Reversible epigenetic control of CD146 phenotypic states in MDA-MB-231 breast cancer cells.

Abstract

e13010 Background: Previous studies have demonstrated epigenetic regulation of the CD146 ( MCAM ) gene, whose protein product plays a key role in epithelial–mesenchymal transition (EMT). Analysis of MCAM promoter methylation in the triple-negative (TN) breast cancer cell line MDA-MB-231 revealed heterogeneous CpG methylation, suggesting the presence of distinct subclonal populations. This study aimed to investigate the relationship between subclonal CpG island methylation and CD146 expression and to evaluate its relevance across breast cancer molecular subtypes defined by PAM50 classification. Methods: CD146 high and CD146 low subpopulations were isolated by fluorescence-activated cell sorting (FACS). Methylation of 22 CpG sites within the MCAM promoter was assessed using bisulfite sequencing (BGS). CD146 expression was analyzed at the mRNA (RT-PCR) and protein (Western blot) levels. In parallel, bioinformatic analyses of TCGA BRCA clinical datasets were performed, including methylation profiling, differential expression analysis, and correlation analyses between MCAM methylation and EMT-related genes ( SNAI1, TWIST1 ) across luminal A (n = 65), luminal B (n = 27), and basal-like (n = 23) subtypes. Results: CD146 high and CD146 low clones exhibited opposing expression profiles, confirmed by FACS, RT-PCR, and Western blot. Differential methylation between these populations was identified at four CpG sites within the analyzed promoter region, indicating their regulatory relevance. Clinical data analysis revealed strong subtype-specific differences in MCAM methylation. The basal-like subtype showed the lowest methylation levels (mean β = 0.35 ± 0.03), with MCAM expression significantly associated with TN status (p < 0.05). The highest methylation levels were observed in the luminal B subtype (mean β = 0.38 ± 0.04), where significant correlations were detected between MCAM methylation and EMT- related genes methylation (r > 0.4). Such correlations were absent in the basal-like subtype. Conclusions: CD146 expression in MDA-MB-231 cells is epigenetically regulated through promoter methylation. Our findings suggest that this mechanism is part of a broader, subtype-dependent epigenetic program. Consistent with previous reports linking transient CD146 loss to increased cancer cell invasiveness, our results support the concept that dynamic, subclonal DNA methylation changes contribute to tumor heterogeneity and selection of highly invasive cell populations. MCAM promoter methylation may therefore represent a potential biomarker for identifying aggressive breast cancer subtypes.