Original article
Human TLR gene family members are differentially expressed in patients with urothelial carcinoma of the bladder,☆☆

https://doi.org/10.1016/j.urolonc.2017.07.029Get rights and content

Highlights

  • TLR family members were differentially expressed in tumor tissues.

  • There was no correlation between their expressions in urine and tissue samples.

  • The levels of proinflammatory cytokines were elevated in urine samples of UC patients.

Abstract

Purpose

Toll-like receptors (TLRs) have an important role in the activation of both innate and adaptive immunity in response to pathogens and endogenous danger signals from damaged or dying cells. The aim of this study was to determine the relationship between urothelial carcinoma (UC) and TLR expression.

Basic procedures

Real-time polymerase chain reaction evaluation was made of the messenger RNA expression of TLRs 1–10 in 24 UC samples and 46 nontumoral bladder tissue samples. The levels of proinflammatory cytokines (IL-1β, IL-6, and IL-8) in the urine samples were also determined with enzyme-linked immunosorbent assay.

Main findings

TLR2–7 and TLR10 expressions were significantly higher in UC than in the control group (P<0.05 for all comparisons). No concordance was found between matched tumor tissue and urine samples in terms of TLR expression. IL-1β, IL-6, and IL-8 levels were significantly higher in urine specimens of patients with UC (P = 0.033, P = 0.001, and P = 0.008, respectively).

Principal conclusions

The results of this study demonstrated that the TLR gene expression profiles reflect the heterogeneity within UC. These results might also prompt further investigation to better understand the role of the TLR gene family expression in the tumor progression of UC.

Introduction

Urothelial carcinoma (UC) comprise 95% of malignant tumors of the bladder and is the second cause of death among urogenital tumors. According to the Turkish Ministry of Health, UC is the third most common cancer type in males but is not in top 10 of cancer types in female patients [1].

Certain risk factors such as smoking and occupational factors are responsible for the development of UCs although the etiology of the disease is still unclear. Abnormal molecular events such as mutations in certain tumor supressor genes in normal urothelial cells are involved in the conventional carcinogenesis model of UC [2], [3].

UCs are classified morphologically and clinically into noninvasive tumors and muscle-invasive tumors. Studies have shown that there are 2 different molecular pathways involved in the subtypes of UC. The most important genomic alterations in low-grade noninvasive UCs have been found in the genes involved in FGFR3, HRAS, and mTOR pathways [4], [5]. In contrast, the genetically altered genes in high-grade noninvasive tumors and invasive tumors are specific tumor supressor genes such as TP53 and RBL1 [2], [4]. The tumor microenvironment also plays a critical role in multiple stages of tumor progression, especially immune-escaping, distant metastasis, and chemotherapy resistance. There is increasing evidence that there are alterations in the expression profiles of Toll-like receptors (TLRs) which recognize danger-associated molecular patterns derived from cancer cells in the tumor microenvironment [6], [7]. Taken together, these studies demonstrate that it may be important to investigate TLR expression in normal urothelium and in their malignant counterparts in tumor tissue.

TLRs are type I transmembrane receptors composed of the extracellular leukine-rich repeat domain and the cytoplasmic Toll/IL-1 receptor domain. Different TLRs recognize various pathogen-associated molecular patterns such as lipopolysaccharide, single-stranded RNA, DNA contained CpG motif, and danger-associated molecular patterns. TLR3, 7, 8, and 9 are localized in the endosomal compartment, whereas the remainder are localized on the plasma membrane of the cell. TLRs are also expressed in cancer cells and may stimulate important molecules such as the antiapoptotic proteins that are involved in cancer cell proliferation [6], [7], [8], [9]. In addition, the ligands derived from cancer cells may increase TLRs expression, and then the cells start to release certain chemokines and cytokines as important components of the tumor microenvironment. Immune cells activated by these cytokines can release other cytokines that cause immune tolerance, limiting antitumor immunity [6], [7], [10]. Therefore, it has been suggested that alterations to the expression pattern of TLR could be a novel potential therapeutic target in cancer.

TLRs have been extensively studied in different cell types and tissues but very limited information is available on urothelial tissue. The aim of this study was to examine TLR expression in nonmalignant urothelium and UC. It was also aimed to determine whether urine-based TLR expression analysis might reflect the TLR expression status in corresponding tumor tissues.

Section snippets

Urothelial tissue and urine samples

The study included 24 consecutive patients who underwent transurethral resection (TUR) of bladder tumors at our instution between 2012 and 2014. All primary tumor samples from the patients were histologically verified as UC and were classified according to WHO and WHO/ISUP [11]. Patients with hematuria who underwent TUR were also studied and a group of 46 patients with no cellular events of inflammatory reaction and no tumor cells during histopathological evaluation of the urothelium were

Patient and tumor characteristics

The mean age of the UC group (1 female and 23 males) and the noncancerous group (34 females and 12 males) was 64.91 ± 12.36 years (range: 45–90 years) and 52.21 ± 17.35 years (range: 24–81 years), respectively. Of the 24 UC patients, 13 (54.2%) were smokers and there was only 1 patient with a positive family history of UC.

The tumor characteristics are summarized in Table 1. None of the patients were found to have lymph node involvement. In the pathological examination of the pT1 tumors,

Discussion

TLRs are expressed in a variety of immune cells, including macrophages, natural killer cells, B cells, and dendritic cells. Recent studies have reported that TLRs are also expressed in both normal epithelial cells and tumor cells [6], [12], [13]. TLR family members exhibit distinct patterns of distribution in tissues [14], [15]. At present, only limited information is available related to the expression of TLRs in urothelial tissue. One clear result of all these studies are that TLR4 and TLR5

Conclusions

In summary, the TLR gene expression profiles reflect the heterogeneity within UC and the expression patterns of specific TLRs might be involved in the pathogenesis of UC. However, our study shows that the TLR family members cannot be used as biomarkers in UC. Further studies are needed to determine reliable biomarkers which could be used for the management of UC in routine clinical practice.

Acknowledgments

The authors want to acknowledge the support of Caroline Jane Walker for language editing. We also thank M. Rasid Bakir, MSc, for statistical analysis.

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    This work was supported by Pamukkale University Scientific Research Unit (Grant no. 2012SBE015).

    ☆☆

    This work was partly presented during the V. International Congress of Molecular Medicine, 20–22 May 2015, Izmir, Turkey.

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