Prostate cancer (PCa) is one of the most common malignant tumors of the male urinary system, and its incidence and mortality rates have increased considerably in recent years [1]. The etiology and pathogenesis of PCa remain unclear, although several factors such as age, family history, ethnicity, and dietary and genetic mutations are known to be related to PCa risk [2]. Classifications based on clinicopathological features, such as prostate cancer-specific antigen, Gleason score, and tumor, node and metastasis (TNM) system, are considered as the accepted practice standards for determining tumor stage for PCa [3]. Unlike PCa, benign prostatic hyperplasia (BPH) is characterized by stromal and epithelial cell proliferation in the prostate in elderly males. Prostate inflammation is responsible for the development of BPH [4] and linked to the pathogenesis with PCa pathogenesis [5]. The inflammatory response in these patients is characterized by the accumulation of immune cells, mainly T and B cells, and macrophages into the prostate tissue, and their activation results in the release of inflammatory cytokines [4, 5, 6]. Prostate-specific antigen (PSA), a glucoproteinase produced by both normal and malignant cells of the prostate gland, is a useful serum biomarker for diagnosing and controlling PCa [2]. Increased PSA levels can indicate reliable sign of prostate cancer aggressivity [7]. In addition, the inflammatory cytokine interleukin 6 (IL-6) secreted by immune cells is known to promote the release of PSA in PCa patients [6].

Prostate cancer patients have elevated numbers of peripheral circulation regulatory (Treg) T [8] and myeloid-derived cells, which are linked to poor outcome in PCa [9]. Among myeloid-related markers, CD13 and C-kit receptor (CD117) play key roles in normal hematopoiesis and are detectable in neoplastic human tissues and at sites of prostate [10, 11, 12]. The CD13/Aminopeptidase N membrane metallopeptidase participates in negatively regulating several signalling molecules, such as the toll-like receptor (TLR) 4 and Janus kinase/signal transducers and activators of transcription (JAK/STAT) [13].

Moreover, immunological investigations have indicated that abnormal expression of TLR4 and JAK/STAT pathways is linked to tumor development, cell migration, immune invasion, and progression of various human diseases, including PCa and BPH [14, 15]. TLR4 activation is triggered by bacterial lipopolysaccharide to elicit an inflammatory response in immune cells [16], and its levels are upregulated in PCa [14]. TLR4 deficient mice fail to respond to viral and bacterial infection [17]. Several investigations have indicated that polymorphisms within the TLR4 gene are associated with PCa [14, 16]. The TLR4 c.1063 A$>$G single nucleotide polymorphism (SNP) was shown to attenuate TLR4 activation and inflammatory responses [18]. Similarly, JAK2 has recently been considered a regulator of the immune response in the pathogenesis of PCa and BPH [15]. The rs10429491 SNP in JAK2 has been reported to be associated with PCa risk [19].

The association between CYLD variants and the risk of PCa and BPH is not well known, although its expression levels regulate PCa progression [20]. CYLD is a deubiquitinating enzyme that functions as a negative regulator of immune reactions and tumor cell proliferation in PCa [21]. CYLD is known to inhibit the survival, glucose uptake, and growth of prostate tumors [22]. Mutations in CYLD lead to cyclindroma disease and myeloma [23].

In this study, we determined the contribution of CYLD, JAK2, and TLR4 gene variants to PCa and BPH risk and their associations with PSA levels, immunophenotype, and clinicopathological features in Vietnamese men.

Our study provides evidence for the association of CYLD sequence variants (c.2351-47, c.2351-46, and rs1971432171) with PCa risk in the Vietnamese study population. In this study, PCa-sensitive SNPs were identified for the first time. Mutations in the CYLD gene are known to be linked to abnormal cellular function in mice [26] and humans [23, 25] and the development of cyclindroma disease and myeloma [23]. Downregulated expression of CYLD is also associated with PCa progression [20]. In contrast, a rs12324931 SNP in CYLD is associated with the risk of inflammatory bowel disease [27]. In our recent study, p.W736G in the CYLD gene was indicated as the pathogenic variant in patients with polycythemia vera [28]; however, it was not found in all cases in this study as well as in patients with leukemia, including 352 patients with myeloid leukemia, and 145 patients with lymphocytic leukemia (unpublished data).

Moreover, PCa cases carrying the CT genotype of CYLD c.2351-47 had an expanded number of activated NK cells and an increased risk of PSA levels of more than 100 ng/mL (Fig. 6 and Table 4). High pretreatment serum PSA levels are associated with worse PCa outcome [29]. CYLD functions as a negative regulator of the immune response, including the activation of NK cells [26], and its expression is inhibited by high glucose [30]. Moreover, CYLD inhibits cancer cell proliferation, glucose uptake, and growth of prostate tumor cells [22]. A recent study indicated that the release of cytokines and chemokines by activated NK cells induces the recruitment of accessory immune cells such as T cells [31]. In addition, high PSA levels are linked to inflammatory responses [6]. Evidence suggests that activation of NK cells in CYLD-sensitive PCa patients is associated with the release of serum PSA.

Functional studies of the CYLD gene revealed that it is a negative regulator of proinflammatory gene expression through TLR4 signalling [32]. Unlike CYLD, carriers of TLR4 rs2149356 were associated with significantly elevated risks of both PCa and BPH compared with healthy controls. In contrast, the c.331-206 SNP in this gene was associated with a reduced risk of prostatic cancer. Changes in TLR4 expression are linked to prostate cancer risk and tumorigenesis by inducing inflammation [5]. Consistently, the rs2149356 SNP was found in Caucasian and South Asian patients with prostate cancer [33]. In contrast, the TLR4 rs11536889 SNP in the 3’ UTR is associated with the risk of prostate cancer in Korea [16] and Sweden [34], but not in other populations [35].

The effects of JAK2 expression on prostate cancer cell function have been revealed in a recent study [36]. Unlike this study, a known rs10429491 SNP in JAK2 is associated with PCa risk [19]. Importantly, carriers of the CC genotype of JAK2 rs4495487 were associated with higher levels of PSA in PCa patients, but not in BPH patients (Table 4).

Growing evidence indicates that chronic inflammation, which results from the infiltration of immune cells into prostate tissue and blood circulation, increases the risk of high-grade PCa development [8, 37]. Similarly, PCa patients have an elevated number of circulating and tumor-infiltrating Tregs [8]. Recently, NK cells isolated from the peripheral blood of patients with PCa displayed increased expression of the surface antigens CD56, CD9, and CD49a [38]. In contrast, we observed that the number of CD56${}^{+}$CD25${}^{+}$expressing cells was higher in patients with PCa than in patients with BPH and healthy controls. Loss of CYLD expression leads to NK cell activation [26]. Moreover, activated NK cells were expanded in PCa patients carrying the CT genotype of the CYLD c.2351-47 variant compared to those with the wild-type genotype (Fig. 6). Consistently, there was no significant difference in the percentage of NK cells and their activation between BPH patients and healthy controls [39]. CD25${}^{+}$NK cells display functional and metabolic activities under regulatory T cell-mediated suppression in cancer patients [40].

Lastly, CD13${}^{+}$CD117${}^{-}$ cells were recruited into the circulation in patients with PCa but not in patients with BPH and healthy controls. CD13 and CD117 are expressed in human prostate and neoplastic tissues [10, 11, 12]. CD13 is also highly expressed in myeloid cells [13], and its activation is mediated through the TLR4 signalling pathway to elicit an inflammatory response [13]. CD13 expression is elevated in several JAK2-positive blood cancers, such as polycythemia vera [28, 41]. Unlike activated NK cells, the number of CD13${}^{+}$CD117${}^{-}$ cells was unaltered in patients with CYLD variants.

Prostate cancer carriers of the CYLD c.2351-47 variant were associated with the recruitment of activated NK cells into the blood circulation and had serum PSA more than 100 ng/mL. Therefore, the CYLD c.2351-47 variant may be a significant risk factor for prostatitis in PCa patients.

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

LVD, PTH, CVM, and NTX conceived and designed the study. LVD, PTH, NTN, DTT, TTPT and NTMC conducted the experiments; LVD, PTH, NHH, NTT, CVM, and NTX analyzed the data. CVM wrote section ‘1’; NTN wrote section ‘2’; and NTX wrote sections (‘3’-‘5’). All the authors contributed to the revision, read and approved the final version of the manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All patients and volunteers provided written informed consent to participate in this study. Person care and experimental procedures were performed according to the Vietnamese law for the welfare of humans and were approved by the Research Ethics Committee, Military Hospital 103, no. 86/CNChT-HDDD.

We thank all patients for their participation.

This research is funded by the Vietnam Academy of Science and Technology (VAST) under grant number NCXS.01.03/23-25.

The authors declare no conflict of interest.