Epigenetic screening of salivary gland mucoepidermoid carcinoma identifies hypomethylation of CLIC3 as a common alteration.
OBJECTIVE
The role of promoter methylation in the development of mucoepidermoid carcinoma (MEC) has not been fully explored. In this study, we investigated the epigenetic landscape of MEC.


METHODS
The Illumina HumanMethylation27 BeadChip array and differential methylation analysis were utilized to screen for epigenetic alterations in 14 primary MEC tumors and 14 matched normal samples. Bisulfite sequencing was used to validate these results, with subsequent quantitative Methylation-Specific PCR (qMSP) to validate chloride intracellular channel protein 3 (CLIC3) in a separate cohort. Furthermore, CLIC3 immunohistochemical (IHC) staining was performed in another separate cohort of MEC. Finally, clinical and pathological characteristics were statistically analyzed for correlation with methylation status of CLIC3 and CLIC3 IHC H-scores by Wilcoxon rank sum, Kruskall-Wallis, and X(2) test tests.


RESULTS
We obtained 6 significantly differentially methylated gene candidates demonstrating significant promoter hyper- or hypo-methylation from the array data. Using bisulfite sequencing, we found one gene, CLIC3, which showed differential methylation between MEC tumor and normal samples in a small validation cohort. qMSP analysis of the CLIC3 promoter in a separate validation set showed significantly lower methylation level in tumor than in normal. The level of CLIC3 methylation in MECs was not statistically correlated with clinical or pathological characteristics. However, IHC staining intensity and distribution of CLIC3 were significantly increased in MECs, compared with those of normal salivary gland tissues.


CONCLUSIONS
Hypomethylation of CLIC3 promoter and its overexpression are significant events in MEC. Its functional role and potential therapeutic utility in MEC are worthy of further exploration.
Department of Oral and Maxillofacial Surgery, Shengjing Hospital of China Medical University, Shenyang, Liaoning, China; Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, Baltimore, MD, USA. Electronic address: wangzm@sj-hospital.org.
Zhiming
Z
Wang
Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, Baltimore, MD, USA.
Shizhang
S
Ling
Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, Baltimore, MD, USA.
Eleni
E
Rettig
Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, Baltimore, MD, USA.
Ryan
R
Sobel
Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, Baltimore, MD, USA.
Marietta
M
Tan
Department of Oncology Biostatistics, Johns Hopkins University, Baltimore, MD, USA.
Elana J
EJ
Fertig
Department of Oncology Biostatistics, Johns Hopkins University, Baltimore, MD, USA.
Michael
M
Considine
Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Adel K
AK
El-Naggar
Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, Baltimore, MD, USA.
Mariana
M
Brait
Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, Baltimore, MD, USA; Milton J. Dance Jr. Head and Neck Center at the Greater Baltimore Medical Center, Baltimore, MD, USA.
Carole
C
Fakhry
Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, Baltimore, MD, USA; Milton J. Dance Jr. Head and Neck Center at the Greater Baltimore Medical Center, Baltimore, MD, USA. Electronic address: pha1@jhmi.edu.
Patrick K
PK
Ha
D000328:Adult; D000368:Aged; D018277:Carcinoma, Mucoepidermoid / Q000235:genetics* / Q000378:metabolism; D016022:Case-Control Studies; D018118:Chloride Channels / Q000235:genetics*; D019175:DNA Methylation; D057890:Epigenomics / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D012468:Salivary Gland Neoplasms / Q000235:genetics* / Q000378:metabolism; D018709:Statistics, Nonparametric
C117019:CLIC3 protein, human; D018118:Chloride Channels
Array; Bisulfite sequencing; CLIC3; DNA methylation; H-score; Head and neck cancer; Immunohistochemistry; Methylation-specific PCR; Mucoepidermoid carcinoma