Abstract
Interleukins are a group of cytokines that contribute to growth and differentiation, cell migration, and inflammatory and anti-inflammatory responses by the immune system. In this study we examined genetic variation in genes from various anti-inflammatory and pro-inflammatory interleukins to determine association with colon and rectal cancer risk and overall survival. Data from two population-based incident studies of colon cancer (1555 cases and 1956 controls) and rectal cancer (754 cases and 954 controls) were utilized. After controlling for multiple comparisons, single nucleotide polymorphisms (SNPs) from four genes, IL3, IL6R, IL8, IL15, were associated with increased colon cancer risk and CXCR1, and CXCR2 were significantly associated with increased rectal cancer risk. Only SNPs from genes within the IL-8 pathway (IL8, CXCR1, and CXCR2) showed a significant association with both colon and rectal cancer risk. Several SNPs interacted significantly with IL8 and IFNG SNPs and with aspirin/NSAID, cigarette smoking, estrogen use and BMI. For both colon and rectal cancer, increasing numbers of risk alleles were associated with increased hazard of death from cancer; the estimated hazard of death for colon cancer for the highest category of risk alleles was 1.74 (95% CI 1.18–2.56) and 1.96 (95% CI 1.28–2.99) for rectal cancer. These data suggest interleukin genes play a role in risk and overall survival for colon and rectal cancer.
Keywords: Interleukins, colon cancer, rectal cancer, single nucleotide polymorphisms, inflammatory
Introduction
Interleukins are a type of cytokine that control growth and differentiation, cell migration and inflammatory and anti-inflammatory responses by the immune system (1). The balance between inflammatory and anti-inflammatory actions is essential for proper control of the immune response and protection against underlying tissue damage. However, different factors such as bacterial or viral infections, diet, individual genetics, and additional environmental factors may disrupt this balance and lead to a heightened state of inflammation which may cause tissue damage which may be acute in nature or lead to chronic conditions (2). Inflammation in the intestinal tract in particular has also been associated with chronic inflammatory bowels diseases such as Crohn’s disease, ulcerative colitis, as well as colon and rectal cancers (1–3).
Pro-inflammatory interleukins generally include IL-1, IL-2, IL-6, IL-8, IL-12, IL-15, and IL-17 while anti-inflammatory interleukins include IL-4 and IL-10. Additionally, IL-3 supports differentiation and proliferation of various immune cells thereby playing a significant role in immune responses. Genetic polymorphisms in a key pro-inflammatory interleukin within the gut, IL-6, have been associated with inflammatory bowel disease (4) as well as colon and rectal cancers (5). Chronic inflammation in the gut is linked to the activation of macrophages and T cells releasing pro-inflammatory cytokines including interleukins such as IL-1beta, IL-6, and IL-8 as well as TNF-alpha and IFN-gamma (4). Of particular note, signaling by the interleukin IL-8 has been shown to promote malignant progression of tumors (6) and has been associated with inflammatory pathways and gastric cancer (7). IL-8 can bind to two different forms of the IL-8 receptor: either IL-8 receptor alpha also called CXCR1 or IL-8 receptor beta also called CXCR2. Polymorphisms in the genes coding for IL-1 or the IL-1 receptor antagonist have been associated with chronic inflammatory diseases including ulcerative colitis (8, 9), Crohn’s disease (10) as well as survival and recurrence in colorectal cancer (11, 12). IL-17 is also associated with inflammatory bowel disease (13). Research of chronic inflammation and host defense indicates that IL-17 works in conjunction with IL-23 to contribute to the pathogenesis observed in inflammatory diseases (14). Polymorphisms in the human IL-23 receptor have already been highlighted in the work on inflammatory bowel disease (15–17).
Improper regulation of anti-inflammatory interleukins is also implicated in cancer and inflammatory disease within the gut. IL-10 is an anti-inflammatory interleukin, the absence of which has been associated with an inflammatory condition and several inflammatory diseases as well as cancers (1, 3, 18, 19). IL-4 plays a role in anti-inflammatory mechanisms and researchers have assessed the association of polymorphisms in IL-4 with risk of developing colorectal cancer in a Korean population (20).
Thus far, few studies have evaluated genetic variation within the pro-inflammatory and anti-inflammatory interleukins. The interrelated actions and balance between inflammatory and anti-inflammatory interleukins may influence risk of developing colon and rectal cancer. We investigated the genetic variation in IL10, IL15, IL1A, IL1B, IL1RN, IL2, IL3, IL4, IL6R, IL8, CXCR1, CXCR2, and IL17A in relation to the risk of developing colon and rectal cancer. Environmental influences which may impact responses to inflammatory cytokines such as aspirin/NSAID use, cigarette smoking, and estrogen use also were evaluated.
Methods
Data come from two population-based case-control studies of colon cancer (cases n=1,555; controls n=1,956) and rectal cancer (cases n=754; controls n=959). Colon cancer cases were identification between October 1, 1991 and September 30, 1994 and included people living in the Twin Cities Metropolitan Area, Kaiser Permanente Medical Care Program of Northern California (KPMCP) and a seven-county area of Utah (21). The rectal cancer study used identical data collection methods as the colon study, except cases came from the entire state of Utah. Cases also were population-based incident cases of the rectosigmoid junction or rectum who were diagnosed between May 1997 and May 2001 in Utah and KPMCP (22). For both studies, cases were between 30 and 79 years old at time of diagnosis with adenocarcinoma, English speaking, mentally competent to complete the interview, had no previous history of CRC, and no known familial adenomatous polyposis, ulcerative colitis, or Crohn’s disease. Controls were matched to cases by sex and by 5-year age groups. At KPMCP, controls were randomly selected from membership lists; in Utah, controls 65 years and older were randomly selected from the Health Care Financing Administration lists and younger controls were randomly selected from driver’s license lists. Controls were selected from driver’s license and state-identification lists in Minnesota. Details of the study have been previously reported (21, 22).
Interview Data Collection
Data were collected by trained and certified interviewers using laptop computers. All interviews were audio-taped and reviewed for quality control purposes (23). The referent period for the study was two years prior to diagnosis for cases and prior to selection for controls.
Tumor Registry Data
Tumor registry data were obtained to determine disease stage at diagnosis and months of survival after diagnosis. Disease stage was categorized centrally by one pathologist in Utah using the sixth edition of the American Joint Committee on Cancer (AJCC) staging criteria. Local tumor registries also provided information on patient follow-up including vital status, cause of death, and contributing cause of death.
Tumor Marker Data
We have previously evaluated tumors for CpG island methylator phenotype (CIMP), microsatellite instability (MSI), TP53 mutations, and KRAS2 mutations (24–27) and were therefore able to evaluate genes in relation to tumor molecular phenotype. Details for methods used to evaluate epigenetic and genetic changes have been described (24–27). Given the rarity of MSI+ rectal tumors (28) we are unable to evaluate that phenotype among rectal cancers.
TagSNP Selection and Genotyping
TagSNPs were selected using the following parameters: LD blocks were defined using a Caucasian LD map and an r2=0.8; minor allele frequency (MAF) >0.1; range= −1500 bps from the initiation codon to +1500 bps from the termination codon; and 1 SNP/LD bin. All markers were genotyped using a multiplexed bead-array assay format based on GoldenGate chemistry (Illumina, San Diego, California). A genotyping call rate of 99.85% was attained. Blinded internal replicates represented 4.4% of the sample set; the duplicate concordance rate was 100%. Individuals with missing genotype data were not included in the analysis for that specific marker. We evaluated associations with candidate genes, including IL1A, IL1B, IL1RN, IL2, IL3, IL4, IL6R, IL8, CXCR1, CXCR2, IL10, IL15, IL17A. Table 1 contains information about all SNPs included on the platform.
Table 1.
Summary of SNPs
| Gene | Location | Coordinate | tagSNP | Major/Minor Allele |
MAF | FDR HWE |
|---|---|---|---|---|---|---|
| IL6R | 1q21 | 152648673 | rs1386821 | A/C | 0.19 | 0.973 |
| 152655820 | rs4075015 | T/A | 0.42 | 0.963 | ||
| 152670960 | rs7549250 | T/C | 0.42 | 0.683 | ||
| 152682401 | rs4845623 | A/G | 0.42 | 0.509 | ||
| 152693594 | rs8192284 | A/C | 0.40 | 0.580 | ||
| 152696716 | rs11265618 | C/T | 0.18 | 0.895 | ||
| 152703008 | rs4509570 | C/G | 0.23 | 0.579 | ||
| 152704520 | rs2229238 | C/T | 0.19 | 0.234 | ||
| IL10 | 1q31-q32 | 205006527 | rs3024505 | C/T | 0.14 | 0.973 |
| 205008152 | rs3024498 | A/G | 0.28 | 0.997 | ||
| 205008487 | rs3024496 | T/C | 0.48 | 0.963 | ||
| 205010591 | rs3024493 | G/T | 0.14 | 0.963 | ||
| 205010856 | rs1554286 | C/T | 0.19 | 0.997 | ||
| 205011268 | rs1518111 | G/A | 0.21 | 0.997 | ||
| 205013257 | rs1800871 | C/T | 0.23 | 0.982 | ||
| 205013520 | rs1800896 | A/G | 0.48 | 0.954 | ||
| 205015988 | rs1800890 | T/A | 0.39 | 0.997 | ||
| IL1A | 2q14 | 113251301 | rs3783546 | G/C | 0.30 | 0.997 |
| 113253694 | rs17561 | G/T | 0.31 | 0.997 | ||
| 113256443 | rs2856838 | C/T | 0.39 | 0.997 | ||
| 113260048 | rs3783521 | C/T | 0.30 | 0.997 | ||
| 113260905 | rs1878321 | T/C | 0.31 | 0.997 | ||
| IL1B | 113304773 | rs1143643 | G/A | 0.35 | 0.997 | |
| 113306861 | rs1143634 | C/T | 0.23 | 0.997 | ||
| 113306938 | rs1143633 | G/A | 0.35 | 0.997 | ||
| 113310858 | rs1143627 | T/C | 0.34 | 0.997 | ||
| 113312300 | rs1143623 | G/C | 0.28 | 0.997 | ||
| IL1RN | 2q14.2 | 113590938 | rs4251961 | T/C | 0.38 | 0.997 |
| 113593250 | rs2637988 | A/G | 0.39 | 0.979 | ||
| 113595768 | rs3213448 | G/A | 0.12 | 0.020 | ||
| 113597418 | rs315936 | C/T | 0.25 | 0.997 | ||
| 113603929 | rs408392 | G/T | 0.27 | 0.997 | ||
| 113605371 | rs380092 | A/T | 0.32 | 0.997 | ||
| 113605532 | rs452204 | G/A | 0.39 | 0.952 | ||
| 113606775 | rs315952 | T/C | 0.30 | 0.901 | ||
| 113607057 | rs315951 | G/C | 0.30 | 0.920 | ||
| 113607883 | rs9005 | G/A | 0.29 | 0.997 | ||
| 113608612 | rs397211 | T/C | 0.29 | 0.997 | ||
| 113608995 | rs4252042 | G/A | 0.11 | 0.997 | ||
| 113609245 | rs315949 | C/T | 0.41 | 0.997 | ||
| CXCR2 | 2q35 | 218698831 | rs4674258 | C/T | 0.49 | 0.901 |
| 218708979 | rs1126579 | C/T | 0.47 | 0.954 | ||
| CXCR1 | 218735133 | rs1008563 | C/T | 0.44 | 0.997 | |
| 218735217 | rs1008562 | C/G | 0.47 | 0.901 | ||
| 218737177 | rs16858808 | C/T | 0.03 | 0.997 | ||
| 218737969 | rs1805038 | C/T | 0 | |||
| 218738088 | rs16858811 | T/G | 0.03 | 0.948 | ||
| IL8 | 4q13-q21 | 74824888 | rs4073 | T/A | 0.46 | 0.997 |
| 74825533 | rs2227307 | T/G | 0.45 | 0.997 | ||
| 74826774 | rs2227543 | C/T | 0.42 | 0.997 | ||
| IL2 | 4q26-q27 | 123591426 | rs2069776 | T/C | 0.27 | 0.682 |
| 123592583 | rs2069772 | A/G | 0.28 | 0.997 | ||
| 123595585 | rs2069778 | C/T | 0.16 | 0.874 | ||
| 123597430 | rs2069762 | T/G | 0.30 | 0.997 | ||
| IL15 | 4q31 | 142785143 | rs12508866 | T/C | 0.23 | 0.997 |
| 142789922 | rs1519551 | A/G | 0.45 | 0.997 | ||
| 142792798 | rs17461269 | T/A | 0.34 | 0.997 | ||
| 142818672 | rs13117878 | C/T | 0.48 | 0.979 | ||
| 142822580 | rs12498901 | G/C | 0.14 | 0.997 | ||
| 142856755 | rs6850492 | G/A | 0.43 | 0.973 | ||
| IL3 | 5q31.1 | 131423014 | rs181781 | G/A | 0.10 | 0.682 |
| 131424377 | rs40401 | C/T | 0.22 | 0.963 | ||
| 131424575 | rs35482671 | A/G | <0.01 | 0.997 | ||
| IL4 | 132040624 | rs2227284 | C/A | 0.26 | 0.997 | |
| 132041078 | rs2227282 | C/G | 0.26 | 0.997 | ||
| 132041198 | rs2243263 | G/C | 0.11 | 0.509 | ||
| 132041862 | rs2243268 | A/C | 0.14 | 0.997 | ||
| 132046068 | rs2243290 | C/A | 0.14 | 0.997 | ||
| IL17A | 6p12 | 52051957 | rs10484879 | G/T | 0.23 | 0.997 |
Minor Allele Frequency (MAF) and FDR-adjusted Hardy-Weinberg Equilibrium (FDR HWE) based on white control population.
Statistical Methods
Statistical analyses were performed using SAS® version 9.2 (SAS Institute, Cary, NC). We report odds ratios (ORs) and 95% confidence intervals (95%CIs) assessed from multiple logistic regression models adjusting for age, study center, race/ethnicity, and sex. SNPs were assessed assuming a co-dominant model and based on this initial assessment, those which appeared to have a dominant or recessive mode of inheritance were evaluated with those inheritance models in subsequent analysis. Main effect model p values were based on a 1-df Wald test statistic and interaction p values were determined using a likelihood-ratio test comparing a full model that included an ordinal interaction term with a reduced model without an interaction term.
Tumor markers were defined by specific alterations detected; CIMP+, any KRAS2 mutation, any TP53 mutation, and among colon cases, MSI+ or a combination of CIMP+/MSI+. Population-based controls were used to assess associations for the population overall when examining multiple outcomes defined by tumor status. A multinomial generalized estimating equation (GEE) approach was used to account for correlation introduced by including subjects multiple times and was implemented in SAS using the GENMOD procedure as described by Kuss and McLerran (29). We also report 1-df Wald p values from multiple logistic regression models comparing cases with and without a particular tumor phenotype. The minimal adjustments were controlled for in all models.
Survival-months were calculated based on month and year of diagnosis and month and year of death or date of last contact. Associations between SNPs and risk of dying of colorectal cancer were evaluated using Cox proportional hazards models to obtain multivariate hazard rate ratios (HRRs) and 95% confidence intervals. In addition to the minimal adjustments tumor molecular phenotype, and AJCC stage were also adjusted for when to estimating HRRs. To summarize risk associated with overall survival for multiple variants across the pathway we created a summary polygenic score that was based on all at-risk genotypes for rectal cancer. The score for each SNP was based on the inheritance model and its associated risk. For the co-dominant or additive model a score of zero, one, or two was assigned which directly correlates to the number of high-risk alleles. After assigning a score for each SNP previously identified as being significant after adjustment for multiple testing, the scores were summed across SNPs to generate an individual polygenic summary score. Individuals missing SNP data were dropped from the analysis. Trend p values are based on likelihood-ratio tests comparing full models including ordinal score terms with reduced models without score terms; Wald p values are also based on 1-df tests.
Adjusted multiple-comparison p values, taking into account tagSNPs within the gene, were estimated using the step-down Bonferroni correction (i.e., Holm method) based on the effective number of independent SNPs as determined using SNPSpD (http://genepi.qimr.edu.au/general/daleN/SNPSpD/) on the full sample of cases and controls (30, 31).
Results
The population was 55.69% male and 69.66% age 60 and over. The majority of participants were non-Hispanic white (90.07%), with 4.94% being Hispanic and 3.69% being African American. We examined 69 SNPs in 13 genes within the cytokine pathway in relation to colon and rectal cancer risk (Table 1). Of these, 15 SNPs in nine genes were significantly associated with either colon or rectal cancer. For colon cancer, one SNP in IL3 (rs181781), two in IL6R (rs4845623 and rs7549250), one in IL8 (rs4073), one in IL10 (rs3024505) and two in IL15 (rs13117878 and rs1519551) were significantly associated with cancer risk. When rectal cancer was examined, one SNP in IL1B (rs1143623), one in IL4 (rs2243263), two in CXCR1 (rs1008562 and rs1008563), one in CXCR2 (rs1126579), one in IL10 (rs1800871), and two in IL15 (rs12508866 and rs17461269) were significantly associated with risk. After adjustment for multiple comparisons 6 SNPs in six genes remained significantly associated with either colon or rectal cancer; IL3 (rs181781), IL6R (rs4845623), IL8 (rs4073), IL15 (rs13117878), remained significantly associated with increased colon cancer risk and CXCR1 (rs1008562), and CXCR2 (rs1126579) remained significantly associated with increased rectal cancer risk (Table 2).
Table 2.
Adjusted significant SNPs in candidate interleukin genes
| Controls | Cases | OR (95% CI) | Wald P | Holm P | ||
|---|---|---|---|---|---|---|
| Colon Cancer | ||||||
| IL3 | rs181781 | 0.0249 | 0.0408 | |||
| GG/GA | 1945 | 1536 | 1.00 | |||
| AA | 10 | 19 | 2.42 (1.12,5.22) | |||
| IL6R | rs4845623* | 0.0046 | 0.0278 | |||
| AA/AG | 1641 | 1242 | 1.00 | |||
| GG | 315 | 313 | 1.29 (1.08, 1.53) | |||
| IL8 | rs4073** | 0.0114 | 0.0149 | |||
| TT | 576 | 396 | 1.00 | |||
| TA/AA | 1380 | 1159 | 1.21 (1.04, 1.41) | |||
| IL15 | rs13117878 | 0.0091 | 0.0362 | |||
| CC/CT | 1520 | 1151 | 1.00 | |||
| TT | 436 | 402 | 1.23 (1.05, 1.44) | |||
| Rectal Cancer | ||||||
| CXCR1 | rs1008562 | 0.0010 | 0.0029 | |||
| CC | 283 | 187 | 1.00 | |||
| CG | 466 | 356 | 1.18 (0.94, 1.49) | |||
| GG | 206 | 211 | 1.57 (1.20, 2.05) | |||
| CXCR2 | rs1126579*** | 0.0012 | 0.0014 | |||
| CC | 290 | 192 | 1.00 | |||
| CT | 470 | 359 | 1.18 (0.94, 1.49) | |||
| TT | 199 | 201 | 1.56 (1.19, 2.05) | |||
Odds Ratios (OR) and 95% Confidence Intervals (CI)adjusted for age, sex, study center, and race/ethnicity.
Similar associations for IL6R rs8192284 (r2=0.83).
Similar associations for IL8 rs2227307 (r2=0.94).
Associations of similar magnitude (in opposite direction) for IL8RB rs4674258 (r2=0.81).
Only SNPs from genes within the IL-8 pathway (IL8, CXCR1, and CXCR2) showed a significant association with both colon and rectal cancer risk, but different SNPs showed significance for each cancer type. Several SNPs within the IL8 gene and IL-8 receptor genes CXCR1 and CXCR2, were associated with increased risk of colon or rectal cancer. For colon cancer risk, the variant allele for IL8 rs4073 was associated with increased risk (OR 1.21, 95% CI 1.04–1.41; Wald p value 0.0114, Holm p value 0.0149). The CC genotype for rs1008562 in CXCR1 was associated with increased risk of rectal cancer compared to the CG or GG genotypes (OR 1.18, 95% CI 0.94–1.49 and OR 1.57, 95% CI 1.20–2.05, respectively; Wald p value 0.0010, Holm p value 0.0029). The CXCR2 rs1126579 TT genotype was also associated with increased risk of rectal cancer compared to CC or CT genotypes (OR 1.18, 95% CI 0.94–1.49 and OR 1.56, 95% CI 1.19–2.05, respectively; Wald p value 0.0012, Holm p value 0.0014).
Interactions between cytokine SNPs and factors associated with colon and rectal cancer risk were examined; those with adjusted p values of <0.05 are shown (Table 3). There were no significant findings after controlling for multiple testing for interaction with NSAIDS, smoking, or BMI. However, when recent estrogen use was examined one SNP in IL10 (rs1554286 Holm p value 0.0376), two SNPs in IL1B (rs1143623 Holm p value 0.0375 and rs1143627 Holm p value 0.0375), and one SNP in CXCR2 (rs1126579 Holm p value 0.0393) interacted significantly for colon cancer whereas one SNP for IL1A (rs3783546 Holm p value 0.0317) and two SNPs for IL8 (rs2227543 Holm p value 0.0407 and rs4073 Holm p value 0.0407) had significant interactions for rectal cancer. There were additional SNPs from interleukin genes with significant raw p-values for estrogen use but after controlling for multiple testing they did not remain significant.
Table 3.
Interaction between aspirin/NSAID use, cigarette smoking, estrogen exposure, and BMI and SNPs in candidate interleukin genes.
| Controls | Cases | Controls | Cases | Interaction P | Holm P | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| N | N | OR | (95% CI) | N | N | OR | (95% CI) | |||
| No Recent Estrogen Exposure | Recent Estrogen Exposure | |||||||||
| Colon Cancer | ||||||||||
| IL10 (rs1554286)* | 0.0094 | 0.0376 | ||||||||
| CC | 318 | 297 | 1.00 | 246 | 139 | 0.49 | (0.37, 0.67) | |||
| CT | 173 | 134 | 0.83 | (0.63, 1.09) | 109 | 73 | 0.58 | (0.40, 0.83) | ||
| TT | 32 | 18 | 0.56 | (0.31, 1.04) | 6 | 10 | 1.40 | (0.49, 3.97) | ||
| IL1B (rs1143623) | 0.017 | 0.0375 | ||||||||
| GG | 259 | 259 | 1.00 | 196 | 124 | 0.50 | (0.36, 0.69) | |||
| GC | 216 | 167 | 0.75 | (0.58, 0.98) | 147 | 75 | 0.41 | (0.28, 0.58) | ||
| CC | 48 | 23 | 0.47 | (0.28, 0.81) | 18 | 22 | 0.97 | (0.50, 1.89) | ||
| IL1B (rs1143627) | 0.0125 | 0.0375 | ||||||||
| TT | 197 | 208 | 1.00 | 155 | 103 | 0.50 | (0.35, 0.71) | |||
| TC | 249 | 199 | 0.73 | (0.56, 0.96) | 175 | 86 | 0.37 | (0.26, 0.53) | ||
| CC | 77 | 42 | 0.48 | (0.31, 0.74) | 31 | 33 | 0.77 | (0.45, 1.34) | ||
| CXCR2 (rs1126579) | 0.0331 | 0.0393 | ||||||||
| CC | 147 | 107 | 1 | 87 | 66 | 0.85 | (0.55, 1.31) | |||
| CT/TT | 376 | 342 | 1.28 | (0.96, 1.72) | 274 | 156 | 0.65 | (0.46, 0.92) | ||
| Rectal Cancer | ||||||||||
| IL1A (rs3783546)** | 0.0127 | 0.0317 | ||||||||
| GG/GC | 156 | 124 | 1.00 | 214 | 150 | 0.75 | (0.53, 1.07) | |||
| CC | 13 | 17 | 1.63 | (0.76, 3.51) | 35 | 11 | 0.33 | (0.16, 0.70) | ||
| IL8 (rs2227543) | 0.0312 | 0.0407 | ||||||||
| CC | 71 | 50 | 1.00 | 72 | 60 | 1.02 | (0.60, 1.71) | |||
| CT/TT | 98 | 91 | 1.36 | (0.86, 2.18) | 177 | 101 | 0.70 | (0.44, 1.11) | ||
| IL8 (rs4073) | 0.0376 | 0.0407 | ||||||||
| TT | 62 | 39 | 1 | 63 | 49 | 1.07 | (0.60, 1.89) | |||
| TA/AA | 107 | 102 | 1.54 | (0.95, 2.51) | 186 | 112 | 0.82 | (0.50, 1.34) | ||
Odds Ratios (OR) and 95% Confidence Intervals (CI) adjusted for age, sex, study center, and race/ethnicity.
Similar associations for IL10 rs1518111 (r2=0.86 with rs1554286).
Similar associations for IL1A rs3783521 (r2=0.97).
IL1A codes for IL-1alpha and IL1B codes IL-1beta both of which are cytokines that bind specifically to the IL-1 receptor; the IL1RN gene codes for an antagonist which binds to the IL-1 receptor, thus we examined interactions between SNPs in these genes. There were no significant interactions between SNPs within this pathway for colon or rectal cancer after adjusting for multiple comparisons. IL-8 can bind to two different forms of the IL-8 receptor: either IL-8 receptor alpha also called CXCR1 or IL-8 receptor beta also called CXCR2. Therefore interactions between SNPs in these genes were examined. For colon cancer, one IL8 SNP (rs4073) interacted with two CXCR1 SNPs (rs1008563 and rs1008562) and one CXCR2 SNP (rs1126579) (Table 4). No interactions were detected for rectal cancer within this pathway. Interferon gamma is a well-known pro-inflammatory cytokine and interactions between other cytokines or receptors within the pro-inflammatory pathway were examined. For colon cancer, two SNPs from the IL8 gene (rs4073 and rs2227543) interacted significantly with one SNP from the IFNG gene (rs2069718) after multiple comparison adjustment (Table 4). For rectal cancer, similar interactions between SNPs within the pro-inflammatory pathways were tested; however there were no significant interactions after adjusting for multiple comparisons. Anti-inflammatory interleukins were also tested for interactions and no significant interactions were observed for colon or rectal cancer.
Table 4.
Associations between candidate interleukin genes in colon cancer
| Colon | ||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Controls | Cases | Controls | Cases | Controls | Cases | Interaction P | Holm P | |||||||||||
| N | N | OR | (95% CI) | N | N | OR | (95% CI) | N | N | OR | (95% CI) | |||||||
| IL8 (rs4073)* | ||||||||||||||||||
| TT | TA | AA | ||||||||||||||||
| CXCR1(rs1008563) | CC | 185 | 152 | 1 | 290 | 254 | 1.06 | (0.81,1.39) | 149 | 118 | 0.92 | (0.67,1.28) | ||||||
| CT | 286 | 185 | 0.77 | (0.58,1.02) | 486 | 389 | 0.96 | (0.75,1.24) | 208 | 182 | 1.03 | (0.77,1.39) | ||||||
| TT | 104 | 59 | 0.68 | (0.46,1.00) | 181 | 136 | 0.9 | (0.66,1.23) | 66 | 76 | 1.34 | (0.90,1.99) | 0.0067 | 0.0233 | ||||
| CXCR1(rs1008562) | CC | 153 | 100 | 1 | 262 | 195 | 1.14 | (0.83,1.56) | 112 | 118 | 1.55 | (1.08,2.24) | ||||||
| CG | 292 | 182 | 0.95 | (0.69,1.30) | 498 | 418 | 1.29 | (0.97,1.71) | 215 | 186 | 1.31 | (0.95,1.81) | ||||||
| GG | 130 | 114 | 1.36 | (0.95,1.95) | 195 | 167 | 1.34 | (0.96,1.85) | 96 | 70 | 1.11 | (0.74,1.65) | 0.0233 | 0.0447 | ||||
| CXCR2(rs1126579)** | CC | 158 | 98 | 1 | 266 | 200 | 1.21 | (0.88,1.65) | 112 | 119 | 1.63 | (1.13,2.35) | ||||||
| CT | 289 | 187 | 1.04 | (0.76,1.42) | 500 | 420 | 1.36 | (1.02,1.80) | 220 | 187 | 1.36 | (0.99,1.87) | ||||||
| TT | 129 | 111 | 1.41 | (0.99,2.02) | 191 | 163 | 1.41 | (1.02,1.96) | 91 | 70 | 1.23 | (0.82,1.84) | 0.0265 | 0.0265 | ||||
| IFNG (rs2069718) | ||||||||||||||||||
| IL8 (rs4073)*** | TT | 187 | 148 | 1 | 272 | 180 | 0.83 | (0.62,1.11) | 117 | 68 | 0.71 | (0.49,1.03) | ||||||
| TA | 297 | 282 | 1.2 | (0.91,1.57) | 474 | 364 | 0.97 | (0.75,1.25) | 186 | 137 | 0.9 | (0.66,1.23) | ||||||
| AA | 157 | 116 | 0.91 | (0.66,1.25) | 186 | 180 | 1.19 | (0.88,1.61) | 80 | 80 | 1.19 | (0.81,1.75) | 0.0204 | 0.0357 | ||||
| IL8(rs2227543)*** | CC | 219 | 184 | 1 | 327 | 236 | 0.85 | (0.66,1.11) | 152 | 95 | 0.69 | (0.50,0.96) | ||||||
| CT | 291 | 266 | 1.11 | (0.86,1.44) | 448 | 345 | 0.93 | (0.73,1.19) | 182 | 139 | 0.9 | (0.67,1.21) | ||||||
| TT | 131 | 96 | 0.88 | (0.63,1.22) | 157 | 143 | 1.09 | (0.81,1.47) | 49 | 51 | 1.22 | (0.78,1.89) | 0.0207 | 0.0357 | ||||
Odds Ratios (OR) and 95% Confidence Intervals (CI) adjusted for age, sex, study center, and race/ethnicity.
Similar associations for IL8 rs2227307 (r2=0.94)
Associations of similar magnitude (in opposite direction) for IL8RB rs4674258 (r2=0.81).
Similar associations for IL8 rs2227307 (r2=0.94 with rs4073 and r2=0.84 with rs2227543).
Associations between tumor markers and SNPs from interleukin genes were assessed (Table 5). For colon cancer, four SNPs from IL1RN (rs452204, rs315952, rs4251961, and rs315949) and one SNP from IL1B (rs1143623) were significantly associated with CIMP+ and/or MSI+ tumor markers when compared to other tumor molecular phenotypes. KRAS2 mutations were associated with one IL17A SNP (rs10484879) for colon cancer and one IL1B SNP (rs1143633) was associated for both colon and rectal cancer. Also for rectal cancer, one SNP from IL8 (rs2227543) was associated with TP53 mutations after adjustments for multiple comparisons.
Table 5.
Associations between candidate interleukin genes and tumor markers
| Heterogeneity P Values |
||||||
|---|---|---|---|---|---|---|
| Controls | Cases | OR | (95% CI) | Wald P | Holm P | |
| Colon | CIMP+ and/or MSI+ |
|||||
| IL1RN (rs452204)1 | ||||||
| GG/GA | 1668 | 314 | 1 | 0.0005 | 0.0024 | |
| AA | 288 | 35 | 0.63 | (0.44,0.89) | ||
| IL1RN (rs315952)2 | ||||||
| TT/TC | 1767 | 327 | 1 | 0.0464 | 0.0478 | |
| CC | 189 | 22 | 0.62 | (0.40, 0.95) | ||
| IL1RN (rs4251961) | ||||||
| TT | 776 | 116 | 1 | 0.0004 | 0.0022 | |
| TC/CC | 1180 | 233 | 1.4 | (1.12, 1.75) | ||
| IL1RN (rs315949) | ||||||
| CC | 702 | 109 | 1 | 0.0015 | 0.0062 | |
| CT | 930 | 163 | 1.17 | (0.92, 1.50) | ||
| TT | 324 | 77 | 1.6 | (1.19, 2.14) | ||
| IL1B (rs1143623) | ||||||
| GG/GC | 1811 | 337 | 1 | 0.0012 | 0.0037 | |
| CC | 145 | 12 | 0.45 | (0.26, 0.79) | ||
| KRAS2 Mutations |
||||||
| IL17A (rs10484879) | ||||||
| GG | 1201 | 239 | 1.00 | 0.0111 | 0.0111 | |
| GT/TT | 772 | 114 | 0.76 | (0.61, 0.95) | ||
| IL1B (rs1143633)3 | ||||||
| GG | 819 | 123 | 1.00 | 0.0072 | 0.0217 | |
| GA/AA | 1137 | 224 | 1.30 | (1.04, 1.62) | ||
| Rectal | ||||||
| IL1B (rs1143633)3 | ||||||
| GG | 428 | 88 | 1.00 | 0.0054 | 0.0151 | |
| GA | 398 | 73 | 0.89 | (0.65, 1.21) | ||
| AA | 133 | 12 | 0.44 | (0.24, 0.79) | ||
| TP53 Mutations |
||||||
| IL8 (rs2227543) | ||||||
| CC | 330 | 113 | 1.00 | 0.0016 | 0.0021 | |
| CT | 474 | 134 | 0.82 | (0.63, 1.06) | ||
| TT | 155 | 30 | 0.58 | (0.38, 0.88) | ||
Models are adjusted for age, study center, race/ethnicity, and sex.
Odds Ratios (OR) and 95% Confidence Intervals (CI) compare cases with tumor molecular phenotype to controls.
Heterogeneity p values based on comparison of cases with tumor molecular phenotype to cases without.
Similar associations for IL1RN rs2637988 (r2=0.88).
Similar associations for IL1RN rs315951 (r2=0.99).
Similar associations for IL1B rs1143643 (r2=0.99).
We also examined whether SNPs in the cytokine pathway were associated with overall survival rates from colorectal cancer (Table 6). For colon cancer, two SNPs in IL1B were associated with overall survival. For rectal cancer, SNPs in IL1A, IL3, and CXCR2 were associated with overall survival. CXCR2 rs1126579 was associated with both risk and overall survival for rectal cancer. Summary scores were generated to reflect the combined effect of risk alleles on overall survival of rectal disease. Increasing numbers of risk alleles were associated with increased hazard of death from cancer. The estimated hazard of death for rectal cancer for the highest category of risk alleles was 2.04 (95% CI 1.38–3.00).
Table 6.
Colorectal Overall Survival
| Death/ Person Years |
HRR | (95% CI) | Trend P | Wald P | Holm P | ||
|---|---|---|---|---|---|---|---|
| Colon Cancer | |||||||
| IL1B | rs1143623 | ||||||
| GG | 162 / 4625 | 1.00 | 0.0073 | 0.0220 | |||
| GC/CC | 147 / 3518 | 1.37 | (1.09, 1.72) | ||||
| rs1143627 | |||||||
| TT | 126 / 3682 | 1.00 | 0.0232 | 0.0465 | |||
| TC/CC | 183 / 4466 | 1.31 | (1.04, 1.65) | ||||
| Rectal Cancer | |||||||
| IL1A | rs3783546* | ||||||
| GG | 73 / 2099 | 1.00 | 0.0204 | 0.0191 | 0.0478 | ||
| GC | 72 / 1831 | 1.08 | (0.77, 1.52) | ||||
| CC | 26 / 359 | 2.07 | (1.28, 3.36) | ||||
| IL3 | rs181781 | ||||||
| GG | 128 / 3475 | 1.00 | 0.0253 | 0.0196 | 0.0321 | ||
| GA | 36 / 761 | 1.31 | (0.90, 1.92) | ||||
| AA | 7 / 53 | 2.47 | (1.11, 5.53) | ||||
| IL8RB | rs1126579** | ||||||
| CC | 38 / 1109 | 1.00 | 0.0228 | 0.0233 | 0.0277 | ||
| CT | 75 / 2077 | 1.14 | (0.76, 1.71) | ||||
| TT | 58 / 1087 | 1.61 | (1.05, 2.46) | ||||
| Summary Score*** | |||||||
| (0–1) | 54/1833 | 1.00 | 0.0003 | ||||
| (2–2) | 54/1405 | 1.36 | (0.92, 2.00) | ||||
| (3–6) | 63/1051 | 2.04 | (1.38, 3.00) | ||||
Hazard Rate Ratios (HRR) and 95% Confidence Intervals (CI) adjusted for age, study center, race/ethnicity, sex, AJCC stage, and tumor molecular phenotype.
Similar associations for IL1A rs3783521 (r2=0.97).
Similar associations for IL8RB rs4674258 (r2=0.81).
Discussion
Chronic inflammatory pathways in the gut have been implicated in the etiology of different cancer types including colon and rectal cancers (1–3). Our study further supports the roles of pro-inflammatory and anti-inflammatory cytokines in risk and overall survival for colon and rectal cancers. Previous studies of colorectal cancer are often not large enough to distinguish between colon and rectal cancer. However, recent publications indicate differences in genetic variations associated with risk and overall survival between colon and rectal cancer (5, 32–34). The majority of risk alleles for both colon and rectal cancer in this study are within the pro-inflammatory classification.
Constitutive expression of pro-inflammatory interleukins leads to an inflammatory state within the gut and is thought to increase risk for the development of colon and rectal cancer (2). The pro-inflammatory interleukins analyzed in this report included IL-1, IL-2, IL-8, IL-12, IL-15, and IL-17 while anti-inflammatory interleukins included IL-4 and IL-10. The IL1RN gene codes for the IL-1 receptor antagonist (IL-1RA) and would be considered anti-inflammatory. IL-3 was considered separately as it supports differentiation and proliferation of various immune cells, thereby playing a significant role in immune responses. Of the interleukins genes tested, we are not aware of any previous reports of associations with SNPs from IL2, IL3, IL4, IL15, or IL17 with colon or rectal cancer. There are, however, reports of genetic associations between IL1A, IL1B, IL1RN, IL2, IL4, IL10, IL17, and inflammatory bowel disease (35–39). Associations between SNPs in interleukin genes and risk or survival have been reported with conflicting results. For example, the IL8 rs4073 SNP was significantly associated with reduced risk in one study (40) although two additional studies indicated no affect on colorectal risk (12, 41). This SNP was significantly associated with increased colon cancer risk in the current study. A third study indicated that patients with IL8 rs4073 had an increased time to recurrence of advanced colorectal cancer (42), but we found no significant associations between this SNP and overall survival for either colon or rectal cancer. Studies analyzing associations between risk or survival and SNPs in interleukin genes such as IL1B, IL1RA, IL10 have also reported conflicting results; some SNPs being associated with increased risk or survival while others associated with a lower risk or survival for colorectal cancer (11, 12, 42–44).
The SNP associations for the cytokines examined in this study were not significantly modified by several of the environmental influences tested, including aspirin/NSAID use or cigarette smoking. However, there is evidence to suggest that estrogens may influence inflammation-related mechanisms (45, 46) which is supported by our data. Estrogen use was associated with increased risk for the TT genotype of one IL10 SNP (rs1554286) in colon. The variant GC or CC genotypes of IL1B (rs1143623) and TC or CC genotypes of IL1B (rs1143627) appear to be protective among non-recent estrogen users for colon cancer. One SNP from the IL-8 interleukin pathway also interacted with recent estrogen use; the CT/TT genotypes of CXCR2 (rs1126579) were protective for colon cancer among recent users. For rectal cancer, recent estrogen use interacted with several SNPs from interleukins genes. Among recent users, a decreased cancer risk was seen for the CC genotype of IL1A (rs3783546), CT/TT genotype of IL8 (rs2227543) and TA/AA genotypes of IL8 (rs4073).
One SNPs from IL-8 receptor gene CXCR2 (rs1126579), were associated with both risk and overall survival of rectal cancer. In addition, this SNP also interacted with one SNP from IL-8, IL8 (rs4073). For CXCR2 (rs1126579), two copies of the variant allele increases colon cancer risk among those with the TT genotype for IL8 (rs4073). The functional effects of this SNP is unknown at the present time. However, the SNP is found at the 3’ end of each respective gene and could potentially alter translation and stability of RNA. No reports of the functional affect of this SNP is currently available, however, given the results from multiple previous studies and our own results, it merits further investigation.
The study has many strengths in that it is a large study with the power to evaluate colon and rectal cancer separately; there is increasing evidence that these represent distinct cancer sites. The data were collected in a very rigid manner using strict quality control measures. We used a tagSNP approach to characterize the genetic variation within the candidate genes. Our analysis was hypothesis driven, and included evaluation of candidate genes and interaction of those genes with lifestyle and genetic factors that had a biological basis. However, we have made numerous comparisons, and although we have adjusted for multiple comparisons, findings could be spurious. Additionally, we lack information on functionality of these SNPs. Assessment of functionality of SNPs associated with colon and rectal cancer is needed.
In summary, genetic variations in multiple interleukin genes were associated with risk for colon or rectal cancer. Few associations were modified by lifestyle factors such as aspirin/NSAID use, cigarette smoking, or obesity; however, several associations were modified by current estrogen use. Genetic variation within interleukins also impacted overall survival after diagnosis. As a whole, our data suggests that interleukin genes play a role in risk and overall survival for colon and rectal cancer and future studies to gain further insight into specific mechanisms are warranted.
Acknowledgements
This study was funded by NCI grants CA48998. This research also was supported by the Utah Cancer Registry, which is funded by Contract #N01-PC-67000 from the National Cancer Institute, with additional support from the State of Utah Department of Health, the Northern California Cancer Registry, and the Sacramento Tumor Registry. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official view of the National Cancer Institute. We would like to acknowledge the contributions of Dr. Bette Caan, Donna Schaffer, and Judy Morse at the Kaiser Permanente Medical Care Program in Oakland, California;, Sandra Edwards, Roger Edwards, and Leslie Palmer at the University of Utah; and Drs. Kristin Anderson and John Potter at the University of Minnesota for data management and collection.
Abbreviations
- SNPs
Single Nucleotide Polymorphisms
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