Impacts of Interleukin-10 Promoter Genotypes on Prostate Cancer

Prostate cancer (PCa) is a multifactorial disease influenced by genetic, environmental, and immunological factors. Genetic polymorphisms in the interleukin-10 (IL-10) gene have been implicated in PCa susceptibility, development, and progression. This study aims to assess the contributions of three IL-10 promoter single nucleotide polymorphisms (SNPs), A-1082G (rs1800896), T-819C (rs3021097), and A-592C (rs1800872), to the risk of PCa in Taiwan. The three IL-10 genotypes were determined using PCR-RFLP methodology and were evaluated for their contributions to PCa risk among 218 PCa patients and 436 non-PCa controls. None of the three IL-10 SNPs were significantly associated with the risks of PCa (p all > 0.05) in the overall analyses. However, the GG at rs1800896 combined with smoking behavior was found to significantly increase the risk of PCa by 3.90-fold (95% confidence interval [95% CI] = 1.28–11.89, p = 0.0231). In addition, the rs1800896 AG and GGs were found to be correlated with the late stages of PCa (odds ratio [OR] = 1.90 and 6.42, 95% CI = 1.05–3.45 and 2.30–17.89, p = 0.0452 and 0.0003, respectively). The IL-10 promoter SNP, A-1082G (rs1800896), might be a risk factor for PCa development among smokers and those at late stages of the disease. These findings should be validated in larger and more diverse populations.


Introduction
Prostate cancer (PCa) is the second most common cancer among men worldwide and is a leading cause of cancer-related mortality.The incidence of PCa is increasing in developed countries.It is estimated that the number of PCa cases in men in the United States will reach 299,010, accounting for 29% of all cancers in 2024 [1].Globally, there were approximately 1,466,680 new PCa cases in 2022 [2].PCa is difficult to diagnose early.Studies have found that PCa may be related to family history, race, occupation, cadmium exposure, vasectomy, obesity, alcohol consumption, and other factors, with genetic factors possibly being the most important, though largely unrevealed [3][4][5][6][7].
The vast majority of PCas are adenocarcinomas originating from glandular cells that line the prostate gland and its tubes.Prostate carcinogenesis is a multi-stage process that begins with the development of low-grade prostatic intraepithelial neoplasia (PIN), followed by localized adenocarcinoma, and ultimately advances to metastatic disease.The pathophysiology of PCa involves complex interactions between genetic, epigenetic, hormonal, and environmental factors.Numerous genetic and epigenetic alterations have been documented in prostate tumors [8].The most common PCa genetic alterations are translocations involving androgen-regulated promoters and the ETS family of transcription factors, such as ERG and the ETV genes [9], among which the TMPRSS2:ERG fusion occurs in approximately 50% of localized PCa tumors [10,11].Other frequent somatic genetic alterations include MYC amplification and mutations in pathways of androgen receptor (AR), PI3K-PTEN, TGF-β/SMAD4, DNA repair, and epigenetic regulators and chromatin remodelers [8].Because a normal prostate needs androgen and its receptor for homeostasis, targeting the AR pathway via androgen deprivation therapy (ADT) is the standard of care for PCa.Resistance to ADT results in castration-resistant PCa (CRPC) and metastatic CRPC (mCRPC), which accounts for most of the PCa death.
We have previously reported a genome-wide association study of PCa [12] together with several candidate genes [13][14][15][16][17] among Taiwanese; however, the genetic contributions to PCa susceptibility and prognosis in Taiwanese individuals are far from satisfying and need further investigation.Interleukin-10 (IL-10) is a pivotal cytokine involved in immune regulation, exerting dual roles in both promoting and inhibiting immune responses.Its impact on tumorigenesis is paradoxical, as it can both facilitate and suppress tumor development [18].IL-10 can inhibit tumorigenesis by enhancing CD8+ T cell activity and suppressing pro-inflammatory cytokines such as IL-6 and IL-23 [19].Conversely, IL-10 can hinder antigen presentation and suppress interferon gamma (IFN-γ), thereby potentially dampening anti-tumor immunity [19].In the context of PCa, elevated IL-10 levels in both tissue and serum have long been recognized as prognostic indicators of PCa aggressiveness and progression [20][21][22][23][24][25].Over-expression of IL-10 has been associated with PCa development in several studies among different populations [20][21][22][23][24][25]; however, the genotype-phenotype correlation is not well known.The IL-10 gene comprises five exons and is located on chromosome 1q31-32 [26].Three commonly studied SNPs in the promoter region of the IL-10 gene, rs1800896, rs3021097 and rs1800872, have been reported to modulate its expression [27,28].
In this study, we examined the associations of three IL-10 promoter SNPs, rs1800896, rs3021097 and rs1800872, with the risk and aggressiveness of PCa in Taiwanese.In addition, we investigated the combinative effects of IL-10 genotype with age and smoking behavior on PCa risk and aggressiveness.Finally, we performed a comprehensive literature review of the associations of rs1800896 with PCa risk.

Recruitment of PCa Cases and Healthy Controls
In this study, 218 patients diagnosed with PCa were recruited from the outpatient clinics of the general surgery at China Medical University Hospital, Taichung, Taiwan.The recruitment was facilitated by the hospital's Tissue Bank.During the same period, 436 healthy volunteers without PCa were recruited as controls.These controls were matched by age and were selected through initial random sampling from the Health Examination Cohort of the same hospital.The details of the inclusion and exclusion criteria have been published previously [12,13].Briefly, the exclusion criteria for the control group included a history of malignancy, metastasized cancer of known or unknown origin, and any familial or genetic diseases.Additionally, control subjects lacking the specified demographic data were excluded from the study.All participants were Taiwanese citizens and the population in Taiwan is racially homogeneous.The study design and protocols have been approved and supervised by the Institutional Review Board of China Medical University Hospital (CMUH110-REC3-005).Several selected characteristics including age, smoking behaviors, family history, disease stages, and pathologic grades are summarized in Table 1.

IL-10 Genotyping Methodology
Genomic DNA was extracted from the peripheral blood of each participant as our published routine protocol [29,30].The genotypes of IL-10 rs1800896, rs3021097, and rs1800872 were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology, as we have previously described [31].The PCR primer sequences and respective restriction endonuclease for each DNA product of IL-10 rs1800896, rs3021097, and rs1800872 SNPs are summarized in Table 2.The locations of the investigated IL-10 polymorphic sites are summarized in Figure 1.

Selection of the Literature
Published articles were chosen from PubMed via EndNote, updated to 1 July 2024.The exclusion criteria were as follows: (a) articles published in non-SCI journals; (b) articles without a well-defined investigated population; (c) articles investigating small sample populations with insufficient analyzing power (fewer than 150 controls or cases, or fewer than 400 total samples); and (d) articles where the genotypic distribution does not fit the Hardy-Weinberg equilibrium (HWE).

Polymorphic Cites (rs Numbers) Primer Sequences Endonuclease Polymorphic Pattern
Digested Adduct Size (bp) F and R stands for forward and reverse primers, respectively.

Selection of the Literature
Published articles were chosen from PubMed via EndNote, updated to 1 July 2024.The exclusion criteria were as follows: (a) articles published in non-SCI journals; (b) articles without a well-defined investigated population; (c) articles investigating small sample populations with insufficient analyzing power (fewer than 150 controls or cases, or fewer than 400 total samples); and (d) articles where the genotypic distribution does not fit the Hardy-Weinberg equilibrium (HWE).

Statistical Analysis
We used Student's t-test to compare the mean age and the Pearson's Chi-square test to compare the genotype frequency between the PCa case and non-PCa control groups.The association between IL-10 genotypes and PCa risk was determined using a multivariable logistic regression analysis.The tests were two-sided, and any p-value less than 0.05 was considered to be statistically significant.

Demographics and Lifestyles for the PCa Case and Non-PCa Control Groups
The distributions of demographic characteristics, including age, smoking behaviors, and family history for the 218 PCa patients and 436 non-PCa healthy controls, are compared in Table 1.Additionally, the disease stages and pathologic grades of the PCa cases are also shown in Table 1.The mean age ± standard deviation of the cases and controls were 63.6 ± 6.9 and 63.9 ± 6.6, respectively (p = 0.58).About 81% of the cases are ever smokers, compared to 77% for the controls (p = 0.27).Among the PCa cases, 7.8% and 1.8% had a family history of any cancer in their first-and second-degree relatives, respectively; 71.1% and 28.9% were in the early and late stages, respectively; and 12.9%, 40.8%, and 46.3% of the PCa cases were of well-, moderately, and poorly differentiated grades, respectively (Table 1).

Contributions of IL-10 Genotypes to PCa Risk
Table 3 reveals the distributions of IL-10 genotypes among the PCa patients and non-PCa control subjects.All the three IL-10 genotypes fit well with the Hardy-Weinberg equilibrium in the controls (p = 0.3428, 0.2680, and 0.6034, respectively).The distributions of the three genotypes were not significantly different between the cases and controls (Ptrend = 0.1755, 0.8296, and 0.6547, respectively) (Table 3).There were suggestive associations

Statistical Analysis
We used Student's t-test to compare the mean age and the Pearson's Chi-square test to compare the genotype frequency between the PCa case and non-PCa control groups.The association between IL-10 genotypes and PCa risk was determined using a multivariable logistic regression analysis.The tests were two-sided, and any p-value less than 0.05 was considered to be statistically significant.

Demographics and Lifestyles for the PCa Case and Non-PCa Control Groups
The distributions of demographic characteristics, including age, smoking behaviors, and family history for the 218 PCa patients and 436 non-PCa healthy controls, are compared in Table 1.Additionally, the disease stages and pathologic grades of the PCa cases are also shown in Table 1.The mean age ± standard deviation of the cases and controls were 63.6 ± 6.9 and 63.9 ± 6.6, respectively (p = 0.58).About 81% of the cases are ever smokers, compared to 77% for the controls (p = 0.27).Among the PCa cases, 7.8% and 1.8% had a family history of any cancer in their first-and second-degree relatives, respectively; 71.1% and 28.9% were in the early and late stages, respectively; and 12.9%, 40.8%, and 46.3% of the PCa cases were of well-, moderately, and poorly differentiated grades, respectively (Table 1).

Contributions of IL-10 Genotypes to PCa Risk
Table 3 reveals the distributions of IL-10 genotypes among the PCa patients and non-PCa control subjects.All the three IL-10 genotypes fit well with the Hardy-Weinberg equilibrium in the controls (p = 0.3428, 0.2680, and 0.6034, respectively).The distributions of the three genotypes were not significantly different between the cases and controls (P trend = 0.1755, 0.8296, and 0.6547, respectively) (Table 3).There were suggestive associations between the rs1800896 genotypes and PCa risk: the homozygous variant GG and heterozygous variant AG genotype were associated with elevated risks of PCa (adjusted OR = 1.18 and 1.98, 95% CI = 0.79-1.68 and 0.85-3.75,p-value = 0.3933 and 0.1451, respectively) (Table 3).

Contributions of IL-10 Alleles to PCa Risk
The allelic frequencies of the three IL-10 SNPs among the PCa patients and non-PCa healthy controls were also examined (Table 4).In line with the findings in Table 3, the rs3021097 and rs1800872 allelic frequencies was not significantly different between the PCa case and non-PCa control groups (p = 0.6106 and 0.3885, respectively) (Table 4).However, there was a suggestive association between the variant G allele of rs1800896 and an increased risk of PCa (OR = 1.33, 95% CI = 0.97-1.83,p = 0.0906).

Association of IL-10 rs1800896 Genotypes with PCa Risk Stratified by Clinicopathologic Characteristics
We further analyzed the association of rs1800896 genotypes with PCa risk stratified by clinicopathologic characteristics including age, smoking behavior, and disease stage (Table 5).Interestingly, a significant association was observed in smokers, but not in nonsmokers.The ORs (95% CI) for the heterozygous variant genotype (AG) and homozygous variant genotype (GG) were 1.46 (0.96-2.24) and 3.90 (1.28-11.89),p = 0.0983 and 0.0231, respectively.In addition, the AG and GGs were associated with a significantly increased risk of PCa in late-stage patients (adjusted OR = 1.92, 95% CI = 1.09-3.68,p = 0.0452; and adjusted OR = 5.81, 95% CI = 2.54-14.77,p = 0.0003, respectively), but not in earlystage patients.

Discussion
In recent years, new advances in molecular and genetic studies have demonstrated a causal relationship between chronic infection, chronic inflammation, and PCa [32,33].Chronic inflammation has been hypothesized to be a cause of PCa, contributing to carcinogenesis during both disease initiation and subsequent progression [34,35].Over-expression of IL-10 has been associated with elevated PCa risk [20][21][22][23][24][25].Elevated IL-10 levels have been detected in the serum of PCa patients, correlating with poor prognosis and higher Gleason scores [22][23][24].In addition, the expression of IL-10 is significantly higher in PCa tumor tissues than in normal prostates and benign prostate lesions and correlates with a high grade and stage of prostate carcinoma [21,36].IL-10 may be produced by either tumor cells themselves or by immune cells [21,[37][38][39][40][41].IL-10 is predominantly expressed in monocytes and Th2 lymphocytes [42,43].IL-10 promotes tumor growth in PCa by suppressing the antitumor immune response through its effects on immune cells [44] and by directly acting on the PCa cells [45].Its suppression of the antitumor immune response includes the suppression of myeloid and T effector cell function [46,47].IL-10 can upregulate the expression of PDL1 on myeloid cells [48], whereas PDL1 binds to its receptor PD1 on T cells, inhibiting T cell function and hence its antitumor immune response [49].IL-10 can also directly act on prostate cancer cells.For example, it can induce neuroendocrine differentiation, inhibit AR activity, and upregulate PDL1 in PCa cells, thereby promoting PCa progression [50].
In this study, the association of three commonly studied IL-10 promoter SNPs and PCa risk was examined among a Taiwanese population containing 218 PCa cases and 436 non-PCa healthy controls.None of the SNPs were significantly associated with the risks of PCa, although a suggestive association between rs1800896 and the risk of PCa was observed (Tables 3 and 4).In the literature, the rs1800896 AA genotype has been associated with a higher risk of developing PCa in Croatian [51] and Indian populations [52].However, these two articles had limitations: one had a small sample size (control:case = 120:120), and the distribution of genotypes in the controls in the other did not fit HWE.On the contrary, it has been reported that the rs1800896 AA genotype was associated with decreased risks of PCa in USA [53], UK [54], and Indian populations [55].Several other studies of populations of European ancestry [56][57][58][59][60][61][62] and a study of Chinese populations [63] did not find significant associations (Table 6).These heterogeneous results may be due to small sample sizes, diverse populations, differing genetic architecture, and a range of environmental exposures.Regarding the biochemical mechanisms underlying our observed association, the literature has consistently demonstrated that individuals with the IL-10 rs1800896 (A-1082G) variant AG and GGs have elevated serum levels of IL-10 compared to those with the wild-type AA genotype [64][65][66][67].Moreover, the rs1800896 SNP is located within the transcriptional factor binding sites and the variant G-allele has higher transcriptional activity than the A-allele in various cell lines [68][69][70][71].These data strongly support our observations that the variant genotypes AG and GG of this SNP correlate with late stages of PCa and significantly increase the risk of PCa in smokers, because IL-10 is a pro-tumorigenic cytokine in PCa and the variant genotypes increase IL-10 level, thus promoting PCa development and progression.
To investigate how these SNPs modulate IL-10 expression, Reuss et al. [68] performed a thorough study characterizing the three IL-10 promoter SNPs on transcriptional activity and their bindings to transcriptional factors using luciferase reporter gene and electrophoretic mobility shift assays (EMSA).They found that the rs1800896 (A-1082G) lies within an ETS-consensus binding site and that the A-allele specifically binds to an ETSfamily transcriptional factor SPI1/PU.1, whereas the G-allele does not.They reasoned that the differential SPI1 binding accounts for the different transcriptional activity and that SPI1 binding to the A-allele negatively regulates IL-10 expression.The negative transcriptional regulation by SPI1 binding has also been reported for other genes [72,73].In addition, several studies have shown that another transcription factor, Sp1, only bound to the G allele in response to inflammatory stimulation, resulting in a much larger increase in IL-10 mRNA and protein levels in B-cell lines with the GG than the AA genotype [69][70][71].It appears that both positive and negative transcriptional regulation contribute to the higher transcriptional activity of the G-alleles of rs1800896.
The associations of the other two SNPs, rs3021097 (T-819C) and rs1800872 (A-592C), with IL-10 expression were inconsistent in the literature [74,75].They are not located within transcription factor binding sites and do not bind to transcriptional factors in EMSA experiments [68].It is thus not surprising that we did not observe any significant associations of these two SNPs with PCa risk.
There have been lots of epidemiological studies investigating the associations between IL-10 promoter SNPs, which may alter the function of this cytokine itself and its related cellular behavior and lead to the development of human disorders.The rs1800896 has been reported to be associated with oral cancer [76,77], nasopharyngeal carcinoma [78], papillary thyroid cancer [79,80], gastric cancer [81][82][83], lung cancer [84], breast cancer [85], renal cell carcinoma [86,87], cervical cancer [88,89], lymphoma [90,91], and leukemia [92,93].The rs3021097 was reported to be associated with gastric cancer [84,94,95], renal cell carcinoma [96], lung cancer [97], cervical cancer [98], bladder cancer [99], and leukemia [28,100].The rs1800872 was associated with oral cancer [101], esophageal cancer [102], gastric cancer [103], lung cancer [84], breast cancer [104], cervical cancer [105,106], lymphoma [107], and leukemia [100].Future larger studies would be necessary to clarify the roles of these SNPs in cancer predisposition due to their modest effect size.In addition, collecting comprehensive clinical and pathological data for all cases, along with full follow-up and periodic assessment of therapeutic responses, is crucial for precise genomics.Recruiting appropriately matched control subjects in terms of age, gender, and lifestyle behaviors is extremely important.Furthermore, a proper combination of multiple SNPs in a single candidate gene, such as IL-10 rs1800896, rs3021097, and rs1800872 in this study, can provide informative coverage of the entire gene.
IL-10 is a major suppressor of immune response and plays important roles in a variety of human diseases, thus becoming an attractive therapeutic target [108,109].In an IL-10 transgenic mouse model, high expression of IL-10 failed to control an immunogenic lung tumor; however, administering an anti-IL-10 antibody significantly inhibited tumor growth [110].Likewise, IL-10 knock-out mice are resistant to UV-induced skin carcinogenesis, correlating with a potent Th1 response in these mice [111].Ruffell et al. [112] showed that blocking IL-10 signaling using an IL-10 receptor (IL-10R) monoclonal antibody enhanced paclitaxel and carboplatin efficacy in a breast cancer model.Blocking IL-10 could also suppress the metastatic behaviors of colorectal cancer cells [113] and promote gastrointestinal cancer cell death [114].These data support the concept that IL-10 suppresses the anti-tumoral immune response, and blocking IL-10 can strengthen the anti-tumoral immune response and inhibit tumor growth.However, it should be cautioned that IL-10 plays a dual role in human carcinogenesis depending on specific cell types and contexts.It can inhibit certain cancers, and a PEG-conjugated human IL-10 has been on clinical trial for pancreatic cancer [109,115].Future studies should test the clinical potential of targeting IL-10 and its downstream molecules in the context of PCa and investigate the pleiotropic effects of IL-10 in different cancers.
The well-established risk factors for PCa include age, ethnicity, family history, genetic predisposition, diet, and environmental factors [116][117][118][119][120][121].We performed stratified analyses on age and did not find significant differences in the associations of these SNPs with PCa risks in young and old aged groups.Smoking is inversely associated with overall PCa incidence, but may lead to more aggressive PCa tumors and worse prognosis of PCa patients [122][123][124].Our stratified analyses demonstrated that the variant genotypes at rs1800896 increases the likelihood of PCa among smokers (Table 6), and are associated with increased risks of late-stage PCa (Table 5).In clinical practice, determining IL-10 genotypes alongside smoking history may aid in developing precise personalized screening and prevention strategies against aggressive PCa.The molecular mechanisms underlying the associations of the IL-10 rs1800896 variant genotypes with PCa risk in smokers and with aggressive PCa remains unclear and warrants future investigation.

Conclusions
In conclusion, this is the first report of three IL-10 promoter SNPs in relation to PCa susceptibility in Taiwanese.We found significant associations of the IL-10 rs1800896 variant genotypes with PCa risk in smokers and with aggressive PCa.Further studies are necessary to validate the IL-10 genetic variants as PCa predisposition markers in diverse populations and investigate the molecular mechanisms underlying the observed associations.

Table 1 .
Demographics of prostate cancer cases and control subjects.

Table 3 .
Distributions of interleukin-10 genotypic frequencies among prostate cancer patients and healthy controls.
OR: odds ratio; CI: confidence interval; a data based on Chi-square test with Yates' correction; b adjusted for age; P trend : p-value based on trend analysis; P HWE : p-value based on Hardy-Weinberg Equilibrium.

Table 4 .
Distributions of allelic frequencies for interleukin-10 polymorphisms in prostate cancer patients and control groups.
a p-value based on Chi-square test with Yate's correction.

Table 5 .
Association of interleukin-10 rs1800896 genotypes with prostate cancer risk stratified by clinicopathologic characteristics.

Table 6 .
Concise summary of the literature for association between interleukin-10 rs1800896 genotypes and prostate cancer.