Functional variants of the chitinase 3‐like 1 gene are associated with clinicopathologic outcomes and progression of prostate cancer

Abstract Chitinase 3‐like 1 (CHI3L1 or YKL40) is a secreted glycoprotein highly expressed in advanced stages of several cancer types, including prostate cancer (PCa). Impacts of genetic variants of CHI3L1 on PCa development have not yet been investigated. The most common well‐studied genetic variations are single‐nucleotide polymorphisms (SNPs). Therefore, the objective of this study was to explore associations of CHI3L1 SNPs with both the susceptibility to PCa and its clinicopathological development. Three promoter SNPs, rs6691378 (−1371, G>A), rs10399805 (−247, G>A) and rs4950928 (−131, C>G), and one non‐synonymous SNP, rs880633 (+2950, T>C), were analysed using a TaqMan allelic discrimination assay for genotyping in a cohort of 701 PCa patients and 701 healthy controls. Results indicated that there were no significant associations of PCa susceptibility with these four CHI3L1 SNPs. However, among elderly PCa patients (aged >65 years), it was observed that polymorphic variants (GA + AA) of CHI3L1 rs6691378 and 10399805 were significantly linked to reduced risks of several clinicopathological characteristics, including a high Gleason grade, advanced pathologic T stage and tumour cell invasion. Moreover, analyses of The Cancer Genome Atlas database revealed that CHI3L1 expression levels were elevated in PCa tissues compared with normal tissues. Interestingly, higher CHI3L1 expression levels were found to be associated with longer progression‐free survival rates in PCa patients. Our findings indicated that levels of CHI3L1 may influence the progression of PCa, and the rs6691378 and 10399805 SNP genetic variants of CHI3L1 are linked to the clinicopathological development of PCa within a Taiwanese population.


| INTRODUC TI ON
Every year, over 34,700 men succumb to prostate cancer (PCa) in the United States, positioning it as the second leading contributor to cancer-related fatalities among males on a global scale. 1 While early-stage PCa can be effectively managed through a radical prostatectomy (RP) and radiotherapy, the same cannot be said for metastatic PCa.[5] Nonetheless, conventional clinicopathological parameters often fall short in accurately predicting prognoses.For instance, elevated serum PSA levels can be observed in cases of PCa and also in benign conditions like prostatic hyperplasia and prostatitis. 6The PSA test suffers from both limited sensitivity and specificity.Furthermore, the significance of PSA declines in the later stages of the disease. 7Hence, given the imperative of an early PCa diagnosis to prevent metastasis and facilitate timely treatment, it is crucial to conduct research aimed at identifying novel and efficient predictive biomarkers.
The chitinase-3-like-1 (CHI3L1) protein, also known as YKL40, is categorized as a secretory glycoprotein and is a member of glycoside hydrolase family 18 (GH18) of chitinases.CHI3L1 is overexpressed in many human cancer types such as lung, liver, breast, colorectal, ovary and cervical cancers.][10][11][12][13] In PCa, elevated serum CHI3L1 levels were also documented in individuals with primary PCa compared with those with benign prostate hyperplasia (BPH), suggesting a potential role for CHI3L1 in influencing the development of PCa. 14 Additionally, higher serum CHI3L1 levels were linked to shorter overall survival (OS) and early mortality among metastatic PCa patients undergoing ADT 15 and docetaxel chemotherapy. 16Most importantly, there is a proposition that CHI3L1, compared with the widely used PSA, could provide more informative insights into predicting tumour burdens and the potential for metastasis. 17e human CHI3L1 gene is located on chromosome 1q31-1q32, and several variants of the CHI3L1 gene were identified.For example, single-nucleotide polymorphisms (SNPs) located in promoter (rs4950928, rs10399931 and rs10399805), exon (rs880633) and intron (rs2071579, rs1538372, rs2275353 and rs946259) regions of the CHI3L1 gene were identified as being associated with its serum levels within the general population.These associations were observed at or below significant genome-wide association levels. 18Therefore, the CHI3L1 rs4950928 C allele was reported to be associated with risk of rectal cancer and increased serum levels of CHI3L1 in Egyptians. 19Moreover, the CHI3L1 rs880633 C allele was also correlated with the risk of liver cancer and higher serum CHI3L1 levels compared with the rs880633 T allele. 20

| Healthy volunteers and patients with PCa
This retrospective study involved two cohorts: a group of 701 Taiwanese PCa patients and a matched set of 701 healthy male controls with the same ethnic background and residing in a similar geographic area.PCa diagnoses were histologically confirmed in all cases following a robotic-assisted laparoscopic RP at Taichung Veterans General Hospital (Taichung, Taiwan) between 2012 and 2018.Demographic characteristics and medical details of patients were collected from their medical records at the time of the PCa diagnosis.These details included PSA values, pathologic Gleason risks of several clinicopathological characteristics, including a high Gleason grade, advanced pathologic T stage and tumour cell invasion.Moreover, analyses of The Cancer Genome Atlas database revealed that CHI3L1 expression levels were elevated in PCa tissues compared with normal tissues.Interestingly, higher CHI3L1 expression levels were found to be associated with longer progression-free survival rates in PCa patients.Our findings indicated that levels of CHI3L1 may influence the progression of PCa, and the rs6691378 and 10399805 SNP genetic variants of CHI3L1 are linked to the clinicopathological development of PCa within a Taiwanese population.

K E Y W O R D S
cancer incidence, chitinase 3-like 1, clinicopathologic progression, prostate cancer, singlenucleotide polymorphism grade, pathologic T (tumour) and N (node) staging, invasion areas of tumour cells (such as seminal vesicle, perineural and lymphovascular involvement) and the D'Amico classification.Prior to blood sampling, informed consent was obtained from each participant, and the study was approved by the ethics committee of Taichung Veterans General Hospital (ethics approval no.CE19062A).

| Blood sample collection and genomic DNA extraction
Peripheral blood of recruited subjects was aseptically collected through venipuncture and preserved in tubes containing EDTA for the purpose of DNA purification.DNA was extracted using a QIAamp DNA Blood Mini Kit from Qiagen (Valencia, CA, USA).
Extracted DNA was dissolved in Tris-EDTA (TE) buffer, consisting of 10 mM Tris and 1 mM EDTA at pH 7.8.Subsequently, the DNA purity was assessed using a Nanodrop-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) to determine the ratio of absorbances at 260 and 280 nm.The final DNA preparations were then stored at a temperature of −20°C in preparation for a subsequent real-time polymerase chain reaction (PCR) analysis.

| Selection and determination of CHI3L1 SNPs
In total, four CHI3L1 genetic variants were selected for analysis.These four SNPs were previously implicated in impacting various aspects related to cancer.They were associated with influencing the occurrence, severity, or progression of different cancer types.
0][21][22] The allelic discrimination of these four CHI3L1 SNPs including rs4950928 (assay ID: C_27832042_10), rs6691378 (assay ID: C_29933614_10), rs880633 (assay ID: C_11891591_1) and rs10399805 (assay ID: C_29969647_10) was evaluated by utilizing the TaqMan SNP Genotyping Assay.This analysis was conducted using the ABI StepOnePlus™ Real-Time PCR System, a product of Thermo Fisher Scientific.This approach allows the differentiation of allelic variants at these specific positions within the CHI3L1 gene, facilitating the genotyping of these SNPs.
Detailed processes regarding DNA genotyping were published in our previous study. 23

| Bioinformatics analysis
The UCSC Xena database (https:// xena.ucsc.edu/ ) facilitated access to clinical data and messenger (m)RNA sequencing information from prostate adenocarcinoma (PRAD) samples sourced from The Cancer Genome Atlas (TCGA).Within this context, we conducted a comparative analysis of CHI3L1 gene expression levels across a range of clinical features, including Gleason scores, clinical stages, pathological tumour sizes, lymph node statuses and the presence of distal metastases.For two-group comparisons, we employed the Wilcoxon signed-rank test, while clinical features with more than two groups were subjected to the Kruskal-Wallis test, followed by post hoc Dunn's tests.To explore the association between CHI3L1 and patients' progression-free survival (PFS), we applied a log-rank test, and high and low CHI3L1 expression groups were determined based on the median cut-off point of CHI3L1.

| Statistical analysis
To assess the association between CHI3L1 genotypic frequencies and clinicopathologic features, we employed multivariate logistic regression models.These models were utilized to calculate odds ratios (ORs), adjusted ORs (AORs) and corresponding 95% confidence intervals (CIs).All statistical analyses were conducted with the SAS software program (vers.9.1, 2005; SAS Institute, Cary, NC, USA).The threshold for statistical significance was set to p < 0.05.

| Demographic characteristics of recruited PCa patients
Demographic and clinicopathological characteristics of 701 PCa patients who received an RP are shown in Table 1.Our study cohort predominantly consisted of older individuals, with 57.8% being over the age of 65 years.Our recruited cohort was consistent with previous reports which indicated that nearly 60% of PCa cases are diagnosed in patients over the age of 65 years. 24The majority of patients exhibited early-stage tumours (clinical T1 or T2 stage, 86%), falling within Gleason grade groups 1 or 2 (60.1%) and presenting with perineural invasion (73.5%).Lymph node metastasis (N0) was absent in 91.4% of cases, as were lymphovascular invasion (84.2%) and seminal vesicle invasion (78.7%).Categorizing patients according to the D'Amico risk classification, over half of the PCa patients (50.4%) were classified as having a high risk (>50% likelihood) of experiencing recurrence within 5 years following treatment.

| Impacts of CHI3L1 genetic polymorphisms on the PCa incidence
We then proceeded to investigate potential correlations between the Genotypic distributions of these four CHI3L1 SNPs in the control group conformed to Hardy-Weinberg equilibrium (χ 2 value = 0.089, p = 0.765 for rs4950928 C>G; χ 2 value = 0.358, p = 0.549 for rs6691378 G>A; χ 2 value = 0.061, p = 0.805 for rs880633 T>C, and χ 2 value = 2.048, p = 0.152 for rs10399805 G>A).Employing AORs with 95% CIs calculated through multiple logistic regression models with age as a covariate, we examined associations between CHI3L1 SNPs and PCa incidence.Notably, in the context of the recruited Taiwanese population, our analyses revealed no significant links between CHI3L1 SNPs and PCa occurrence.This outcome held true whether assessed through a dominant or codominant model, as detailed in Table 2.

| Impacts of CHI3L1 genetic polymorphisms on CHI3L1 expression
We subsequently assessed the association between CHI3L1 polymorphisms and CHI3L1 gene expression in whole blood tissues using samples from healthy individuals sourced from the Genotype-Tissue Expression (GTEx) database.Individuals carrying the polymorphic A allele of rs10399805 (Figure 1A) and rs6691378 (Figure 1B) all exhibited lower CHI3L1 expression compared with those with wildtype homozygous genotypes.Abbreviation: PSA, prostate-specific antigen.

| Correlations of CHI3L1 expression levels with clinicopathologic features and prognoses of PCa patients
To perform a more comprehensive analysis of CHI3L1 expression levels in both normal and PCa tissues and to explore potential correlations of CHI3L1 levels with the progression and prognosis of PCa, we made use of TCGA-PRAD dataset.Our examination of the dataset revealed that CHI3L1 expression levels were notably higher in tumour tissues compared with noncancerous tissues (Figure 2A, Furthermore, when examining survival data, a Kaplan-Meier plot showed that PCa patients from TCGA-PRAD dataset who exhibited CHI3L1 low tumours experienced shorter progression-free survival (PFS) times compared with those with CHI3L1 high tumours (Figure 2F).

| DISCUSS ION
CHI3L1/YKL40 genetic variants were demonstrated to impact its messenger (m)RNA expression and exhibit strong associations with various diseases, including asthma, Alzheimer's disease (AD), atopy and hypertension.For instance, Tsai et al. found that the rs10399931 GG genotype was linked to heightened serum YKL40 levels and increased severity of lung obstruction in asthma patients from southern Taiwan using steroids. 25Additionally, Dai et al. revealed that polymorphisms in the CHI3L1 gene (such as rs4950928 and rs10399931) were connected with the AD risk and prognosis, potentially influencing CHI3L1's expression in plasma. 26Sohn et al.
reported an association between the rs10399805 polymorphism in the promoter region of the CHI3L1 gene and atopy.Individuals with the TT genotype exhibited a 2.5-fold increase in CHI3L1 mRNA expression in peripheral blood cells compared with those with the CC genotype. 27Moreover, the rs10399805, rs4950928 and rs2297839 genotypes in the CHI3L1 gene were identified as stable biomarkers for predicting a hypertension risk. 28 Note: ORs with their 95% CIs were estimated by logistic regression models.Abbreviation: PSA, prostate-specific antigen.
rs10399805, were associated with the development of cervical precancerous lesions and invasive cancer.mRNA expression in peripheral blood cells compared with those carrying the CC/GG genotype. 27Those findings suggested that the T/A allele of rs10399805 may contribute to promotion of CHI3L1 expression.Indeed, the T/A allele of rs10399805 was associated with elevated serum CHI3L1 levels in patients with atopy 27 and hypertension. 28However, this correlation was observed not to be significant in patients with coronary artery disease. 31In contrast, data extracted from the GTEx database showed decreased CHI3L1 expression in whole blood tissues among individuals carrying the polymorphic A allele of rs10399805.3][34] In line with these previous studies, our findings indicated that CHI3L1 transcripts in PCa were significantly higher compared with those in noncancerous tissues or matched normal tissues from TCGA-PRAD dataset.
Contrary to the common trend of poor prognostic impacts associated with high CHI3L1 expression, our results surprisingly revealed Our current study still has some limitations that need to be considered.First, our study only recruited a Taiwanese population.
Including other ethnic populations in future studies will allow for comparisons and validation of the findings across different racial groups.Additionally, owing to the relatively small sample size, the frequencies of some homozygous variants were low in subgroups and therefore may limit the statistical power and precision of the Furthermore, Su et al. indicated a significant association of the homozygous phenotype (AA) for the minor allele of CHI3L1 rs10399805 and rs6691378 with a lower risk of developing lymph node (LN) metastasis in Taiwanese with oral cancer. 21Despite several studies having investigated the clinical significance and functional role of CHI3L1 in PCa, the effects of CHI3L1 genetic variants on PCa have not been explored.In this study, our aim was to examine associations of SNPs within the CHI3L1 gene with the risk of the clinicopathological development of PCa in a Taiwanese population.
These included three promoter SNPs of rs6691378 (−1371, G/A), rs10399805 (−247, G/A) and rs4950928 (−131, C/G), and one nonsynonymous SNP of rs880633 (2950, T/C).These specific SNPs were chosen based on information from the Chinese HapMap dataset, which is focused on the Han Chinese population in Beijing, China.

[
T/C] and rs10399805 [G/A]) within the CHI3L1 gene and PCa occurrence.We initially examined genotype frequencies of these SNPs across the entire recruited population.As illustrated in Table 2, predominant distribution frequencies of CHI3L1 rs4950928, rs6691378, rs880633 and rs10399805 SNPs in PCa patients included homozygous C/C, G/G and G/G for rs4950928, rs6691378 and rs10399805 loci, respectively, and heterozygous T/C for the rs880633 locus.
left panel) and to corresponding matched normal tissues (Figure 2A, right panel).Nevertheless, our investigation identified no significant correlations between elevated CHI3L1 expression levels and various clinicopathological features, including the Gleason score (Figure 2B), clinical stage (Figure 2C), pathological T stage (Figure 2D) and lymph node (Figure 2E, left panel) or distal metastasis (Figure 2E, right panel).

22
Despite these findings, clinical implications of CHI3L1 SNPs in PCa remain largely unexplored.These SNPs potentially lead to expression and functional alterations of CHI3L1, which could influence PCa progression.Herein, we found for the first time that genetic variants in CHI3L1 play pivotal roles in shaping clinicopathological characteristics of PCa within a Taiwanese population.PCa is mainly a disease of seniors aged 60-70 years. 1 This study presents findings that individuals aged over 65 years, possessing the mutated base A variant of CHI3L1 rs6691378, exhibited a notably reduced risk of developing high Gleason grade (3-5), advanced T stage (3 or 4) and seminal vesicle invasion under a dominant genetic model (GA + AA).Furthermore, our investigation revealed that elderly PCa patients carrying the GA or AA genotype of rs10399805 similarly demonstrated significantly diminished risks of developing a high Gleason grade, advanced T stage, and seminal vesicle and perineural invasion.These results were similar to observations of our previous study, which indicated that oral cancer patients who had at least one mutant A allele of CHI3L1 rs6691378 and rs10399805 had a significantly lower frequency of developing lymph node metastasis. 21These insights shed light on the potential influence of specific CHI3L1 genetic variants on disease progression and clinical characteristics of PCa among older individuals.

TA B L E 5
Odds ratios (ORs) and 95% confidence intervals (CIs) of the clinical status and CHI3L1 rs6691378 genotypic frequencies in 405 prostate cancer patients aged >65 years.Both rs6691378 and rs10399805 SNPs are situated within the promoter regions of the CHI3L1 gene.Rehli et al. identified various transcription factor-binding sites in the CHI3L1 gene promoter, including C/EBP-and AML-1-binding sites within the region spanning positions −234 to −252 relative to the ATG start site of transcription. 30Notably, rs10399805 resides at position −247 and was reported to influence CHI3L1's transcriptional activity.For instance, Sohn et al. conducted an in vitro promoter assay using THP-1 cells which revealed that a C/G to T/A conversion at the rs10399805 SNP led to an increase in reporter gene expression.Furthermore, the −247T allele exhibited heightened affinity for C/EBP, as determined by an electrophoretic mobility shift assay.In an in vivo context, individuals with the TT/AA genotype exhibited elevated CHI3L1 risk profiles.In addition to rs10399805, there is currently no reported correlation between the rs6691378 SNP and serum CHI3L1 levels in any disorder.To address this gap, we conducted preliminary assessments of the association between CHI3L1 rs6691378 polymorphisms and CHI3L1 gene expression from the GTEx database, where we observed decreased CHI3L1 expression in whole blood tissues among individuals carrying the polymorphic A allele of CHI3L1 rs6691378.Nevertheless, correlations of rs10399805 and rs6691378 SNPs with serum CHI3L1 levels in patients with PCa warrant further investigation in future work.
that elevated CHI3L1 expression was correlated with a longer PFS in PCa patients.This observation suggests that CHI3L1 might play a tumour-suppressive role in the context of PCa.Notably, recent research highlighted the dual nature of CHI3L1 in cancer.For instance, in GBM stem-like cells (GSCs) with methylated O6methylguanine-DNA methyltransferase promoter (MGMT-m), CHI3L1 functioned as a tumour suppressor gene, sensitizing GSCs' response to temozolomide (TMZ) by activating DNA damage responses (DDRs).In contrast, in MGMT promoter-unmethylated (MGMT-um) GSCs, it promoted tumorigenesis and contributed to TMZ resistance by inhibiting DDRs.36This indicates that the methylation status of the MGMT promoter might influence CHI3L1's function in human cancers.
results.Therefore, conducting larger independent cohorts from different medical centres can provide more robust and reliable findings regarding the impact of CHI3L1 SNPs on the risk and development of PCa.Moreover, our current study only indicated the impacts of CHI3L1 rs10399805 and rs6691378 SNPs on CHI3L1 gene expression in whole blood tissues among healthy individuals based on the GTEx database.To further validate the influence of CHI3L1 SNPs on CHI3L1 expression in PCa patients, mRNA and DNA should be collected simultaneously from the same samples from PCa patients in future work.In summary, our study identified distinct allelic effects of CHI3L1 SNPs (rs10399805 and rs6691378) within a Taiwanese population, that impact the clinicopathologic development of PCa.Furthermore, we uncovered a prognostic role for CHI3L1 in PCa using clinical samples.Our results suggest that the promoter SNPs, rs10399805 and rs6691378, might influence CHI3L1 gene expression, subsequently modulating PCa progression.These genetic variants could potentially serve as critical markers to predict the aggressiveness and prognosis of PCa.AUTH O R CO NTR I B UTI O N S Yu-Ching Wen: Conceptualization (equal); data curation (equal); funding acquisition (equal); writing -original draft (equal).Chia-Yen Lin: Data curation (equal); resources (equal).Kuo-Hao Ho: Data curation (equal); software (equal).Yung-Wei Lin: Conceptualization (equal); methodology (equal).Chi-Hao Hsiao: Conceptualization (equal).Shian-Shiang Wang: Data curation (equal); resources F I G U R E 1 Impacts of chitinase 3-like 1 (CHI3L1) rs6691378 and rs10399805 polymorphisms on CHI3L1 expression.The Genotype-Tissue Expression (GTEx) Portal (https:// www.gtexp ortal.org/ home/ ) provided validated results regarding CHI3L1 expression based on different genotypes.Violin plots show that CHI3L1 rs10399805 (A) and rs6691378 (B) mutations were associated with lower CHI3L1 expression levels in whole blood.
Distributions of demographical characteristics of 701 patients with prostate cancer.
TA B L E 1 Odds ratios (ORs) and 95% confidence intervals (CIs) of the clinical status and CHI3L1 rs4950928 and rs6691378 genotypic frequencies in 701 patients with prostate cancer.Odds ratios (ORs) and 95% confidence intervals (CIs) of the clinical status and CHI3L1 rs880633 and rs10399805 genotypic frequencies in 701 patients with prostate cancer.
29thin the CHI3L1 gene was identified as being correlated with the risk, progression and OS rate of patients afflicted with hepatocellular carcinoma (HCC).Notably, patients carrying the CC genotype of rs880633 displayed elevated serum CHI3L1 levels, whereas individuals with the TT genotype exhibited the lowest serum CHI3L1 levels.29Furthermore,two CHI3L1 SNPs, namely rs6691378 and TA B L E 3 TA B L E 4