Unmasking of epigenetically silenced genes and identification of transgelin as a potential methylation biomarker in breast cancer

Abstract

Tumor suppressor genes (TSG) are frequently silenced in cancer by epigenetic mechanisms, including promoter DNA hypermethylation and repressive chromatin formation by means of histone deacetylation. 5-aza-2'-deoxycytidine (AZA) and Trichostatin A (TSA) are DNA methyl-transferase and histone deacetylase inhibitors, respectively, and are used as anti-cancer agents for induction of epigenetically suppressed genes. In this study, in an attempt to unmask epigenetically suppressed potential TSGs in breast cancer, two breast carcinoma cells and one non-tumorigenic breast cell line were treated with AZA and TSA, either separately or in combination, and with DMSO as control. Afterwards, highthroughput expression profiling revealed significantly affected genes and pathways in response to epigenetic induction in each cell line. Analysis of 32 candidate genes highlighted Transgelin (TAGLN) as a putative TSG that is frequently downregulated by promoter DNA hypermethylation in breast cancer cell lines, and in 61.9% of normal-paired breast tumors according to bisulfite sequencing; and in 63.02% of unpaired breast tumor tissues as determined by bioinformatics analyses of public microarray data. Both relapse-free and overall survivals of patients were more favorable with lower TAGLN methylation. Moreover, TAGLN promoter methylation levels diagnosed tumors tissues with 83.14% sensitivity and 100% specificity. qRTPCR and IHC experiments demonstrated that, TAGLN was persistently downregulated in breast cancer cell lines in comparison to non-tumorigenic cells; and in three independent sets of breast tumor tissues, compared to normal tissues. Furthermore, TAGLN expression was associated with good prognostic factors. Functional analyses in breast cancer cells revealed negative effect of transgelin on colony formation abilities of cells, and analyses with epithelial-to-mesenchymal (EMT) markers implicate an association of transgelin with EMT status of breast cancer. In short, TAGLN downregulation by promoter DNA hypermethylation in breast cancer could serve as a growth advantage to the breast cancer cells, while it could be used as a diagnostic or prognostic biomarker for breast cancer.