Epigenetic silencing of CRABP2 and MX1 in head and neck tumors

Abstract

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease affecting the epithelium of the oral cavity, pharynx and larynx. Conditions of most patients are diagnosed at late stages of the disease, and no sensitive and specific predictors of aggressive behavior have been identified yet. Therefore, early detection and prognostic biomarkers are highly desirable for a more rational management of the disease. Hypermethylation of CpG islands is one of the most important epigenetic mechanisms that leads to gene silencing in tumors and has been extensively used for the identification of biomarkers. In this study, we combined rapid subtractive hybridization and microarray analysis in a hierarchical manner to select genes that are putatively reactivated by the demethylating agent 5-aza-2'-deoxycytidine (5Aza-dC) in HNSCC cell lines (FaDu, UM-SCC-14A, UM-SCC-17A, UM-SCC-38A). This combined analysis identified 78 genes, 35 of which were reactivated in at least 2 cell lines and harbored a CpG island at their 5' region. Reactivation of 3 of these 35 genes (CRABP2, MX1, and SLC15A3) was confirmed by quantitative real-time polymerase chain reaction (PCR; fold change, >or=3). Bisulfite sequencing of their CpG islands revealed that they are indeed differentially methylated in the HNSCC cell lines. Using methylation-specific PCR, we detected a higher frequency of CRABP2 (58.1% for region 1) and MX1 (46.3%) hypermethylation in primary HNSCC when compared with lymphocytes from healthy individuals. Finally, absence of the CRABP2 protein was associated with decreased disease-free survival rates, supporting a potential use of CRABP2 expression as a prognostic biomarker for HNSCC patients.

Figure 1

Flowchart for the identification of differentially methylated genes in HNSCC cell lines. FaDu cell line was treated with 5Aza-dC, and purified mRNA was used to construct RaSH cDNA libraries. A set of 151 nonredundant genes was used to prepare an enriched cDNA platform for microarray analysis. A total of 48 genes were reactivated in at least two cell lines. From them, 35 genes harboring a CpG island located at their 5′ region were submitted to qRT-PCR. Up-regulation of gene expression by 5Aza-dC was confirmed for three genes. Bisulfite sequencing revealed three differentially methylated genes.

Figure 2

A representative result of bisulfite sequencing. Each panel represents a schematic representation of the genome structure of each gene including their 5′ CpG islands. Exons and untranslated regions are represented by filled or open boxes, respectively. The transcription initiation site is represented by +1. Expanded view shows the position of CpG islands and the region analyzed by bisulfite sequencing. Vertical marks represent individual CpG dinucleotides and their spacing accurately reflects the CpG density of the region. MSP primers are represented by horizontal arrows in the panels. Primer sets M and U were designed for the same CpG dinucleotide (indicated by an asterisk). Methylation profiles of the treated (5Aza-dC) and untreated (mock) cell lines are indicated in the lower part of the panels. Each row represents one sequenced clone, and open and filled circles represent unmethylated and methylated CpG dinucleotides, respectively. (A) Bisulfite sequencing of CRABP2 in the FaDu cell line. (B) Bisulfite sequencing of MX1 in the FaDu and UM-SCC-14A cell lines. (C) Bisulfite sequencing of SLC15A3 in the FaDu and UM-SCC-38A cell lines.

Figure 3

MSP analysis of CRABP2 and MX1 genes. Representative results of MSP analysis of CRABP2 and MX1 genes in the FaDu cell line and five different HNSCC samples. Methylated tumors are indicated with asterisks. The lanes indicated by M and U correspond to the products amplified by MSP primer sets specific for methylated and unmethylated DNA, respectively. In vitro methylated DNA (IVD) and normal human peripheral lymphocytes (NL) were used as positive and negative methylation controls, respectively. (A) MSP of CRABP2 R1. (B) MSP of CRABP2 R2. (C) MSP of MX1.

Figure 4

CRABP2 protein immunostaining patterns. Representative immunostainings of (A) morphologically normal epithelium and (–BD) HNSCC samples for CRABP2. Chromogenic detection (brown precipitate) counterstained with hematoxylin. Original magnifications: A, x200 (A); B–D, x400. Staining was scored as negative (B), weakly positive (C), or strongly positive (D).

Figure 5

Kaplan-Meier disease-free survival estimates from 73 patients for CRABP2 expression. Continuous and dashed lines depict patients with positive (weak and strong) or negative CRABP2 expression (P = .0531), respectively.