Expression of toll-like receptors in non-endemic nasopharyngeal carcinoma

Toll-like receptors (TLRs) are a family of transmembrane receptors that play an important role in innate immune defence. TLRs recognize the receptor-specific pathogen-associated molecular patterns (PAMPs) of microbes, and respond by activating immune cells against them [1]. TLRs also recognize the endogenous damage-associated molecular patterns (DAMPs) released from injured tissues, and TLR pathways have been shown to maintain tissue homeostasis by regulating wound healing processes and apoptosis [2‐4]. In humans, ten different TLRs (TLR1-10) have been characterized [1]. For TLR1-9, several specific ligands have been identified, whereas the ligand for TLR10 remains elusive [1, 5]. TLR1, TLR2, TLR4, TLR5, and TLR6 were originally characterized as exclusively expressed on the cell surface and TLR3, TLR7, TLR8, and TLR9 as almost exclusively expressed in intracellular compartments such as endosomes [1]. The subcellular localization of TLR10 has not yet been characterized [5]. Nevertheless, recent findings suggest that TLR localization may be altered across cell types and in response to stimulation or disease [5]. Many studies have shown changes in TLR expression with oncogenic transformation [6]. However, the actual function of cancer-associated TLR modulation remains controversial. TLR stimulation may have anti- or pro-tumoral effects depending on the TLR receptor and cancer type [6]. Over the past decade, interest in the role of TLRs in tumorigenesis has increased, and numerous preclinical and clinical trials are ongoing to develop TLR agonists for cancer therapy [7].

Nasopharyngeal carcinoma (NPC) is a highly invasive malignant tumour arising from the mucosal epithelium of the nasopharynx. NPC has marked geographical disparities in incidence, with the highest occurrence in Southeast Asia and lowest in Europe and North America [8]. NPC is subdivided into three major histological types: keratinizing squamous cell carcinoma (KSCC), non-keratinizing carcinoma, and basaloid squamous cell carcinoma [9]. Non-keratinizing carcinoma can be further subdivided into undifferentiated and differentiated types [9]. The etiology of NPC is poorly understood, but epidemiological studies indicate that both genetic and environmental factors contribute to its development [10]. In high-incidence endemic areas, more than 95% of NPC tumours show non-keratinizing histology with Epstein-Barr virus (EBV) association, while in low-incidence non-endemic areas, the histology and viral status are more diverse. In the latter areas, KSCCs and human papillomavirus (HPV)-positive tumours are additionally found [11‐13]. Although it is commonly accepted that EBV and HPV can contribute to carcinogenesis, these viruses alone seem insufficient for malignant transformation [14‐16]. Recent studies indicate that the host’s immunological responses to viruses and precancerous lesions have an important role in cancer development, and that tumour progression could partly be due to a failure in the innate immune response [16]. In HPV-positive oropharyngeal squamous cell carcinoma (OPSCC), strong expression of TLR5 and low expression of TLR7 has been associated with poor disease-specific survival [17]. Furthermore, the intensity of TLR4 expression has been significantly lower in laryngeal papillomas transforming into laryngeal squamous cell carcinoma than in papillomas without malignant transformation [18]. In NPC, the importance of TLRs in tumour immunity has been demonstrated in studies from endemic areas, where certain sequence variants in TLR genes were associated with increased NPC risk [19‐22]. However, data on TLR expression in NPC is scarce and limited to endemic cases.

The purpose of this whole population-based study on NPC was to characterize the expression of six TLRs of interest for malignant transformation and immune activation. We studied the expression of TLR1, TLR2, TLR4, TLR5, TLR7, and TLR9 in Finnish patients and related the findings to histopathological subtypes, viral status, and survival.

TLR signalling has been associated with tumour development [25]. The expression pattern and the function of TLRs in tumour progression seem to be cell-type specific and relate to different conditions, such as infections [5]. In our whole population-based study of NPC in Finland, we examined the expression of TLR1, TLR2, TLR4, TLR5, TLR7, and TLR9 in 150 tumours and analysed their pattern of expression according to EBV and HPV status, and clinical outcome. To our knowledge, this is the first study on TLRs in non-endemic NPC published to date. A few studies, conducted in high-incidence areas, have reported that genetic polymorphisms of TLR3, TLR4, TLR9, and TLR10 are associated with a risk of developing NPC in endemic populations [19‐22], but outcome results related to TLR expression are lacking worldwide.

The present study demonstrated that the expression patterns of TLR2 and TLR5 were related to the viral status while both TLRs were expressed significantly less in EBV-positive than in HPV-positive or EBV/HPV-negative NPC. These expressions were also related to well-established prognostic factors such as age and histology [23], which tend to dilute the independent prognostic significance of TLR2 and TLR5 in multivariable Cox regression analysis. However, in Kaplan-Meier analysis, the patients with strong TLR5 expression had worse survival compared to those with mild or negative expression (Fig. 3). This is in line with clinical studies on OPSCC and oral tongue squamous cell carcinoma (OTSCC), which report the association of poor DSS with strong expression of TLR5 [17, 26]. The exact effects of TLR-mediation on tumour growth are not known, but several in vitro studies on other types of carcinomas have shown that activation of TLR5 can promote tumorigenesis. For example, such activation has enhanced the proliferation of gastric cancer cells, and the migration and invasion of salivary gland adenocarcinoma [27, 28].

In contrast to TLR5, the patients with positive TLR7 expression had better DSS and OS than the patients with no TLR7 expression, and TLR7 was found to be a significant prognostic factor in multivariable Cox regression analysis. The finding that the patients with mild TLR7 expression had slightly better 5-year survival than the patients with strong TLR7 expression was not expected, but a similar result has been reported in patients with oral squamous cell carcinoma [29]. However, that report did not mention the survival rates of the patients with TR7-negative tumours. Nevertheless, it described variable TLR7 expression patterns when comparing between normal, dysplastic, and carcinoma tissues [29]. In our study, TLR7 expression in NPC samples was prominent on nuclear membranes, while in benign tissues TLR7 was expressed in the cytoplasm and in the nuclei. Another example of a defensive marker with derangement of function, in both over- and underexpression, is matrix metalloproteinase-8 [30].

Our findings suggest that viruses, or tumours related to specific viruses, or simply malignant neoplasms evoke distinct immunological reactions, but the hosts’ responses vary because of yet unknown causes. This might partly explain the differences in the effectiveness of oncological treatments. However, according to the hypothesis proposed by Wee et al. [31], causation may also be the reverse. They suggested that genetic TLR polymorphisms, especially in X-chromosome-linked TLR8, affect the innate immune response and make certain populations and individuals more vulnerable to infection-related cancers [31]. In NPC, altered TLR function could allow the virus to enter the nasopharyngeal mucosa and cause persistent infection finally resulting in a carcinogenic process [31]. We did not evaluate TLR8 expression in our tumour cells, but we found the usual but unexplained male preponderance in EBV- and HPV-positive patients (74 and 76% were males, respectively). In line with this, there were significantly more women in the virus-negative group (56%) compared to the EBV-positive (p = 0.001) and the HPV-positive (p = 0.020) groups. This suggests that men are more susceptible to virus-related NPC than women even in a non-endemic low-incidence population.

We studied TLR expression by immunohistochemistry to determine the expression sites of TLRs in carcinoma cells. As stated in a recent review article by Hamonic et al. [5], comprehensive understanding of the changing localizations of TLRs could aid us in understanding the basis of cancer immunology and possibly in developing new treatment modalities. We found that both in the benign adenoid control tissues and in the NPC samples, TLR1, TLR2, TLR4, and TLR5 were expressed diffusely in the cytoplasm instead of the cell surface, where they have been reported to localize when activated by pathogen-associated molecular patterns [32]. Interestingly, TLR4 expression was detected in the nuclei of benign controls but not in the nuclei of NPC cells. In benign samples, TLR5 was expressed exclusively in the basal layer of the nasopharyngeal epithelium as reported also for the normal oral mucosa [33], while in NPC the expression was diffuse. Similar to NPC, Pimentel-Nunes et al. have reported changing TLR localizations in gastric carcinogenesis [34]. They found that the normal gastric mucosa expressed TLR2, TLR4, and TLR5 in a polarized manner in the apical and particularly the basolateral membrane. By contrast, in metaplasia, dysplasia, and adenocarcinoma, these TLRs were expressed throughout the cytoplasm with no apparent polarization [34]. Also in previous studies on OPSCC and OTSCC, TLR5 has been localized in the cytoplasm rather than the membranes of the neoplastic cells [26, 35]. These findings suggest that activation of TLRs in abnormal locations may be related to carcinogenetic processes. We can only speculate whether TLRs have different functions in benign and malignant tissues, or if they become activated in different cellular compartments by PAMPs or DAMPs. Further research is needed to describe the mechanisms and causal connections between these phenomena.

In the present study, the retrospective nature of patient data carries limitations regarding the history of smoking habits and alcohol consumption. This limited our possibilities to evaluate the impact of these known carcinogens on the differences in expression of the studied TLRs. However, the uniform nationwide health care system in Finland enabled us to collect complete treatment and follow-up data, and a high proportion of diagnostic histopathological samples for TMA. In fact, histopathology was available for 150 (72%) of a total of 207 patients. While the size of the Finnish population limited the number of cases, we can nevertheless attest that the present cases represent a truly whole population-based material for the NPC patients treated during this 20-year period [36].

Our study is the first to evaluate TLR expressions in NPC of a non-endemic area. We found that TLRs were highly expressed in NPC, and intensity of TLR2 and TLR5 expressions were related to viral status. Expressions of TLR2 and TLR5 were strong in virus-negative cases, which will logically lead us to further studies on other possible factors causing NPC. In addition, TLR7 seems to be an independent prognostic factor of non-endemic NPC.