UROLOGICAL ONCOLOGY IL-6 Genomic Variants and Risk of Prostate Cancer Mohammad Taheri1, Rezvan Noroozi2,3, Azadeh Rakhshan4, Molud Ghanbari5, Mir Davood Omrani1*, Soudeh Ghafouri-Fard2* Purpose: To evaluate the role of Interleukin-6 (IL-6) single nucleotide polymorphisms in prostate cancer (PCa) and benign prostate hyperplasia (BPH). Materials and Methods: We genotyped two IL-6 intronic variants (rs1800795 and rs2069845) in PCa cases, BPH cases and healthy men referred to Labbafinejad and Shohadaye Tajrish Medical Centers using tetra ARMS-PCR method. Results: The study included 130 PCa cases, 200 BPH cases and 200 healthy men. The C allele of rs1800795 was associated with PCa risk in the assessed population (OR (95% CI) = 1.45 (1.06-1.98)). However, the frequency of rs2069845 variants was not significantly different between PCa, BPH and control groups. The A C haplotype (rs2069845 and rs1800795 respectively) was associated with PCa and BPH risk (OR (95% CI) = 1.67 (1.12- 2.48); OR (95% CI)= 1.78 (1.25 – 2.54)). Besides, the A G haplotype (rs2069845 and rs1800795 respectively) has a protective effect against both PCa and BPH in the assessed population (OR (95% CI) = 0.63 (0.46-0.87); OR (95% CI)= 0.6 (0.45-0.79)). Conclusion: Consequently, the results of the current study provide further evidence for contribution of IL-6 in prostate cancer. Keywords: IL-6; prostate cancer; benign prostate hyperplasia INTRODUCTION Prostate cancer (PCa) and benign prostate hyperpla-sia (BPH) are two androgen-dependent patholog- ical conditions with shared inflammatory elements as well as common genetic and epigenetic changes(1). Both diseases have been associated with BK virus (BKV) in- fection(2). Expression of certain matrix nuclear proteins can differentiate these two conditions(3). The high prev- alence of these disorders among aged males has surged researchers to find genetic susceptibility loci(4-6) with possible application as biomarkers or therapeutic tar- gets(7). A recent meta-analysis of literature has shown the age-standardized rate of prostate cancer was 9.11 in Iran(8). Inflammatory responses have a well-document- ed role in cancer pathogenesis through modulation of tumor microenvironment, distortion of cytokine balance and production of reactive oxygen species(9). Among cy- tokine, the role of interleukin (IL)-6 in prostate cancer pathogenesis has been vastly evaluated. Multiple lines of evidence point to its role in this type of malignan- cy. First, serum IL-6 levels have been correlated with PCa burden as defined by serum prostate specific an- tigen (PSA) levels or clinically apparent metastases(10). Moreover, its higher levels might be an indicator of ir- responsiveness to hormone ablation therapy(11). IL-6 act 1Urogenital Stem Cell Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 3Phytochemistry Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 4Cancer Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 5Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. *Correspondence: Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Emails: Soudeh Ghafouri-Fard: s.ghafourifard@sbmu.ac.ir. Mir Davood Omrani: davood_omrani@yahoo.co.uk Received & Accepted as a paracrine factor that modulates PCa autophagy and neuroendocrine differentiation(12). The regulatory role of IL-6 on is exerted through the AMPK/mTOR path- way(13). The role of IL-6 in induction of cell prolifera- tion and prevention of apoptosis is exerted through vari- ous cancer-associated signal pathways such as the Janus tyrosine family kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, the extra- cellular signal-regulated kinase 1 and 2 (ERK1/2)-mi- togen activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3-K) pathway(9). Ex- perimental studies have shown that IL-6 induces and/or augments the conversion of prostate cancer cells from an androgen-dependent to an androgen-independent phenotype(14). Functional variants within IL-6 coding gene including the rs1800795 and rs2069845 single nu- cleotide polymorphisms (SNPs) have been previously shown to alter circulating IL-6 levels(15-17). Moreover, the C allele of rs1800795 has been associated with in- creased risk of PCa in an American population but the association did not remain significant after accounting for multiple tests(18). The GG genotype of this SNP has been associated with an increased risk of metastasis of primary breast cancer(19). Considering the role of IL-6 in PCa pathogenesis as well as the presence of com- Urology Journal/Vol 16 No. 5/ September-October 2019/ pp. 463-468. [DOI: 10.22037/uj.v0i0.4543] mon inflammatory mechanisms in PCa and BPH, we aimed at evaluation of the associations between two functional polymorphisms within this gene (rs1800795 and rs2069845) and risk of PCa and BPH in an Irani- an population. The current study is the first association study of IL-6 polymorphisms in Iranian patients with PCa and BPH. MATERIALS AND METHODS Patients The appropriate sample size was calculated for rs2069845 with minor allele frequency of 0.25 as- suming the study power of 70% and significance level of 5% to be 120 cases and 120 controls. The current case-control study recruited 130 newly diagnosed PCa cases, 200 newly diagnosed BPH cases and 200 healthy men referred to Labbafinejad and Shohadaye Tajrish Medical Centers. The diagnosis was established based on pathological examination of biopsied samples. The study protocol has been approved by ethical committee of Shahid Beheshti University of Medical Sciences. All study participants signed the informed consent forms. Control subjects were selected from men seeking rou- tine health assessment during 2016 and were matched to patients. Men attributed to control group had no history of lower urinary tract symptoms, inflammato- ry disease of prostate, prostate enlargement or family history of PCa. Controls had normal PSA levels. PCa or BPH was diagnosed through evaluation of clinical prostate biopsies by an expert pathologist especially in BPH patients with high PSA levels (4.0 ng/ml or more). Exclusion criteria were inadequate pathologic sample, history of former malignancies in other organs and previous chemo-radiotherapy. Blood samples were col- lected from patients in EDTA tubes before commence- ment of any therapy such as surgery, radiotherapy, and chemotherapy. Clinicopathological data were collected through filling questionnaires and assessment of medi- cal reports. Genotyping Genomic DNA was extracted from blood samples of all study participants using standard salting out method. The rs2069845 and rs1800795 intronic variants within IL-6 gene were genotyped using tetra-primer ARMS- PCR technique and were visualized after staining on 2% agarose gel. The amplification program was started with denaturation step at 95 ºC for 5 minutes followed by 35 cycles of 95 ºC for 45 seconds, specific annealing temperatures for 35 seconds, and 72 ºC for 35 seconds and a final extension step in 72 ºC for 10 minutes. Ten percent of samples were sequenced using ABI 3730xl DNA analyzer (Macrogen, Korea) to confirm the results of tetra-primer ARMS-PCR. The nucleotide sequences of primers used for genotyping are shown in Table 1. Table 1. The nucleotide sequences of primers used for genotyping (SNP: single nucleotide polymorphism, bp: base pair). SNP Primer sequence Tm Annealing temperature PCR product size (bp) rs2069845 Forward inner primer (G allele): 5′- GTTTCCCAGTCCTCTTTACACCAACG 66 °C 62 °C 197 bp (G allele) Reverse inner primer (A allele): 5′- 66 °C 292 bp (A allele) TTTATGATCTGTTGAAAGACCACTGACCT Forward outer primer: 5′- 66 °C 434 bp CATCCTGCCTCTGCCATTTCTACTTAA (two outer primers) Reverse outer primer: 5′- 66 °C ATTCTGACATCTGAGATAATGCCTGG rs1800795 Forward inner primer (C allele): 5′- 68 °C 61 °C CACTTTTCCCCCTAGTTGTGTCTTCCC 206 bp (C allele) Reverse inner primer (G allele): 5′- 68 °C 155 bp (G allele) ATTGAGCAATGTAACGTCCTTTAGCTTC Forward outer primer: 5′- 68 °C 306 bp (two outer primers) CAATACATGCCAATGTGCTGAGTCACTA Reverse outer primer: 5′- AGAATGATCCTCAGTCATCTCCAGTCCT 68 °C Variables Prostate cancer group BPH group Controls Age (mean ± SD) 66.54 ± 9.5 67.96 ± 3.97 64 ± 5.12 BMI (mean ± SD) 25.06 ± 2.14 24.97 ± 3.47 25.7 ± 1.2 Prostate weight (gr) (mean ± SD) 58 ± 98.31 61.87 ± 29.52 - PSA (ng/mL) (mean ± SD) 9.13 ± 9.28 8.94 ± 7.2 < 4 <4 25 (19.23%) 39 (19.5%) 200 (100%) 4-10 73 (56.15%) 94(47%) 0 >=10 32 (24.61%) 67 (33.5%) 0 Smoking Never smoker (%) 69 (53.1%) 121 (60.5%) 124 (62%) Current or former smoker (%) 61 (46.9%) 79 (39.5%) 76 (38%) Gleason score <=6 68 (52.3%) - - >6 62 (47.7%) - - Abbreviations: SD: standard deviation, PSA: prostate specific antigen, BMI: body mass index). Table 2. Demographic and clinical data of study participants (BPH: benign prostate hyperplasia, IL-6 variants in prostate cancer-Taheri et al. Urological Oncology 464 Vol 16 No 04 September-October 2019 465 Statistical analysis The agreement of genotype frequencies with the Hardy– Weinberg equilibrium was assessed using Chi-square test. The associations between genotype frequencies and PCa or BPH were evaluated in three inheritance models including recessive, dominant and co-domi- nant using Pearson's chi-square test. The P values were corrected through multiplying by the number of SNPs. P values less than 0.05 were regarded as significant. The linkage between rs1800795 and rs2069845 var- iants were assessed using D’ and r values. Haplotype block frequencies and their associations with PCa and BPH were computed using Partition-Ligation–Expec- tation-Maximization (PL-EM) algorithm(20) (SNPana- lyzer 2.0 software) with supposition of 0.01 minimum frequencies for blocks. The results were stated as Odds ratios (OR) and 95% confidence interval of OR (95% CI), P-value and Bonferroni adjusted P-values. Patients were matched to control group in variables such as BMI and smoking history. RESULTS Demographic and clinical data of study participants Table 2 shows the demographic and clinical data of PCa, BPH and healthy subjects participated in the study. PCa and BPH patients were age-matched (P = 0.061). The three study groups were not significantly different in smoking (P = 0.39) and BMI (P = 0.79). Genotyping The distributions of alleles and genotypes of the as- sessed SNPs were in accordance with HWE in the three study groups. Table 3 shows the results of evaluation of HWE. Figure 1 and 2 show the results of ARMS-PCR for genotyping the mentioned SNPs. The C allele of rs1800795 was associated with PCa risk in the assessed population (OR (95% CI) = 1.45 (1.06- 1.98), Adjusted P = 0.04). However, the frequency of rs2069845 variants was not significantly different be- tween PCa, BPH and control groups. (Table 4) We also assessed the frequencies of IL-6 haplotypes in the three study groups and found significant over-pres- entation of A C haplotype (rs2069845 and rs1800795 respectively) in both PCa and BPH groups compared with control subjects (OR (95% CI)= 1.67 (1.12- 2.48), Adjusted P = 0.04; OR (95% CI)= 1.78 (1.25 – 2.54), Adjusted P = 0.006 respectively). Besides, the A G haplotype (rs2069845 and rs1800795 respectively) has been shown to exert protective effect against both PCa and BPH in the assessed population (OR (95% CI)= 0.63 (0.46-0.87), Adjusted P = 0.02; OR (95% CI)= 0.6 (0.45-0.79), Adjusted P = 0.001 respectively). Table 5 shows the detailed information of haplotype analy- Table 3. Exact test for Hardy-Weinberg equilibrium. SNP rs2069845 P-value rs1800795 P-value AA AG GG GG GC CC PCa 46 69 15 0.15 39 55 36 0.08 BPH 78 91 31 0.60 61 91 48 0.22 Control 82 97 21 0.32 77 87 36 0.19 Abbreviations: SNP: single nucleotide polymorphism, BPH: benign prostate hyperplasia, PCa: prostate cancer Figure 1. The results of ARMS-PCR for genotyping the rs1800795 and rs2069845 SNPs. Figure 2. The results of ARMS-PCR for genotyping the rs2069845 SNPs. IL-6 variants in prostate cancer-Taheri et al. sis) No strong linkage disequilibrium (LD) has been ob- served between the analyzed polymorphic sites (LD analysis, D′ = 0.14; r = 0.01; P = 0.5). DISCUSSION The role of IL-6 in the pathogenesis of PCa has been extensively evaluated. However, associations between genomic variants of IL-6 gene and risk of PCa have not been assessed in different populations. In the present study we assessed associations between two functional variants of IL-6 and prostate disorders in a cohort of Iranian patients with prostate disorders. We found sig- nificant over-presentation of the rs1800795 C allele in PCa patients compared with healthy subjects. We did not find any difference in allele or genotype frequencies of this SNP between BPH patients and controls which might rule out its contribution in the pathogenesis of BPH despite its putative role in PCa. Although the C allele of rs1800795 has been associated with higher circulating IL-6 levels in human subjects(15,17,21), the association between this allele and PCa risk in Amer- ican patients did not remain significant after multiple testing correction. However, authors suggested further evaluation of the association between this genomic var- iant and PCa risk(18). Consequently, our results provide additional support for their observation. Moreover, the C allele of rs1800795 has been associated with higher concentrations of circulating C reactive protein (CRP) (16), which has been regarded as a negative predictor of survival in PCa (22). So this polymorphism might exert its effects in PCa pathogenesis through multiple mech- anisms including alterations in IL-6 and CRP levels. However, despite previous studies demonstrated higher IL-6 in carriers of minor allele of rs2069845(17), we did not find significant difference in allele and genotype frequencies of rs2069845 between PCa, BPH and con- trol groups. Notably, we found significant over-presentation of A C haplotype (rs2069845 and rs1800795 respectively) in both PCa and BPH groups compared with control sub- jects. On the other hand, the A G haplotype (rs2069845 and rs1800795 respectively) has been shown to exert protective effect against both PCa and BPH in the as- sessed population. Such data further supports the sig- nificance of rs1800795 and unimportance of rs2069845 variants in conferring PCa or BPH risk. However, the implication of other functional variants within these haplotypes cannot be ruled out. As no differences have been found in haplotype frequencies between BPH and PCa groups, assessment of haplotypes cannot differen- tiate between these two conditions. Although most of previous studies have demonstrated the usefulness of IL-6 concentrations as predictive bi- omarkers in PCa patients, some inconsistencies exist. For instance, Nakashima et al. reported serum IL-6 lev- el as a major prognostic factor for prostate cancer and its extent of disease(23). In line with their study, Alcover et al. highlighted the effectiveness of IL-6 in predict- ing the biochemical progression of prostate cancer (25). On the other hand, Pierce et al. failed to detect any as- sociation between circulating IL-6 concentration and PCa risk and proposed that rs1800795 may alter PCa risk through other mechanisms(19) among which might be modulation of CRP levels. Alternatively, they sug- gested that the variability in IL-6 levels or the insuf- ficiency of a single assessment of IL-6 as an indicator of long-standing blood levels might result in failure of Table 4. Association analysis of rs2069845 and rs1800795 polymorphisms and risk of PCa and BPH (P*: Adjusted P value). SNP Model Sample size (%) PCa vs. Control BPH vs. Control PCa vs. BPH PCa (%) BPH (%) Control (%) OR (95% CI) P P* OR (95% CI) P P* OR (95% CI) P P* rs2069845 Allele G vs. A 99 (38) 153 (38) 139 (35) 1.15 (0.83-1.60) 0.38 0.77 1.16 (0.87-1.55) 0.30 0.61 1.00 (0.0.72-1.37) 0.96 1.00 161 (62) 247 (62) 261 (65) Co-dominant GG vs 15 (12) 31 (15.5) 21 (10.5) 1.26 (0.6-2.70) 0.59 1.00 1.59 (0.82-2.94) 0.33 0.66 1.54 (0.85-2.78) 0.35 0.7 AA AG vs 69 (53) 91 (45.5) 97 (48.5) 1.26 (0.79-2.04) 0.99 (0.64-1.49) 1.11 (0.75-1.64) AA Dominant GG+AG 84 (64.6) 122 (61) 118 (59) 1.27 (0.8-2.00) 0.31 0.61 1.09 (0.73 -1.62) 0.68 1.00 1.17 (0.74-1.85) 0.51 1.00 vs AA 46 (35) 78 (39) 82 (41) Recessive GG vs 15 (12) 31 (15.5) 21 (10.5) 1.11 (0.55-2.24) 0.77 1.00 1.56 (0.86-2.83) 0.14 0.27 0.71 (0.37-1.38) 0.31 0.62 AG +AA 115 (88.5) 169 (84.5) 179 (89.5) rs1800795 Allele C vs G 127 (49) 187 (47) 159 (40) 1.45 (1.06-1.98) 0.02 0.04 1.33 (1.00-1.76) 0.05 0.09 1.09 (0.8-1.49) 0.6 1.00 133 (51) 213 (53) 241 (60) Co-dominant CC vs 36 (27.7) 48 (24) 36 (18) 1.96 (1.09-3.57) 0.08 0.16 1.69 (0.97.-2.94) 0.16 0.32 2.44 (1.37-4.35) 0.74 1.00 GG CG vs 55 (42.3) 91 (45.5) 87 (43.5) 1.25 (0.75-2.08) 1.32 (0.85-2.08) 1.3 (0.87-1.92) Dominant CG+CC 91 (70) 139 (69.5) 123 (61.5) 1.46 (0.91-2.34) 0.11 0.23 1.42 (0.94-2.16) 0.09 0.18 1.02 (0.63-1.66) 0.92 1.00 vs GG 39 (30) 61 (30.5) 77 (38.5) GG Recessive CC vs 36 (27.7) 48 (24) 36 (18) 1.74 (1.03-2.95) 0.04 0.07 1.44 (0.89-2.34) 0.14 0.28 1.21 (0.73-2.00) 0.45 0.9 CG+GG 94 (72.3) 152 (76) 164 (82) rs2069845 rs1800795 PCa BPH Control PCa vs. Control BPH vs. Control PCa vs. BPH OR (95% CI) P P* OR (95% CI) P P* OR (95% CI) P P* A G 0.35 0.31 0.47 0.63 (0.46-0.87) 0.005 0.02 0.6 (0.45-0.79) 3.6 E-4 0.001 1.06 (0.77-1.46) 0.73 1.00 G C 0.22 0.16 0.22 1.07 (0.75 – 1.53) 0.71 1.00 0.89 (0.64 – 1.24) 0.5 1.00 1.2 (0.83– 1.72) 0.33 1.00 A C 0.27 0.31 0.18 1.67 (1.12- 2.48) 0.01 0.04 1.78 (1.25 – 2.54) 0.001 0.006 0.94 (0.65 – 1.35) 0.73 1.00 G G 0.16 0.22 0.13 1.23 (0.75- 2.01) 0.41 1.00 1.64 (1.07- 2.49) 0.02 0.08 0.75 (0.47 – 1.19) 0.22 0.87 Table 5. The frequencies of haplotype blocks in the three study groups (P*: Adjusted P value). IL-6 variants in prostate cancer-Taheri et al. Urological Oncology 466 Vol 16 No 04 September-October 2019 467 detecting the expected association(18). Taken together, the rs1800795, or another variant in LD with it might confer PCa risk possibly through mod- ulation of IL-6 RNA and protein levels or even other independent mechanisms. Considering the short plas- ma half-life of IL-6(25) and the presence of a circadian rhythm for this cytokine due to the circadian alterations of cortisol(26), we propose assessment of genomic vari- ants within this gene as an alternative to evaluation of its serum concentrations. Such studies would elaborate the role of IL-6 in PCa risk and pave the way for design- ing personalized therapeutic options. Our study had some limitations including sample size. 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