**5. Epigenetic changes in laryngeal cancer**

Laryngeal cancers are the most frequent cancers of the head and neck region and their occurrence is strongly associated with the exposure to cigarette smoke and the consumption of strong alcohols. Almost all laryngeal cancers are squamous cell carcinomas (Paluszczak et al, 2011).

The chondroitin/dermatan sulfate fine chemical structure is altered in laryngeal carcinomas (Kalathas et al, 2009) as well as in most cancers. In healthy larynx, chondroitin/dermatan sulfate (C-6 and C-4 sulfated) in the cartilaginous parts is present in greater amounts compared to cancer. Moreover, the decrease in cancer is more abrupt in C-6 sulfation; C-4 sulfation is diminished gradually to the advanced stages of cancer. These alterations may be due to differential biosynthesis of core protein precursors, to differences in the substrates

fundamental pathways has been reported in NPC, such as apoptosis, DNA damage repair, tumor invasion and metastasis. Many TSGs were aberrantly methylated in their 5'CpG islands: 84% of the RASSF1A, 80% of the RARβ2, 76% of the DAP-kinase, 46% of the p16, 89,7% of the CDH13, 65% of the CHFR, 50,9% of the RASSF2A (Li et al, 2011). DNA methylation may play an important role in the maintenance of specific EBV latency programmes in the nasopharyngeal carcinoma cells. Methylation of both viral and cellular genes may be involved in the transformation of nasopharyngeal epithelial cells. Induction of epigenetic alterations in certain cellular genes was proposed as one of the mechanisms for

Loss of heterozygosity at 3p21 is common in various cancers including nasopharyngeal carcinoma (NPC). BLU is one of the candidate tumor suppressor genes (TSGs) in this region. Ectopic expression of BLU results in the inhibition of colony formation of cancer cells, suggesting that BLU is a tumor suppressor. Qiu and colab. (2004) identified a functional BLU promoter and found that it can be activated by environmental stresses such as heat shock, and is regulated by E2F. The promoter and first exon are located within a CpG island. BLU is highly expressed in testis and normal upper respiratory tract tissues including nasopharynx. However, in all seven NPC cell lines examined, BLU expression was downregulated and inversely correlated with promoter hypermethylation. Biallelic epigenetic inactivation of BLU was also observed in three cell lines. Hypermethylation was further detected in 19/29 (66%) of primary NPC tumors, but not in normal nasopharyngeal tissues. Treatment of NPC cell lines with 5-aza-20-deoxycytidine activated BLU expression along with promoter demethylation. Although hypermethylation of RASSF1A, another TSG located immediately downstream of BLU, was detected in 20/27 (74%) of NPC tumors, no correlation between the hypermethylation of these two TSGs was observed (Qiu et al, 2004). Aberrant "epigenetic code" of cell signaling facilitates the subsequent selection of genetic mutations of certain signaling pathways in the initiation and progression of NPC. As more epigenetic alterations of cell signaling genes are found, we will obtain systematic understanding of the molecular features of NPC. Study of epigenetically silenced cell signaling regulators in NPC will lead to the further development of clinical strategies of NPC prevention and therapy. Moreover, promoter methylation of cell signaling regulators could serve as diagnostic biomarkers for NPC risk assessment, early detection, and

Laryngeal cancers are the most frequent cancers of the head and neck region and their occurrence is strongly associated with the exposure to cigarette smoke and the consumption of strong alcohols. Almost all laryngeal cancers are squamous cell carcinomas (Paluszczak et

The chondroitin/dermatan sulfate fine chemical structure is altered in laryngeal carcinomas (Kalathas et al, 2009) as well as in most cancers. In healthy larynx, chondroitin/dermatan sulfate (C-6 and C-4 sulfated) in the cartilaginous parts is present in greater amounts compared to cancer. Moreover, the decrease in cancer is more abrupt in C-6 sulfation; C-4 sulfation is diminished gradually to the advanced stages of cancer. These alterations may be due to differential biosynthesis of core protein precursors, to differences in the substrates

enhancing the transformation of nasopharyngeal epithelial cells by EBV infection.

prognosis (Li et al, 2011).

al, 2011).

**5. Epigenetic changes in laryngeal cancer** 

pool, and to differential expression of the enzymes involved in chondroitin/dermatan sulfate biosynthesis.

The purpose of Kalathas and colab. (2010) was to examine the expression of the various chondroitin/dermatan sulfate synthesizing and modifying enzymes in laryngeal cartilage in healthy, macroscopically normal and cancerous specimens by RT-PCR analysis and western blotting. Furthermore, methylation specific PCR (MSP) was used to find out if DNA methylation is a regulative mechanism of their expression in laryngeal cancer. C4ST1 gene expression was very low in healthy specimens (20 times lower compared to the GAPDH gene), and increased in patients' specimens as indicated by both RT-PCR and western blotting. Its expression seemed to be controlled via methylation of a CpG island, since hypomethylation of the gene was observed in the pathologic samples compared to the macroscopically normal samples. D4ST1 gene was about equally expressed with the GAPDH gene and possessed its highest expression in the healthy tissues. In cancer, its expression was decreased 4 to 5 times and it was about equal between normal and pathologic samples. The CpG island near the promoter region was fully unmethylated therefore it did not affect the enzyme expression. DSE expression was not detected in the macroscopically normal samples, and the highest levels of it were observed in the pathologic samples, as indicated by RT-PCR, being about 10-times more compared to healthy. DSE expression seemed to be controlled by methylation of the promoter region in certain samples; the pathologic samples were hypomethylated compared to the macroscopically normal. The differential modification of the various glycosaminoglycans during cancer reflected differential expression of the enzymes involved in their biosynthesis. In their study, the clearest observations in laryngeal cancer were the significant decrease of CHSY3, CHST3 and D4ST1, and the significant increase of DSE. DSE is responsible for the epimerization of glucuronic acid in dermatan sulfate chains, which in addition require D4ST1 for their sulfation. The differential expression of only these two enzymes, which are highly responsible for the biosynthesis of dermatan sulfate, a glycosaminoglycan with tumor-inhibitory activity, indicates that a simple imbalance in enzymes' expression may affect tumor progression.

Different environmental factors are able to modulate the epigenetic information. It is widely accepted that the aberrant DNA methylation changes can be induced by both cigarette smoke and alcohol. Alcohol consumption and smoking induced the hypermethylation of p15 in the upper respiratory tract cells (Chang et al, 2004), while the hypermethylation of MGMT, p16 and DAPK was associated with tobacco-chewing induced oral cancers (Vuillemont et al, 2004). A correlation was also found between cigarette smoking and the hypermethylation of CDH1, RARbeta and FHIT (Van Engeland et al, 2003). Alcohol consumption was associated with the methylation of APC, p14, p16, MGMT, RASSF1A and hMLH1 (Van Engeland et al, 2003). Since alcohol and tobacco are the major risk factors for the development of laryngeal squamous cell carcinomas (LSCC), one can predict the frequent occurrence of the methylome aberrations in LSCC patients.

Since Slaughter's proposal of a genetic field defect concept for the explanation of the local relapse occurrence, much evidence has accumulated for its confirmation. The last ten years brought evidence that the genetic changes in the field are frequently accompanied by epigenetic aberrations. The epigenetic field defect was observed for oesophageal (Oka et al, 2009), lung (Guo et al, 2004) or stomach (Ushijima, 2007) cancers. Similarly, epigenetic

Epigenetics in Head and Neck Squamous Cell Carcinoma 187

and not necessarly to the hardness of the wood. Woo dust is a complex mixture of compounds including a wide variety of biologically active substances, also genotoxic and carcinogenic compounds (Hanahan & Weinbrg, 2000). The particulate nature of the wooddust exposure plays a role in generating reactive species of oxygen (ROS) within cells and inducing DNA damage and evoking an inflammatory response (Bornholdt & Saber, 2007). Multiple mechanism of carcinogenesis have been proposes to be involved in the development of sinonasal cancer related to wood-dust exposure, but there is very little experimental or human data in the literature. The published findings have been based on a relatively limited number of cases, mostly involving adenocarcinomas. In these studies, high frequencies of DNA copy number chamges as detected by comparative genomic hybridization have been detected (Ariza et al,, 2004 and Korinth et al, 2005), while the mutation rates reported for the KRAS gene (Fratini et al, 2006 and Yom et al, 2005) and the p53 tumor suppressor gene have been lower (Perrone et al, 2003 and Licitra et al, 2004)). Initially, KRAS and HRAS mutations were found to be quite frequent in sinonasal cancer, with implications for histogenetic and prognostic significance (Yom et al, 2005), but recent studies show that tumors with KRAS mutation might represent only a small proportion of all sinanasal carcinomas (Bornholdt et al, 2008). Most of the studies having as subject p53 mutation have concentrated on the intestinal type of adenocarcinoma, and the numbers of cases studied have been rather low. In these studies, a variable occurrence of p53 mutations has been reported (18-60%) (Licitra et al, 2004 and Perrone et al, 2003). Some of the studies have also examined the accumulation of p53 in the cell nucleus in adenocarcinoma type of

sinonasal carcer. The accumulation of p53 may reflect a p53 mutation.

inactivation can lead to disease (Ellen et al, 2009).

With the exception of chromium, which forms DNA adducts, most carcinogenic metals are weak mutagens and act by epigenetic mechanisms (Arita & Costa 2009). Nickel, a metal linked to occupational and environmental exposures, has carcinogenic effects, despite the fact that it is not known to be a mutagen (Kasprzak et al, 2003; Chen et al, 2006; Ellen et al, 2009). *In vitro* and *in vivo* experiments reveal that nickel compounds silence gene expression by causing DNA methylation, an effect explained by the ability of nickel ions to substitute magnesium in the DNA phosphate backbone and to increase heterochromatin condensation (Arita & Costa 2009). The ability of Ni2+ ions to displace Mg2+ and cause chromatin condensation, establishing dense regions of heterochromatin that prevent accessibility to the respective genomic region, was also revealed by Ellen and colleagues (2009) with atomic force microscopy and circular dichroism spectropolarimetry. Chromatin condensation could in addition trigger DNA methylation in the compacted region, which also affects gene expression. When the silenced chromosomal region contains genes that are relevant to cancer initiation or progression, such as tumor suppressor genes or senescence genes, their

Nickel compounds also induce global changes in histone acetylation, methylation, and ubiquitylation. Kang et al. (2004) linked nickel to hypoacetylation to apoptosis for the first time, when they found hypoacetylation and demethylation of histones as potential mechanisms leading to apopotosis. Golebiowski and Kasprzak (2005) reported decreased acetylation of histones H2A, H2B, H3 and H4, in a time- and concentration-dependent manner, in human and rat cell lines exposed to nickel. Chen et al. (2006) revealed that nickel decreased a specific histone demethylase and by this mechanism, it increased global H3K9 mono- and dimethylation in several cell lines, and strongly suggested that this increased methylation causes the silencing of a transgene. This effect was dependent, *in vitro*, on iron

changes in normal mucosa cells derived from surgical margins were detected in head and neck carcinomas (Martone et al, 2007). However, so far such changes have not been observed specifically in laryngeal cancers. In Paluszczak's paper (2011) evidence of a widespread occurrence of the aberrations in the profile of DNA methylation in laryngeal cancer patients is presented. Less than ten percent of cancer cases did not show any epigenetic changes in the normal mucosa samples. Gene methylation frequency was only slightly lower in the normal epithelium of epiglottis or trachea than in tumor cells. However, it should be taken into account that all the patients were exposed to similar laryngeal cancer risk factors. As discussed earlier, tobacco and alcohol are associated with the aberrations in the DNA methylation profile. The long-term exposure of patients to these factors could be responsible for the common appearance of epigenetic defects in a large field of upper respiratory tract mucosa. The existence of the epigenetically changed field in laryngeal cancers seems to be confirmed especially by such cases where lack of gene methylation in tumor cells was accompanied by the presence of hypermethylation in the normal epithelial cells although the percentage of patients with such gene methylation pattern was rather low.
