justin G Sibert1, John D Potter1,2, Yutaka Yasui1 and Cornelia M Ulrich1,2

1 Fred Hutchinson Cancer Research Center, Seattle, Washington, 98109, USA
2 Department of Epidemiology, University of Washington, Seattle, Washington, 98195, USA

Correspondence: Cornelia Ulrich, Cancer Prevention Research Program, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, M4-B402, Seattle, WA 98109–1024, USA. E-mail: nulrich{at}fhcrc.org


    Abstract
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Background Transforming growth factor-ß1 (TGFb1) is a multifunctional signalling molecule with a wide array of roles. Animal experiments suggest that TGFb1 plays a biphasic role in carcinogenesis by protecting against the early formation of benign epithelial growths, but promoting malignant transformation of those growths that do develop. A polymorphism in the signal peptide sequence of the TGFß1 gene (L10P) has been associated with increased levels of plasma TGFb1 in individuals with the P allele.

Methods We investigated whether this polymorphism was associated with the risk of colorectal adenomatous or hyperplastic polyps in a case-control study of individuals from Minnesota. Risk of colorectal polyps was evaluated separately for individuals with adenomatous polyps (n = 513) and hyperplastic polyps (n = 191) relative to polyp-free controls (n = 606) using logistic regression analysis.

Results No overall association was seen between the L10P polymorphism and risk of colorectal adenomatous polyps. The age- and sex-adjusted odds ratios (OR) of developing colorectal hyperplastic polyps were 1.0 (95% CI: 0.7, 1.4) and 0.7 (95% CI: 0.4, 1.1) for individuals with the LP and PP genotypes, respectively, compared with individuals with the LL genotype. When stratified by smoking, evidence for a decreased risk of hyperplastic polyps associated with the P allele was seen only among ever smokers (P for trend = 0.05).

Conclusions Whereas adenoma risk did not vary by TGFß1 L10P genotype, these results suggest that the L10P variant allele may have a protective role in the development of colorectal hyperplastic polyps, possibly consistent with its role as an inhibitor of epithelial growths.


Keywords Colorectal cancer, colorectal adenoma, transforming growth factor beta, polymorphism, smoking, aspirin, non-steroidal anti-inflammatory drugs

Accepted 19 December 2003

Adenomatous, and possibly hyperplastic polyps, are considered precursors of colorectal adenocarcinoma.1–3 Although genetic and environmental risk factors for adenomatous and hyperplastic polyps have been described,1,4–7 specific growth factors or cytokines that contribute to the development of these polyps have not been identified.

Transforming growth factor-ß (TGFb) is a dimeric polypeptide growth factor that has been implicated in epithelial carcinogenesis.8,9 Three TGFb isoforms in mammals (TGFß1, TGFß2, TGFß3) act through the same receptor signalling system.9,10 In non-transformed epithelial cells, TGFß is multifunctional. It can act as a cell-cycle regulator11 and plays an important role in the maintenance of the extracellular matrix.9 TGFß is capable of promoting angiogenesis and can directly induce this process in vivo.8,12 In addition, it strongly regulates immune function, and can inhibit the proliferation and activation of immune cells.13

Many tumour cells overexpress TGFß and are resistant to its growth-inhibiting effects.8,9 It is believed that this increased production of TGFß inhibits local inflammatory responses, thereby allowing cancer cells to escape host immunosurveillance.8,9 Additionally, increased levels of plasma TGFß1 in cancer patients and overexpression of TGFß1 in tumour cells have been shown to correlate with increased tumour vascularity and a poor prognosis.9 TGFß1 can induce the expression of cyclooxygenase-2,14 which has been implicated in colon carcinogenesis.15

Given the multiple actions of TGFß, its role in carcinogenesis remains unclear. Whereas TGFß inhibits the growth of normal epithelial cells, resistant cancer cells that overexpress TGFß are able to become more invasive, recruit new vasculature, and more easily metastasize.8,9 This ‘biphasic’ role of TGFß in the development of cancer was clearly demonstrated in a study in which the overexpression of TGFß1 was targeted to keratinocytes of mice that were treated with topical chemical carcinogens.16 TGFß1 inhibited the development of benign papillomas in these mice, further evidence of its role as an inhibitor of cellular proliferation; however, those skin lesions that did form were much more likely to undergo malignant progression due in part to the capacity of TGFß1 to promote angiogenesis and enhance tumour cell invasiveness.16,17 This study suggests that TGFß1 might protect against the early formation of epithelial growths, such as colorectal adenomatous and hyperplastic polyps. However, individuals with elevated levels of TGFß1 might be more likely to experience progression from polyp to adenocarcinoma compared with individuals with lower TGFß1 levels.

The L10P polymorphism in the signal peptide sequence of the TGFß1 gene has been associated with increased levels of TGFß1 protein18–20 and mRNA19 in individuals with the variant allele. Recently, the P allele has been shown to result in a 2.8-fold increase in TGFß1 section compared with the L allele in vitro.21 The 10PP genotype has previously been found to be associated with both a reduced risk of breast cancer among elderly white women,22 and an increased risk of invasive breast cancer among a large cohort of European women.21 We evaluated the association of colorectal adenomatous and hyperplastic polyps risk with the TGFß1 L10P polymorphism in a case-control study of individuals from Minnesota. Given the role of TGFß1 in inhibiting early lesions, we hypothesized that individuals with the variant allele would be at a reduced risk of colorectal adenomatous and hyperplastic polyps.


    Materials and Methods
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Study subjects
Study recruitment for this case-control study has been described previously.23 Briefly, individuals with colorectal adenomatous or hyperplastic polyps and polyp-free control subjects were part of a study of colorectal polyps in Minnesota. All subjects were recruited through a large multiclinic private gastroenterology practice in metropolitan Minneapolis which, at the time, performed approximately 60% of all colonoscopies in the area. Patients between the ages of 30 and 74 years who were scheduled for a colonoscopy between April 1991 and April 1994 were eligible for the study (eligibility criteria below). Patients were recruited prior to colonoscopy so as to blind patients and recruiters to the final diagnosis. This study was approved by the institutional review boards of the University of Minnesota, each endoscopy site, and the Fred Hutchinson Cancer Research Center. Written informed consent was obtained from each study participant.

Eligibility criteria for cases and controls were: resident of Twin Cities metropolitan area; English-speaking; no known genetic syndrome associated with predisposition to colonic neoplasia; no individual history of cancer (except non-melanoma skin cancer); no history of inflammatory bowel disease. Cases were identified as meeting all of the eligibility criteria and having a first diagnosis of colon or rectal adenomatous or hyperplastic polyp at the time of the colonoscopy. The participation rate for all colonoscoped patients was 68%.

Questionnaires were mailed to eligible subjects and collected the day of the procedure along with a blood draw. Colonoscopy findings were recorded on standardized sheets. Patients for whom the colonoscopy did not reach the caecum were eliminated from the study. All removed polyps were examined histologically by the study pathologist using diagnostic criteria established by the National Polyp Study.24 Patients were assigned to one of four groups based on the colonoscopy and pathology findings: adenomatous polyp cases (≥one adenomatous polyp and no hyperplastic polyps, n = 438); hyperplastic polyp cases (≥one hyperplastic polyp and no adenomas, n = 219); adenomatous and hyperplastic polyp cases (≥one adenomatous polyp and ≥one hyperplastic polyp, n = 138); and controls (no polyps, n = 708).

Data collection
Data on usual physical activity, smoking habits, anthropometric measurements, medical information including family history of cancer and polyps, demographic information, and reproductive history (women only) were obtained from participants by self-administered questionnaires. When data were incomplete, study staff followed-up with participants by phone. Dietary intake was assessed using an adaptation of the Willett semi-quantitative food frequency questionnaire.25–27 Data on smoking history included current and past smoking status, age when smoking began, average number of cigarettes smoked daily, and years since quitting smoking. Pack-years of smoking were calculated by multiplying the number of years smoked by the number of current (or past, for those who quit) cigarettes smoked daily divided by 20.

Genotyping
Several sequence variations in the TGFß1 gene have been identified, including two common polymorphisms (–509C– > T and 869T– > C [L10P]) that have been associated with serum concentrations of TGFß1.18–20,28 Because these polymorphisms are reported to be in linkage disequilibrium in three populations,29–31 we chose to genotype for the L10P polymorphism.

TGFß1 exon 1 (L10P) genotyping was performed at the Core Laboratory of the Public Health Sciences Division of the Fred Hutchinson Cancer Research Center (JB) using a Taqman assay.22 Included in each batch of samples were control DNAs representing all TGFß1 (L10P) genotypes and at least two negative controls. For quality control purposes, genotyping was repeated for a random selection of 100 samples. There were no discrepancies between the two results.

Statistical data analysis
Because the TGFß1 L10P genotype frequencies vary by race, the analysis was restricted to Caucasians (97% of the population). Standard techniques for case-control studies were used. To estimate the association between TGFß1 L10P genotype and the risk of colorectal polyps, unconditional logistic regression models were used to obtain maximum likelihood estimates of odds ratios (OR), and 95% CI. The following covariates have been identified as potential confounding factors in previous analyses of this population4,6,23 and were evaluated for inclusion into the model using the likelihood ratio test: age, sex, alcohol intake (g/day), smoking (pack-years), ever use of hormone replacement therapy among women (yes/no), regular use of aspirin (at least once per week), regular use of non-steroidal anti-inflammatory drugs (NSAIDs) (at least once per week), family history of colon cancer (yes/no), hours of physical activity, and the dietary intake variables kilocalories (kcal), percentage kcal from fat, folate, vitamin B6, and vitamin B12. After evaluation, the following variables were included in the multivariate model: age, sex, alcohol intake (g/day), smoking (pack-years), ever use of hormone replacement therapy among women (yes/no), regular use of aspirin (at least once per week), and regular use of NSAID (at least once per week). Adjustment for the remaining variables did not alter the results.

Polyp risk was evaluated separately for individuals with ‘any adenomatous polyp’, ‘only hyperplastic polyps’, ‘only adenomatous polyps’, and ‘both adenomatous and hyperplastic polyps’, relative to polyp-free controls. Because the genotype effects on the odds of developing a specific type of polyp appeared similar for ‘any adenomatous polyp’, ‘only adenomatous polyps’, and ‘both adenomatous and hyperplastic polyps’, the results are presented for the genotype effects on the odds of developing: (1) ‘any adenomatous polyp’ rather than being polyp-free, and (2) ‘only hyperplastic polyps’ rather than being polyp-free.

To assess the monotone relationship between selected ordinal categorical variables and the risk of colorectal polyps across genotypes, a test for trend was used. We also evaluated effect modification of the L10P polymorphism by stratification on the variable in question (e.g. smoking), using one group as the common reference and comparing OR associated with the polymorphism across the levels of the variable in question. Statistical significance was determined by testing for different slopes across genotypes for each level of the potential effect modifier. All statistical tests were two-sided and all analyses were performed with SAS 8.0 (SAS Institute, Cary, NC).


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The study population has been described previously.4,6,23 Selected characteristics are presented in Table 1. Briefly, controls were younger than individuals with adenomatous polyps and were more likely to be female. Individuals with adenomatous or hyperplastic polyps were more likely to smoke compared with controls. Regular use of aspirin and other NSAIDs was somewhat more common in controls than cases. The TGFß1 exon 1 L10P polymorphism was in Hardy–Weinberg equilibrium in all groups, with an allele frequency of 0.39 for the P (proline) allele in polyp-free controls.


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Table 1 Characteristics of the study population

 
The 10LP and 10PP TGFß1 genotypes were not significantly associated with the risk of adenomatous or hyperplastic polyps, relative to the 10LL (wildtype) genotype (Table 2); the association between the 10PP genotype and the risk of hyperplastic polyps was weakly inverse. There were no significant differences in the ORs of developing adenomatous or hyperplastic polyps when stratified by the location of the largest adenoma, tumour size, or the number of polyps (data not shown).


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Table 2 Association between TGFß1 genotype and adenomatous and/or hyperplastic polyps

 
Associations between TGFß1 L10P genotype and the risk of hyperplastic polyps, stratified by variables related to polyp development, are shown in Table 3. Only gene–environment interactions with a hypothesized biological mechanism were considered. The risk of hyperplastic polyps did not appear to vary largely within strata of age, gender, or regular aspirin or NSAID use. As reported earlier, smoking was associated with an increased risk of hyperplastic polyps.5–7,32–35 Among ever smokers, the presence of the TGFß1 P allele was inversely associated with hyperplastic polyp risk (LL genotype: OR = 3.7, 95% CI: 2.0, 6.9; LP genotype: OR = 3.2. 95% CI: 1.7, 6.0; PP genotype: OR = 1.8, 95% CI: 0.8, 4.0); relative to never-smokers with the LL genotype. P for trend for ever smokers: 0.05).


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Table 3 Association between TGFß1 L10P genotype and risk of hyperplastic polyps, stratified by factors associated with polyp development

 

    Discussion
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To our knowledge this is the first reported evaluation of the association between the risk of adenomatous or hyperplastic polyps and polymorphisms in the TGFß1 gene. We had hypothesized that individuals with the TGFß1 10PP genotype might be at a reduced risk of colorectal polyps given that this genotype has been associated with increased levels of TGFß1 protein and mRNA, and overexpression of TGFß1 in animal models has been shown to reduce the occurrence of benign epithelial growths.16 We observed a weak inverse association between the risk of colorectal hyperplastic polyps and the TGFß1 10PP genotype (OR = 0.7, 95 % CI: 0.4, 1.1), but found no overall association between the TGFß1 polymorphism and the risk of colorectal adenomatous polyps. In addition, the risk of hyperplastic polyps associated with smoking varied by genotype: whereas heavy smoking was associated with an increased risk of hyperplastic polyps overall, smokers with a P allele appeared to be at a somewhat decreased risk relative to wildtype smokers.

The variant allele of the TGFß1 L10P polymorphism has been associated with increased levels of TGFß1 protein and mRNA,18–20 and has been shown to result in a 2.8-fold increase in TGFß1 secretion in vitro.21 Two case-control studies have previously linked the TGFß1 L10P polymorphism to breast cancer risk. Ziv and colleagues found that elderly white women (median age of enrollment: 70 years) with the 10PP genotype were at statistically significantly lower risk of developing breast cancer than women with the 10LL or 10LP genotype.22 However, a large European study found that women with the 10PP genotype were at a significantly higher risk of invasive breast cancer compared with women with the 10LL or 10LP genotype.21 Although apparently conflicting, these two studies may be consistent with the biphasic role of TGFß1 in cancer progression. The observation that women with the variant 10PP genotype were diagnosed at an older age in the study by Ziv et al. supports the role of TGFß1 as an inhibitor of early growths. However, the observation by Dunning et al. that this same 10PP genotype confers higher risk of invasive breast cancer21 may be further evidence that once a tumour is initiated, increased levels of TGFß1 might act to promote malignant progression.

As previously reported from studies of this and other populations, smoking is strongly associated with an increased risk of hyperplastic polyps.5–7,32–35 Yet, the specific mechanisms by which smoking might contribute to the development of hyperplastic polyps are unclear. One possible mechanism involves DNA adduct formation and activating point mutations in the proto-oncogene k-ras by carcinogens in cigarette smoke.36 In normal epithelial cells, TGFß is capable of overriding mitogenic Ras signals, thus inhibiting proliferation37,38; however, when an oncogenic ras mutation occurs (as could be mediated by cigarette carcinogens), hyperactivated Ras becomes capable of blocking TGFß's antiproliferative signals.38 If individuals with elevated levels of TGFß have an increased capacity to inhibit Ras-induced proliferative signals, this could possibly explain our observation that smokers with the TGFß1 10PP genotype appeared to have a somewhat decreased risk of hyperplastic polyps compared with smokers with the 10LP and 10LL genotypes.

One limitation of the study is that the small number of individuals with hyperplastic polyps reduced our statistical power in many stratified analyses. It is therefore possible that the observed relationship between smoking, the L10P polymorphism, and risk of hyperplastic polyps is a spurious, false-positive finding. Given the evidence of the biphasic role of TGFß1 in carcinogenesis, and oncogenic molecular changes induced by smoking, our findings seem biologically feasible. However, because most of our findings did not reach statistical significance, they must be considered as possibly attributable to chance. Another possible limitation is that adenoma cases included individuals with concurrent hyperplastic polyps. However, the risk patterns for individuals with both adenomas and hyperplastic polyps were similar to those with only adenomatous polyps.

In summary, the results of this case-control study indicate that the TGFß1 L10P polymorphism is not associated with the risk of colorectal adenomatous polyps, but may be linked to reduced risk of hyperplastic polyps. To further evaluate the biphasic role of TGFß1 in colorectal carcinogenesis, studies should examine if individuals with the TGFß1 10PP genotype are overrepresented among colon cancer cases. Because the variant allele increases TGFß1 secretion, it is possible that individuals with the variant allele may be at decreased risk of developing colorectal polyps, but at increased risk of progression of those polyps to colorectal carcinoma.8


KEY MESSAGES

  • A polymorphism in the TGFß1 gene (L10P) is associated with increased levels of TGFß1 protein and mRNA in individuals with the variant allele.
  • In a case-control study of individuals from Minnesota with colorectal adenomatous and hyperplastic polyps, compared with polyp-free controls, the TGFß1 10PP genotype was not associated with risk of adenomatous polyps, but weakly inversely associated with hyperplastic polyp risk.
  • Smoking was a strong risk factor for hyperplastic polyps, and the reduced risk of these polyps associated with the variant P allele was seen only among ever smokers.

 


    Acknowledgments
 
We thank Mari Nakayoshi for word processing assistance. Sources of financial support: NIH R01CA89445 and NIH R01CA59045.


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