Differential Epigenetic Regulation of TOX Subfamily High Mobility Group Box Genes in Lung and Breast Cancers

Abstract

Aberrant cytosine methylation affects regulation of hundreds of genes during cancer development. In this study, a novel aberrantly hypermethylated CpG island in cancer was discovered within the TOX2 promoter. TOX2 was unmethylated in normal cells but 28% lung (n = 190) and 23% breast (n = 80) tumors were methylated. Expression of two novel TOX2 transcripts identified was significantly reduced in primary lung tumors than distant normal lung (p<0.05). These transcripts were silenced in methylated lung and breast cancer cells and 5-Aza-2-deoxycytidine treatment re-expressed both. Extension of these assays to TOX, TOX3, and TOX4 genes that share similar genomic structure and protein homology with TOX2 revealed distinct methylation profiles by smoking status, histology, and cancer type. TOX was almost exclusively methylated in breast (43%) than lung (5%) cancer, whereas TOX3 was frequently methylated in lung (58%) than breast (30%) tumors. TOX4 was unmethylated in all samples and showed the highest expression in normal lung. Compared to TOX4, expression of TOX, TOX2 and TOX3 in normal lung was 25, 44, and 88% lower, respectively, supporting the premise that reduced promoter activity confers increased susceptibility to methylation during lung carcinogenesis. Genome-wide assays revealed that siRNA-mediated TOX2 knockdown modulated multiple pathways while TOX3 inactivation targeted neuronal development and function. Although these knockdowns did not result in further phenotypic changes of lung cancer cells in vitro, the impact on tissue remodeling, inflammatory response, and cell differentiation pathways suggest a potential role for TOX2 in modulating tumor microenvironment.

ANK1 Methylation regulates expression of MicroRNA-486-5p and discriminates lung tumors by histology and smoking status

“…Cells were transfected with ANK1 -specific siRNA (siANK1), miR-486-5p mimic, or scrambled controls from Applied Biosystems using Lipofectamine 2000 (Invitrogen, Santa Clara, CA) as described [38]. Four different siRNAs targeting different sequences of ANK1 (Fig.…”

TOX2 regulates human natural killer cell development by controlling T-BET expression

“…18 The high mobility group box domain of TOX2 shows a high amino acid homology (.92%) to the other members, whereas the regions outside this domain are less conserved. A previous study of the rat ortholog GCX-1 showed exclusive expression of TOX2 in reproductive tissues, suggesting a specific role in follicular development.…”

Epigenetic Repression of CCDC37 and MAP1B Links Chronic Obstructive Pulmonary Disease to Lung Cancer

“…Studies from our group and others have shown that genes with constitutively lower expression and promoter activity are more prone to becoming methylated during cancer development within that organ. 28,32 These prior findings and concepts appear operative for regulating expression of CCDC37 and MAP1B during the development of COPD and lung cancer.…”