Background
Renal cell carcinoma (RCC) is a cancer of increasing incidence and mortality [
1]. At the time of the diagnosis, up to one third of the patients have metastasized disease and a half of the remaining patients will experience a recurrence after an initially curative treatment [
2]. Despite the many well-known prognostic factors for the disease, the behaviour of RCC is very difficult to predict.
Toll-like receptors (TLRs) are pattern recognition receptors that detect both microbe- and host-derived molecular patterns. Thus far, at least 13 mammalian TLRs have been recognized, each of them responding to a different ligand. The subcellular expression sites of the various TLRs also vary. TLRs 1, 2 and 4 are expressed and bind their ligands on the cell surface while the TLR9 subfamily (including TLRs 3, 7, 9 and 13) reside in intracellular vesicles. Ligand binding to TLRs activates transcription factors, such as NF-kappaB and the eventual outcome of TLR activation is an immune reaction, characterized by increased production of inflammatory mediators. Specifically, TLR9 is a receptor for both microbial and vertebrate DNA. The intracellular expression of TLR9 and also possibly the other endosomal TLRs is thought to evade self-recognition of DNA and RNA [
3‐
7].
It is now well established that TLR9 is also expressed in various cancer cells, including breast, brain, ovarian, gastric, lung and prostate cancer cells [
8‐
11]. Furthermore, in clinical breast, ovarian and prostate cancer specimens, increased TLR9 expression was associated with decreased tumour differentiation [
10‐
13]. It has also been demonstrated that stimulation of TLR9-expressing cancer cells with synthetic TLR9-ligands increases their in vitro invasion which is associated with the down-regulation of tissue inhibitor of metalloproteinases-3 (TIMP3) and the up-regulation of matrix metalloproteinase-13 (MMP-13) activity. Although bacterial DNA, similar to the synthetic CpG-sequence containing TLR9-ligands, also induces invasion in TLR9 expressing cancer cells in vitro, the natural TLR9-ligand that might induce invasion for example in breast cancers, remains unknown [
10,
11].
In the normal kidney, TLR9 expression has been detected in the renal tubules and interstitial tissue, while the tubulointerstitial and glomerular expression has been detected in lupus nephritis [
14]. Previously, TLR9 has been associated with renal disease, such as glomerulonephritis [
15] and lupus nephritis [
16]. To our knowledge, there are no previous studies of TLR9 expression in RCC. However, the efficacy of a synthetic TLR9-agonist has been studied in a clinical trial in advanced metastatic RCC. This compound was found to have only modest antitumour activity [
17].
The aim of this study was to investigate TLR9 expression in RCCs and to evaluate the prognostic significance of TLR9 immunostaining in RCCs.
Discussion
We demonstrate here for the first time that TLR9 is frequently expressed in RCCs. Although there was no association between the immunoexpression of TLR9 and histological subtype, stage or grade of RCC, cytoplasmic TLR9 expression was a statistically significant prognostic factor in RCC specific survival in both univariate and multivariate analyses and TLR9 expression was an independent marker of better prognosis in RCC. Our findings thus suggest that the lack of TLR9 confers aggressive behaviour of renal carcinoma cells. The significance of nuclear TLR9 expression remains obscure, but it may also represent unspecific staining.
Expression of TLR9 has been previously detected in various cancer cell lines and in various clinical cancer specimens. Synthetic TLR9-ligands induce cancer cell invasion
in vitro and high TLR9 expression has been associated with poor differentiation of various cancers, suggesting that high TLR9 expression or naturally existing DNA-ligands might induce TLR9-mediated invasion, and thus contribute to worse outcomes in cancers with higher TLR9 expression. In this light, our finding demonstrating the lack of TLR9 expression as a poor prognosis marker is RCC is surprising. So far, the association between TLR9 and clinopathological parameters and the survival of cancer patient has been evaluated in only a few studies. In breast cancer it has been demonstrated that immunoexpression of TLR9 is significantly increased in high-grade tumours compared with lower-grade tumours [
12,
22]. Similarly, it has been shown that recurrent breast carcinomas exhibit a significant increase in the mRNA levels of TLR9 in cancer cells [
23]. However, a remarkable percentage (57.5%) of recurrent breast tumours was shown to express TLR9 by fibroblast-like cells and these tumours have reported to have low probability of metastasis [
23]. It has also been demonstrated that cell surface stimulation of TLR9 promotes cell proliferation and survival in hepatocellular carcinoma [
24]. In neuroblastoma, TLR9 expression has been found to correlate inversely with disease stage [
25] whereas in glioma, TLR9 expression has shown to be significantly higher in high grade tumours compared to low-grade gliomas and TLR9 immunoexpression has been reported to be a statistically significant marker of poorer prognosis in glioma [
26]. Thus, the contribution of either high or low TLR9 expression to the pathophysiology of cancer may be highly tumour specific.
Upon the recognition of DNA, TLR9 recruits specific intracellular adaptor proteins to initiate signalling pathways and the eventual outcome is an immune reaction characterized by the increased production of inflammatory mediators like interferon and other inflammatory cytokines [
3,
27]. RCC is generally renowned of its immunogenic nature. RCC can allure different effector cells of both the innate and adaptive immune system including natural killer (NK) cells, dendritic cells (DC) and various T cells [
28]. A variety of tumour-associated antigens (TAAs) which can evoke tumour-specific T-cell-defined immune responses in cancer patients has been detected in RCC tumours [
29]. More importantly, immunotherapy with interferon alpha (IFN-α) or interleukin 2 (IL-2) can produce even complete and durable response in advanced RCC [
30] and tumour vaccines have shown to have some response, too [
31]. Rare cases of spontaneous regression of metastases in RCC caused probably by immunologic mechanism have been reported [
32]. Thus, the prognostic significance of TLR9 expression in RCC may be associated with immune responses to the tumour cells. Hypothetically, in the absence of RCC TLR9 expression, such responses are not evoked and they are less susceptible to immunosurveillance and they can progress. These issues warrant further investigation.
Low oxygen environments can be created by various pathophysiological conditions, including infection, inflammation, tissue injury, and solid tumours [
33]. Hypoxia is one of the significant features of solid tumours, including kidney tumours. Hypoxia and the compensatory hyperactivation of angiogenesis are thought to be particularly important in RCC [
34]. In hypoxia, an increased expression of various TLRs including TLR9 has been demonstrated [
35,
36] and this induction of TLRs has shown to be coordinated by the hypoxia inducible factor 1 (HIF-1) [
35]. Whether or not the absence of TLR9 in RCC is regulated by hypoxia and HIF-1 and thereby, increase the aggressive behaviour of the tumour cells also warrant further investigation.
Acknowledgements
The authors wish to thank Ms Mirja Vahera, Ms Erja Tomperi, Ms Mirja Mäkeläinen for their skilful technical assistance, and Pasi Ohtonen, M. Sc. for his invaluable assistance with statistical analyses. This study was funded by grants from the Finnish Cancer Foundation (HR), the Finnish Urological Association (HR) and Päivikki and Sakari Sohlberg Foundation (TKP, MHV).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HR performed statistical analyses and drafted the manucript. PH evaluated the immunohistochemical staining. SK revised the manuscript. KSV carried out immunohistochemical studies. TKP conceived of the study. KSS revised the manuscript. MHV participated in the design of the study, evaluated the immunohistochemical staining and revised the manuscript. All authors read and approved the final manuscript.