Laryngeal cancer (LC) is a second most common cancer of Head and Neck Cancer (HNC) group which develops from laryngeal squamous cell epithelium. It comprises 40% of all HNC and 1–2% of all human malignancies . The most important risk factors in LC development are tobacco smoking and excessive alcohol consumtion comprising very important synergistic effect [2, 3]. In past 20 years there is a slight decrease in the overall incidence of LC due to global anti-smoking compaign especially in USA and European countries. Unfortunatelly it has no significant influence on overall patient survival which is not markedly improved as there is still a great number of local and regional recurrences, advanced metastases and secondary primary tumors . Patient prognosis depends mostly on staging status at presentation [5–7]. Last few decades it became obvious that chronic inflammation predisposes to different forms of cancer. Pathogens as bacteria and viruses activate innate immune mechanisms which enables their elimination from the organism . Some pathogens can change innate immune mechanisms and lead to the chronic inflammation. Chronic inflammation has protumorigenic effect and enhances the risk of tumorigenesis . Important examples are H. pylori infection and development of gastric carcinoma , Hepatitis B and C virus infection and hepatocellular carcinoma , Human papilloma virus in development of oropharyngeal carcinoma [12, 13]. The profile of expression of different molecules engaged in activation and modulation of immune response and inflammation, their enhanced accumulation and decreased activation determine the balance between protumorigenic and antitumorigenic microenvironment . Cytokines control the inflammation through activation of different signal pathways by inducing protumorigenic immunity (IL -12, TRAIL, IFNγ), tumor development (IL-6, IL-17, IL-23), tumor growth and survival (TRAIL, FasL, TNF-α, EGFR, TGF-β). Generally, we can say that carcinoma is result of the change in signal pathways which participate in the cell proliferation regulation. Key factors in relationship between chronic inflammation and carcinogensis are transcription factors (NF-κB i STAT3) and proinflammation cytokines (IL-1β, IL-6, IL-23 i TNF-α) .
Innate immunity mechanism is the first line of organism defence and it is regulated by germline-encoded pattern recognition receptors (PRRs) expressed by cells, including macrophages, monocytes, dendritic cells, neutrophils and epithelial cells. The most studied PRRs are membrane-bound Toll-like receptors (TLRs) which recognize pathogen-associated molecular patterns (PAMPs) and intracellular NOD-like receptors (NLRs) which recognize PAMPs as well as danger-associated molecular patterns (DAMPs).
Activation of those receptors lead to complex signal pathway activation with resulting transcriptional factors NF-κB i AP-1 activation and synthesis and producing of proinflammatory cytokines and chemokines. Activation of NLRs results in inflammasomes formation which enables inactive to active caspase transformation and pro-IL-1β maturation. NLRs and TLRs pathways are connected so activation of TLRs pathway enhance producing of pro-IL-1β which mature by NLRs signaling pathway activation .
There are two genes, ASC/TMS1 (Apoptosis-associated speck like protein containing a caspase recruiting domain/Target of methylation induced silencing-1) gene and MyD88 (Myeloid differentiation primary response 88) gene, which both encode adaptor molecules with important roles in signal pathways activated by organism response to inflammation or injury.
Adaptor molecule ASC is involved in signal pathway characterized by formation of molecular platforms inflammasomes which activate NLRs and control maturation and secretion of proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18).
From all NLRs NLRP3 is currently the most fully characterized in creation of NLRP3 inflammasome. NLRP3 inflammasome consists of NLRP3 scaffold, the ASC adaptor molecule and caspase-1. It is activated upon exposure to specific ligands like pathogens, toxins, crystals, PAMPs and DAMPs. Upon NLRP3 activation, NLRP3 oligomerization leads to the PYD domain clustering homotypic interaction with the PYD domain of adaptor molecule ASC, and interaction of the CARD domain of adaptor molecule ASC with CARD domain of the procaspase-1. Procaspase-1 permits autocleavage and formation of the active caspase-1 which leads to the maturation and secretion of interleukin-1β (IL-1β) [8, 15, 16].
From the literature it is known that the mechanisms involved in NLRP3 activation are not well established and still intensely debated but mostly acceptable are three main models. First model explain that extracellular ATP stimulates opening of ATP-dependant ion channel of the cell membrane and in that way pore formation in the cell membrane allows direct NLRP3 inflammasome activation by NLRP3 cytosol agonists. Second model propose as activators particulate structures (urates, silica, asbestos) which activate phagocytes and by lysosomal damage release lysosomal contents consequently activating NLRP3 inflammasome formation. According to third model all NLRP3 agonists trigger the generation of reactive oxygen species (ROS) and this pathway activate NLRP3 inflammasome formation .
On the other hand, adaptor molecule MyD88 has a key role in TLRs signaling pathway and enables signal transduction cascade with final activation of transcription factor NF-κB and secretion of proinflammatory cytokines like interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and interleukin-12 (IL-12). The signal transduction begins by biding adaptor protein MyD88 with intracellular TLR domain, activation of kinase IRAK-4, 2, and 1 (IL-4,2,1 receptor associatetd kinase) and TRAF6 (receptor associated factor 6) phosphorylation, which subsequently activate TAK1 (TGF-β activated kinase 1). TAK1 phosphorylates IKKβ leading to degradation of IκB inhibitory protein dissociating it from NF-κB, thus allowing NF-κB to translocate to the nucleus and finally activate transcription .
It is well known that some epigenetic mechanisms, like DNA methylation, can change gene expression by methylation of CpG islands in the regulatory regions of the genome. As those islands, rich with CG dinucleotides (> 55%) are situated in regulatory gene regions they affect the transcription of genes. If CpG islands are unmethylated genes are expressed and if they are hypermethylated the expression of the genes is repressed (gene silencing). The inflammation induce DNA methyltransferase (DNMT) dependent DNA methylation which subsequently leads to silencing of the genes associated with carcinogenesis .
In this study, we hypothesized that the changes in the methylation status of promoter regions of ASC/TMS1 and MyD88 genes, responsible for activation and regulation of inflammation in healthy and laryngeal cancer tissue, might be related with development and progression of the laryngeal cancer. The aim of our study is to investigate is there a difference in the methylation status of this genes in healthy and tumor tissue and does it correlate with protein expression.
In recent literature the methylation status of this genes has not yet been analyzed in laryngeal cancer and the pyrosequencing method used to determinate methylation status of this genes has not been used in other cancers studies.