Vol 19 No 1 January-February 2022 138 UROLOGICAL ONCOLOGY The Role of Kallikrein10 (KLK10) Polymorphism in Prostate Cancer Susceptibility Seda Gulec Yılmaz1, Faruk Yencılek2 , Asıf Yıldırım3, Fatma Tuba Akdeniz1, Altay Burak Dalan1, Zerrin Barut4, Turgay Isbır1* Purpose: The present study aims to investigate the potential role of Kallikrein 10 (KLK10) genotype and allele frequencies in predisposition to prostate cancer. Materials and Methods: KLK10 (rs7259451) gene polymorphisms were determined by real-time polymerase chain reaction analysis in patients with prostate cancer (n = 69) and controls (n = 76). Results: KLK10 gene frequencies were significantly different in the case and control groups (P = .028). GG carri- ers were significantly higher in the control group (P = .034), whereas TT carriers were higher in the prostate cancer group (P = .033). Furthermore, The patients with GG genotype had the lowest PSA levels while TT carriers had the highest (P = .005). Conclusion: According to the results, we suggested that carrying variant T allele and also carrying homozygote TT genotype could be a potential risk, while ancestral homozygote GG genotype and G allele are risk reducing factors for prostate cancer. Keywords: KLK10 gene; polymorphism; prostate cancer; PSA; rs7259451 INTRODUCTION Prostate cancer is the most common cancer among men aged over 40 years. Moreover, after lung can- cer, it is the second most common mortality factor in cancer-related deaths. Incidence rates of prostate can- cer have more than doubled in the last decades, because of great improvements in diagnostic assets and high- tech screening methods(1). Although prostate cancer is a multifactorial disease with several potential risk factors, such as smoking, obesity, age, diabetes and en- vironmental changes, recent advances in genetics have contributed in understanding its pathological metabo- lism. However, prostate cancer etiology is still vague and investigations about genetic polymorphisms enable to display individual differences and predispositions (2). Thanks to recent improvements in genetic analysis, many genes and their variations have been demonstrat- ed in cancer research. There are many polymorphisms associated with prostate cancer tendency(3,4). These ge- netic relations help to understand the molecular basis of the disease as well as provide a clinical diagnostic utility. The protein function and effectiveness could be altered by genomic variations, called polymorphism. These variations have different impacts on cancer and cancer prognosis. They also give rise to predispositions via initiating a physiological process indirectly(5,6). 1Department of Medical Biology, Faculty of Medicine, Yeditepe University, Istanbul, Turkey. 2Department of Urology, Faculty of Medicine, Yeditepe University, Istanbul, Turkey. 3Department of Urology, Göztepe Education and Research Hospital, Istanbul, Turkey. 4Department of Basic Medical Science, Faculty of Dentistry, Antalya Bilim University, Antalya, Turkey *Correspondence: Turgay Isbır; Department of Medical Biology, Faculty of Medicine, Yeditepe University, Istanbul, Turkey. Yeditepe Universitesi Yerleskesi, Inonu Cad. 26 agustos Yerleskesi, 34755 Kayısdagı-Atasehir, Istanbul, Turkey. Tel: +90 5332823726/ +90 2165780000-1263, E-mail: turgay.isbir@yeditepe.edu.tr. Received August 2020 & Accepted March 2021 Human kallikrein-related peptidases, catalyse peptide bond hydrolysis, are a member of the serine peptiases family. Kallikrein 3 (KLK3), the most well- known family member, named as Prostate-Specific-An- tigen (PSA), is a well-known kallikrein (KLK) and has great importance in prostate cancer prognosis and high PSA levels could lead the biochemical failure which predisposes the patients to metastasis(7). Another family member, called Kallikrein10 (KLK10), is a protein that is involved in steroid hormone stimulation via affecting hormone-receptor complexes. The human tissue KLK is located on chromosome 19q13.4 and encoded by ster- oid hormone-regulated genes. It has been demonstrat- ed that dysregulation of KLK expression is associated with multiple diseases, such as cancer(8). Human KLK was determined as eligible biomarker for diagnosis and prognosis for many cancer types, such as ovarian, breast and prostate. Investigations have fo- cused on understanding the relationship between pros- tate cancer and the polymorphisms of several KLK genes. Therefore, this study is aimed to investigate the potential role of KLK10 genotype and allelic frequen- cies in the tendency to prostate cancer (PC). MATERIALS AND METHODS The study population consisted of 69 patients who re- Urology Journal/Vol 19 No. 1/ January-February 2022/ pp. 41-44. [DOI: 10.22037/uj.v18i.6425] curred from Yeditepe University Urology Department and 76 age-matched healthy controls. Control group consisted of healthy individuals with age 40 -80 years, they were not diagnosed with this disease, following a clinical examination. The patients' group consisted of individuals who had prostate cancer with age range be- tween 40-80 years old. The diagnosis of prostate can- cer was demonstrated by the clinical, and pathological examinations. The tumor differentiation status was evaluated using Gleason score criteria. The clinical ex- aminations classified T -stage as early -stage (T1 and T2) and late -stage (T3 and T4). Pathologic T -stage was classified as T2a, T2b, T2c, T3a, and T3b. All pa- tients and control groups gave their informed consent following a detailed explanation of the protocol of the study. Blood samples from all individuals were col- lected in tubes containing EDTA. Clinical and demo- graphic information’s of patients and controls obtained from hospital records. Genomic DNA extraction from 350µl of whole blood was performed by Invitrogen iPrep PureLink gDNA blood isolation kit (Invitrogen, Life Technologies, Carlsbad, California, USA). DNA samples were measured with NanoDrop 2000 (Ther- moscientific, Waltham, Massachusetts, USA). Deter- mination of KLK10 gene (rs7259451) polymorphism was performed in Applied Biosystems 7500 Fast Real- Time PCR instrument (Applied Biosystems, Foster City, CA, USA) by using TaqMan Genotyping Assay and TaqMan Genotyping Master Mix (TaqMan Re- agents, Applied Biosystems, Foster City, CA, USA). The reactions were carried out with primer sequence as 5’-TAAGGCAAGACTCAGGATAAAACAC[G>T] GTGGTGTGGCCGGGAGCGGTGGCTC-3’. Due to the Minor allele frequency (MAF) analysis, allele fre- quencies considered as G wild type and T mutant form. Statistical analyses were performed using SPSS Ver. 23 software (SPSS Inc, Chicago, IL, USA). The signif- icant difference between groups was examined by Stu- dent’s t and one way Anova tests, also demographic and clinic data were compared by Chi square and Fisher’s exact tests. Risk estimations were examined with odds ratio (OR) at 95% confidence interval (CI). P < 0.05 is denoted as statistically significant. RESULTS Demographic and clinical properties are summarized in Table 1. There were no differences between the pa- tients and controls in mean ages. There were also no significant difference regarding BMI and smoking hab- its between the groups, although diagnosis for diabetes, Coronary Artery Disease (CAD) and hypertension were significantly higher in the PC group (P < .001). The pa- tients with PC had significant a statistically higher level of PSA when compared to the control group (P = .006). The genotypic and allelic frequencies of KLK10 in PC KLK10 in prostate cancer- Güleç Yılmaz et al. Urological Oncology 42 Table 1. The comprehensive comparison of sensitivity Parameters Prostate cancer (n=69) Control (n=76) p-Value Age (years), mean±SD 68.52 ± 7.36 66.49 ± 8.45 0.867 Body mass index (kg/m2), mean±SD 27.01 ± 3.71 27.28 ± 3.55 0.773 Smoking status n (%) 52 (76.5%) 16 (23.5%) 0.415 Diabetes Mellitus n (%) 6 (66.7%) 3 (33.3%) < 0.001* CAD n (%) 1 (25%) 3 (75%) < 0.001* Hypertension n (%) systolic blod pressure >140 17 (81%) 3 (19%) < 0.001* diastolic blod pressure >90 PSA (ng/mL) mean±SD 25.94 ± 41.14 3.0 3± 2.66 0.006* Gleason score, mean±SD 7.74±0.88 - - Pathological T-stagen (%) T2a 9 (13 %) - - T2b 10 (14.5 %) - - T2c 29 (42 %) - - T3a 11 (15.5 %) - - T3b 10 (15 %) - - Clinical T-stage n (%) Early (T1+T2) 61 (88,4 %) - - Late (T3+T4) 8 (11.6 %) - - *P values less than 0.05 denoted statistical significance. .χ2: Chi square used for comparison of patients with PC and control group; student t test is used for comparing quantitative data Abbreviations: CAD:Coronary Artery Disease, PSA: Prostate-spesific antigen, n: number of individuals, SD: standart deviation. Prostate cancer (n=69) n (%) Control (n=76) n (%) P value χ2 OR 95 % CI Genotype P = .028* χ2 =7.178 GG 26 (37.7 %) 42 (55.3 %) .034* 4.489 0.489 0.252-0.951 GT 35 (50.7 %) 32(42.1 %) .298 1.081 1.415 0.735-2.727 TT 8 (11.6 %) 2 (2.6 %) .033* 4.524 4.852 0.993-23.703 Allelic count n (%) G Allele 87 (63.1 %) 64 (64%) 0.033* 4.524 0.206 0.042-1.007 T Allele 51 (36.9 %) 36 (36%) 0.034* 4.489 2.043 1.051-3.970 Table 2. Kallikrein10 (KLK10) rs7259451 genotypic and allelic frequencies in prostate cancer and control group. *P values less than 0.05 denoted statistical significance. χ2: Chi square and Fisher's exact test used for comparison of patients with PC and control group; Abbreviations: n:number of individuals; χ2:Chi-Square; OR:odds ratio; CI:Confidence interval Vol 19 No 1 January-February 2022 138 and control groups are given in Table 2. There were significant differences between the groups in the fre- quency of KLK10 genotypes (P = .028). The frequency of the GG homozygote genotype was significantly high- er in the control group than the patient group and those with GG genotype were ~2 fold likely to be healthy control than patients (χ2 = 4.489, %95 CI= 0.252-0.951, OR= 0.489, P = .034). There were no statistically sig- nificant correlations between the groups regarding the GT heterozygote genotype (P = .298), however, the TT homozygote genotype was significantly higher in the patient group when compared to the control group (P = .033). Ancestral G allele frequency was significant- ly higher in controls than patients with PC(P = .033), while mutant T allele frequency was significantly high- er in the patient group (P = .034). Sixty-four percent of healthy control participants were carrying the G allele, whereas 36% of them had the T allele. As it is shown in Table 3, although there was no statis- tically difference between KLK10 genotypes regarding prostate volume (P = .236), the homozygote mutant al- lele (TT) group displayed the highest prostate volume. According to PSA levels genotype groups are signif- icantly different. The patients with KLK10 GG geno- type carriers had the lowest and TT carriers had highest PSA levels (P = .005). While there was no statistical difference in allele distributions (P = .486), we found that T allele carriers had higher PSA levels than G allele carriers (Table 3). DISCUSSION In the last decades, polymorphism, expression and ge- nome-wide studies have undergone a great improve- ment; however, there is no particular evidence to iden- tify prostate cancer susceptibility. On account of this, new studies should be performed in population-based case-control studies. Owing to its tumor suppressing effect, the KLK10 gene accounts for the prediction of cancer prognosis(9). The human KLK10 gene and its potential role have been investigated in various cancer types in different populations. The present study aims to investigate the associations between KLK10 poly- morphism and prostate cancer in a Turkish population. KLK genes and KLK proteins have structural char- acteristics, such as localization on the same chromo- somal domain. They also have analogue translational sites, stop and start codons. KLK genes have five peer exons and four codons with no association with other genes located on the same gene region. Altered KLK gene expressions vary in different cancer types; more- over, it has been asserted that KLK proteins participate in proliferation, angiogenesis and metastasis(8,10). Sev- eral studies indicated that the KLK protein family, the best known of which is KLK3, also known as PSA, is widely used for prostate cancer diagnosis(10). KLK fam- ily members has been investigated as novel serum bio- markers, although there is unclear evidence regarding KLK gene expression and protein levels(11). Bayani et al. (2008) demonstrated that dysregulated KLK expres- sion levels in breast, ovary and prostate are associated with increased KLK protein levels. They indicated that unbalanced translocations associated with altered pro- tein levels. Furthermore, they showed that there was a relation between cancer progression and KLK protein levels(12). Although there have been several investigations into the KLK protease family, the association between physio- logical role and genetic variations is still unclear. An- gelopoulou et al. (2009) demonstrated KLK mRNA expressions of cancer tissues. They found that KLK9 and KLK10 have the highest expression levels among cancerous mammary tissues. Thus, it has been suggest- ed that KLK9 and KLK10 participate in proteolytic cas- cades(13). KLK proteins are initially translated as prepro- enzymes that carry signal peptide on N-terminus and mature active enzyme takes place after a short propep- tide. The signal cleaves from propeptide domains, and forms a mature enzyme complex. Human KLKs par- ticipate in different biological processes, such as reg- ulating neural development, regulating blood pressure, semen liquefaction and also cell proliferation(14). There- fore, several studies have been conducted on the role of human KLKs in diverse cancer types. Yousef et al. (2005) showed the relation between KLK10 and endo- crine related malignancies in silico analyses. Although KLK genes had different expression profiles in various tissues, KLK10 gene expression significantly downreg- ulated in ovarian, breast, testicular and prostate cancer lines. Consequently, the KLK10 gene represented a tu- mour suppressor function, especially in endocrine-re- lated malignancies and KLK10 could be considered as a cancer biomarker gene(15). The potential biomarker role of KLK10 gene was documented in human breast, ovary and prostate cancer cell lines by Sidiropoulus et al. (2005). They investigated epigenetic alterations on the tumor suppressor role of KLK10 mRNA expres- sion. However, various mechanisms account for down- regulating gene expressions; hypermethylation of CpG islands on the KLK10 gene could explain the specific tumor suppressing mechanism of KLK10 expression profile in cancer cells(16). Another study demonstrat- ed the tumor biomarker role of altered KLK10 serum level in patients with various malignancies. Although the relation between gene regulation and serum protein level is still unclear, Luo et al. (2001) showed a positive correlation with serum KLK10 level and ovarian cancer severity(17). * P = values less than 0.05 denoted statistical significance. Student t test and one way ANOVA test used used for comparison of genotypes and alleles Abbreviations: PSA: Prostate-spesific antigen; SD: standart deviation Prostate Volume (mL) mean±SD PSA (ng/mL) mean±SD GG 45.05 ± 29.79 26.39 ± 7.16 GT 39.00 ± 13.28 P = .236 40.49 ± 13.57 P = .005* TT 47.77 ± 19.11 81.08 ± 70.79 G allele 42.29 ± 17.00 P = .972 33.44 ± 15.80 P = .486 T allele 43.07 ± 15.38 40.54 ±3 5.69 Table 3. KLK10 ( rs7259451) genotype variations and prostate volume and PSA levels in patient with prostate cancer KLK10 in prostate cancer- Güleç Yılmaz et al. Vol 19 No 1 January-February 2022 43 Urological Oncology 44 The relation between single nucleotide polymorphisms in human KLK10 gene and endocrine-related malig- nancies, such as prostate, testicular, breast and ovarian cancer, was investigated by Bharaj et al. (2002). Five coding regions of KLK10 gene sequencing analysis performed in different human tumour tissues were ob- tained from cancer patients and the sequence analysis showed that the mutant variant was significantly higher in prostate tumours than adjacent normal tissues. Thus, they identified human KLK10 gene polymorphisms at codon 50 associated with PC risk(18). In silico analysis of KLK10 single nucleotide poly- morphisms (SNPs) demonstrated that KLK10 expres- sion could be altered by intronic SNPs via regulating transcription factors. Intronic SNPs could change not only gene expressions, but also hormone response el- ement binding domains. Batra et al. (2010) performed KLK10 gene sequencing to analyse possible associa- tion between KLK10 and cancer survival. The analysis showed that KLK10 rs 7259451 polymorphism located on the 5’UTR intronic region where upstream of andro- gen response elements (AREs) clustered. In addition, they asserted that intronic SNPs could regulate trans- lation via epigenetic factors, such as hypermethylation and microRNA alterations(19). Their results support the present study as regard the importance of KLK10 (rs 7259451) gene polymorphism on PC susceptibility. CONCLUSIONS Although the present study has limitations, such as small sample size, to the best of our knowledge, it was the first in vivo study which investigated the association between PC susceptibility and KLK10 intronic SNP. Our results implicated that homozygote ancestral GG genotype and carrying G allele could be protective from PC. Besides not only carrying mutant homozygote gen- otype TT, but also having T allele, could be a potential risk factor for PC. CONFLICT ON INTEREST The authors declare that they have no conflict of inter- est. REFERENCES 1. Siegel RL, Miller KD, Jemal A. CA Cancer J Clin 2016; 66: 7–30. 2. Cintra HS, Pinezi JC, Machado GD, et al. 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