Introduction
Glioblastoma (glioblastoma multiforme; GBM) is the most aggressive primary brain tumor in the adult nervous system and is associated with a poor prognosis(d1) and always casts devastating effects on patients’ life expectancy and quality although they account for only < 2% of all human cancer types (b1). Relative survival estimates for glioblastoma are quite low and only approximately 4.5% of patients survive 5 years after diagnosis(d2 ). According to WHO classification, gliomas grouped into 4 histologic grades based on the degree of differentiation, anaplasia, and aggressiveness as WHO Grade I-IVtumors. Grade I corresponding to a benign tumor and grades II to IV increasing in malignancy. Grade III and Grade IV gliomas are considered malignant and have a v ery poor prognosis. the mean survival of GBM (grade IV) patients remains dismally at approximately 12 months despite intensive and comprehensive therapies (i-Louis).
There is accumulating evidence to suggest that deregulated NF-KB activity is a hallmark of most human cancers [a-8, 9].Recent studies suggest an important role for NF-kB signaling in GBM and implicate NF-KB activation as an important driver of the malignant phenotype that confers a negative prognosis in patients with GBM.(d-7-9)Immunohistochemical staining analysis for the p65 subunit of NF-KB with a p65-specific antibody, Western blot analysis of frozen tissue derived from tumors(d-17) and using primary cultures derived from GBMs revealed increased expression of p65 in glioma cells compared to normal brain(d-18)Although NF-kB has been shown to play a major role in cancer development by inducing and maintaining a proinflammatory microenvironment,constitutive NF-kB activation appears to promote tumor initiation and progression through mechanisms such as cell proliferation, apoptosis, angiogenesis, tumor metastasis, and reprogramming of metabolism(e-9)However, aberrant constitutive activation of NF-κB occurs in most glioblastomas,(f-10) suggesting additional mechanisms of NF-κB activation. The transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB) plays a crucial role in the regulation of immune and inflammatory responses through regulating the expression of inflammatory cytokines and chemokines and, in turn, induced by them [a-5-7]. The molecular pathogenesis of glioma is thought to involve multiple genetic alterations that result in aberrant activity of pathways involved in proliferation, cell cycle regulation, and apoptosis(d-4-5). A promoter region polymorphism of NFKB1 gene encoding the p50 subunit of NF-KB regulates gene activity.(ref?)
Toll-like Receptors(TLR) belong to the superfamily of pattern recognition receptors that classically activate and mediate inflammatory responses in innate immune cells by recognizing invading pathogens. A total of 13 mammalian TLR orthologs (10 in humans and 12 in mice) have been described so far(c-14,15). The expression and function of these receptors are also found not just in immune cells but also in non-immune cells. (c-17). TLRs recognize conserved molecular signatures on microorganisms called pathogen-associated molecular patterns and rapidly alert the host to potential dangers(c-15) Apart from their role in host defense mechanisms, especially TLRs 2 and 4 have critical functions in pathologies like cancers(c-20,21) Recent studies have directly demonstrated that NF-κB plays a role in TLRs-induced tumorigenesis when TLRs are activated by their ligand (15, 18). According to et al, TLR2 expression in the tumor microenvironment remarks that immune surveillance is activated against the altered epithelial cells, whereas TLR2 expression by malignant keratinocytes may be a sign of apoptosis resistance as a prosurvival mechanism [13]. TLR4 promotes tumor cell adhesion and invasion in a murine model by acting NF-kappa B [16], and the silencing of TLR4 increases tumor progression and metastasis in a murine model of breast cancer [17]. The TLR2 Arg753Gln, TLR4Asp299Gly and TLR4Thr399Ile, missense single-nucleotide polymorphisms, has been the most widely discussed.
Based on the cooperation between NF-KB and TLRs, we suspect that the presence of a polymorphism in these genes could increase the glioma risk.The aim of the present study, therefore, was to investigate the role of the NF-KB -94ins/del ATTG, TLR2 Arg753Gln, TLR4 Asp299Gly and TLR4 Thr399Ile polymorphisms in glioma and to clarify whether or not genetic alterations by this way affect the disease’s clinical outcome.
Material and Methods
Subjects
A group of 120 (mean age, 53.8 ± 5.1 years) patients with Glioma diagnosis followed in the Department of Neurosurgery of Cerrahpasa Medical School and a control group consisted of 225( mean age, 54.3 ± 4.4 years) healthy individuals were enrolled in the study. There was no gender bias in the analysis (data not shown).The patient pool included 53 diffuse astrocytomas,26 mixed type astrocytoma,7 oligodendrogliomas,2 gangliogliomas,1 pilocytic astrocytoma(A total of 89 High -grade Glioma) and 15 glioblastoma multiforms, 4 anaplastic ependymoma and 12 anaplastic astrocytomas (A total of 31 Low -grade Glioma). Each subject was of Turkish origin and completed a health questionnaire to provide details of medical history.Patients with pathologically confirmed metastatic brain tumors or prior cancer history were excluded during the recruitment stage.The study was approved by Institutional Ethical Committee of Cerrahpasa Medical School, Istanbul University, and proceeded in agreement with the Helsinki Declaration. Submission of the individuals to the study was conditioned by an obtained written informed consent form regarding the use of their samples for this research.
DNA extraction
3 ml of venous blood samples were taken from patients and controls in the Department of Neurosurgery between June-September 2016 and stored at −20◦C until DNA extraction. Genomic DNA was extracted from blood using Roche DNA purification kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. The DNA concentration and the purity measurement was applied by NanoDrop™ spectrophotometer and DNA samples with 260/280 OD ratio of 1.8±0.2 were included in the study.
Genotype Detection
A281/285 base pair(bp) fragment encompassing the-94 ins/del ATTG polymorphic site in NFKB1 gene, a 111 bp fragment encompassing the codon 753 in TLR2 gene, a 468 bp fragment encompassing the codon 299 and a385bp fragment encompassing the codon 399 in TLR2 gene were amplified by polymerase chain reaction using a Gene AmpPCR System 2700 (Applied Biosystems).For primer sequences and PCR conditions, see Figure 1.
Genotyping for these 4 polymorphisms were performed by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) analysis. After confirmation of successful PCR amplificationby2% agarose gel electrophoresis, PCRproductswere digested for 4 hours with 3 units of restriction endonuclease enzyme at 37 oC (Fermentas) and electrophoresed on4%agarose gel and visualized under UV illumination.Used restriction enzymes were PfIMI for NFKBI, Msp I for TLR2-753, Nco I for TLR4-299 and Hinf I for TLR4-399.The PCR product of NFKBI (281/285 bp) was cleaved into 3 fragments of 281 bp, 240 bp and 45 bp. Homozygosity for the common allele (represented by del/del genotype) appeared as 281bp bands, while the homozygosity of the variant allele (represented by ins/ins genotype) appeared as 240 bp and 45 bp bands, respectively and heterozygous genotype(ins/del) appears as bands, 281 bp, 240 bp and 45 bp.
The PCR product of TLR2-753(111 bp) was cleaved into 2 fragments of 111 bp and 93 bp. Homozygosity for the common allele (represented by A/A genotype) appeared as 111 bp bands, while the homozygosity of the variant allele (represented by G/G genotype) appeared as 93 bp bands and heterozygous genotype(A/G) appears as bands, 111bpand 93 bp.
The PCR product of TLR4-299(468 bp) was cleaved into 2 fragments of 249 bp and 226 bp. Homozygosity for the common allele (represented by A/A genotype) appeared as 249 bp bands, while the homozygosity of the variant allele (represented by G/G genotype) appeared as 226 bp bands and heterozygous genotype(A/G) appears as bands, 249bp and 226 bp.
The PCR product of TLR4-399(385 bp) was cleaved into 3 fragments of 385 bp, 365 bp and 20 bp. Homozygosity for the common allele (represented by C/C genotype) appeared as 385 bp bands, while the homozygosity of the variant allele (represented by T/T genotype) appeared as bands, 365 bp and 20 bp. Heterozygous genotype(C/T) appears as bands, 385 bp, 265 bp and 20 bp. For restriction enzyme digestion patterns See Figure 2.
Data analysis
2.5 Statistical analysis
GraphPad Prism 5 program and SPSS software version 18 (?) were used for the analysis of the patients and control values. Hardy-Weinberg equilibrium (HWE) was tested by chi-square analysis. Genotype and allele frequencies were compared between the cases and the controls by chi-square analysis. Odds ratio (OR) and respective 95% confidence intervals (CIs) were reported to evaluate the effects of any difference between allelic and genotype distribution. A two-sided p-value ≤ 0.05 was considered statistically significant. The confidence in the results will generally be weaker if it is conducted as part of a multiple comparison analysis, rather than a single comparison analysis. Associations of p olymorphisms were assessed using logistic regression analysis and Spearman rho correlation test
Discussion
About half of all brain tumors are gliomas and the deadliest human brain cancer, GBM, has a median survival of around one year from the time of diagnosis for several decades (e-1). Accelerating chemo/radiotherapeutic resistant cases and relapses, the underlying molecular and cellular mechanisms need to be urgently elucidated.
Numerous studies support the concept that many types of malignancies, including glioblastoma, have strong associations with NF-KB induced pathways in case of tumor cell proliferation and invasion. Interestingly, accumulating evidence suggests that NF-KBs function as double-edged swords, with both pro- and anti-tumor consequences. In the early stages, the normally regulated NF-KB induces cellular apoptosis in response to oncogene activation or cellular stress and thus functions as a tumor suppressor [6]. During tumor progression, instead, increased levels of inflammatory cytokines in the NF-κB regulating pathways or accumulated defects in the solid tumor microenvironment will contribute to aberrant NF-KB activity [a-30, 31]. Inhibition of NF-KB activity, for example, with the ubiquitin-editing protein A20(m-52), KLF6 deletion(f) or inhibitor of growth protein 4(m-39)has been demonstrated to reduce brain tumor growth, invasion and angiogenesis [m-54] whereas Pin1(m-53), Epidermal growth factor(m-4) or platelet-derived growth factor(m-47) contributes to constitutive NF-KB activation so the tumor development. A NFKB1 polymorphism, namely the -94insertion/deletion ATTG (rs28362491), is potentially functional and the most widely investigated [x-21]. This modification in the promoter region of the NFKB1 gene occurs between two important regulatory elements activator protein 1 and κB binding site, respectively. The deletion of four bases (ATTG) changes the mRNA stability and modifies translation efficiency by reducing or preventing the binding to nuclear proteins and leads to lower transcript levels of the NFKB1 gene [x-21, 22]. Since the location of this modification is proximal to binding sites that are important to promoter regulation, the ATTG deletion allele leads to a very reduced promoter activity and is involved in lower levels of p50 protein. Therefore, the different level of the protein subunits between the carriers of -94del allele and -94ins allele may be expected to contribute to inter-individual variations in cancer risk. Using a reporter assay, a previous study reported that the -94ins/del ATTG polymorphism has a regulatory effect on NFKB1 gene expression and that the activity of the ins allele is higher than del allele by 2 fold.[w-6]. In this study, we analyzed 120 glioma cases and 225 controls.The results in Table 2 revealed a significant association between insertion allele carriers and the enhanced cancer risk. We found that ins/ins genotype and ins allele frequency were significantly higher in the glioma patients compared to control subjects(p=0.003) NFKB1 -94ins/del ATTG polymorphism is associated with non-small cell lung cancer(w), bladder cancer(q-12), colorectal cancer(x), papillary thyroid Carcinoma(x-54), breast cancer(x-52) but not hepatocellular cancer(q-30).The probable mechanism behind the observed association may be linked to the enhanced expression and activity of p50. NFKB1 insertion allele is associated with the increased promoter activity and enhanced NFKB1 mRNA expression(ref).This association might influence glioma cancer development. Similar results such as in our previous report were determined in an Asian population and not in a Caucasian population. Yang et al similarly found a significant association between the NFKB1-94ins/del ATTG polymorphism and ovarian, oral, and prostate cancer(ref)
TLR2 and TLR4 have been under investigation for expression on glioma cells, and their contribution to tumor development has been mostly described as a tumor promoting. TLR2 expression levels, for instance, are reported to be elevated in glioma patient samples(i-vinnakota) and human glioblastoma U87 cells(i-haghparast) with low survival rates.TLR2 is mentioned to be an invasion-promoting factor in MDA-MB-231 breast cancer cells via activating NF-KB(b-10). On the other hand, host genetic factors are considered to be involved in tumor progression and the toll-like receptors genes have been shown to be polymorphic.
A non-synonymous arginine-to-glutamine substitution occurs in the intracellular Toll/interleukin 1 receptor domain of the TLR2 gene. A gene function report suggested that the TLR2 Arg753Gln polymorphism may disturb tyrosine phosphorylation, dimerization with TLR6, and MyD88 recruitment with an effect on NF-KB activation [u-20, 30]. Yet, the role of TLR2 in controlling the invasion of glioma still remains unknown. The TLR4 expression is also detected in U87 and U118 glioma cell lines(i-tewari2012). Meanwhile, in vitro and animal model have shown the two most-studied non-synonymous SNPs of TLR4: an A to G transition (rs4986790), resulting in Aspartate-Glycine substitution at position 299 (Asp299Gly) and C to T transition (rs4986791), resulting in threonine-isoleucine substitution at position 399 (Thr399Ile), to cause approximately 50 % reduction in TLR4 expression on the membrane surface of innate immune cells and altered GPI binding, induce excessive production of pro-inflammatory cytokines and LPS hyporesponsiveness [r-11, 13, 14]. The present study was designed to detect the TLR2 and TLR4 polymorphisms in glioma. TLR4 AA genotype and A allele were associated with a significant risk of glioma whereas the GG genotype for TLR2 rs5743708, has a protective role in glioma. Davododi et al, instead, observed that variant TLR4Asp299Gly and TLR4Thr399Ile alleles, as well as the TLR2Arg753Gln allele, are not associated with risk of colorectal cancer(ref). Interestingly, TLR2Arg753Gln was associated with prostate cancer(v-118), TLR4Asp299Gly was associated with gastric and colorectal cancer (v-109-114) whereas TLR4Thr399Ile was associated with gastric cancer(v-128) but not with colorectal cancer(v-116). In addition, Asp299Gly but not Thr399Ile reported affecting TLR4 signaling(y). Nevertheless, the way that TLR4 affects the tumor development is still needed to be elucidated.
According to our study results indicate that the ins/ins genotype of NFKB -94ins/delATTG, and the AA genotype of TLR4Asp299Gly are risk factors for glioma and people carrying ins allele have an approximately 1.47 times susceptibility risk of glioma.The TLR4 Thr399Ile polymorphism is not a risk factor. However, GG genotype of TLR2Arg753Gln seems to be protective against glioma As it is a well- known fact that some variants and /or genes working together increase the risk.We, therefore, analyzed the relationship between the disease and combined gene analysis. According to combined genotype analysis, combined genotype of TLR2Arg753Gln- NFKB1 and TLR4Asp299Gly-NFKB1 and TLR4Thr399Ile-NFKB1 – were a risk for glioma development.
Combined genotype analysis showed that del/ins-GG genotype of TLR2Arg753Gln-NFKB1, del/ins+GG genotype of TLR4Asp299Gly-NFKB1, del/ins-CC genotype of TLR4Thr399Ile-NFKB1 were risk factors for glioma development.
However, this observation needs to be interpreted with caution because it was not been documented in previous immunogenetic studies of all TLR2, TLR4 and NF-KB polymorphisms, and glioma from other countries.