Epigenetic Biomarkers in Head and Neck Cancer

Head and neck cancers (HNCs) are the most prevalent and aggressive type of cancers. Genetic, epigenetic, environmental and viral risk-factors are associated with HNC carcinogenesis. Persistent infection of oncogenic human papillomaviruses (HR-HPVs) represent distinct biological, molecular and epigenetic entities in HNCs. There are three main epigenetic mechanisms that regulate transcription, these are DNA methylation, histone modifications and alteration in non-coding RNA networks, which can dissected to identify innovative and accurate epigenetic biomarkers for diagnosis and prognosis of HNC patients. Due to the lacunae of accurate distinctive biomarkers for the definite diagnosis of HNC, the identification of predictive epigenetic markers is necessary that might modify or increase HNC patient’s survival. In this mini review, we briefly summarize the current knowledge of different epigenetic biomarkers in HNC. DOI : 10.14302/issn.2572-3030.jcgb-18-2428 Corresponding author: Shilpi Gupta, National Institute of Cancer Prevention and Research (ICMR), I -7, Sector 39, NOIDA, Uttar Pradesh 201301, INDIA, Email: shilpimicrobio@gmail.com, drsgupta03@gmail.com


Introduction
Head and neck cancers (HNCs) are the 6 th most common and heterogenous group of malignancies with about 780,000 new cases being diagnosed every year and more than 350,000 deaths reported annually [1].
Head and neck carcinogenesis is mainly caused by chronic use of tobacco products, excessive alcohol intake and persistent infection with oncogenic human papillomaviruses (HR-HPVs).
HPV-positive HNCs represent distinct biological, molecular entities in comparison to the HPV-negative and tobacco induced HNCs.HR-HPV infection significantly effect HNC prognosis.The HPV infected HNC patients are mainly non-tobacco smoking, younger age population with better prognosis than those with HPV-negative HNC patients [2][3][4][5][6].These strong evidences establish the distinct molecular and clinical role of HPV as a unique viral biomarker for HNC diagnosis and prognosis.These distinct entities can be due to epimutations inspiring further investigation on molecular and epigenetic modifications to modify HNC patient's outcome.
It seems that HNC is as much a disease of misdirected epigenetic factors as that of genetic factors, which has emerged into one of the "hallmarks of cancers".Various scientific studies have suggested that epigenetic alterations such as DNA methylation and the post translational modifications of histone proteins and/ or mis-expression of non-coding RNAs (ncRNAs), are frequently associated with initiation, progression, differentiation and drug resistance in HNCs [7].
Therefore, these epigenetic markers are emerging as Epigenetic alterations are also involved in cellular plasticity during cancer progression, and further contribute to formation of cancer stem-like cells or cancer stem cells (CSCs) [10,11].In this mini review, we explore different epigenetic mechanisms that are known to be altered during HNC carcinogenesis.Writers (Histone Modifiers)

Methylation Specific Biomarkers
The structure of chromatin is dynamic and its fundamental unit is the nucleosome, which consists of Over-expression of Polycomb complex member, enhancer of zeste homolog 2 (EZH2) in tongue cancer, correlates with reduced overall survival and was found to be of great prognostic importance [20].In another study, SUZ12 overexpression in HNCs correlated with cervical node metastasis along with reduced survival.It was further shown that the knockdown of SUZ12 inhibited cell proliferation and migration [21].In an independent study, mutations and copy number variations (CNVs) in MLL1, MLL2, MLL3, KDM6A and KDM6B were found to be a contributing factor in HNC progression [22].Number of missense, nonsense and frameshift mutations in MLL2 and EZH2 are associated with HNCs [23].Increased expression of histone deacetylase-2 (HDAC-2) associated with metastasis along with poor survival and further, served as a good prognostic marker for HNC patients [24].These studies suggest that treating HNC patients by targeting and    [35].These researches highlight the potential role of piRNA-mediated epigenetic regulation in cancer-related processes in human somatic cancers including HNCs [36].Several studies have correlated piRNA expression to clinical parameters associated with HNCs [37].Dysregulation of piRNAs was found to be associated with overall survival and can be utilized as a signature in HNC patients [38].Since piRNAs are emerging players in HNCs, a comprehensive characterization of piRNAs can be used to develop effective therapeutic strategies for HNC.cancer pateints [56,57].Moreover, higher expression of SNORA42 (Box H/ACA) served as an essential prognostic marker for cancer patients [58].Chamorro-Petronacci and co-workers (2018) reported that 14 snoRNAs were downregulated and 1 snoRNA was overexpressed in HNC patients [59].These emerging findings highlight the potential role of snoRNAs in carcinogenesis and further point towards the possible role of snoRNAs as unique molecular markers and drug targets for the treatment of HNCs [60].

MiRNAs
important and reliable tools for early diagnosis and prognosis of HNC.In addition, dysregulation in any of these 3 distinct and mutually reinforcing epigenetic mechanisms lead to mis-regulation of gene expression, resulting in the development of HNCs [7-9].Epigenetic alterations causing aberrant transcriptional activity, provide selective advantage to the tumor through the silencing of tumor suppressor genes or activation of oncogenes and/or mutation effecting DNA repair genes.
165bp of DNA strand wrapped around the histone octamer core, comprising of two copies of 4 globular histone proteins (H2A, H2B, H3, and H4).Histone proteins play an essential role in regulating the epigenetic state of the cell [19].The posttranslational modification (PTM) of histones and DNA is an important and frequently observed in HNCs.These histone alterations are closely related to DNA methylation, leading to carcinogenesis.Chromatin protein marks which cooperate with DNA methylation marks in maintaining gene expression, are altered in various cancer cells including HNCs.Expression of the genes coding for polycomb and trithorax group members (which frequently play roles during development to establish chromatin protein marks) are known to be overexpressed in HNCs as shown in Figure 1 (GEPIA).
modulating different epigenetic genes (responsible for control of cell survival, proliferation, migration, differentiation and apoptosis) may improve the efficacy of conventional HNC therapy.Readers (Chromatin Remodelers) ATP-dependent chromatin remodelers are responsible for opening and closing of chromatin.In the form of multimeric protein complexes, they catalyze the nucleosome sliding or repositioning, ejection and unwrapping.The chromatin remodeling enzymes are classified into different families on the basis of sequence similarities in the ATPase domain and differential combination of accessory protein domains.These are categorized in four types; (i) SWI/SNF (mating type SWItch/Sucrose Non-Fermenting) family, (ii) ISWI (Imitation SWItch) family, (iii) CHD (Chromodomain Helicase DNA-binding) family and (iv) INO80 (INOsitol requiring) family.The chromatin remodelling enzymes consist of domains such as the bromodomain (recognition of acetylated lysines) [25], Chromodomain and PHD finger (recognition of methylated-lysines) [26], and HAND-SANT-SLIDE domains for DNA binding [27], Helicase SANT associated (HSA) domain, play an important role in recognition of various histone modification marks and catalyze nucleosome sliding or ejection.The HSA domain overlaps with the DNA binding domain known as DBINO (DNA binding domain ofIno80)[28,29].A number of genes coding for chromatin remodelling proteins show altered expression in various cancers.In Figure2, meta-analysis using GEPIA for genes encoding for chromatin remodelling proteins, show majority of these genes over-expressed (BRG1, BAF47, BAF57, BAF53a, BAZ1A, CHRAC1, CHRAC17, CHD4, RUVBL1 AND RUVBL2) in HNCs.It is expected that over-expression of chromatin remodelling enzymes may lead to global changes in nucleosome occupancy profiles altering gene expression patterns.Altered expression of Remodelling and Spacing Factor-1 (RSF-1) has been analysed in several tumors including HNCs.Gain-of-function of RSF-1 leads to aggressive tumor Freely Available Online www.openaccesspub.org| JCGB CC-license DOI : 10.14302/issn.2572-3030.jcgb-18-2428Vol-1 Issue 2 Pg.no.-44

Figure 2 .Figure 1 .
Figure 2. Comparison of expression (transcripts per million) for Chromatin Remodelling family members in normal versus the head and neck cancer samples.Analyzed using the data from GEPIA miRNAs are the class of small, single-stranded ncRNAs, of about 22 nucleotides in size[39].miRNAs constitute the largest class of non-coding RNA molecules that have either tumor suppressor or oncogenic effects and control various physiological and pathological processes such as cell growth, proliferation, apoptosis, and differentiation wherein each miRNA has the potential to target multiple mRNAs or gene targets.A number of studies have demonstrated that some miRNAs affect gene expression and dysregulate signaling networks involved in HNC pathogenesis [40, 41].Earlier, it was analysed that in majority of cases, 4 of the most important tumor suppressor miRNAs (miR-34b, miR-137, miR-193a, and miR-203), which are loated in close proximity to the CpG islands, were silenced through hypermethylation [42].On the other hand, upregulation of oncogenic miR-21 was associated with Freely Available Online www.openaccesspub.org| JCGB CC-license DOI : 10.14302/issn.2572-3030.jcgb-18-2428Vol-1 Issue 2 Pg.no.-46 increase nodal metastasis and invasiveness inHNCs[43][44][45].An early research revealed that polymorphisms in miR-146, miR-149, miR-196 and miR-499 were found to increase the risk of non-smoker HPV-induced HNC patients[46].miR-146 polymorphism is associated with an increased risk of HNCs[47].Overexpression of miR-21, miR-181b, miR-184 and miR-345 has been associated with malignant transformation during HNC[48].Higher expression of miR-21, miR-34c, 184 and miR-155 is known to play a role in anti-apoptotic and proliferative processes leading to downregulation of SOX7 protein and overexpression of c-Myc, Cyclin D1 and Bcl-2 gene[49].DNA hypomethylation induced upregulation of miR-663, leads resistance to chemotherapy in breast cancer cells[50] and this has been exploited to serve as a promising non-coding biomarker for HNC.Comprehensive profiling of global miRNAs levels in HNCs is yet to be investigated.These studies are essential for stratification of HNC subtypes on the basis of HNC subsites, HPV status and mechanism along with detailed functions of miRNAs for development of effective therapeutic evaluations.The Small Nucleolar RNAs (snoRNAs) snoRNAs are a type of linear transcripts with 60-300 nt size.They are primarily classified into two types of snoRNAs; (i) C/D box and (ii) H/ACA box [51-54].snoRNAs are linked with a set of core proteins interacting partners known as sno-ribonucleoproteins (RNPs) to form stable and functional snoRNP particles [55].It has been shown that SNORD78 (Box C/D) is upregulated in lung and prostate

Freely
Available Online www.openaccesspub.org| JCGB CC-license DOI : 10.14302/issn.2572-3030.jcgb-18-2428Vol-1 Issue 2 Pg.no.-47 cells [63].Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was shown to be overexpressed in HNCs and have been further shown to play an essential role in maintaining epithelial-mesenchymal transition (EMT)-mediated migration in HNCs [64] hence serving as a unique prognostic biomarker.HOTAIR, a lncRNA act as a biomarker for aggressive HNCs as its overexpression is associated with bad prognosis in different subtypes of HNCs [65].Since the role of lncRNAs have been well-characterized in HNC carcinogenesis, they can be used as novel molecular markers for HNC diagnosis and further carry a potential therapeutic advantage for aggressive HNC Concluding Remarks Growing knowledge on epigenetic alterations such as DNA methylation, histone modifications, and various ncRNA signalling pathways in HNCs may help in the understanding of disease pathogenesis and offer to identify novel unique epigenetic biomarkers for disease diagnosis, prognosis and treatment response for highly heterogenous group of HNCs.Specific targeting of these epigenetic alterations will help in developing novel epigenetic therapeutic approaches to enhance chemo-radio sensitivity and efficacy in HNCs.