Epigenetic screening of salivary gland mucoepidermoid carcinoma identifies hypomethylation of CLIC3 as a common alteration

Abstract

Objectives 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 X2 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.

Prognostic value of CLIC3 mRNA overexpression in bladder cancer

“…CLIC3 and Rab25 synergistically promote late endosomal/lysosomal integrin cycling and drive cancer progression (Dozynkiewicz et al, 2012). In addition, CLIC3 is highly expressed in mucoepidermoid carcinoma of the salivary gland and promotes tumor progression (Wang et al, 2015). Our study found that CLIC3 is closely linked to BC and analyzed its possibility as a prognostic marker.…”

“…CLIC3 can drive cancer progression through glutathionedependent oxidoreductase activity (Hernandez-Fernaud et al, 2017). CLIC3 is highly expressed in a variety of tumors, such as mucoepidermoid carcinoma (Wang et al, 2015) and malignant pleural mesothelioma (Tasiopoulou et al, 2015). However, the expression of CLIC3 in BC has not yet been reported.…”

New Developments in Salivary Gland Pathology: Clinically Useful Ancillary Testing and New Potentially Targetable Molecular Alterations

“…85,86,90,91,97,148 Despite the latter, it has been shown that some CRTC1-MAML2 fusion-positive MECs have increased EGFR signaling via amphiregulin (AREG), and that may stimulate growth of MEC. 149 This could provide support for testing/determination of MEC fusion status, if elevated levels of AREG and subsequent activation of AREG-EGFR signaling prove to be a pathway for targeting with anti-EGFR therapy. 150 In MEC, the reported rate of HER2 overexpression appears to be low.…”

Epigenetic alterations in salivary gland tumors

“…Hyper-or hypomethylation of DNA may induce the development of tumors by regulating their components, such as proteins of the extracellular matrix, and it can cause chromosomal instability (Dawson & Kouzarides, 2012;Sharma et al, 2010). Epigenetic alterations, isolated or combined, may be involved in the development and progress of SGT (Aniello et al, 2006;Augello et al, 2006;Binmadi et al, 2018;Daa et al, 2008;Durr et al, 2010;Ge et al, 2011;Guimarães et al, 2016;Hu et al, 2011;Kaira et al, 2013;Ling et al, 2016;Nikolic et al, 2018;Schache et al, 2010;Shao et al, 2011Shao et al, , 2012Stephens et al, 2013;Tan et al, 2014;Wagner et al, 2017;Wang et al, 2015;Williams et al, 2006;Xia et al, 2018Xia et al, , 2013Zhang et al, 2007Zhang et al, , 2014.…”

“…Although most studies involve malignant salivary gland tumors, it has been demonstrated that epigenetic alterations may also play a key role in benign salivary gland tumors (Nikolic et al, 2018). Different models and methods have been employed to study these alterations, from cancer stem cell models to tissue from the human biopsy with analysis in genomic level showing the epigenetic landscape of SGT (Nikolic et al, 2018;Sharma et al, 2010;Wang et al, 2015;Zhang et al, 2007). Although there are some studies focused on the comprehensive role of epigenetic alterations in salivary gland tumors, there is no review that synthesizes current literature on this genetic mechanism.…”