DNA Methylation of Tumor Suppressor and Metastasis Suppressor Genes in Circulating Tumor Cells

Abstract

BACKGROUND Circulating tumor cells (CTCs) are associated with prognosis in a variety of human cancers and have been proposed as a liquid biopsy for follow-up examinations. We show that tumor suppressor and metastasis suppressor genes are epigenetically silenced in CTCs isolated from peripheral blood of breast cancer patients. METHODS We obtained peripheral blood from 56 patients with operable breast cancer, 27 patients with verified metastasis, and 23 healthy individuals. We tested DNA extracted from the EpCAM-positive immunomagnetically selected CTC fraction for the presence of methylated and unmethylated CST6, BRMS1, and SOX17 promoter sequences by methylation-specific PCR (MSP). All samples were checked for KRT19 (keratin 19, formerly CK-19) expression by reverse-transcription quantitative PCR. RESULTS In CTCs of patients with operable breast cancer, promoter methylation of CST6 was observed in 17.9%, BRMS1 in 32.1%, and SOX17 in 53.6% of patients. In CTCs of patients with verified metastasis, promoter methylation of CST6 was observed in 37.0%, BRMS1 in 44.4%, and SOX17 in 74.1%. In healthy individuals, promoter methylation of CST6 was observed in 4.3%, BRMS1 in 8.7%, and SOX17 in 4.3%. DNA methylation of these genes for both operable and metastatic breast cancer was significantly different from that of the control population. CONCLUSIONS DNA methylation of tumor suppressor and metastasis suppressor genes is a hallmark of CTCs and confirms their heterogeneity. Our findings add a new dimension to the molecular characterization of CTCs and may underlie the acquisition of malignant properties, including their stem-like phenotype.