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Epigentics mostly refers to promoter hypermethylation, although other less abundant alterations such as histone deacethylation, global genomic hypomethylation and histone remodelling are present. DNA hypermethylation represents the covalent addition of a methyl group to a cytosine nucleotide resulting in 5-methylcytosine. This modification is catalysed by the DNA methyltransferase enzyme family (DNMTs) with S-adenosyl-methionine acting as a methyl donor11. Cytosine methylation occurs in CpG dinucleotides which have an asymmetrical distribution throughout the genome. However, a small proportion of the CpG dinucleotides is clustered together in a 500 base pair long regions, called ï¿½CpG-islandsï¿½, where they take up more than half of the nucleotides. These CpG-islands are known to be located in promoter regions of approximately 50% of mammalian genes18.
The promoter sequence is a gene control region where general transcription factors and RNA polymerases bind, before the DNA transcription is initiated. Deletions, loss-of-function mutations and promoter hypermethylation can all lead to loss or decreased gene expression. According to Knudsonï¿½s two-hit-hypothesis, a tumor suppressor gene is silenced when both alleles are knocked out. Promoter hypermethylation can cause loss of heterozygosity by inducing the second hit, after a mutational first hit. In sporadic cancer, two point mutations are rarely responsible for bi-allelic inactivation of tumor suppressor genes, while promoter hypermethylation of both alleles is more common11,19. This indicates that aberrant hypermethylation is an early event in the development of tumors16.
Under physiological conditions there is a basic methylation pattern, sometimes referred to as ï¿½methylotypeï¿½20. In the basic pattern the majority of genes have promoter regions with unmethylated CpG-islands, whereas methylation of CpG dinucleotides outside the CpG-islands is abundantly present. This is thought to be part of a natural defence mechanism which safeguards the integrity of the genome during replication, on one hand by imposing transcriptional repression on large parts of mainly noncoding DNA, which may contain harmful sequences, and on the other hand supporting transcription of coding DNA through gene promoter hypomethylation3.
Compared to normal cells, there is a significant shift in the basic pattern of DNA methylation in neoplastic cells. Promoter regions are no longer hypomethylated, but increased CpG methylation does occur in promoters of specific genes, mainly involved in DNA repair, apoptosis, cell cycle regulation and tumor suppression. Simultaneously, loss of methylation in otherwise silenced regions takes place, a process named ï¿½global hypomethylationï¿½, which is characterised by an increase in overall gene expression level due to weakened transcriptional repression. These changes indeed affect genetic stability and contribute to cancerization of the cell. Aberrant promoter hypermethylation is present is almost all types of cancer, including HNSCC, and the pattern of methylation appears to be tumor type specific16,21. In addition to that, the possible reversibility of abnormal methylation patterns makes DNA hypermethylation an appealing target for new cancer specific therapy. In fact, several chemotherapeutic agents have been found to possess demethylating properties which can reverse the transcriptional silencing of oncogenes22.
Promoter hypermethylation in oral and oropharyngeal squamous cell carcinoma
Epigenetic DNA alterations play a prominent role in cancer, including OSCC, the main focus of this review. Numerous studies have investigated epigentic alterations in OSCC and have found that promoter hypermethylation of multiple genes is highly prevalent, although various methylation rates for each gene have been reported. The silenced genes are typically tumor suppressor genes. Table 1 is a summary of candidate genes with substantial evidence for being hypermethylated in OSCC, and their reported clinicopathological associations. The inclusion criterion was as follows; hypermethylation, proven in more than one study was considered as substantial evidence. Furthermore, we made a selection of candidate genes and classified them according to their potential biomarker application as indicated by reported associations (table 2). The main emphasis of this review is the p16INK4a oncogene, because its role has been well-studied in oral and oropharyngeal squamous cell carcinoma (table 1). We also discuss the hypermethylation of the p14ARF oncogene.
The proteins p16INK4a and p14ARF are two alternative splice variants of the CDKN2A gene, located on chromosome 9p21. Both proteins function as inhibitors of cell cycle progression (figure 1). The
p16INK4a protein promotes senescence and differentiation by interfering in the retinoblastoma (Rb) pathway. It prevents entry into S phase by inhibiting the CDK4/6-cyclin D1 complexes, thereby preventing the phosphorylation of Rb proteins. As a result, E2F transcription factors are inactivated, as the Rb-E2F complex remains intact. Accordingly, dysfunctional p16INK4a results in uncontrolled cell cycle progression. The p14ARF protein activates the tumor suppressor gene p53 by inhibiting MDM2, an ubiquitin ligase that marks p53 for degradation. Overexpression of p14ARF enhances the p53 function which in turn leads to cell cycle arrest or apoptosis in cells3,52.
In the last decade, aberrant promoter hypermethylation of p16 and p14 has been observed in oral and oropharyngeal cancer tissue (table 1) as well as premalignant oral lesions53-57 and histologically healthy mucosa surrounding the tumor38,39,58. More important, p16 and p14 gene inactivation is primarily due to promoter methylation30. In one of the earliest studies focussing on OSCC as a distinct entity, Wu et al (1999), demonstrated that the vast majority of the tumors (>80%) had loss of p16 expression34. Interestingly, p16 promoter hypermethylation appeared to be more common than point mutation (23% and 7%, respectively). In a Brazilian cohort of 45 patients with resected primary OSCC tumors, Crodeiro-Silva at al. (2012) investigated the methylation status of four genes and found high rates of hypermethylation for CDKN2A (p16 and p14), EDNRB, RUNX3 and SFN. They also reported more CDKN2A and EDNRB promoter hypermethylation in subjects with lymph node metastases40. In an Indian cohort, Kaur et al. (2010) selected four genes and evaluated their methylation status in a sample of 92 OSCC patients39. EDNRB, KIF1A, p16 and DCC were found to be highly methylated in tumor tissue, and p16INK4a methylation was associated with nodal involvement. In another study a semi-quantitative approach (pyrosequencing) was adopted in order to quantify the promoter hypermethylation of five genes in OSCC samples and analysed whether there is a correlation between the quantitative methylation index and clinicopathological variables from the patients33. No such correlation was observed, methylation of the genes p16INK4a, CYGB and CYCA1 however, was highly tumor specific, because healthy resections margins contained significantly less abnormal methylation for these genes. Also, hypermethylation of ECAD and RARï¿½ was observed in tumor tissue and adjacent healthy mucosa. No significant hypermethylation is observed in healthy control tissue33,39.
Several studies have evaluated the presence of promoter hypermethylation in oral premalignant lesions. In an early study, loss of p16 function was reported in a small number of patients (17/37) with leukoplakia, accounting for 5 out of 8 patients who developed malignant transformation53. Also, increasing p16 methylation rates were reported for mild to severe dysplastic lesions, 30% and 82% respectively54. In patients with severe dysplastic epithelial lesions, Kresty et al (2002) detected a p16 methylation rate of 57%, while p14 was methylated in 3.8% of the samples55. An association was found for p16 hypermethylation with loss of heterozygosity and lesions of the tongue and floor of the mouth. Two studies investigated the prognostic significance of p16 hypermethylation in oral epithelial dysplasia56,57. In the first study, a significant proportion of patients with malignant transformation of epithelial dysplasia had p16 hypermethylation compared to patients with no malignant transformation (57% vs 8%, p=0.002). However, p16 did not correlate with time of onset of transformation56. Cao et al (2009), supported these findings when they described a significantly higher progression rate for oral dysplasia to OSCC in p16 hypermethylated cases (43.8% vs 17.4%; OR=3.7)57. This effect was more evident in patients aged above 60 years (OR=12.0, p=0.013) and subjects with moderate epithelial dysplasia (OR=15.6, p=0.022). These findings suggest that p16 hypermethylation is a powerful marker for selecting patients with precancerous lesions who are at risk for progression to malignant disease.
Although not fully convincing due to small sample size, the feasibility of p16 hypermethylation in surgical resection margins was confirmed by Goldberg and colleagues demonstrating hypermethylation in margins of three patients with SCC of the tongue59, and in a later study, positive margins were reported in 4 OSCC patients of which two developed a local recurrence23. Recently, a prospective study including a larger number of Indian patients with SCC of the tongue, showed that 43% (13/30) of histologically tumor free margins contained p16 hypermethylation and further analysis showed a 6.3 fold increased risk for local recurrence for this margins58. Still, the p16 methylation status did not affect the overall survival rate. A more recent study in OSCC resection margins also could not establish a correlation between p16 hypermethylation and overall survival42. To our knowledge these two studies are the first to evaluate the significance of p16 hypermethylation as a predictive and prognostic marker in surgical resection margins. Further research is needed to investigate whether intraoperative p16 hypermethylation analysis in surgical margins results in more accurate resection with less recurrence compared to the conventional histopathological assessment.
The fact that aberrant promoter hypermethylation of p16 and p14 is detected in both peritumoral tissue and premalignant lesions, suggests that these epigenetic alterations are an early event, making tissue more prone to cancerization. These findings are in concordance with the concept of ï¿½field cancerizationï¿½, originally proposed by Slaughter et al. in 1953 to explain the high recurrence rate of head and neck cancers60. They hypothesised that multiple acquired genetic defects in large patches of mucosa in the upper aerodigestive tract, make morphologically normal epithelium prone to dysplastic or malignant transformation3,60. This so called ï¿½fieldsï¿½ are not limited to the boundaries of the malignancy, but extend into surgical resection margins and increase the risk of local relapse or a second primary tumor.