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Summer Research Fellowship Programme of India's Science Academies

Analysis of TLR4 gene expression in oral epithelial dysplasia and squamous cell carcinoma

Shaista Haider

Jamia Millia Islamia, Jamia Nagar, Okhla, New Delhi 110025

Dr. Mausumi Bharadwaj

Scientist G (Director Grade & Head), Molecular Biology Group, ICMR - National Institute of Cancer Prevention and Research 201301

Abstract

Cancer of the oral cavity is the 11th most common malignancy in the world. But in developing countries, it is a major health problem. It includes the cancers of tongue, lip, gingiva, mouth floor, parotid and salivary glands. The main risk factors are tobacco and alcohol consumption. Although early diagnosis is relatively easy, presentation with advanced disease is not uncommon. Data shows annual incidence is high around the world, which is over 300,000 diagnosed cases, and the annual mortality is about 145,000 deaths. Toll-like receptors (TLRs) are transmembrane proteins, expressed on immune cells that recognize Pathogen Associated Molecular Patterns (PAMP) which are exogenous and endogenous molecules on pathogens. Once activated, they initiate cascade of signalling pathways leading to the release of cytokines and chemokines, followed by recruitment of immune cells and further cytokine production, the production of angiogenic mediators and growth factors which are a leading cause of tumor progression. As a member of pattern recognition receptors, toll-like receptor 4 (TLR4) plays a pivotal role in tumor immune microenvironment and has been increasingly investigated. TLR4 shows pro-tumorigenic effects mainly due to its expression on tumor cells where it mediates resistance of tumor cells to damage induced by cytotoxic T lymphocytes. The purpose of this study is to check the expression of TLR4 in oral carcinogenesis with the help of Immunohistochemistry. Immunohistochemistry (IHC) determines the presence of antigens by means of antigen-antibody interaction. Previous studies showed that TLR4 is highly expressed in cancerous tissues than normal controls. In these studies, the expression level of TLR4 increased significantly from mild to severe dysplasia as compared to controls.

Keywords: Toll-like receptors (TLRs), nuclear factor, kappa-light-chain-enhancer of activated B cells (NF-κB), oral squamous cell carcinoma (OSCC), Immunohistochemistry (IHC)

Abbreviations

OSCC Oral squamous cell carcinoma 
 TLRToll-like receptors 
SNP Single nucleotide polymorphism 
PCR Polymerase Chain Reaction
PBS Phosphate-buffered saline 
TBS Tris-buffered saline 
APS Ammonium Persulfafte
TEMED Tetramethylethylenediamine 
 DAMPDamage-associated molecular pattern 
PAMP ·         Pathogen-associated molecular pattern 
 BSABovine Serum Albumin
 TETris- ethylenediaminetetraacetic acid 
 DAB3, 3’- Diaminobenzidine  
TRIF TIR-domain-containing adapter-inducing interferon-β 
 TRAMTRIF-related adaptor molecules 
 IRF3Interferon response factor-3 
HPV Human papillomavirus 
EBV Epstein-Barr virus 
HSV Herpes-Simplex Virus 
 MHCMajor Histocompatibility Complex 
 VEGF·         Vascular endothelial growth factor 
PDGF Platelet-derived growth factor 
 FGFFibroblast growth factor 
 TNMTumour, lymph node, metastasis 
 AJCAmerican Joint Committee 
LPS Lipopolysaccharide 
HSP Heat-shock proteins  

INTRODUCTION

Background

Cancer of the oral cavity is the 11th most common malignancies in the world [1]. Cancer is defined as uncontrolled proliferation of cells that invade other tissues and cause damage to the surrounding. Oral cancer, a part of head and neck cancer, is a malignant neoplasia which arises in the oral cavity includes the cancer of lip, cheek, tongue, floor of mouth, gingiva and throat. It is associated with different levels of differentiation and a propensity for lymph node metastasis. Oral epithelial cell dysplasia is the earliest form of precancerous lesions that is associated with high potential to turn into malignance. It is the manifestation and continuous clinical change that develops with time and progresses towards cancer. According to epidemiologic research data, epithelial lesions can be one of the major causes of malignancies [2]. When the abnormal cells invade the underlying tissues there is a high risk of progression towards malignancy. The gross genomic aberrations in DNA ploidy status have promise can be used as a predictor of malignant progression. Now days, the degree of dysplasia is considered as the best tool to predict the potential progression of oral lesions [3].

Toll-like receptors (TLRs) are transmembrane proteins, expressed on immune cells that recognize pathogen-associated molecular patterns (PAMP) exogenous and endogenous molecules on pathogen [4]. These proteins are the components of innate immune system and initiate intra-cellular signaling to elicit inflammatory response towards any infection. In various studies they have been described as promoters of cell proliferation, invasiveness, and angiogenesis in a variety of cancers. Once activated, they initiate cascade of signaling pathways leading to the release of cytokines and chemokines, followed by recruitment of immune cells and further cytokine production, the production of angiogenic mediators and growth factors, and finally promotes the cause of tumor progression [5], [6]. TLR4 predominantly shows pro-tumorigenic effects and is highly expressed in cancerous tissues than normal control cells [7]. It is expressed on tumor cells to mediate resistance to themfrom the damage induced by cytotoxic T lymphocytes (Tc cells), the triggered expression of TLR4 on immune cells which in turn activate NF-κB signaling by tumor cells and playrole in tumor development and progression [8], [9].

Immunohistochemistry (IHC) determines the presence of antigens by means of antigen-antibody interaction. The site of antibody is identified by direct labelling of the primary antibody or by labelling the secondary antibody. Immunohistochemistry is used to distinguish undifferentiated oral neoplasms of different origins through the detection of tumor antigens using known antibodies[10]. With increase in the number of marker proteins being recognized, IHC is used as molecular markers to determine metastatic tumors in the field of cancer detection [11]. Thus, immunohistochemistry plays important role in diagnosis, investigation, and determination of the behavior and pathogenesis of oral tumors. The aim of the present study was to analyze the expression of TLR4 gene in oral pre-cancer, cancer and controls cases.

Objectives of the Research

1)      Preparation of formalin-fixed paraffin-embedded tissue sections for checking the expression of targeted proteins.

2)      To find out the expression of TLR-4 gene in oral pre-cancer, cancer and control tissue sections by Immunohistochemistry.

 3)      Statistical analysis of above findings.

LITERATURE REVIEW

Oral Cancer

Cancers are a huge accumulation of disorders that affect unusual cell extension with the potential to overrun or reach to different portions of the body. The chance of carcinoma rises significantly with age; also various carcinomas happen more regularly in advanced nations.

Types of Cancer

Cancers occur due to abnormal proliferation of different types of cells in the body. It may be either benign or malignant.

benign tumor remains confined to its original location and do not invade or spread to nearby tissues. On the other hand, a malignant tumor is capable to invade surrounding normal tissue and metastasize throughout the body via the circulatory or lymphatic systems, known as metastasis.

There are four main types of cancers:

Carcinoma: It is the most common type of cancers. It is the cancer of epithelial tissues which originate in the skin, lungs or tissues that line internal organs.

Sarcoma: It is the solid tumor of connective tissues that arise in bones, muscles, cartilages and blood vessels.

Lymphoma and myeloma: These are the cancers of immune system. Lymphoma originates from the cells of lymphatic system, while myeloma starts from plasma cells.

Leukemia: It is the cancer of bone marrow in which too many immature white blood cells (WBCs) are formed.

Epidemiology of Cancer

It is the study of determinants, distribution and frequency of occurrence of cancer in specific population at specific tim

Global Scenario

New global cancer data has shown that the global cancer burden has increased to 18.1 million cases and 9.6 million cancer deaths [1]. According to International Agency for Research on Cancer (IARC) estimate every 1 in 5 men and 1 in 6 women is at the risk of developing cancer worldwide and every 1 in 8 men and 1 in 11 women are at the risk of death due to cancer. Growing and aging global population, increase in exposure to cancer risk factors and change in life style are involved in driving this global burden of cancer (Fig. 1)

Screenshot (23)_1.png
    Global scenario of Cancer Incidence and Mortality

    Indian Scenario

    In India cancer burden has increased to 1.16 million cases and 7.8 lakhs deaths. The risk of developing cancer in males is 9.81% while that in females is 9.42%. The risk of death due to cancer in males is 7.34% and in females the risk is 6.28%.

    Epidemiology of oral cancer

    Global scenario

    A total of 354,864 new cases and 177,384 cases of death due to oral cancer have been estimated globally. have been reported [12].

    Screenshot (24).png
      Bar chart of region-specific incidences for Cancers of Lip and Oral Cavity

      Indian scenario

      Among males, oral cancer is the most commonly diagnosed cancerwhile in female’s breast cancer the most frequently diagnosed cancer followed by the cancer of lip and oral cavity. Total of 119,992 new cases have been reported. The risk of developing new cases is 11.42%. Reports show 92,011 (16.1%) new cases in males and 27,981 (4.8%) new cases in females. Total of 72,616 death cases have been reported with the risk of 10.09% in both the sexes.

      Signs and Symptoms

      • Patches on the lining of the mouth or tongue
      • Mouth ulcers or sores that do not heal
      • Chronic swelling
      • A lump or thickening of the skin or lining of the mouth
      • Pain when swallowing
      • Jaw pain or stiffness
      • Hypersensitivity
      • A sensation that something is stuck in the throat
      • Painful tongue
      • Sore throat
      • Jaw pain and stiffness
      • Hoarse voice
      • Chronic pain in the neck or ear
      • Loose teeth with no apparent reason
      • Poorly fitting dentures

      Stages of Cancer

      Staging of oral cancer means the assessment of the clinical extent of the disease by visual examination, biopsies and imaging investigation of different parts of mouth.

      TNM staging of oral cancer [12]

      TNM stands for Tumor Node Metastasis, where

      ·         T describes the size and depth of primary tumor,

      ·         N gives the idea about the spreading of cancer to the lymph nodes, and

      ·         M describes if the cancer has spread to other parts of the body

      Primary Tumor (T)

      There are 4 stages,

      Different stages of Primary Tumor
      TX Primary tumor cannot be assessed 
       T0No evidence of primary tumor 
       TisCarcinoma in situ 
      T1 The tumor is 2 cm or lesser in dimension and 5 mm or less in depth and is contained within the oral cavity 
      T2 The tumor is in between 2-4 cm in dimension 
      T3 The size of tumor is more than 4 cm and id deeper than 10 mm 
      T4a (lip)The tumor is growing further and is invading the surrounding tissues like floorof mouth, skin, cortical bones and into the muscles of tongue, maxillary sinus and facial skin 
      T4b (lip and oral cavity)The tumor spread further and invades near masticator space, the jaws and jaw muscles, base of skull and encases the internal carotid artery 

      Regional lymph nodes (N)

      Stages of regional lymph nodes
      NX Regional lymph nodes can’t be assessed 
      N0 No metastasis in regional lymph node 
      N1 Lymph node has been metastasized on the same side of neck. The size of node is not greater than 3 cm 
      N2a Size of metastasized lymph node is between 3-6 cm, has not spread into the surrounding tissues 
      N2b Metastasis in more than one lymph node as the same side of cancer, size is not more than 6 cm 
      N2c Metastasis in the nodes of both the sides, none is more than cm in dimension, that cancer has not spread to the tissues surrounding the lymph nodes 
      N3a Size of lymph node containing the cancer cells is greater than 6 cm, not spread into surrounding tissues 
      N3b Different lymph nodes contain cancer which has spread into surrounding tissues 

      Distant metastasis

      Stages of distant metastasis
      cM0 No metastasis 
      cM1 Metastasis to different parts of body 
      cM2 Distance metastasis has been microscopically confirmed 

      Stage 0: It also known as carcinoma in situ or pre-cancer stage. The cancer cells are attached to the linings of oropharynx or mouth and have not spread yet.

      Stage 1: It is the earliest stage of invasive cancer. The tumor is 1 inch or less and has not spread to the nearby tissues or lymph nodes.

      Stage 2: The tumor is 1-2 inches and is not deeper than 10mm. It has not yet spread to nearby lymph nodes or any organs.

      Stage 3: The tumor is more than 2 inches and has not spread yet. If it has spread then to only on the same side of lymph node.

      Stage 4: It is the advanced stage of cancer. The cancer has grown and spread to the surrounding area and to the rest of the body.

      Screening of Dysplasia and Oral Cancer

      The diagnosis in the oral cavity is a complicated process because the potential malignant lesions mimic the cancer in early stage [13].

      There are two approaches in early detection oral dysplasia and cancer:

      • Oral cancer screening program: to identify patients with suspicious lesson
      • Specific diagnostic tools: to determine the stage of cancer

      A biopsy is required to determine the exact makeup of a suspicious looking area. Diagnostic tools are needed for early detection of oral dysplasia and malignancies. The tests include visual examinations, brush biopsy, scalpel biopsies, histopathology, CT scans, magnetic resonance imaging (MRI), positron emission tomography (PET), autofluorescence, toluidine blue staining, DNA analysis, salivary proteomics, spectroscopy, analysis of biomarkers, etc. Advancement in imaging techniques is helpful in more accurately screening of the problem.

      Light based oral screening

      • VEL scope: The working principle isbased on the excitation and emission at particular wavelength. The device uses visible light at 430 nm for excitation of certain compound and the emission is observed.
      • Vizilite: The particular area is rinsed with acetic acid and the oral cavity is examined with a chemiluminescent light stick.
      • Microlux: It is similar to Vizilite and the source of light is battery –powered fibroblastic visible light.

      Diagnostic tests

      Brush biopsy: It is a sensitive technique where a brush is used to collect cells from oral epithelium. It is a painless approach to examine any suspicious lesion and the specificity is over 90%.

      Scalpel biopsy: Tissue sampling by scalpel biopsy is thought to be the keystone for the diagnosis of premalignant and malignant oral diseases. An oral biopsy is an invasive method and involves both psychological implications for the patient as well as possible often technical difficulties for the health practitioner. For extensive lesions, the most appropriate and representative areas must be selected to avoid diagnostic errors.

      Vital staining: Toluidine Blue (TB) solution is used for the identification of early OSCC and high-grade dysplasia. 1% aqueous TB solution is helpful in differentiating areas of carcinomas in situ (invasive carcinoma) from normal tissue. TB is highly sensitive and moderately specific for malignant lesions.

      Laser capture microdissection: It provides the ideal method to extract the cells from the specimen. LCM when combined with IHC staining provides more accurate microdissection of tissues. It is used as molecular diagnostic tool to establish protein fingerprint for early detection of OSCC.

      DNA analysis: It measures the ploidy level of suspicious cells. Genomic instability contributes to the development of cancer microenvironment and the dysplastic lesion may progress to cancer. Up to 100% sensitivity and specificity has been reported.

      Saliva testing: It is a cost-effective approach to collect large number of samples non-invasively. It is used to measure specific macromolecules and for the analysis of genomics and proteomics of the patient such as cytokines, growth factors, metalloproteinase, DNA transcript and mRNA expression.

      Lab-on-a-chip: It is also known as micro-total-analysis system, where a chip is used to detect the dysplastic lesions and cancerous tissues with the help of transcription profile as well as the membrane-associated cell proteins that are singularly expressed by cancerous cells.

      Microscopy: It is used for diagnostic differentiation of the biochemical composition of cell. Poh et al examined oral mucosal biopsies with the help of fluorescence visualization, and loss of heterozygosity. Multispectral digital microscope (MDM) is also utilizes as a tool for the detection of oral neoplasia. It acquires in-vivo images by narrow-band (NB) reflectance, fluorescence and orthogonal Polaroid reflectance (OPR) for proper evaluation of dysplasic lesions.

      Spectroscopy: It is done by exciting the sample by specific wavelength of light and detection of the fluorescence produces by the endogenous fluorophore molecules and monitoring the therapeutic response of the tissues. A change in absorption or emission spectra is because of changes in blood content and altered tissue metabolism. Diffuse reflectance spectroscopy (DRS) is helpful in the detection of (pre)malignant tissue alterations.

      Tomography: Optical coherence tomography (OCT) is a non-invasive imaging technique which builds cross-sectional image of tissue to detect areas of inflammation, dysplasia and any architectural changes in malignant cells.

      Treatment

      For the treatment of early stages of cancer surgery and radiotherapy are most commonly used either in single modalities or in combination. The choice of the treatment is based on the age of patient, location of tumor, associated illness and the availability of expertise. New techniques, e.g. 3D conformal radiotherapy and intensity modulated radiotherapy, will help in minimizing the side effects by selectively targeting the lesions.

      For the aggressive tumors in advanced stage a combined approach integrating surgery, radiotherapy, and chemotherapy is preferred. For the patient with deep infiltrative tumors and bone infiltration, surgery is followed by postoperative radiotherapy. For those who are not medically fit for surgery radiotherapy alone or in combination with chemotherapy is approached.

      After the treatment is completed the patient should be followed up at regular intervals to check for any recurrence symptoms should be encouraged about the health issues regarding the use of tobacco or alcohol.

      Major Risk Factors of Oral Cancer

      Tobacco, smoking and alcohol use are among the major risk factor. Smokers are 3 times more likely than nonsmokers to develop mouth cancer, and people who smoke and drink alcohol have up to 30 times higher risk than those who do not smoke and drink. HPV infection is also involved in causing cancer of different types. In 2018, 177,000 deaths occurred out of 355,000 people diagnosed positive. Even though smoking cessation campaigns have decreased the rate of tobacco induced tumors, the rate of HPV related cancers has offset the gains. The global incidence of 2/3rd of oral cancer occurs in low-and middle-income countries, half of those cases are in South Asia. India alone accounts for 1/5th of all oral cancer cases and 1/4th of all oral cancer deaths.

      SCC is the highest prevalence lesion among other oral malignancies in epidemiological studies. It is an attribute or exposure of an individual that increases the susceptibility towards developing a disease. Various factors have been shown to be significantly associated with oral cancers:

      Chemical factors

      Tobacco: Tobacco in various forms like chewing, smoking, etc., shows carcinogenic activity. Cigarettes, cigars, and bidi etc., are the most commonly used form smoking.

      Alcohol: Alcohol is shown to be the major risk factor in OC in various studies. Individuals consuming more alcohol are at higher risk of developing oral cancer. Alcohol has additive effect and studies haveshown that it facilitates the entry of carcinogens into the exposed cells and alter the metabolism of oral mucosal cells [14].

      Areca Nut Chewing: Areca nut also known as betel nut, is considered as a type 1 carcinogen. It is chewed in raw, dried, or roasted form, or as part of betel quid, by millions of people in Asia and among Asians worldwide. The inclusion of tobacco in the betel quid adds significantly to its property of carcinogenicity.

      Biological factors

      Viruses: Viruses are inert outside the cells and do not have their replication and protein synthesizing machinery. They are capable to hijack the host cell machinery and modify the DNA and chromosomes in the cells which further may lead to malignant transformation.

      HPV: Human papillomavirus (HPV) is a group of more than 150 types of viruses. Infection with any type can cause different form of cancers such as cancer of mouth, cervix, throat, etc.HPV related oropharyngeal cancer in the tonsils and the base of the tongue has become more common in recent years. HPV-16 is the most commonly detected virus in head and neck squamous cell carcinoma (HNSCC) in 90-95% cases, followed by HPV-18, HPV-31, and HPV-33 (Fig. 3).

      HPV genome.png
        The HPV genome

        Functions of HPV Genes : HPV genome is composed of 8 kb and has been divided into early (E) and late genes (L). Their function is as follows:

        Functions of HPV Genes 
        E1 Maintenance of viral genome as episome
        E2 Amplification of viral genome
        E4 Play role in viral genome amplification
        E5 Promote the growth of virus by forming complex with EGFR, PDGFR and CSF-1R
        E6 Binds to and degrade p53, inhibits Bak and Bax, activates telomerase, stabilize Src-family kinases
        E7 Binds to and degrades Rb protein, activates histone deacetylase, NF-kB transcription complex, etc.
        L1 Encodes the major capsid protein and allows the assembly of full virus
        L2 Encodes the minor capsid protein, virus-like particles are formed in its absence

        EBV: Epstein–Barr virus (EBV) infection causes oral hairy leukoplakia and lymphoproliferative disease in immunosuppressed patients. Prevalence studies have shown presence of EBV in OSCC patients, but casual association is still unclear.

        HSV: Herpes simplex virus (HSV), HSV-1 also known as "oral herpes" is commonly associated with sores around the mouth and lip and is supposed to be the causative agent of OC. A population-based study has showen that HSV-1 enhances the development of OSCC in HPV infected patients and in individuals with cigarette smoking habits.​

        Candida: Infection with candida has an increased risk of dysplasia and malignancies with higher proliferative phenotype. It is shown to have a role in initiation of OC.

        Genetic factors

        • Genetic susceptibility: 10% of all the cancers are affected by genomic organization of the individual. Genetic component play role in the development of OC. Evaluation of specific polymorphism in the major genes involved in oral carcinogenesis is the major area of study. Glutathione S-transferase M1 (GSTM1) null genotype is shown to be the most persistent marker to study polymorphic susceptibility in head and neck cancer including OC.
        • Proto-oncogenes, Oncogenes and Genetic alterations: Genetic alterations represent molecular basis of carcinogenesis including point mutations, amplifications, rearrangements, and deletions which may lead to carcinogenesis. Aberrant expression of oncogenes such as epidermal growth factor receptor (EGFR), c-myc, K-ras, int-2, Parathyroid adenomatosis 1 (PRAD-1) and B-cell lymphoma (bcl) have been reported in OC progression and advancement.
        • Tumor suppressor genes: Inactivation of p53 is most commonly found in all the cancers.  Loss of chromosome 9p21 occurs in the majority of invasive tumors in head and neck cancer. Loss of protein p16 and chromosome 17p has been observed in premalignant lesions as well as human cancers including OC.
        • Genomic instability: Loss of heterozygosity and microsatellite instability have been observed in several cancers including OC. Loss of function of the tumor suppressor p53 results in uncontrolled cell division and progressive genomic instability. Chromosome 9p21 containing p16, 3p14 containing FRA3B are frequently lost in early stage of tumorigenesis. Variations in different chromosomes (3p, 9p, 17p and 18q) are associated with the development and progression of HNSCC.
        • Epigenetic alterations: Methylation, caused by DNA methyl transferases, is the most important epigenetic modification which play major role in tumorigenesis. These alterations may inactivate DNA repair enzymes and may aid in genomic instability. Abnormal hypermutation in p16 has been observed in HNSCC.

        TLRs

        Toll-like receptors (TLRs), a group of pattern-recognition receptors (PRRs), are transmembrane microbial sensing proteins, expressed on immune cells, such as B-lymphocytes, monocytes and to some extent dendritic cells (DCs) as well as epithelial cells, that recognize pathogen-associated molecular patterns (PAMP) exogenous and endogenous molecules on pathogen. These proteins are the components of innate immune system and initiate intra-cellular signaling to elicit inflammatory response towards any infection. These proteins are immensely conserved from Drosophila to humans and share similarity in their structure as well as in their functionality.In various studies they have been described as promoters of cell proliferation, invasiveness, and angiogenesis in a variety of cancers [15]. Once activated, they initiate cascade of signaling pathways leading to the release of cytokines and chemokines, followed by recruitment of immune cells and further cytokine production, the production of angiogenic mediators and growth factors, and finally promotes the cause of tumor progression. TLRs are also involved in the induction of apoptosis through the expression of anti-apoptotic proteins and apoptosis inhibitors. Though the role of TLRs in cancer is yet to be fully known, recent studies suggest that TLRs have dual role in malignant cells and cancer: abnormally high concentration and dosage of TLRs manifest an anti-cancer effect whereas low dosage of TLRs enhance cancer growth [16].

        tlr.jpg
          Pro- and anti-Tumor Pathways of TLRs

          Role of TLRs in inflammation and carcinogenesis

          TLRs are expressed in immune cells as well as in tumor microenvironment (TME) and may lead to tumor exacerbation. TLRs not only induce apoptosis, but also release signaling molecules such as cytokines and chemokine in the tumor microenvironment, followed by recruitment of more immune cells to further release of aberrant pro-inflammatory cytokines, chemokines, pro-angiogenic factors and growth factors and ultimately lead to tumor progression. A complex interaction between malignant cells and immune cells in the tumor microenvironment contributes to the heightened immune reaction and aid in anti-tumor response in TLR signaling pathway [17].

          Because of their role in inflammation, tissue regeneration and fibrogenesis, TLRs are the likely contributors to mediate the effects of innate immune system on carcinogenesis. Because of their ability to enhance antigen presentation, TLRs have become potent targets in infectious disease and in neoplastic disease treatment. The downstream signaling pathways of TLRs require two major adapter proteins, MyD88 and TRIF (TIR domain-containing adapter inducing IFN-β), TIRAP (TIR domain-containing adapter protein or Mal) and TRAM (TRIF-related adapter molecule). Interaction of the TIR domains on TLRs and adapter molecules triggers a kinase cascade that elicits the release of transcription factors like NF-κB, AP-1, IRF3 and IRF7. Certain adapter proteins are activated in response to stimulation of some TLRs, whereas other TLRs activate multiple adapter molecules. This in turn specify the immune response to different signaling molecules i.e., ligands, e.g. quick antiviral response by IRF-3 activation through TRIF following TLR3 stimulation, and two-phase NF-κB activation subsequent to TLR4 ligation [18].

          Historical-timeline-for-cytokines-TLRs-inflammation-and-cancer-1-Virchow-1-2.png
            Historical timeline for cytokines, TLRs, inflammation and cancer

            TLR4 and oral cancer

            TLR 4 is a transmembrane protein encoded by TLR4 gene. Its stimulation and activation lead to an intracellular signaling pathway NF-kB and inflammatory cytokine production which is necessary for the activation of potent immune response. It recognizes lipopolysaccharide (LPS) component present in many Gram-negative bacteria as well as in some Gram-positive ones. Beutler’s group clearly demonstrated that TLR4 is the key sensor for LPS. LBP protein recognizes and binds to LPS and forms LPS-LBP complex and assists the interaction of LPS and a glycosylphosphatidylinositol-anchored protein CD14 that binds the LPS-LBP complex and facilitates the transfer of LPS to the TLR4/MD-2 receptor complex and modulates LPS recognition. LPS binding further stimulates the dimerization of TLR4/MD-2. There is a conformational change in TLR4 which help in recruitment of different adaptor proteins, necessary for downstream signaling (Fig 6) [19].

            Screenshot (18).png
              Overview of LPS/TLR4 signaling

              TLR 4 signaling has been divided into two types: MyD88-dependent and MyD88-independent (TRIF-dependent) pathways.

              The MyD88-dependent pathway: regulated by two adaptor-associated proteins: Myeloid Differentiation Primary Response Gene 88 (MyD88) and TIR Domain-Containing Adaptor Protein (TIRAP). MYd88 has two domain, TIR domain and death domain (DD). Upon LPS stimulation, MyD88 recruits and activates a death domain-containing kinase, IL-1 receptor-associated kinase-4 (IRAK-4) which is important for transmitting TLR signals, including the induction of proinflammatory cytokines. MyD88 signaling also activates IL-1 Receptor-Associated Kinases (IRAKs) and TNF Receptor-Associated Factor 6 (TRAF6). TRAF6 is an adaptor molecule that leads to the activation of MAPK cascades (Mitogen-Activated Protein Kinase) and IKK (IκB Kinase). IKKs signaling pathway leads to the induction of the transcription factor NF-κB, on the other hand activation of MAPK signalingleads to the activation of second transcription factor AP-1. TIRAP-MyD88 regulates early NF-κβ activation and production of proinflammatory cytokines, such as IL-12.​

              The MyD88-independent pathway: TRIF is the key adaptor protein that mediates MyD88-independent signaling. Studies with TRIF-deficient macrophages have demonstrated that TRIF plays a vital role in the activation of transcription factor IRF3, and the late-phase activation of NF-κB and MAPK. TRIF recruits TRAF3 to activate IRF3, subsequently TRAF3 gets associated with TANK (TRAF family member-associated NF-kB activator), TBK1 (TANK binding kinase 1) and IKKi to mediate downstream signaling. Together with NF-κB, IRF3 triggers the transcription of target genes, e.g., Type I interferons. Induction of Type I interferons and interferon-inducible genes are pre-dominant in anti-viral and anti-bacterial immune responses (Fig 7) [20].

              RESULTS AND DISCUSSION

              Expression Profile of TLR 4 in Oral Pre-Cancer, Cancer and Control Subjects

              Screenshot (30).png
                MyD88 dependent pathway(A) and Independent pathway(B)

                MATERIALS AND METHODS

                Clinical Specimens

                The present study included 20 oral parafilm embedded tissue specimens. Out of which, 5 samples from pre-cancerous patients, 10 from cancerous and 5 subjects were considered as controls. Tissue specimens were taken in 10% formalin solution. The tissue was cut into thin sections (~ 5 µm thick) using a microtome and was placed on poly L-lysine (Sigma, St. Louis, MO, USA) coated slides and processed for conventional histological assessment by H & E staining and Immunohistochemistry (IHC). All the collected clinical specimens were confirmed histo-pathologically prior to including in the study. All samples were collected from collaborating hospitals of ICMR- NICPR, Noida. The procedure of IHC was done according to the protocol used in our laboratory.

                Expression of TLR4 Gene by Immunohistochemistry

                Paraffin-fixed slides having tissues of oral cavity were heated for a short period of time and deparaffinizing was carried out by washing the slides twice in xylene for 15 minutes each, followed by acetone washing for 8 minutes and then washing in 100% ethanol for 8 minutes. The slides were then washed under running tap water for 30 minutes. The slides were incubated in 12% blocking solution (44mL of methanol and 5 mL of H₂O₂) for 10 minutes followed by 3 times of washing in 1X TBS (Tris base and NaCl maintained at pH 7.4) solution in water-bath shaker for 15 minutes each at 37⁰C. The epitopes were unmasked by immersing the slides in 10 mM citrate buffer and heating in the microwave oven for 10 minutes at 800W and for 5 minutes at 400W. The slides were then washed 3 times in 1X TBS solution at 37⁰C with an interval of 15 minutes each. The slides were incubated overnight with primary antibody prepared in 1% BSA solution (1: 50) in a humidified chamber at 4⁰C. The slides were then given 3 washing in 1X TBS the next day with an interval of 15 minutes each. Enough amount of secondary antibody (horseradish peroxidase) was added to the slides and the slides were washed in 1X TBS for 3 times with an interval of 15 minutes each. Substrate buffer was prepared using 3, 3’-diaminobenzidine (DAB) and was added to the tissues in dark, allowing the color to develop. The slides were initially washed with running water and any excess solution was blotted dry. The slides were finally counter-stained with haematoxylin for approximately 10 seconds and then washed in running water and then kept for drying. The slides were mounted using DPX solution and were observed under the microscope.

                Scoring of Immunohistochemical Staining

                Immunohistochemical scoring of our targeted protein has followed by scoring criteria. In brief, the positive stain cell percentage ranging from 0 to 100%. The ranging criteria from 1 to 4+ as 0 to 10% = 0; 10 to 30% = 1; 30 to 50% = 2; 50 to 70% = 3 and 70 to 100%= 4. Protein expression was also validate according to cell staining graded 0–3 =‘0’ count -ve staining; ‘1’ count for weak; ‘2’ for modest and ‘3’ for strong staining.

                The MyD88-independent pathway: TRIF is the key adaptor protein that mediates MyD88-independent signaling. Studies with TRIF-deficient macrophages have demonstrated that TRIF plays a vital role in the activation of transcription factor IRF3, and the late-phase activation of NF-κB and MAPK. TRIF recruits TRAF3 to activate IRF3, subsequently TRAF3 gets associated with TANK (TRAF family member-associated NF-kB activator), TBK1 (TANK binding kinase 1) and IKKi to mediate downstream signaling. Together with NF-κB, IRF3 triggers the transcription of target genes, e.g., Type I interferons. Induction of Type I interferons and interferon-inducible genes are pre-dominant in anti-viral and anti-bacterial immune responses (Fig 7) [20].

                TRIF is the key adaptor protein that mediates MyD88-independent signaling. Studies with TRIF-deficient macrophages have demonstrated that TRIF plays a vital role in the activation of transcription factor IRF3, and the late-phase activation of NF-κB and MAPK. TRIF recruits TRAF3 to activate IRF3, subsequently TRAF3 gets associated with TANK (TRAF family member-associated NF-kB activator), TBK1 (TANK binding kinase 1) and IKKi to mediate downstream signaling. Together with NF-κB, IRF3 triggers the transcription of target genes, e.g., Type I interferons. Induction of Type I interferons and interferon-inducible genes are pre-dominant in anti-viral and anti-bacterial immune responses (Fig 7) [20].

                TLR 4 protein expression in oral pre-cancer, cancer and control samples by Immunohistochemistry

                Expression of TLR4 was observed as light or dark brown granules in cytoplasm or on cell membrane shown in Fig 8. Dark brown staining corresponds to higher expression of TLR4. Immunohistochemistry results showed that the expression level of TLR4 was higher in pre-cancer, cancer tissues as compare to controls.

                The MyD88-independent pathway: TRIF is the key adaptor protein that mediates MyD88-independent signaling. Studies with TRIF-deficient macrophages have demonstrated that TRIF plays a vital role in the activation of transcription factor IRF3, and the late-phase activation of NF-κB and MAPK. TRIF recruits TRAF3 to activate IRF3, subsequently TRAF3 gets associated with TANK (TRAF family member-associated NF-kB activator), TBK1 (TANK binding kinase 1) and IKKi to mediate downstream signaling. Together with NF-κB, IRF3 triggers the transcription of target genes, e.g., Type I interferons. Induction of Type I interferons and interferon-inducible genes are pre-dominant in anti-viral and anti-bacterial immune responses (Fig 7) [20].

                TRIF is the key adaptor protein that mediates MyD88-independent signaling. Studies with TRIF-deficient macrophages have demonstrated that TRIF plays a vital role in the activation of transcription factor IRF3, and the late-phase activation of NF-κB and MAPK. TRIF recruits TRAF3 to activate IRF3, subsequently TRAF3 gets associated with TANK (TRAF family member-associated NF-kB activator), TBK1 (TANK binding kinase 1) and IKKi to mediate downstream signaling. Together with NF-κB, IRF3 triggers the transcription of target genes, e.g., Type I interferons. Induction of Type I interferons and interferon-inducible genes are pre-dominant in anti-viral and anti-bacterial immune responses (Fig 7) [20].

                Expression profile of TLR 4 in oral pre-cancer, cancer and control subjects
                TLR4 Expression Weak (%) Medium (%) High (%) p-Value
                Normal n=5 4(80) 1(20) 0(0) Reference
                Pre- Cancer (%) n=5 1(20) 3(60) 1(20) 0.2059 16(0.7210-355.08)
                Cancer (%) n=10 1(10) 4(40) 5(50) *0.0332 36.00(1.770-732.11)

                TLR 4 showed higher expression in oral cancer cases as compared to controls. Analyzing the expression of TLR 4 in pre-cancer, cancer and normal cases it was observed that cancerous cases showed the highest expression (50%) as compared to pre-cancer (20%) and controls (5%). This correlation showed the significant association between cases and controls (p=0.0332, OR=36.00, 95% CI=1.770 to 732.11) as shown in the Table .

                DISCUSSION

                Oral cancer is the cancer of oral cavity occurring as a result of a progressive accumulation of genetic aberrations and epigenetic changes that enables them to escape from normal cellular and environmental controls. Normal cells are constantly subjected to signals that develop a degree of autonomy from these signals, resulting in uncontrolled growth and proliferation of cells of lip, cheek, tongue, floor of mouth, gingiva and throat. Current evidences suggest that TLR play a crucial role in the activation of innate immune response against invading pathogens, production of cytokines, and development of adaptive immune responses. In contrast to their protective role against pathogen infections, TLRs are expressed on malignant cells contribute to tumor progression. Ligation of TLR4 expressed on tumor cells can also induce chronic inflammation in the tumor microenvironment known to be a tumor-promoting factor [21]. Because activated NF-κB shows antiapoptotic properties, high levels of its expression in tumor cells are associated with tumor progression and induction of chronic inflammation in the tumor microenvironment. When the expression levels of TLR4 in situ were correlated with tumor differentiation, we found that TLR4 is expressed in lesser quantity in poorly differentiated tumors and is highly expressed in well-differentiated and moderately well-differentiated tumors. Thus, in vivo activation of the tumor-associated TLR4 is likely to induce maximized chronic inflammation, promote tumor growth, and protect tumor cells from apoptosis.

                Numerous studies have been reported regarding the high expression of TLR 4 in various cancers, such as colon, liver, pancreatic, ovarian and oral cancer. Szczepanski M, et al. reported the high level of TLR4 protein expression in head and neck squamous cell carcinoma and poor expression in normal mucosa [22]. Kotrashetti VS conducted a pilot study in 2013 to analyze the expression of TLR4 in various grades of oral squamous cell carcinoma by immunohistochemical study and observed that TLR4 expression was observed in 96.29% of oral squamous cell carcinoma and 96.42% in oral epithelial dysplasia, and hence concluded that TLR4 expression increases with increasing degrees of oral epithelial dysplasia. It was suggested that TLR4 activation may promote cancer metastasis and high expression of TLRs may play a role in tumorigenic activity and survival [23]. Sun et al. conducted a study to correlate the expression of TLR4 with tumor differentiation and found that high expression of TLR4 were observed in high grade cancer where the tumors are well differentiated whereas poorly differentiated tumors exhibit weak expression of TLR4 [24]. Similar results were also reported in our present study that TLR4 was strongly expressed in cancerous and pre-cancerous oral cases while low level expression of TLR4 was observed in normal cases.

                CONCLUSION

                The present study clearly showed that the TLR-4 gene was strongly expressed in cancerous cases as compare to normal control and gives the indication this gene may help in the progression of disease. So the further study needs to validate in more samples to analyze the role of TLR 4 gene in up or down-regulating protein expression which may help in the progression of squamous cell carcinoma of oral mucosa.

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                ACKNOWLEDGEMENTS

                The satisfaction that accompanies the successful completion of any task would be incomplete without the mention of people who made it possible. I thank almighty for giving me the support and blessing to complete the project. I consider myself as a very lucky individual as I was provided with an opportunity to be a part of it. I am also grateful for having a chance to meet so many wonderful people and professionals who led me though this internship period.

                First and foremost, I would like to express my deep sense of gratitude to Indian Academy of Sciences (IASc-INSA) for providing me a golden opportunity for the fellowship and such a great platform to do a fruitful summer project. The AuthorCafe platform was extremely user-friendly and helped me in compiling my report.

                I express my heartful thanks to my professor, Dr. Mohammad Mahfuzul Haque, HOD of Department of Biotechnology, Jamia Millia Islamia, New Delhi, for his recommendation letter.

                I would like to express my gratitude to Dr. Mohammad Askandar Iqbal, Assistant professor, Department of Biotechnology, Jamia Millia Islamia, New Delhi, for taking part in useful decision and giving necessary advices and guidance and for recommending me to carry out this two-month project.

                I am deeply indebted to Dr. Mausumi Bharadwaj, Scientist G, National Institute of Cancer Prevention and Research, ICMR, Noida for the invaluable help, stimulating suggestions and encouragement and helped me complete my work.

                It is my radiant sentiment to place on record my best regards, deepest sense of gratitude to Ms. Upma Sharma and Mr. Mohammad, PhD student at NICPR, for their immense support, precious guidance, motivation and genuine encouragement throughout the period of my project which were extremely valuable for my study both theoretically and practically. Their constant guidance and advice played the vital role in making the execution of the report.

                I acknowledge Ms. Heena, Mr. Rajeshwar, Dr. Vineeta, Dr. Ayaz Shahid, Dr. Shilpi, Mrs. Manikankana, Mr. Mabood, who not only taught me the techniques but also refined my work professionly.

                Above all I am deeply indebted to my parents and my sister Firdous Haider for their moral support and encouragement, without the help of whom this project would not have been possible.

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