Oral squamous cell carcinoma (OSCC) is the sixth most common type of cancer and has a restricted prognosis. The etiology of OSCC cancer is multifactorial but notably related to tobacco and alcohol chronic consumption. Infection and chronic inflammation may have an impact in the development or, more probably, on OSCC progression and aggressiveness. And this interaction may be triggered by Toll-Like receptors (TLR) through the recognition of a variety of toxic irritants and pathogens that are commonly and chronically in contact with the oral cavity. This paper carried out a critical revision on the possible relationship of OSCC carcinogenesis and TLR. Currently, there is evidence that TLR2, TLR3, TLR4 and TLR9 have an impaired expression in OSCC tumor cells that can be related to tumor growth, aggressiveness, invasion and apoptosis resistance. Moreover, that the disturbed TLR function may also be related to others molecules that act together with these receptors, such as c-IPA2 and CD-14, correlated respectively to TLR3 and TLR4 function. Besides, tumor cells are able to modulate the expression and function of TLR proteins in different kinds of immune cells, such as natural killer lymphocytes, myeloid dendritic cells and regulatory T-lypmhocyte, resulting in impaired immune anti-tumor defense. Finally, all the observed changes might be induced through inherited SNP in TLR genes.
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Keywords: toll-like receptors, chronic inflammation, oral cancer
Oral squamous cell carcinoma (OSCC) is the sixth most common type of cancer and has a restricted prognosis for over 50% of cases diagnosed annually. The etiology of OSCC cancer is multifactorial, and the most important risk factors are the tobacco and alcohol chronic consumption (1, 2). Other factors such as human papillomavirus (HPV) infection, diet and nutrition, immunosuppression, mate drinking, socio-economic status may also be involved (3).
Infection and inflammation may have an impact in the development of OSCC (4). This relationship was originally described Virchow in the 19th century. However, current genetic evidence revealed that inflammatory cells and cytokines are more likely to contribute to tumor growth and progression than act as a direct tumor initiator or an effective host anti-tumor response (5).
Actually, many cancers arise from sites of chronic inflammation related to infectious agents, e.g. gastric cancer and Helicobacter pylori, cervical cancer and HPV, hepatocarcinoma and hepatitis C (4, 6). The oral cavity and the upper aero-digestive tract are chronically exposed to a variety of toxic irritants and pathogens. Regarding the infectious agents, for example, the DNA of HPV16 can be detected in up to 72% of oropharyngeal cancers (4, 6). And chronic opportunistic candidal infection may be associated with oral cancer pathogenesis (7) +ref.Marilu2012.
The chronic inï¬‚ammation related to cancer is more commonly associated with microbial infection as described above, but also can be a result of chronic inflammation of autoimmune diseases (8), such as oral lichenoid lesion, one of the most common oral premalignant diseases (9-12).
Altogether the toxic irritants from alcohol and tobacco as well as the infectious agents are recognized by Toll-Like receptors (TLR), cell surface receptors of the innate immune system, which recently are thought to be involved with tumor development and progression (13, 14). Additionally, patients with oral premalignant lesions like oral lichenoid lesions present altered immune response as a consequence of TLR-pathway disruption (15).
The transcriptional activation of NF-kB signaling, a TLR pathway, has been identiï¬ed as a major cell signaling involved with inï¬‚ammation and tumor aggressiveness in a number of human cancers including OSCC, since is related to programmed cell death, either as inductor or inhibitor of apoptosis or necrosis. Imbalances in TLR function might be responsible for the disrupted activation of this pathway in OSCC (16).
Moreover, TLR can be modulated by DNA metabolic stress and the consequences of this interaction fall within control of the p53 master regulator, a tumor suppressor protein, which dysfunction has been already correlated with cancer development. Thus, the impairment in both TLR and p53 has many implications for health and disease, especially with malignant diseases (17).
Recent evidence shows that functional TLR are also implicated in a wide variety of tumors, including colon, breast, prostate, lung, gastric, esophageal and nasopharyngeal cancer (18, 19).
This paper carried out a significant and critical review on the possible relationship of OSCC carcinogenesis and immune response, mediated through TLR. To the best of the author's knowledge, all relevant articles published before June 2012 available in the PubMed database are included in this review.
Toll-like receptors and cancer: from tumor etiology and progression to its treatment
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Twenty-seven years ago, the first Toll-genes were discovered in Drosophila melanogaster and associated to dorsal-ventral embryonic polarity. Further studies showed that mutations of proteins involved in Toll-gene signaling pathway decrease survival of that fly after fungal infection, revealing that the Toll-gene has an essential role in the innate immune response. In 1991 it was observed that human interleukin-1 receptor had homology to drosophila Toll-gene, especially regarding the cytoplasmic portion; and the first reported human Toll-like receptor (TLR) was finally described by Nomura and colleagues in 1994 (14).
Nowadays 10 TLRs have been identified in humans. TLR family is one of the largest and best-studied families of pattern recognition receptors (PRR), which recognize, selectively, a large number of varied pathogen-associated molecular patterns (PAMPs) derived from a broad type of microorganisms, triggering the innate immune response (14, 20, 21).
TLRs are also expressed by tumor cells (6, 18, 19). And it was firstly perceived in the 1890s by William Coley, who observed the destruction of tumor cells after injection of streptococcal bacteria. Subsequently, he performed a clinical trial to demonstrate this relationship with inconsistent and dangerous results, which led him to ostracism. In 1998, just four years after the description of the first human TLR, Bruce Buetler and colleagues showed that lipopolysaccharide (LPS), a streptococcal bacteria toxin, activates the immune system through TLR and this interaction can kill cancer cells. Nowadays, Coley is recognized as a pioneer in cancer immunotherapy and the TLR are hot targets for drug development (22).
Since TLR mediate immune response activation (14, 23), the hypothesis of possible antitumor immunity through TLR activation was proposed. Therefore, disrupted TLR function allows tumor cells to avoid recognition by the immune system and hence not be eliminated. However, some cancers also appear to be sustained by chronic inflammation and this kind of stimulation can be also favorable for tumor progression, this duality of function resembles a "double-edge sword" (19, 20, 24).
The relationship between chronic inï¬‚ammation, mediated through TLR, and risk of tumor development and progression is a vast field of investigation with interesting and promising results, even for the comprehension of already identified molecules compromised in cancer. A normal component of chromatin overexpressed in a variety of tumors, high-mobility group protein B1 (HMGB1), interacts with TLR producing chemokines and cytokines that stimulate tumor progression and metastasis. Other endogenous mediators related to TLR stimulation include S100 proteins in melanoma and heat shock proteins (HSP) may promote metastatic tumor progression (23).
The pharmaceutical industry spent millions of dollars developing new substances for cancer treatment through TLR activation, some in phase III clinical trials for skin and lung cancers. Usually, for these types of cancer, targeting TLR, especially TLR7 and TLR9, activates Th1-immune response characterized by an influx of cytotoxic T cells, interferon and other cytokines that can destroy cancer cells and inhibit tumor growth (19, 22, 24)
For OSCC some laboratory investigations were performed in attempt to discover a treatment modality through TLR modulation. Chemopreventive and anti-inflammatory agents have already earned a reputation in cancer treatment and research. MTT proliferation assay revealed that inhibitory substances such as acetylsalicylic acid, celecoxib, dexamethasone (anti-inflammatory drugs), curcumin and EPs7630 (chemopreventive agents) reduced the OSCC proliferation through the reduction of TLR3 expression and, consequent, NF-kB inhibition (16).
Another MTT assay regarding the effect of imiquimod, a TLR7 agonist, on human OSCC cell lines proliferation showed reduced tumor cell proliferation in a dose-dependent manner, suggesting that this TLR7 agonist activates caspase-dependent mitochondrial pathway of apoptosis (25). On the other hand, the interaction TLR7-imiquimod which induces apoptosis in skin squamous cell carcinoma as well, is markedly worse for normal keratinocytes; thus this substance cannot be feasible for OSCC treatment (26).
An alternative substance successfully used as an immunotherapeutic agent against many types of malignancies, the OK-432 - a penicillin-killed and lyophilized preparation of a low-virulence strain of Streptococcus pyogenes - enhanced host immunity, through TLR4 interaction, promoting growth inhibition and apoptosis of head and neck cancer cells (27).
Whatsoever, due to the duality of TLR activation, knowing the TLR role in a specific kind of cancer is crucial to modulate its response promoting only anti-tumor activity for OSCC treatment. This role, based in the literature review, was scrutinized in the next sections.
Toll-like receptor genetic expression in tumor cells of OSCC
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Several studies identified TLR expression in tumor cells and different types of inflammatory cells both derived from OSCC patients, this data is summarized in Table 1.
TLR2 was found higher expressed on keratinocytes from dysplastic epithelium of white-plaque lesions and from OSCC, in an immunohistochemical staining analysis of 50 formalin-fixed paraffin-embedded tissues of each group. The authors suggested that TLR2 expression by malignant keratinocytes may be correlated with apoptosis resistance (28).
An analysis of 20 frozen tissue sections of laryngeal carcinoma samples, through immunohistochemistry and indirect immunofluorescence, also showed elevated expression of TLR2, as well as high levels of TLR3 and TLR4 in the malignant cells (29).
An additional study revealed strong TLR2 and TLR3 mRNA expression, but low levels of TLR4 and TLR9 mRNA expression through quantitative RT-PCR study with eight types of oral cavity, larynx and maxillary sinus malignant cell lines. TLR2 and TLR3 were also highly expressed at the protein level in most cell lines, as evaluated by Western blot and flow cytometry analysis (30).
A human pharyngeal squamous cell carcinoma cell line also exhibited strong mRNA expression of TLR2, TLR3 and TLR5, whereas TLR1 and TLR4 were barely detected in quantitative RT-PCR. And the TLR2 and TLR5 protein were also highly expressed in immunohistochemical analysis of three larynx squamous cell carcinoma tissue samples, whereas TLR3 presented weak staining (31).
The expression of the TLR2 appear to be elevated in OSCC tumor cells and may be correlated with apoptosis inhibition, even though one study with moderately differentiated primary tongue cancer cell line identified weak expression of TLR2 and that this receptor along with TLR5 were functionally expressed in this OSCC cell line, but did not influenced cell proliferation, migration, invasion and angiogenesis (32). This difference may be carefully analyzed and might be related to the different sources for TLR2 expression evaluation. The studies with human oral and laryngeal carcinoma samples found high TLR2 expression even with small sample size. The conflicting result was originated by studies with cultured cell lines, maybe due to different grades of differentiation.
Apart from the studies of SzczepaÅ„ski and colleagues (2007) (29) and Nomi and colleagues (2012) (30), other studies also demonstrated upregulated expression of TLR3. A western blot experiment found overexpression of this receptor in eight cell lines including malignant cells from OSCC and in malignant cells of pharynx and larynx tumors; and in approximately 80% of the 30 analyzed tumors and lymph node metastases of patients with OSCC. These data strongly suggested that the initial upregulation of NF-ÎºB in cells of the head and neck region is TLR3 independent and that this upregulation is an additional mechanism contributing to the constant activity of NF-ÎºB pathway in cancer cells, consequently, with tumor growth. In another words, NF-ÎºB initial stimulation is not a direct consequence of TLR3 stimulation by pathogenic or endogenous agent, since OSCC is frequently associated with chronic inflammations (33).
Overexpression of TLR3, TLR4, TLR7 and TLR9 mRNA (P < 0.05) were detected in esophageal carcinoma through RT-PCR of 20 frozen samples. And the immunohistochemical study of 87 formalin-fixed and paraffin-embedded tissue blocks from esophageal carcinoma showed statistically significant difference in protein expression of only TLR3 and TLR7 in tumor cells. Moreover, the TLR3 expression was significantly associated with depth of invasion and lymph node metastasis and the TLR7 expression in tumor cells was significantly associated with tumor grade. Despite the statistically non-significant difference in this analysis, the TLR4 expression in tumor cells was significantly associated with lymph node metastasis and TLR9 expression was found to gradually increase with worsening histological grade (34).
And another investigation in respect of the TLR3 expression, through a tissue microarray analysis of 153 patient samples revealed low cytoplasmic TLR3 protein expression in 80% of samples while high expression was noted in 20%. High TLR3 expression was signiï¬cantly correlated with patients whose tumors were poorly differentiated and who had perineural invasion, revealing the prognostic signiï¬cance of TLR3 protein expression in OSCC. Moreover, the TLR3 stimulation in a OSCC cell line with polyinosinic-polycytidylic acid (poly I:C), a synthetic double-stranded (ds) RNA that corresponds a TLR3 agonist, activate the NF-kB pathway, leading to IL-6 and CCL5 secretion and promotion of cancer cell migration, implying aggressiveness and invasion in OSCC (6).
Interestingly and contradictory, another studied of TLR3 stimulation in vitro by poly I:C, resulted in increased apoptosis of cancer cells in a dose-dependent manner, suggesting that targets of TLR3 could be one strategy for OSCC treatment (30). Another investigation also found high levels of TLR3 and TLR6 gene transcripts in seven cell lines of nasopharyngeal cancer and interesting results about the potential candidate for cancer treatment through TLR3 activation with poly I:C. This interaction significantly reduced the capacity of these cells to form metastasis in draining lymph nodes of athymic mice, because it downregulated the expression of chemokine receptor CXCR4 in a dose-dependent manner, and inhibited cell migration in response to a CXCR4 ligand, the stromal cell-derived factor-1a (SDF-1a) in chemotaxis assays (35, 36).
The mentioned studies found high expression of TLR3 in malignant cells of squamous cell carcinoma, except the study of Rydberg and colleagues (2009) that observed low TLR3 protein expression, but might be cautiously examined because it was achieved in an evaluation of only three samples of larynx squamous cell carcinoma. However, the conflicting results about TLR3 function in oral cancer, after in vitro stimulation, might have been produced by the different types and stage of differentiation of the malignant cell lines.
As verified by the follow study, which analyzed TLR3 signaling primary and metastatic oral cavity, oropharynx, hypopharynx malignant cell lines. All cell lines express similar levels of TLR3, however in primary cell lines, the treatment with poly I:C causes TLR3-mediated activation of NF-kB signaling pathway and metastatic cell lines, poly I:C was unable to activate prosurvival NF-kB signaling, which leads to enhanced apoptosis in these cells. In this study the authors suggested that this altered phenotype could be related to the desensitization of primary tumor cells exposed to dsRNA in the aerodigestive mucosal microenvironment, as opposed to the sterile nodal site of metastatic cells (37).
Regarding to the TLR4 expression in tumor cells, the studies from Rydberg and colleagues (2009) (31) and Nomi and colleagues (2012) (30) revealed low expression in laryngeal, pharyngeal and oral carcinoma cell lines. Although the study from SzczepaÅ„ski and colleagues (2007) found a positive TLR4 expression in tumor cells, this gene was less expressed in tumor cells than the TLR2 and TLR3; and the inflammatory cell infiltrate of the tumor mass and stroma most frequently expressed the TLR4 (29).
Another study also detected a weak expression of TLR4 and moreover, that it was not a functional receptor in moderately differentiated primary tongue cancer cell line, which may be related to inherited polymorphisms (32) and a study with 78 formalin-fixed and paraffin-embedded samples of laryngeal squamous cell carcinoma showed moderated TLR4 staining in 49% of samples submitted to immunohistochemistry and this expression was not significantly correlated with clinicopathologic variables, such as tumor aggressiveness or overall survival (4).
However a study of oral and larynx tumor cell lines and 39 formalin-fixed and paraffin-embedded samples of oral cavity and larynx tumor tissue samples obtained high levels of TLR4 mRNA and protein expression, through RT-PCR, flow cytometry and immunohistochemistry. Moreover, the highest level of TLR4 mRNA was detected in the well-differentiated tumor cell lines and tumors tissue. The cytoplasmic protein expression of TLR4 was also strong positive in well-differentiated or moderately well-differentiated tumors instead of poorly differentiated tumors, opposing to Bergmann and colleagues (2011) (4) conclusions. Finally, and contradicting the results from Park and colleagues (2010) (32), TLR4 was functionally active on OSCC cells and LPS (TLR4 ligand) promotes tumor cell proliferation and induces tumor cell resistance to drug-mediated apoptosis, especially in larynx well differentiated cell line (38).
Another study also correlated the expression of TLR4-NF-kB pathway molecules and a strong association with the aggressiveness of laryngeal carcinoma in an immunohistochemical analysis of 24 paraffin-embedded tissue samples from patients surgically treated for advanced laryngeal squamous cell carcinoma (39). Thus the expression of TLR4 might be related with tumor aggressiveness and may serve in future as a biomarker for tumor proliferation and aggressiveness.
The other TLR were less explored investigations of the inflammatory and immunologic context of OSCC. The TLR1 expression, described in only one study of OSCC tumor cells, was barely detected in pharyngeal squamous cell carcinoma cell line and the relationship of this receptor remain unknown (31).
The TLR5 generally was strongly expressed in tumor cells, in mRNA and protein levels in pharyngeal carcinoma (31) and OSCC cell lines (32). Although was demonstrated as a functionally expressed receptor little is known about its contribution to OSCC (32).
TLR7 and TLR9 usually showed strong expression in OSCC (32) and laryngeal carcinoma cell lines on protein and molecular level, particularly of TLR 7 and 9 (40), and a possible relationship with tumor growth was suggested.
This relationship of TLR9 was also observed by Min and colleagues (2011), which observed increased TLR9 expression in OSCC, correlated to an elevated level of Ki-67 expression in an immunohistochemical analysis of 60 OSCC formalin-fixed paraffin-embedded tissues that likewise has close correlation with large tumor size and poorly differentiated stage. Additionally, that the activation of TLR9 of OSCC cell lines with CpG oligodeoxynucleotide (CpG-ODN), a TLR9-agonist, could increase its proliferation and inflammatory chemokine release (41). And for Kauppila and colleagues (2011), which observed high TLR9 expression in an immunohistochemical analysis of 85 esophageal adenocarcinoma formalin-fixed paraffin-embedded specimens. The elevated TLR9 expression was correlated with advanced lesions and indicated poor overall survival (42).
The expression TLR6, TLR8 and TLR10 were not investigated in OSCC tumor cells, the first gene transcript was detected in a study of nasopharyngeal carcinoma cell lines but its relationship or function in this tumor was not identified (35).
Much remains to be studied concerning the function of TLR on tumor and normal immunological cells and its correlation with cancer development. On the other hand, is also important to examine the expression of other contributing proteins in the TLR signaling cascade, for example, the adapter protein MyD88 and CD14 that are related with the TLR4-signaling pathway (8). The study of this other molecules was discussed in the next section.
Genetic expression of molecules associated to toll-like receptors in OSCC tumor cells
The CD14 acts as a co-receptor along with the TLR2 and TLR4 in the recognition of bacterial LPS, inducing inflammatory response via NF-ÎºB activation and cytokine secretion. In a tissue microarray study of esophageal cancer samples, the CD14 showed significant up-regulation. This activation in tumor cells may induce proinflammatory factors and the synthesis of immunosuppressive molecules, which enhance the resistance cytotoxic lymphocyte attack and lead to immune evasion (43).
A study of an antiapoptotic protein found that mRNA as well as protein levels of cellular inhibitor of apoptosis protein 2 (c-IAP2) were elevated in nasopharyngeal tumor cell lines and the protein level were also increased in malignant cells than in the lymphoid stroma or in adjacent residual nonmalignant mucosa in the analyzed patient biopsies. Basic science studies showed that the presence or not of this protein can impact on TLR-pathways, especially on LPS interaction with TLR2 and TLR4. However in this analysis the authors found that TLR3 and TLR9 transcripts were detected by RT-PCR in all cultured cells, but the TLR4 was detected only one due to the possible relationship with the expression of the TLR4 and the tumor grade; and focused the combined analysis of c-IAP2 and TLR3 interaction with interesting results. It was demonstrated that TLR3-mediated apoptosis through poly I:C stimulation could be prevented in cases with high c-IAP2 expression on nasopharyngeal tumor cells. Thus, the tumor cell growth was dependent on c-IAP2 presence at a high concentration (44).
This dependence of c-IAP2 and the TLR3-mediated apoptosis could be the reason for the opposed results of the already discussed studies of the effect of TLR3 stimulation with poly I:C (6, 30, 35-37, 45), which do not accessed the c-IAP2 status.
Besides the effect of the entire TLR-pathway, it was postulated that the TLR expression in tumor microenvironment by the inflammatory cells could activate the immune surveillance against the altered cells, as anti-tumor defense, and these aspects were reviewed and discussed subsequently.
Toll-like receptor genetic expression in inflammatory cells of OSCC patients
Tumor cells are able to modulate the expression and function of TLR proteins in different kinds of immune cells, resulting in reduced immune function against tumor cells (46). Currently, the follow studies investigated this relationship in immune cells and OSCC.
NK cells isolated from cancer patients exhibit strongly impaired anti-tumor functions, which is also noted in OSCC. A study of natural killer (NK) immune cells derived from healthy blood donors demonstrated that this cell constitutively express TLR1, TLR2, TLR3 and TLR7. However, flow cytometric analysis showed NK cells incubated with OSCC supernatants presented quickly TLR3 down-regulation, due to the internalization of this receptor. And the activation of TLR3, by poly I:C, even with the influence of the immunosuppressive OSCC microenvironment, significantly increased again the surface expression of TLR3, but further consequences of TLR3 stimulation were not explored in this investigation (45).
An additional study regarding the NK cell activity in OSCC showed that in vitro stimulation of NK cells from healthy blood donors with single-stranded immunostimulatory RNA (ss-isRNA) strongly activated cytotoxicity against tumor cells of OSCC. The immunostimulatory potential of ss-isRNA was mediated through TLR7 up-regulation. The observed overexpression of TLR7 was reduced in the presence of OSCC supernatant, but the authors suggest ss-isRNA as potential immunostimulatory tool against OSCC (47).
Myeloid dendritic cells (MDC), as well as other types of immune cells, present their cellular functions strongly affected by tumor cells. Western-hybridization and flow cytometry experiments revealed detectable expression levels of all human TLR, except TLR4 and TLR9 in MDC isolated from healthy blood donors. The stimulation of these cells with OSCC supernatants generated an increased expression of TLR7, suggesting its participation in tumor promoting events (48).
The stimulation of immune cells through OSCC supernatants caused TLR7 upregulation in MDC (48) and NK cells, in the last case followed by ss-isRNS stimulation (47). However the effects of TLR7 function in immune cells of OSCC patient still require clarification, due to opposed consequences observed in both cited studies. Frenzel and colleagues (2006) reported that the high TLR7 expression by MDC could be related with tumor progression (48), while Pries and colleagues (2008) found that targeting TLR7 activated cytotoxicity against OSCC, suggesting this stimulation for tumor treatment (47).
Stimulation of dendritic cells (DC) by TLR7 or TLR9 agonists in the presence of OSCC-derived supernatants is followed by downregulation of TLR9, in contrast to upregulation of TLR7, also observed in DC from the microenvironment of breast and ovary cancers (24).
Human neutrophils (PMN), engaged in the early phase of anti-tumor response, present decreased expression of TLR2 and TLR6 in a Western blot analysis of 20 OSCC patients' blood samples. These cells also present downregulation of induced myeloid leukemia cell differentiation protein (Mcl-1), an anti-apoptotic protein, and this finding was accompanied with the increased intensity of apoptosis in the PMN. Moreover, the impaired expression of both TLR leads to disturbances in the secretion of different anti-tumor molecules by PMN, such as TNF-related apoptosis-inducing ligand (TRAIL). In summary, these cells fail to produce anti-tumor molecules and are auto-eliminated for the tumor microenvironment (49).
Another type of inflammatory cell but without protective function in relation to cancer immunity is the regulatory T-lymphocyte (T-reg) that in normal conditions maintains the tolerance against own-antigens preventing autoimmunity, but there is some evidence this subset is associated with tumor progression due to the mediated suppressing immune response (50).
Flow cytometry analysis demonstrated significantly increased percentages of circulating T-reg cells in patients with OSCC, which present significantly higher expression of TLR4, TLR6, TLR9 and TLR10, whereas TLR2 was not detectable. TLR4 stimulation with heat shock protein 60 or LPS increased significantly the suppressive function of T-reg cells, contributing with tumor growth through the immunossuppression (51).
More studies are required to elucidate the relation of the complete TLR-signaling pathway and its involvement in tumor and inflammatory cells of OSCC patients. These alterations related to an impaired TLR function may perhaps be a replication of variations on these genes, such as polymorphisms, this type of modification was explored in the next section.
Toll-like receptor polymorphisms and oral cancer
Single nucleotide polymorphisms (SNP) in coding regions of the gene may result in amino acid substitutions altering protein function; in noncoding regions, like introns and promoter regions, can produce altered splicing, reduced mRNA stability, impaired synthesis of transcription factors, that in general, may change the structure of translation initiation codons or the efficacy of enhancer or repressor elements, which may lead to the downregulation of the wildtype transcript (20, 52).
Altogether, these alterations can lead to the development of multifactorial diseases even those with strong environmental component, such as cancer. And have been extensively investigated through population-based association studies, estimating disease risk between carriers and non-carriers of a particular genetic polymorphism. Actually there is strong evidence of TLR genetic polymorphisms impacting on risk of cancer, especially in gastric and prostate cancer (53).
There is some genetic evidence that TLR expression, especially of the TLR3, is impaired in OSCC, and this may suggest a unique interaction of environmental and genetic factors, such as inherited SNP in this gene or in other genes of TLR family. Scarcely any studies identified SNP in TLR correlated with OSCC. In the head and neck region the studies of SNP in TLR are mainly of nasopharyngeal carcinoma risk. These data are summarized in Table 2.
Four TLR3 SNP - 13909C/T, rs3775291; 13766C/T, no rs designation (N.D.); 829A/C, N.D.; 9948C/T, rs5743312 - were genotyped by polymerase chain reaction (PCR)-based DNA direct sequencing in Chinese population with nasopharyngeal carcinoma. The allele C for the SNP 829A/C, at a nocoding region of the gene (intron 1) increased the overall risk of nasopharyngeal cancer in the study population (OR = 1.49, 95%CI = 1.10-2.00, P = 0.0068). When the authors assessed the interaction of these SNPs in NPC risk adjusted for age, gender and viral capsid antigen (VCA)-IgA antibody titers (detect current or recent infection with Epstein-Barr virus (EBV), which may be related with this neoplasia), the risk of development of malignant lesion was significantly lower in individuals who carried the haplotype "ATCT" (OR = 0.028, 95%CI = 0.002-0.341, P = 0.0054), compared to those who carried the most common haplotype "ACCT" (54).
An analysis through PCR-restriction fragment length polymorphism (RFLP) of two TLR4 SNP in coding region (Asp299Gly, rs4986790; Thr399Ile, rs498679) revealed a significant association between the SNP TLR4-rs4986790 and advancement and recurrence of OSCC and survival of these patients, with reduced disease-free survival (HR = 2.37, 95%CI = 1.05-5.33, P = 0.04) and decreased overall survival (HR = 2.00, 95%CI = 1.02-3.92, P = 0.04). For the other SNP, TLR4-rs498679, a similar pattern in relation to the disease-free survival was observable, patients with the mutant variant displayed a significantly higher risk for disease advancement (HR = 4.97, 95%CI = 2.00-12.37, P = 0.0006). Moreover, that OSCC patients treated with surgery and adjuvant systemic therapy (radio and chemotherapy) presenting the wild-type genotype for the SNP TLR4-rs4986790 showed significantly longer period of disease-free survival. Thus TLR4 genotype also seems to have an impact on the success of antitumor therapy (4).
Another study evaluating the above cited TLR4 SNP (D299G, rs4986790; T399I, rs498679) and a SNP from TLR2 (R753Q) through PCR-RFLP found that the differences in genotype and allele distribution of the SNP TLR4-rs4986790 were statistically signiï¬cant between cases and controls (P = 0.007 and P = 0.004, respectively), and that the presence of the mutated allele (CT/TT genotype) of this SNP was associated with increased nasopharyngeal cancer risk (OR = 1.853, 95% CI = 1.184-2.961, P = 0.007). Furthermore, the mRNA expression of IL-1a, TFN-a and IL-10 and protein concentrations of IL-1a and IL-10 were signiï¬cantly increased in patients with the combined CT/TT genotype of the SNP TLR4-rs4986790 compared to those with the wild-type (55).
The SNP (11350G/C, N.D.) in the 3'-untranslated region (3'-UTR) of TLR4 genotyped through PCR-based DNA direct sequencing method is significantly associated with nasopharyngeal cancer in Chinese population (OR =2.21, 95 %CI =1.34-3.64), while the another SNP in this region (11449C/T, N.D.) was not correlated with the development of this malignant lesion not even in the haplotype analysis, suggesting that 11350C variant in the 3'-UTR of exon 4 in the TLR4 may act as a genetic susceptibility factor for nasopharyngeal cancer due to the reduction of mRNA stability (52, 56).
Another study exploring the risk of inheritance of SNP and the development of nasopharyngeal carcinoma, investigated seven SNP in TLR10 gene (rs10856837, rs11466651, rs11466652, rs11466653, rs11096956, rs11096955, rs11466655) through PCR-based DNA direct sequencing in Chinese population. The analysis of association of each SPN separately using did not showed any statistically significant correlation, but haplotypes examination revealed statistically significant association of men with the GCGTGGC haplotype and nasopharyngeal cancer (OR = 1.415; 95% CI = 1.02-1.94; P = 0.005), even with adjustment for age, gender, and VCA-IgA antibody titers. This association may indicate that a mutation in a causal SNP or haplotype modifies immune response to EBV and thus increases the individual risk for nasopharyngeal cancer (57).
The evidence of sequence variants in TLR10 may in indicate that these individual probably present alteration in other genes of the TLR6-TRL1-TLR10 gene, because phylogenetic analysis demonstrated that the TLR1, TLR6 and TLR10 present similar amino acid sequence and are actually in the same chromosome (4p15.1-q12). The alteration of this gene cluster was already described in development of other types of cancer via inflammation process, such as prostate cancer (57, 58). Thus, the investigation of SNP in TLR stills a huge field of investigation.
Apparently the expression of TLR2, TLR3, TLR4 and TLR9 is impaired in OSCC tumor cells, as well as the expression of associated molecules, such as CD14 and c-IAP2. Immune cells such as NK, MDC and PMN from cancer patients exhibit damaged anti-tumor functions, possible mediated to immunosuppressive effects of TLR3, TLR4 and TLR7. These alterations were correlated with many aspects of the OSCC; however were not supposed to contribute with the OSCC tumorigenesis.
High TLR2 expression was correlated with apoptosis resistance (28-31). Upregulated expression of TLR3 was significantly associated with tumor growth, poorly differentiated tumors and invasion (6, 29, 30, 33, 34). Weak TLR4 expression was characteristic of poorly differentiated tumors correlated with proliferation and resistance to drug-mediated apoptosis (4, 29-32, 38, 39). Elevated TLR9 expression was correlated with advanced lesions and indicated poor overall survival (32, 40-42).
Nevertheless great part of the reviewed studies evaluated the expression of TLR in head and neck cancer cell lines and those with patient samples often utilized archival formalin-fixed paraffin-embedded tissues, in some cases with a small sample size and heterogeneous results; all of these compromise a proper evaluation.
Concerning about SNP, for example, some mutations in less explored TLR genes present interesting findings and were not already explored in OSCC, as well as those revised by Kutikhin and colleagues (2011). As illustrated by a SNP in the promoter region of the TLR8 gene (rs3761624) that alters the responsiveness of p53, a well-known human tumor suppressor, in primary human cells, which integrates the p53-mediated DNA damage and innate immune responses (17).
Since TLR are in focus of molecular cancer therapy development, intense and more profound research regarding the TLR involvement in OSCC is encouraged, through researches with human OSCC samples for gene and protein expression along to SNP investigation in these genes.