«Int. J. Mol. Sci. 2012, 13, 2331-2353; doi:10.3390/ijms13022331 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 ...»
Int. J. Mol. Sci. 2012, 13, 2331-2353; doi:10.3390/ijms13022331
International Journal of
Epigenetic Disregulation in Oral Cancer
Massimo Mascolo 1, Maria Siano 1, Gennaro Ilardi 1, Daniela Russo 1, Francesco Merolla 1,
Gaetano De Rosa 1,2 and Stefania Staibano 1,*
Department of Biomorphological and Functional Sciences, Pathology Section, University of Naples “Federico II”, Naples 80131, Italy; E-Mails: email@example.com (M.M.);
firstname.lastname@example.org (M.S.); email@example.com (G.I.); firstname.lastname@example.org (D.R.);
email@example.com (F.M.); firstname.lastname@example.org (G.D.R.) 2 Centro di Riferimento Oncologico di Basilicata (C.R.O.B.) Oncology Research Center of Basilicata, Rionero in Vulture, Potenza 85028, Italy * Author to whom correspondence should be addressed; E-Mail: email@example.com;
Tel.: +39-81-7462368; Fax: +39-81-7463414.
Received: 6 December 2011; in revised form: 9 February 2012 / Accepted: 13 February 2012 / Published: 21 February 2012 Abstract: Squamous cell carcinoma of the oral region (OSCC) is one of the most common and highly aggressive malignancies worldwide, despite the fact that significant results have been achieved during the last decades in its detection, prevention and treatment. Although many efforts have been made to define the molecular signatures that identify the clinical outcome of oral cancers, OSCC still lacks reliable prognostic molecular markers. Scientific evidence indicates that transition from normal epithelium to pre-malignancy, and finally to oral carcinoma, depends on the accumulation of genetic and epigenetic alterations in a multistep process. Unlike genetic alterations, epigenetic changes are heritable and potentially reversible. The most common examples of such changes are DNA methylation, histone modification, and small non-coding RNAs. Although several epigenetic changes have been currently linked to OSCC initiation and progression, they have been only partially characterized. Over the last decade, it has been demonstrated that especially aberrant DNA methylation plays a critical role in oral cancer. The major goal of the present paper is to review the recent literature about the epigenetic modifications contribution in early and later phases of OSCC malignant transformation; in particular we point out the current evidence of epigenetic marks as novel markers for early diagnosis and pro
Keywords: epigenetics; oral cancer; tumor progression; prognosis; molecular therapy
1. Introduction Head and neck cancers constitute the sixth most common malignant tumors worldwide, affecting approximately 650,000 people and causing almost 350,000 cancer deaths per year [1,2]. These malignancies encompass tumors arising from the epithelium of the nasal and oral cavity, paranasal sinus, pharynx and larynx. Oral cancer is the most frequent cancer of the head and neck district, with squamous cell carcinoma being by far the commonest single entity, accounting alone for about 90% of all malignancies of the oral cavity . Due to its related high mortality and low cure rate, oral squamous cell carcinoma (OSCC) represents a major public health problem, with a great individual and socioeconomic impact. In fact, despite of the many advancements made in the field of oral cancer prevention and multimodality treatments, the five-year survival rate for OSCC remains at a disappointingly stable level, almost unchanged over the past 20 years [4,5]. The poor prognosis of OSCC is mainly due to a low response rate to current therapeutic strategies, particularly for tumors diagnosed in advanced stage. This specific subset of patients is characterized by a high occurrence of invasion to surrounding tissues, lymph node and distant metastasis and by a peculiarly high risk of second malignancy during the patient’s lifetime.
Oral carcinogenesis is a multistep process modulated by endogenous and environmental factors.
Among these latter, a major role is played by tobacco and alcohol regular intake , as well as by Human Papillomavirus (HPV) persistent infection [7–11]. These predisposing factors may lead to a wide range of genetic and epigenetic events that promote genomic instability and tumor development and progression. The genetic alterations involved in the development and progression of oral premalignancy and OSCC, are caused by irreversible changes in DNA sequence including gene deletions, amplifications and mutations leading both to oncogenes activation or tumor suppressor genes inactivation [12,13].
Epigenetics is another major player in multistep carcinogenesis of oral cancers. Here we discuss the current literature in the field of the epigenetics of oral cancer, placing a great deal of emphasis on DNA methylation, histone modification and post-transcriptional gene down-regulation by microRNAs.
The emerging but still debated role of high-risk HPV persistent infections in determining a different subset of OSCC will also be discussed, reviewing some of recent publications about this topic and pointing out the relationship between HPV infection and cancer prone epigenetic modifications. We finally aim to highlight the important translational implications of the epigenetic regulation as new diagnostic, prognostic and predictive markers in oral cancer, an actually growing field of research due to the poor reliability of conventional markers in OSCC diagnosis, treatment and follow-up .
high-level expression to complete silencing, depending on the influence of the “epimutations” which interfere with the action of activators and suppressors on specific promoters in the chromatin context . With minor exceptions (T- and B-cells of the immune system), all differentiation processes are triggered and maintained through epigenetic mechanisms. Epigenetic inheritance includes DNA methylation, histone modifications and RNA-mediated silencing. Disruption of any of these three distinct and mutually reinforcing epigenetic mechanisms leads to inappropriate gene expression, resulting in cancer development and other “epigenetic diseases” [16–19] (Table 1).
3. DNA Methylation DNA methylation is the most common and the best-studied epigenetic modification . It is mediated by different DNA methyltransferases (DNMT)  and usually involves lysine and arginine residues on histone tails. The methylation of DNA refers to the covalent addition of a methyl group to the 5-carbon (C5) position of cytosine bases that are located 5' to a guanosine base in a CpG dinucleotide. CpG dinucleotides, scattered throughout the genome, are usually found clustered in 0.5–4 kb regions, named CpG islands, the major part of which localized at the promoter of tumor suppressor genes . CpG islands of growth-regulatory genes promoter regions are often found hypermethylated in tumors, this event causing the transcriptional “silencing” of tumor suppressor genes [23,24], contributing to cancer progression; on the contrary, it has also been described the derepression of proto-oncogenes transcription by hypo/demethylation, this leading to increased mutation rates and to chromosome instability, which constitutes an early hallmark of tumor cells [25–27]. Moreover, the loss of function of tumor-suppressor genes, which often occurs in tumors, has been ascribed more frequently to epigenetic silencing through methylation than to genetic 2334 Int. J. Mol. Sci. 2012, 13 defects [20,28], supporting the hypothesis that epigenetic alterations have a significant role in every step of carcinogenesis. The genes most frequently hypermethylated and silenced in cancer cells reside in chromosome regions commonly showing loss of heterozygosity. The LOH of hypermethylated genes may provide a selective growth advantage to tumor cells, and is often involved in metastatic ability and in tumor neo-angiogenesis .
4. DNA Methylation in Oral Carcinogenesis
Although a clear correlation between the epigenetic-driven deregulation of gene expression and the oral cancer progression is at present not fully demonstrated, hypermethylation and consequent silencing of several tumor suppressor genes, out of a group of more than 40 genes, has been identified in OSCC  (Table 2); the genes found hypermethylated in OSCC cover a wide range of cellular processes, including cell cycle control (p16, p15), apoptosis (p14, DAPK, p73 and RASSF1A), Wnt signalling (APC, WIF1, RUNX3), cell-cell adhesion (E-cadherin), and DNA-repair (MGMT and hMLH1) [30–33].
4.1. CDKN2A The methylation rate of CDKN2A has been widely investigated and reported in the literature.
CDKN2A gene maps on chromosome 9p21 and encodes the cell cycle regulatory protein p16, which inhibits the cyclin-dependent kinase 4 and 6 activity, inducing cell-cycle arrest in the G1 phase.
The reported incidence of hypermethylation of p16 ranges from 23% to 76% in OSCC [41,43,54–58].
Some studies examined also this phenomenon in oral mucosa with different degree of dysplasia (pre-neoplastic lesions, OIN) [59,60] and in normal adjacent mucosa, showing higher values of hypermethylation in OIN compared to normal mucosa, but lower than in the OSCC.
4.2. E-Cadherin and N-Cadherin
CDH1 gene (cadherin 1 type 1) is located on chromosome 16q22.1 and encodes for E-cadherin, a 120-kd single-span transmembrane glycoprotein, with five extracellular and one cytoplasmic domain, interacting with catenins. This molecule is mainly involved in the formation of adhesive junctions in epithelial cells, playing a fundamental role in in many aspects of the establishment and maintenance of intercellular adhesion, cell polarity, intracellular signaling and tissue architecture. Several studies evaluating E-cadherin expression in different carcinomas, documented the crucial role of this molecule during tumor progression and invasion. In fact E-cadherin absence is strictly linked to alterations in cell key functions and motility. In addition it is shown that the loss of its expression is frequently involved in tissue metastasis. Similarly to others malignancies, it was shown a correlation between low expression of E-cadherin and a more aggressive behaviour of OSCC. Hypermethylation of CDH1 was 2336 Int. J. Mol. Sci. 2012, 13 also extensively reported [41,57,61–63]: in these studies the E-cadherin gene hypermethylation frequency ranged between 7% and 46% [30,64–65]. In a recent review, Vered et al. analyzed the recent literature evaluating the immunoexpression of E-cadherin in OSCC, highlighting the confusion existing about the expression of this protein in normal and neoplastic tissue. They stressed the need for a critical review of the IHC-based expression evaluation of this molecule, in order to better define the association between its expression and clinical outcome .
In a recent study, Di Domenico M examined N-cadherin expression, a calcium-dependent adhesion protein, in series of 94 OSCC. Neoplastic tissue showed a significantly higher expression of this protein, almost exclusively cytoplasmic, than normal tissue. In addition tumors with high N-cadherin value were characterized by a more aggressive behaviour. These data suggested that N-cadherin could have a potential role in predicting the biological behavior of SCC .
PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor-suppressor gene located on chromosome 10q23.3, the loss of which expression is thought to be involved in important cellular processes including survival, differentiation, proliferation, apoptosis and invasion. In addition, due to lack of control of the signaling pathways that mediate apoptosis and migration, such as Ras/phosphoinositide 3-kinase (PI3K)/AKT, it plays a fundamental role in tumor cell survival and proliferation and metastasis. PTEN is frequently deficient in several malignancies because of mutations or epigenetic changes [68,69] In addition evidences has also been provided supporting that CpG islands of the PTEN promoter are methylated in several type of human cancers, such as endometrial carcinoma , gastric , non-small-cell lung carcinoma  and cervical cancer .
Kurasawa et al. analyzed the immunohistochemical expression of PTEN in 113 OSCC and 9 OSCC cell-lines . The resulting data showed a significant difference of expression between tumor samples and normal tissues. No mutations were showed, but in 4 out 6 OSCC cell lines a lower expression of PTEN mRNA were observed. Taken together these results supported the hypothesis that PTEN plays an important role in OSCC pathogenesis and that its down-regulation is due to hypermethylation . However, the role of PTEN in OSCC remains uncertain. Squarize et al.
demonstrated that aggressive OSCC did not express PTEN  and Shin et al. showed that the genetic or epigenetic inactivation of PTEN was related to OSCC carcinogenesis , while several authors didn’t support this association [50,74,75]. Several studies have investigated the role of pTEN in OSCC and correlated the abnormal expression of PTEN to the occurrence, development and invasion of OSCC . In a recent review, Diez-Perez et al. report the data relative to a comparison study between oral cancer tissue and normal mucosa, showing a 77.8% reduction of gene expression, due to its promoter methylation .
respond to stress or damage, leading to genomic instability. P53 is mutated in the majority of human cancers, including oral cancers, with frequency ranging from 25% to 69% [77–80]. In several instances, however, p53 shows a loss of function due to epigenetic events, instead of genetic alterations. This is the case of the epigenetic inactivation of p53 non mutated protein by the E6 protein of high-risk HPVs (mostly HPV16 and 18), in OSCC as in some laryngeal cancers (see below for further discussion about this topic).
DAPK1 (death associated protein kinase 1) gene maps on chromosome 9q34.1, it encodes a pro-apoptotic calcium/calmodulin regulated serine/threonine kinase inducing apoptosis (p53-dependent apoptotic checkpoint) [43,58,81]. The reported frequency of DAPK promoter hypermethylation ranges from 18% to 27% [64,82,83].