Implications of Methylation Patterns of Cancer Genes in Salivary Gland Tumors

Abstract

Purpose: We investigated the methylation status and protein expression of four tumor suppressor genes to determine their role in salivary gland tumorigenesis. Experimental Design: We performed methylation-specific PCR and protein analyses of 29 normal salivary glands, 23 benign, and 79 malignant salivary gland neoplasms to determine the pattern and potential diagnostic and/or biological role of the RASSF1, RARb2, DAPK, and MGMT tumor suppressor gene methylation in these tumors. Results: No methylation was detected in the normal tissues. Methylation occurred in 9 of 23 (39.1%) benign tumors; 3 (25.0%) pleomorphic adenomas and 6 (66.7%) Warthin's tumors at the MGMT, DAPK , or RASSF1 genes. Methylation occurred in 33 of 79 (41.8%) malignant tumors; 8 (30.8 %) adenoid cystic carcinomas, 6 (33.3 %) mucoepidermoid carcinomas, 6 (42.9%) acinic cell carcinomas, and 13 (62.0 %) salivary duct carcinomas. RASSF1 and RARb2 represented 75.8% of methylation events occurring most frequently in salivary duct and acinic cell carcinomas. Overall, we found no significant correlation between protein expression and methylation status of individual genes, but observed low or absent protein expression in several methylated tumors. Significant correlations were found between methylation and aggressive malignant phenotypes (P = 0.0004) and age (P = 0.05). Conclusions: (a) Benign and malignant salivary tumors differed in the frequency and pattern of gene methylation; (b) high-grade carcinomas were significantly methylated compared with low-grade phenotypes; (c) RASSF1 and RARb2 were highly methylated in malignant tumors and can be targeted for therapy; and (d) methylation pattern may serve as a diagnostic and biological marker in assessing these tumors.Salivary gland neoplasms are composed of histopathologically and clinically diverse entities of disputed histogenesis and unpredictable behavior (1 -3). Efforts to identify biomarkers that assist in diagnosing these tumors and in explaining their evolution and progression have been unrewarding (4 -6). The underlying reasons for such slow progress in understanding these tumors include rarity, failure to grow cell lines, clinical and methodologic interstudy differences, and the lack of a phenotypic progression model of their evolution. However, recent molecular genetic studies have provided important information on common chromosomal loci and genes alterations in a spectrum of the tumors (7 -15). These data are critical in advancing our understanding of their biology and identifying new targets of potential therapeutic applications.Epigenetic alterations may also play a role in the development of these tumors. A particular advantage of these alterations is that, unlike genetic alterations, they are reversible and can be restored in vitro and in vivo by using demethylating agents in cell lines and tumors (16 -19). Methylation is one of the epigenetic modifications that play an important role in the transcriptional inactivation of tumor suppressor genes in human cancer (20). Several m...

The proliferation marker Chromatin Assembly Factor-1 is of clinical value in predicting the biological behaviour of salivary gland tumours

“…Recent studies evidenced that epigenetic alterations may play a role in the development and progression of SGT (32)(33)(34)(35). However, a relatively limited variety of SGT histotypes and genes analyzed for epigenetic regulation has been examined (36).…”

Immunolocalization of DNMT1 and DNMT3a in Salivary Gland Neoplasms

“…Hypermethylation of the tumor suppressor genes p16INK4a, p14ARF, p27Kip1, E-cadherin, 14-3-3 , RASSF1A, DAPK, MGMT, RAR ␤ 2 and RB1 has been demonstrated in some cases of salivary gland neoplasms [9][10][11][12][13][14]22] . Also, overexpression of various DNMTs in lung cancer may result in promoter hypermethylation of multiple tumor suppressor genes which then leads to poor prognosis [18] .…”

“…Epigenetic silencing of gene expression by promoter CpG island hypermethylation has been shown to be important in the formation of a variety of cancers including oral squamous cell carcinoma [8] . Indeed, several studies have demonstrated hypermethylation events in ACC [9][10][11] , MEC [11][12][13] , PA [11][12][13][14] , malignant PA [10] , salivary duct carcinoma [11] and acinic cell carcinoma [11] .…”

Quantitative Promoter Hypermethylation Analysis of Cancer-Related Genes in Salivary Gland Carcinomas: Comparison with Methylation-Specific PCR Technique and Clinical Significance

“…All of which are known for their ability to suppress vital cellular processes, including cell-cycle regulation, apoptosis, DNA repair, differentiation, and metastasis (34 -38). In a previous study, we used the MSP method to analyze the methylation status of some of these genes in these tumors (39). However, because of the arbitrary and qualitative nature of the MSP technique, the need for more objective and quantitative assessment of methylation has recently been raised (26).…”