Insulin Resistance-Related Genes and Advanced Left-Sided Colorectal Adenoma

Background: Insulin resistance has been linked with colorectal neoplasia through a number of mechanistic and observational studies. Allelic variants of genes encoding components of the insulin pathway, including insulin (INS), insulin receptor (INSR), and insulin receptor substrate-1 and insulin receptor substrate-2 (IRS1 and IRS2) have been associated with hyperinsulinemia and insulin resistance and may, therefore, predict susceptibility to colorectal neoplasia.

Methods: We investigated whether single nucleotide polymorphisms (SNP) in the INS, INSR, IRS1, and IRS2 genes are associated with risk of advanced left-sided colorectal adenoma, a cancer precursor. We analyzed 20 SNPs in a largely Caucasian study population comprising 766 cases with advanced adenomas of the distal colon and 771 controls, all of whom had undergone flexible sigmoidoscopy as part of the screening arm of the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial.

Results: Overall, we found limited evidence for a role of gene variants of the insulin signaling pathway and prevalence of advanced colorectal adenoma. We observed a statistically significant interaction between INSR genotypes and body mass index (BMI) with colorectal adenoma prevalence (P value for global test = 0.003) and suggestion of an interaction between INSR genotypes and glycemic load (P value for global test = 0.06); however, exploration of the interaction of BMI and glycemic load with the individual SNPs in INSR did not suggest a single SNP that may explain the significance of these global tests of interaction and did not yield any consistent patterns.

Conclusion: These findings do not provide strong evidence for associations between polymorphic variation in genes of the insulin signaling pathway and advanced left-sided colorectal adenoma. Evidence for interaction between INSR variants and BMI and glycemic load for risk of advanced left-sided colorectal adenoma requires independent confirmation, and genotyping of INSR across a broader region and at greater density may be necessary to fully elucidate the nature of these interactions. (Cancer Epidemiol Biomarkers Prev 2007;16(4):703–8)

Insulin resistance, or the impaired ability to normalize plasma glucose levels, has emerged as a putative mechanism that links obesity with colorectal malignancy. The occurrence of type 2 diabetes mellitus, a pathologic sequel to chronic insulin resistance, is known to confer increased risk of colorectal cancer (1), and several metabolic consequences of the insulin-resistant state, including hyperinsulinemia, hyperglycemia, hypertriglyceridemia, and increased plasma levels of nonesterified fatty acids have been positively associated with colorectal cancer in prospective studies (2, 3). Indicators of insulin resistance, such as serum levels of insulin, C-peptide (a marker of insulin secretion), glycated hemoglobin, and glucose have been positively associated with colorectal neoplasia in a number of studies (4-8). In addition, circulating levels of insulin-like growth factor I, which are invariably elevated in insulin resistance, have been associated with colon cancer risk (7, 9, 10).

At the molecular level, obesity-induced insulin resistance arises as a result of compromised insulin signaling in liver, muscle, and adipose tissue. This may be due to elevated levels of free fatty acids or cytokines from adipose tissue, such as tumor necrosis factor-α, which can antagonize phosphorylation events at the insulin receptor, thereby abrogating signaling pathways (11). Due to compensatory up-regulation of insulin synthesis by the pancreas, tissues that express insulin receptors, such as colon, may be chronically exposed to increased levels of insulin, free fatty acids, and glucose, with subsequent mitogenic and tumor-promoting effects (12, 13).

Several common genetic variants have been identified within the insulin signaling pathway that have a diabetogenic effect and are also associated with hyperinsulinemia and insulin resistance (14-17). Given the emerging relation between insulin resistance and colorectal cancer, genetic variants that predispose to insulin resistance may also confer increased susceptibility to colorectal neoplasia. However, there are limited data on the association of insulin resistance-related gene polymorphisms and colorectal neoplasia. Carriage of the R allele at the insulin receptor substrate-1 (IRS1) G972R polymorphic locus, which confers increased susceptibility to insulin resistance and type 2 diabetes mellitus (16, 18), was found to be positively associated with colon cancer in a large United States–based investigation (19). The same study also reported a positive association with colon cancer for heterozygosity at the IRS2 G927D locus (19). However, no studies of colorectal neoplasia have evaluated variants of the insulin gene itself or the insulin receptor, a pivotal component of the insulin signaling pathway.

We therefore investigated the association between common allelic variants of four key genes of the insulin signaling pathway: insulin (INS), insulin receptor (INSR), IRS1, and IRS2, and advanced left-sided colorectal adenoma, an established precursor of colorectal cancer, within the context of a large, early cancer detection program. In addition, we evaluated whether the association between single nucleotide polymorphisms (SNP) of these genes and colorectal adenoma was modified by body mass index (BMI) or glycemic load.

The case-control study described here was nested within the screening arm of the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO trial; ref. 20). The PLCO trial is a large, randomized controlled trial designed to test the efficacy of cancer screening and to investigate early markers and etiology of cancer (21, 22). Participants aged 55 to 74 were enrolled at 10 U.S. sites (Birmingham, AL; Denver, CO; Detroit, MI; Honolulu, HI; Marshfield, WI; Minneapolis, MN; Pittsburgh, PA; Salt Lake City, UT; St. Louis, MO, and Washington, D.C.). Those randomly assigned to the screening arm were offered a flexible sigmoidoscopy examination of the distal (left-sided) colorectum (60 cm) at study entry. If neoplastic lesions were detected, participants were referred for subsequent colonoscopic examination. All available medical and pathologic reports on follow-up were obtained and coded by trained medical record abstractors. The institutional review boards of the U.S. National Cancer Institute and the 10 screening centers approved the study, and all participants provided informed consent.

Participants for this case-control study were selected from individuals assigned to the screening arm of the PLCO Trial between September 1993 and September 1999 who had undergone a successful sigmoidoscopy (insertion to at least 50 cm within >90% of mucosa visible or a suspect lesion identified), completed the baseline risk factor questionnaire, and provided a blood sample for use in etiologic studies (n = 42,037). Of these, 4,834 were excluded because of a self-reported history of Crohn's disease, ulcerative colitis, familial polyposis, Gardner's syndrome, colorectal polyps, or cancer (except basal-cell skin cancer). We randomly selected 772 of 1,234 cases with at least one left-sided advanced colorectal adenoma (defined as ≥1 cm, high-grade dysplasia, or villous components) and 777 of 26,651 control participants with a negative sigmoidoscopy examination (i.e., no polyp or other suspect lesion detected), matched to the cases by gender and ethnicity.

Our strategy for selecting SNPs entailed collecting data on polymorphisms from publicly available databases such as dbSNP,⁸

SNP500,⁹ and SNPper¹⁰ and from literature searches on INS, INSR, IRS1, and IRS2. We selected SNPs located within exonic gene regions, putative regulatory regions, and exon-intron junctions with priority given to those linked with hyperinsulinemia and insulin resistance in previous studies. The selected SNPs were verified in a panel of 102 individuals of self-described Caucasian (n = 31), African-American (n = 24), Hispanic (n = 23), and Pacific Rim (n = 24) ethnicity (23) by resequencing ∼300 bp of DNA either side of the putatively polymorphic locus. SNPs with a minor allele frequency (MAF) of 5% or more among Caucasians (which represent 93% of our study population) were chosen for genotyping in the current study. This led to the selection of three SNPs in INS, five SNPs in INSR, and four SNPs in IRS1 and two SNPs in IRS2. Subsequent to this initial selection, a more comprehensive investigation of INSR was undertaken in which we resequenced around a SNP every 5-10 kb across the entire INSR gene region. Using this approach, an additional 34 SNPs with >5% MAF among Caucasians were verified. We applied the haplotype-tagging SNP (htSNP) selection method developed by Clayton et al. (htSNP2 software)¹¹ to exploit the correlation between SNPs and reduce the number of SNPs to be genotyped in our study population. A total of 11 htSNPs were selected, which included the 5 SNPs originally selected for INSR and 6 additional SNPs necessary to predict the 39 common SNPs among the SNP500 Caucasian population with high probability (R_H² = 0.90).

DNA was extracted from buffy coat or whole blood samples using routine methods. Genotyping was performed at the Core Genotyping Facility of the Division of Cancer Epidemiology and Genetics, National Cancer Institute, using TaqMan (Applied Biosystems, Foster City, CA).¹²

For validation purposes, TaqMan assays were initially applied to the 102 individuals with sequence information and were subsequently applied to the PLCO samples only if sequencing and TaqMan results were 100% concordant; otherwise, a new TaqMan assay was designed. For six cases and six controls, no genotype data were available due to insufficient DNA yields, DNA that did not amplify, or failure of unambiguous genotyping. Blinded quality control samples, comprising 40 individuals, were assayed between two and four times, and these genotypes were found to be 100% concordant. All polymorphic loci analyzed in this study had genotypic distributions consistent with Hardy-Weinberg equilibrium among the controls.

At baseline, participants filled out a risk factor questionnaire, including questions on demographic factors, personal and family medical history, physical activity, weight, height, and smoking history. Dietary intake for the year before enrollment was assessed using a 137-item food frequency questionnaire, including an additional 14 questions on supplement use. BMI was calculated as body weight in kilograms divided by the height in square meters. Details concerning the assessment of glycemic load were described elsewhere (24, 25).

Odds ratios (OR) and 95% confidence intervals (95% CI) for the association between genotypes and colorectal adenoma risk were estimated using unconditional logistic regression, with the most common genotype serving as reference. All analyses were adjusted for age, gender, ethnicity, and study center. Additional risk factors for colorectal tumors (smoking, nonsteroidal anti-inflammatory drug use, physical activity, BMI, educational attainment, and history of diabetes) did not change the β-coefficients for any of the genotypes by >10% and were not included as covariates in the analysis. To calculate tests for trend, we assigned the ordinal values 0, 1, and 2 to the most prevalent genotypes in rank order of homozygous for the common allele, heterozygous and homozygous for the rare allele, respectively.

We did gene-specific global tests of association by simultaneously including all of the SNPs in a given gene in the logistic regression model and then comparing it to a null model that includes none of the SNPs (26). In this multivariate analysis, each SNP was coded by two dummy variables corresponding to homozygous and heterozygous variant genotypes. The resulting likelihood-ratio χ² value had 2k degrees of freedom, with k denoting the number of SNPs in a given gene. We also did a global omnibus test for interaction for each individual gene with BMI or glycemic load in relation to colorectal adenoma by simultaneously including all of the cross-product terms of the BMI or glycemic load variables (coded as continuous variables) with the different genotypes for a gene (coded as dummy variables) and comparing it to a null model that includes only main effects for BMI or glycemic load and the genotypes. These multilocus global tests automatically adjust for multiple testing based on the degrees of freedom of the corresponding χ² test. Moreover, the multilocus tests can efficiently capture the multivariate linkage disequilibrium (LD) pattern within a gene and, hence, can be more efficient than tests based on SNPs for detecting associations or interactions when the true causal variant in the region may not have been genotyped.

If the global omnibus test for interaction was significant, we also investigated potential interactions between the individual SNPs and BMI (categorical variable dichotomized at BMI = 25 kg/m², which is the WHO's threshold for defining overweight) or glycemic load (dichotomized using the mean value among the control participants). To assess deviation from a multiplicative interaction model, we used the log likelihood ratio test to compare the fit of the logistic models with and without the interaction terms.

We also investigated the association between specific haplotypes and advanced colorectal adenoma. Haplotype blocks were characterized among the control individuals according to the method of Gabriel et al. (27) and were visualized using Haploview (28). For SNPs within the same block, we estimated haplotype frequencies with the expectation-maximization algorithm (29). We used logistic regression to estimate the associations between each haplotype and colorectal adenoma using the most common haplotype as the reference category. Because of very high LD within blocks, there was little phase ambiguity in haplotype reconstruction, and thus, the related statistical uncertainty was ignored (30).

In this study population, individuals with left-sided advanced colorectal adenoma were slightly older, less well educated, and had a higher prevalence of self-reported diabetes, compared with controls (Table 1).

Overall, there was little evidence for association of any of the four insulin resistance–related genes with prevalence of colorectal adenoma (Table 2). The P values for global association of the SNPs in INS, INSR, IRS1, and IRS2 were 0.08, 0.90, 0.46, and 0.79, respectively. There was some indication of association for a few of the individual SNPs. Homozygosity for the less common T-allele (versus A/A) at the INS IVS1-6 locus was weakly associated with colorectal adenoma, OR, 1.28 (95% CI, 0.86-1.91), although heterozygosity at this locus was inversely associated with risk, OR, 0.79 (95% CI, 0.63-1.00; P trend, 0.03; Table 2). Heterozygosity at the INSR exon 17-4 locus (C/T versus C/C) was positively associated with advanced adenoma risk, OR, 1.27 (95% CI, 1.01-1.61), and the data were suggestive of a significant trend (P = 0.08). Genotypes at two polymorphic loci in IRS1 were weakly associated with adenoma: IVS1 + 12245 (G/G versus CC), OR, 0.35 (95% CI, 0.12-1.01; P trend, 0.05); and IVS1 + 4357 (G/A versus G/G): OR, 1.25 (95% CI, 0.96-1.64; P trend, 0.25). Neither of the IRS2 polymorphisms was associated with colorectal adenoma. Haplotype analysis for each of the four genes did not reveal any statistically significant associations with colorectal adenoma (data not shown). Haplotype frequencies for INS, INSR, IRS1, and IRS2 markers in this study population are available upon request.

We detected a statistically significant interaction between INSR genotypes and BMI in colorectal adenoma risk (P value for global test = 0.003; d.f., 22; Table 3). For some of the SNPs, genotype-specific associations differed according to BMI, although only one of the individual tests for interaction (for INSR IVS14+88G/A) approximated statistical significance (P_interaction = 0.05; data not shown). We observed no statistically significant interactions between BMI and the other genes under investigation, nor any interaction between glycemic load and these genes in relation to colorectal adenoma risk; however, there was the suggestion of an interaction between glycemic load and INSR genotypes in colorectal adenoma risk (P value for global test = 0.06; d.f., 22; Table 3). Restriction of analyses to non-Hispanic white individuals or nondiabetic subjects did not significantly alter any of the results. The association of the studied genetic polymorphisms with colorectal adenoma did not differ following stratification of the analysis by adenoma location (descending and sigmoid colon versus rectum).

Results from this large study of advanced left-sided colorectal adenomas do not provide strong evidence for associations between polymorphisms of the INS, INSR, IRS1, and IRS2 genes and disease risk. However, based on a global test of interaction, we found evidence of effect modification by BMI on the association of INSR gene variants and colorectal adenoma. Because the omnibus test for interaction adjusted for multiple testing, this finding can be considered as robust. However, exploration of the interaction of BMI with the individual SNPs in INSR did not suggest a single SNP that may explain the significance of the global test of interaction. These results instead indicate potential existence of an untyped causal variant(s) within the genetic region that is in LD with several of the SNPs under study. The insulin receptor is encoded by a large gene spanning almost 80 kb of DNA, and although we genotyped SNPs that were broadly spaced across the entire region, our coverage may not have been sufficient to capture all of the genetic variation in this population. The investigation of SNPs at greater density is necessary to further investigate the potential causal variants highlighted by this study and to replicate our finding.

The INSR is an important candidate gene to consider for susceptibility to colorectal neoplasia, given its pivotal role in insulin signaling. In effect, the INSR functions as a molecular switch, integrating external nutritional information into growth and metabolism-related intracellular signals. Several rare mutations have been identified in INSR that confer moderate to severe insulin resistance (31, 32); however, the identification of more common genetic variants that are associated with insulin resistance or type 2 diabetes mellitus has, thus far, proved fruitless (15, 33). Both hyperinsulinemia and obesity are associated with physiologic and biochemical changes at the level of the INSR, such as decreased expression of INSR at the cell surface and reduced tyrosine kinase activity. Furthermore, obesity is associated with elevated levels of adipokines such as tumor necrosis factor-α, which have been linked mechanistically to the development of insulin resistance via interactions at the insulin receptor level (11). It is therefore conceivable that differential effects of INSR variants are observed in overweight versus nonoverweight individuals.

Although we detected only weak associations for several of the SNPs under investigation in this study we feel some of these warrant discussion. The weakly positive association between the INS IVS1-6T/T genotype (also known as −23HphI) and colorectal adenoma was intriguing given that the T allele is in tight LD with the INS 5′-VNTR class III allele, which is associated with increased expression of insulin mRNA and insulin levels, and type II diabetes (14, 34-36). In addition, recent evidence suggests that the IVS1-6 A>T polymorphism itself has functional consequences, and homozygosity for the T-allele has been associated with a 89% increased risk of type II diabetes (37). The IVS1-6T/T genotype may, therefore, be a marker for elevated insulin levels. However, our interpretation is complicated by the apparent inverse association for the more prevalent heterozygous genotype, thus precluding a clear genetic model. Overall, these observations suggest a potential role for this gene in colorectal adenoma risk, but underscore the need for further investigation in a larger population sample.

IRS1 is a substrate for the insulin receptor tyrosine kinase and interacts with downstream effector pathways that modulate glucose metabolism and cell growth. We observed weakly positive associations between two SNPs within intron 1 of IRS1 and advanced colorectal adenoma. No known functionality has been attributed to these SNPs to date, and it is possible that they are in LD with some other causal variant. A nonsynonymous IRS1 SNP, G972R, has been associated with colon cancer previously (19). We were unable to investigate this polymorphism due to technical difficulties with the assay; however, it is possible that the associations observed for the two intronic SNPs in our study were reflective of the moderate LD that exists between these markers and the G972R polymorphism in the SNP500 population.⁹ This argument is countered somewhat by the fact that within the SNP500 Caucasians, the G972R SNP is in LD with the IRS1 Ex1 + 1723 SNP, which was not associated with colorectal adenoma in our study population. It is difficult to reach to any definitive conclusions because the SNP500 Caucasian population comprises only 31 individuals, and secondly, we do not know the LD pattern between our analytic SNPs and the G972R SNP in the study population.

A strength of this study is that all cases had an adenoma of advanced pathology, which are more likely to progress to carcinoma, rendering them an important surrogate end point for colorectal cancer. However, it should be noted that only left-sided adenomas were investigated, and the results may not be applicable to right-sided adenomas, which could potentially differ etiologically. The analysis of multiple SNPs across a gene increases the probability of detecting a signal from a causal variant; however, future work should aim to more comprehensively evaluate SNPs at greater density, thereby improving gene coverage. In addition, other components of the insulin signal transduction pathway, such as those downstream of IRS-1 and IRS-2, are potentially important candidate genes for colorectal neoplasia, especially those that regulate cell growth.

In summary, the findings presented here provide only weak support for a putative role of genetic variants in the insulin signaling pathway in relation to advanced colorectal adenoma risk. However, we present preliminary evidence for an interaction between genetic variation of the INSR and BMI in risk of advanced left-sided colorectal adenoma.