Promoter hypermethylation profile of tumor‐associated genes p16, p15, hMLH1, MGMT and E‐cadherin in oral squamous cell carcinoma

Abstract

Aberrant promoter hypermethylation of tumor-associated genes leading to their inactivation is a common event in many cancer types. Using a sensitive restriction-multiplex PCR method, we studied the promoter hypermethylation profile of the p16, p15, hMLH1, MGMT and E-cad genes in oral squamous cell carcinoma (OSCC) of Indians. We analyzed a total of 51 samples for the p15 tumor-suppressor gene and 99 samples for each of the remaining genes. Our studies indicate an incidence of promoter hypermethylation of 23% each for p16 and p15, 8% for hMLH1, 41% for MGMT and 35% for E-cad. We observed aberrant hypermethylation of the promoter region of at least 1 of these genes in 74.5% of cases (n ‫؍‬ 51) for which all the 5 genes were studied. Abnormal methylation was detected in tumors irrespective of stage and location in the oral cavity, whereas no abnormal methylation was detectable in normal oral squamous tissues obtained from 25 OSCC patients. Oral squamous cell carcinoma (OSCC) is one of the most widely prevalent cancer types in developing countries, including India, and is associated with tobacco and alcohol abuse. 1 Although recent research has given deeper insight into the etiology of the disease, its occurrence as a potentially fatal disease continues unabated. It is therefore essential to identify and develop newer risk markers for diagnosis and therapy of OSCC.Methylation profiling of promoter regions is essential for understanding the regulation of imprinted genes, X-chromosome inactivation and the role of various tumor-suppressor genes in the genesis of different types of cancer. 2,3 Methylation profiling of tumor-suppressor genes is a potentially powerful diagnostic tool for early detection of various types of cancer. 3-5 Assessment of methylation inactivation is important in assigning functional roles of genes and may have implications in the function of other interacting proteins. 3,6 Inactivation of several tumor-associated genes, especially tumor-suppressor genes, has been attributed to aberrant hypermethylation of their promoter regions. 2,3,6 We included in our study 5 tumor-suppressor genes: p16, a CDK inhibitor involved in regulation of the cell cycle by the cyclin D-Rb pathway, control of which is lost in virtually all tumor types; 7 p15, another CDK inhibitor involved in cell-cycle regulation; 8 hMLH1, the human homolog of bacterial MutL, involved in mismatch repair; 9 MGMT, the gene involved in repair of methylated guanosine residues formed due to alkylated carcinogens; and E-cadherin, involved in homotypic epithelial cell-cell adhesion. 10 All of these genes are known to be inactivated by methylation in various cancers. 3,11 Methylation profiling of CpG sites originally involved digestion of sample DNA using a methylation-sensitive restriction enzyme followed by Southern hybridization 7,12 or PCR using primers flanking the restriction site. [13][14][15] Using these assays, methylation patterns could not be determined from very small amounts of DNA and the occurrence of false-negatives due to degra...

Promoter Hypermethylation Profile of Tumour Suppressor Genes in Oral Leukoplakia and Oral Squamous Cell Carcinoma

“…Our studies indicate that the p16 and E-cadherin are the most preferentially targeted genes for methylation in oral squamous cell carcinoma of Indians. There are already reports of p16 and E-cadherin genes suitable for diagnosis of OSCC in individuals at high risk for this disease [23]. The same high percentage methylation in leukoplakia samples suggests that the methylation of these two genes [Table/ Fig-6] may be an early event in the genesis or progression of oral squamous cell carcinoma.…”

E‐cadherin is a selective and strongly dominant prognostic factor in squamous cell carcinoma: A comparison of E‐cadherin with desmosomal components

“…These (unpublished) data do not support the view that promoter hypermethylation is a frequent mechanism of downregulated or heterogeneous ecad expression in HNSCC. [55][56][57][58] In fact, Yeh et al 59 reported a lack of correlation between ecad promoter methylation and aberrant protein expression.…”

Genetic or epigenetic silencing of low density lipoprotein receptor‐related protein 1B expression in oral squamous cell carcinoma

“…(8) Although several genes, for example, p14, p15, p16, RASSF1A, VHL, hMLH1, and MGMT, have been reported to be silenced by aberrant DNA methylation, (9)(10)(11)(12)(13)(14)(15)(16) some of them were infrequently methylated in OSCC compared with other tumors. In order to create the b.est possible panel of markers for the prediction of outcome, sensitivity to chemotherapy and radiation, and disease status OSCC, more candidates for tumor-suppressor genes targeted by promoter methylation will no doubt be tested in this disease.…”