Sequence Variants of Toll-Like Receptor 4 and Susceptibility to Prostate Cancer

Chronic inflammation has been hypothesized to be a risk factor for prostate cancer. The Toll-like receptor 4 (TLR4) presents the bacterial lipopolysaccharide (LPS), which interacts with ligand-binding protein and CD14 (LPS receptor) and activates expression of inflammatory genes through nuclear factor-κB and mitogen-activated protein kinase signaling. A previous case-control study found a modest association of a polymorphism in the TLR4 gene [11381G/C, GG versus GC/CC: odds ratio (OR), 1.26] with risk of prostate cancer. We assessed if sequence variants of TLR4 were associated with the risk of prostate cancer. In a nested case-control design within the Health Professionals Follow-up Study, we identified 700 participants with prostate cancer diagnosed after they had provided a blood specimen in 1993 and before January 2000. Controls were 700 age-matched men without prostate cancer who had had a prostate-specific antigen test after providing a blood specimen. We genotyped 16 common (>5%) single nucleotide polymorphisms (SNP) discovered in a resequencing study spanning TLR4 to test for association between sequence variation in TLR4 and prostate cancer. Homozygosity for the variant alleles of eight SNPs was associated with a statistically significantly lower risk of prostate cancer (TLR4_1893, TLR4_2032, TLR4_2437, TLR4_7764, TLR4_11912, TLR4_16649, TLR4_17050, and TLR4_17923), but the TLR4_15844 polymorphism corresponding to 11381G/C was not associated with prostate cancer (GG versus CG/CC: OR, 1.01; 95% confidence interval, 0.79-1.29). Six common haplotypes (cumulative frequency, 81%) were observed; the global test for association between haplotypes and prostate cancer was statistically significant (χ² = 14.8 on 6 degrees of freedom; P = 0.02). Two common haplotypes were statistically significantly associated with altered risk of prostate cancer. Inherited polymorphisms of the innate immune gene TLR4 are associated with risk of prostate cancer. (Cancer Res 2005; 65(24): 11771-8)

Human Toll-like receptor (TLR) is a type I transmembrane protein with an extracellular domain consisting of a leucine-rich repeat region and an intracellular domain homologous to that of the human interleukin (IL)-1 receptor (1). Lipopolysaccharide (LPS) interacts with ligand-binding protein and CD14, a LPS receptor, to present LPS to TLR4, which then activates the expression of inflammatory genes (e.g., IL-1, IL-6, and IL-8) through either nuclear factor-κB or mitogen-activated protein kinase signaling (2, 3). Human TLR4 is located on chromosome 9q32-q33, has four exons, and is highly expressed in lymphocytes, spleen, and the heart (4). Exposure to bacterial products or proinflammatory cytokines increases TLR4 expression in monocytes and polymorphonuclear leukocytes (2, 5). TLR4-deficient or TLR4-mutant mice showed lower response to viral and bacterial infection than did wild-type mice (6–8), which was related to a subsequent reduction in the innate immune response. Previous work has reported that genetic variation in TLR4 is related to the risk of atherosclerosis (9, 10), septic shock, smallpox, Chlamydia trachomatis, Chlamydia pneumoniae, and Mycobacterium tuberculosis and related to the pathogen recognition of Gram-negative bacteria and respiratory syncytial virus (11–13).

An association study (14) in a Swedish population explored the relationship between TLR4 sequence variations and risk of prostate cancer. In that study, among eight single nucleotide polymorphisms (SNP) studied, a sequence variant (11381G/C) in the 3′ untranslated region (UTR) of TLR4 was associated with prostate cancer risk [GG versus CG/CC: odds ratio (OR), 1.26; 95% confidence interval (95% CI), 1.01-1.57]. The same investigators also found that other SNPs in other genes in the TLR family, which formed TLR6-TLR1-TLR10 gene cluster, were associated with the risk of prostate cancer (15). Chronic inflammation has been associated with some cancers (e.g., cervix, breast, primary liver, and bladder cancers; ref. 16). An emerging body of evidence, including studies on sexually transmitted infections, clinical prostatitis, and genetic and circulating markers of inflammation and response to infection, supports a possible link between chronic intraprostatic inflammation and risk of prostate cancer (17). Therefore, we hypothesized that genetic polymorphisms of TLR4 are associated with the risk of prostate cancer. We did both SNP and haplotype analyses to test this hypothesis.

Study population. In this nested case-control study, incident prostate cancer cases were identified from the ongoing Health Professionals Follow-up Study (HPFS) with follow-up from 1986 to 2000. A total of 51,529 U.S. men ages 40 to 75 years were enrolled in 1986. Every participant completed a mailed questionnaire on demographics, lifestyle, and medical history and a semiquantitative food frequency questionnaire at baseline. Information on exposures and diseases was updated every other year, and diet information was updated every 4 years. Deaths were identified through reports by family members, the follow-up questionnaires, or a search of the National Death Index (18). This study was approved by the institutional review board at the Harvard School of Public Health.

Blood samples were obtained from 18,018 of the participants between 1993 and 1995 and collected in tubes containing sodium EDTA. Samples were shipped by overnight courier and centrifuged; the aliquots, including plasma, erythrocytes, and buffy coat, were stored in liquid nitrogen freezers. We used a QIAamp blood extraction kit (Qiagen, Inc., Valencia, CA) for DNA extraction. All DNA samples were whole genome amplified, and the quality-control samples had 100% genotype concordance rates. Among the men who gave a blood specimen, 95% responded to the 2000 questionnaire and 18 died of prostate cancer before the end of follow-up and were included in the case series.

We identified 700 incident prostate cancer cases and 700 controls (97% were Caucasians). Each case was matched with one control who was alive, had not been diagnosed with cancer by the date of the case's diagnosis, and had a prostate-specific antigen (PSA) test after the date of blood draw. The latter criterion ensured that controls had the opportunity to have an occult prostate cancer diagnosed. All controls had a PSA test within 2.5 years of the date of diagnosis of their matched case. Cases and controls were matched on year of birth (±1 year), PSA test before blood draw (yes/no), time (midnight to before 9 a.m., 9 a.m. to before noon, noon to before 4 p.m., and 4 p.m. to before midnight), season (winter, spring, summer, and fall), and exact year of blood draw, because plasma analyses were being done on the same case-control set.

Laboratory assays. We selected all common SNPs (n = 20) in TLR4 with frequencies greater than 5% from the Innate Immunity in Heart, Lung, and Blood Disease-Programs for Genomic Applications (IIPGA). These SNPs were identified by resequencing the TLR4 gene of 23 unrelated Europeans from Centre du Etude Polymorphisme Humain (CEPH) families. Resequencing of TLR4 included 2.5 kb 5′ of the gene, exons, and 1.5 kb 3′ of the gene. We included a less common nonsynonymous SNP (TLR4_13015) because it causes a change in an amino acid. Laboratory personnel were blind to case-control status. All case-control matched pairs were analyzed together using the Sequenom system. Multiplex PCR were carried out to generate short PCR products (>100 bp) containing one SNP. The details of PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry are available on request. Six control DNA samples were used for optimization. Three SNPs failed the Sequenom assay design due to either high dimer potential for the forward extended primer or other SNPs that might interfere with primer annealing or extension. After genotyping, two other SNPs were removed because of low genotyping success rates (<90%). Finally, a total of 16 SNPs (Fig. 1; Table 1) were genotyped in three plexes at the Harvard Partners Center for Genetics and Genomics (Boston, MA). For each SNP, genotyping data were missing in <5% of the study participants. Sixty-eight quality-control samples were obtained from 18 external participants and each of them had two to six duplicates. These quality-control samples were genotyped together with all other samples in this study. All quality-control samples passed the quality-control test (discordance rate = 0).

Ascertainment of prostate cancer. Investigators reviewed the medical and pathology records for men with prostate cancer reported from the follow-up questionnaire or death certificate to confirm adenocarcinoma of the prostate and to document clinical presentation, stage, and Gleason sum of the tumor. Because the reporting of a prostate cancer diagnosis by these health professionals tends to be accurate, self-reported cases for which we were unable to acquire medical records (7% of this case series) were included. The clinical presentation was categorized into elevated serum PSA concentration only, abnormal digital rectal examination with or without an elevated serum PSA concentration, or other/unknown. The cases were categorized into regionally invasive or metastatic (stage ≥T_3b, N₁, or M₁), organ-confined or minimal extraprostatic extension (T_1b-T_3a and N₀M₀), higher grade (Gleason sum ≥7), and lower grade (Gleason sum <7). Incidental microscopic focal tumors (stage T_1a) were excluded because they are generally indolent and susceptible to detection bias due to differential rates of surgery for benign prostatic hyperplasia. In addition, men with a previous cancer, except nonmelanoma skin cancer, before the date of blood draw were excluded. Confirmed non-T_1a tumors between blood draw and January 31, 2000 were included. In the blood subcohort, 92% of cases were confirmed by medical record and 5% by other corroborating information; only 3% were based on self-report (19). We included the self-reported cases in the analyses because the concordance between self-report and medical record confirmed cases was high (>90%) in this cohort.

Statistical analysis. The Hardy-Weinberg equilibrium (HWE) test was done for each SNP among controls. Haplotype block structure (Fig. 2) was determined by using Haploview (http://www.broad.mit.edu/mpg/haploview/index.php) and Locusview (http://www.broad.mit.edu/mpg/locusview/). The partition-ligation-expectation-maximization algorithm was applied to estimate haplotype frequencies in each block by using the tagSNP program (20). We calculated haplotype frequencies by using both progressive-ligation (as implemented in SAS PROC HAPLOTYPE) and partition-ligation-expectation-maximization algorithms (21, 22). We arbitrarily broke the 16 SNPs into partitions of three to four SNPs each (e.g., SNP1-4, SNP5-8, etc.). In regions of high linkage disequilibrium and limited haplotype diversity, these algorithms yield quite similar results to each other and to other algorithms (e.g., PHASE; ref. 23). In particular, choice of partition does not noticeably change estimates of haplotype frequency (22). Haplotype frequency estimates in TLR4 using partition-ligation and progressive-ligation differed by ≤0.001. Conditional logistic regression models were used to estimate ORs for disease in participants carrying either 1 or 2 versus 0 copies of the minor allele of each SNP and each multilocus haplotype; haplotype trend regression (24) was used to test global association between TLR4 haplotypes and prostate cancer. The type I error rate is controlled by the single multiple-degree-of-freedom test of association between TLR4 haplotypes and prostate cancer. Given a significant global test, haplotype- and SNP-specific tests can provide some guidance as to which variant(s) contributes to the significant global test, although the nominal Ps we present do not control the family-wise error rate for these post hoc comparisons.

Age and family history are known risk factors for prostate cancer (25, 26); body mass index (BMI) is related to risk of prostate cancer although not consistently (27). We evaluated how these factors modified the association between TLR4 SNPs or haplotypes and the risk of prostate cancer by comparing a model with terms for main effects and interaction terms to the model with terms for main effects only using the likelihood ratio test. Because of the role of TLR4 in the innate immune response, aggressiveness of prostate cancer may relate to genetic variations of TLR4. We tested the association between TLR4 haplotypes and aggressiveness of prostate cancer among patients with prostate cancer by using two definitions for tumor aggressiveness (aggressiveness 1: stage T_3b or T₄ or N₁ or M₁ or death due to prostate cancer; aggressiveness 2: stage T_3b or T₄ or N₁ or M₁ or death due to prostate cancer or Gleason sum ≥7). Aggressiveness 1 is useful for participants lacking information for Gleason sum and indicates how far a cancer has progressed independent of grade. Aggressiveness 2 may indicate the potential of the tumor to progress by considering grade information. All analyses were conducted with SAS release 9.0 (SAS Institute, Cary, NC). All statistical tests were two sided.

Sixteen SNPs in TLR4 were genotyped. In the IIPGA data set, the minor allele frequency of SNP11, a nonsynonymous SNP, was slightly less than 5% in the resequencing data of 23 unrelated Europeans from CEPH families but was slightly greater than 5% in our study population. SNP8 was out of HWE among controls (P = 0.0001) and was therefore dropped from all the analyses (Table 1). SNP10, SNP11, SNP13, and SNP15 were out of HWE (P = 0.02-0.03), but we retained them in the analyses as these Ps were marginal and may be chance findings and the internal blinded quality-control specimens did not show evidence of genotyping error.

Only self-reported Caucasians (97% of participants) were included in our analyses, minimizing the likelihood of false-positive findings due to population stratification (28, 29). However, inclusion of non-Caucasians does not change the HWE results among controls.

The study population included 700 incident prostate cancer cases and 700 matched controls. Age and BMI distributions were similar for cases and controls (Table 2). Family history of prostate cancer was significantly different between cases and controls (P = 0.009). The mean age started smoking, lifetime average number of cigarettes per day, and alcohol consumption were similar for cases and controls. Among cases, 80% were in tumor stage T_1b to T_3a, 73% had Gleason grade 5 to 7, 8% had aggressive prostate cancer (based on aggressiveness 1 definition), and 37% had aggressive prostate cancer (based on aggressiveness 2 definition). Eighteen cases died of prostate cancer before January 31, 2000.

Men carrying one copy of the minor allele of SNP1 had an increased risk of prostate cancer (OR, 1.38; 95% CI, 1.10-1.73; Table 3). Men carrying one copy of the minor allele of SNP6 and SNP9, on the contrary, had lower risk of prostate cancer (OR, 0.73; 95% CI, 0.57-0.93; OR, 0.66; 95% CI, 0.51-0.86). Men carrying two copies of the minor alleles of eight SNPs were at statistically significantly lower risk of prostate cancer (SNP3: OR, 0.66; 95% CI, 0.46-0.94; SNP4: OR, 0.59; 95% CI, 0.39-0.90; SNP5: OR, 0.64; 95% CI, 0.45-0.93; SNP7: OR, 0.63; 95% CI, 0.41-0.95; SNP10: OR, 0.64; 95% CI, 0.45-0.91; SNP13: OR, 0.51; 95% CI, 0.28-0.96; SNP14: OR, 0.50; 95% CI, 0.27-0.95; SNP16: OR, 0.38; 95% CI, 0.16-0.92).

Fifteen SNPs spanning TLR4 form one “block” using a modified version of the Gabriel et al. (30) algorithm, where blocks identified with the default settings in Haploview are merged if they have multiallelic D′ greater than 0.8 and the cumulative frequency of common (>5% frequency) haplotypes in the merged block is >80% (31). Six common haplotypes (frequency >5%) were found with an accumulated frequency of 81% in controls (Table 4). The P for the global test of the six common haplotypes was 0.02. Men carrying one copy of the minor Hap1 had a 1.40-fold increased risk of prostate cancer (95% CI, 1.12-1.75). Hap5, on the other hand, was associated with a lower risk of prostate cancer (OR, 0.59; 95% CI, 0.41-0.86).

The associations between TLR4 genotypes and the risk of prostate cancer varied by age (Table 5). SNP4, SNP7, SNP13, and SNP16 variant carriers ages <65 years had a statistically significantly lower risk of prostate cancer than did noncarriers (CC and CT versus TT: OR, 0.64; 95% CI, 0.47-0.86; AA and AG versus GG: OR, 0.63; 95% CI, 0.46-0.85; CC and CG versus GG: OR, 0.66; 95% CI, 0.47-0.94; CC and CA versus AA: OR, 0.59; 95% CI, 0.41-0.86, respectively). BMI and family history were not effect modifiers for the association between TLR4 SNPs and prostate cancer. In addition, the association between TLR4 haplotypes and prostate cancer was not modified by age, BMI, or family history.

Case-only analysis (aggressive versus nonaggressive) was done among prostate cancer patients. None of the SNPs or haplotypes (Table 6) showed a statistically significant association with tumor aggressiveness.

TLR4 is located on the surface of the macrophage and plays an important role in signaling the innate immune response in response to bacterial or viral infection. TLR4 is the pathogen recognition receptor for most Gram-negative bacteria (LPS), many Gram-positive bacteria, mycobacteria (2), spirochetes, viruses, and other ligands (4). Chronic intraprostatic inflammation may increase risk of prostate cancer. Variation in TLR4 may reduce or even block the signaling of the immune response and therefore lower the risk of prostate cancer.

Animal studies showed that TLR4-deficient mice exposed to respiratory syncytial virus had higher levels of infectious virus in their lungs and either were unable to clear the virus or cleared it several days later than wild-type mice (6). For C3H/HeJ mice, the codominant lps^d allele corresponded to a missense mutation in the third exon of the TLR4 gene, resulting in substitution of proline with histidine at codon 712. For C57B/10ScCr mice, a null mutation of TLR4 was observed among mice homozygous for the second lps mutation. These mutations rendered the C3H/HeJ and C57B/10ScCr mice refractory to the toxic effects of lps from Gram-negative bacteria (7, 8). Experimental studies revealed that mutations in TLR4 were related to hyporesponsiveness to viral and bacterial infection and therefore to a reduction in the innate immune response and inflammation.

A previous study (14) in a Swedish population explored the association between genetic variation of TLR4 and the risk of prostate cancer. The authors found that one SNP (11381G/C, equivalent to our SNP12) was associated with the risk of prostate cancer (CC and CG versus GG: OR, 1.26; 95% CI, 1.01-1.57); this SNP was not associated with risk in our study. In addition, the authors did not observe any association between TLR4 haplotypes based on eight SNPs and prostate cancer. The disparity between the Zheng et al. study and our study might be due to differences in the study populations, differences in case detection by PSA, or extent of genotyping. We genotyped more SNPs in TLR4, which provided a more comprehensive assessment of TLR4 genetic variation than the previous study, which was based on genotyping eight common SNPs.

Age modified the association between TLR4 SNPs and prostate cancer, but we observed no effect modification by BMI and family history. Among men ages <65 years, carriers of SNP4, SNP7, SNP13, or SNP16 had a statistically significantly lower risk of prostate cancer. This indicated that TLR4 genetic polymorphisms had a greater influence among younger than older men and is consistent with a greater influence of inherited genetic risk among younger men.

TLR4 haplotypes and risk of aggressive prostate cancer among cases (Table 6) did not differ using either of the two aggressiveness definitions used in this study or the definition of aggressiveness used by Zheng et al. (14).

Men carrying one copy of variant Hap1 had a 1.40-fold increased risk of prostate cancer. In Hap1, SNP1 is the only SNP carrying the variant allele. Men carrying one copy of the minor allele of SNP1 had a 1.38-fold increased risk of prostate cancer, although the OR for homozygous variant was not significantly elevated. Hap5 was associated with a lower risk of prostate cancer, which after comparison with hap4 and hap6 was found to be mainly attributable to variants of SNP13, SNP14, and SNP16. These SNPs located in the 3′ UTR may be associated with lower risk of prostate cancer because they may influence mRNA stability and thereby reduce the innate immune response, inflammation, and subsequent carcinogenesis. SNP1, located in the 5′ UTR, where the promoter or transcription factor binding sites are located, may potentially exert regulator effects and therefore increase cancer risk. Functional assays are needed to elucidate the molecular mechanisms underlying these associations.

Four of the SNPs genotyped were significantly out of HWE in the controls but not in the cases. Because of the large number of loci tested, a conservative significance threshold (α = 0.001) is conventionally used when testing HWE to detect genotyping errors (32). None of these four loci are significant at the Bonferroni-corrected significance level of 0.003 (0.05/15). Even with the less conservative Benjamini-Hochberg step-up procedure to control the false discovery rate (<5%), the null hypothesis of HWE is rejected only for SNP8. Considering none of the SNPs that showed marginal evidence for deviation from HWE in controls (P < 0.05) showed any evidence of deviation in cases (P > 0.10), we do not believe that the marginal HWE tests in controls suggest systematic genotyping errors for these SNPs.

Furthermore, haplotype trend regression conditions on observed genotypes and hence is relatively robust to moderate departures from HWE in haplotype frequencies. We have shown (33)⁴

that the haplotype trend regression is closely related to the prospective likelihood (34, 35); the latter approach has been shown to yield accurate tests and parameter estimates when HWE does not hold (specifically when there is an excess of homozygotes, as is the case for these five SNPs; ref. 36). We also did haplotype analyses excluding the four SNPs showing marginal evidence of departures from HWE. Haplotype frequency and OR estimates for the 11-SNP haplotypes corresponding to the previous 15-SNP haplotypes (e.g., GGATATGA**G*T*G and GGATATGAGAGGTGC) were quite similar (differing by at most 0.005 and 0.03, respectively). The two haplotypes corresponding to the significant haplotypes in the previous analysis were also significant at the 0.05 level.

In recent years, chronic inflammation due to the innate immune response to infection has been suspected to be a risk factor for cancer at many sites (37, 38). However, research on inflammation and prostate cancer is limited. Our findings suggest that inflammation may be associated with risk of prostate cancer, and future investigation of genetic variation in innate immune genes may cast light on the etiology of this enigmatic disease.

Grant support: NIH grants UO1 CA98233 and CA55075.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Pati Soule and Ana-Tereza Andrade for DNA sample extraction and the Partners High-Throughput Genotyping Center (Dr. David Kwiatkowski, Alison Brown, and Maura Regan) for genotyping.