Frequently Methylated Tumor Suppressor Genes in Head and Neck Squamous Cell Carcinoma

Kristi L Bennett,Dominic Smiraglia,Esther Herpel,Rainer Claus,Christoph Plass,Khelifa Arab,Peter Plinkert,Mau-Ting Lin,Matthew Karpenko,Gerhard Dyckhoff

doi:10.1158/0008-5472.can-07-6509

Abstract

Head and neck squamous cell carcinoma (HNSCC) is a very aggressive cancer. In advanced stages, the patient has poor chances of receiving effective treatment, and survival rates are low. To facilitate timely diagnosis and improve treatment, elucidation of early detection markers is crucial. DNA methylation markers are particularly advantageous because DNA methylation is an early event in tumorigenesis, and the epigenetic modification, 5-methylcytosine, is a stable mark. A genome-wide screen using Restriction Landmark Genomic Scanning found a set of genes that are most commonly methylated in head and neck cancers. Five candidate genes: septin 9 (SEPT9), sodium-coupled monocarboxylate transporter 1 (SLC5A8), functional smad-suppressing element on chromosome 18 (FUSSEL18), early B-cell factor 3 (EBF3), and iroquois homeobox 1 (IRX1) were methylated in 27% to 67% of the HNSCC patient samples tested. Furthermore, approximately 50% of the methylated tumor samples shared methylation between two of the five genes (most commonly between EBF3 and IRX1), and 15% shared methylation between three of the five genes. Expression analysis revealed candidate gene down-regulation in 25% to 93% of the HNSCC samples, and 5-aza-2'-deoxycytidine treatment was able to restore expression in at least 2 of 5 HNSCC cell lines for all of the genes tested. Overexpression of the three most frequently down-regulated candidates, SLC5A8, IRX1, and EBF3, validated their tumor suppressor potential by growth curve analysis and colony formation assay. Interestingly, all of the candidates identified may be involved in the transforming growth factor beta signaling pathway, which is often disrupted in HNSCC.

Highlights

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide
The study described here used these Restriction Landmark Genomic Scanning (RLGS) profiles to identify potential novel tumor suppressor genes that are frequently methylated in HNSCC
These profiles were reevaluated to identify the specific genes that are frequently methylated in HNSCC. This analysis yielded 23 different loci among the profiles that had decreased RLGS fragment intensity in >10% of the profiles [examples shown in Fig. 1A and complete list in Table 1; the 10% cutoff provides a significant difference (P < 0.05)]. Eleven of these fragments have been previously cloned, eight of which were found to be associated with a CpG island and a known gene: ALX3, HMX2, SLC5A8, septin 9 (SEPT9), FUSSEL18, early B-cell factor 3 (EBF3), iroquois homeobox 1 (IRX1), and GBX2 (Table 1)

Summary

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. According to the National Cancer Institute’s Surveillance Epidemiology and End Results cancer statistics review, 34,360 individuals were predicted to be diagnosed with HNSCC in 2007. Treatment of HNSCC is complex, Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

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Results

Conclusion