title: 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.
authors.affiliation: Department of Chemistry, University of Athens, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Chimonidou
authors.forename: Areti
authors.initials: A
authors.lastname: Strati
authors.forename: Alexandra
authors.initials: A
authors.lastname: Tzitzira
authors.forename: Georgia
authors.initials: G
authors.lastname: Sotiropoulou
authors.forename: Nikos
authors.initials: N
authors.lastname: Malamos
authors.forename: Vasilis
authors.initials: V
authors.lastname: Georgoulias
authors.forename: Evi S
authors.initials: ES
authors.lastname: Lianidou
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D055332:Cystatin M / Q000235:genetics; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D020868:Gene Silencing; D016147:Genes, Tumor Suppressor; D006801:Humans; D053539:Keratin-19 / Q000235:genetics / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism / Q000473:pathology; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins / Q000235:genetics; D009360:Neoplastic Cells, Circulating / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055760:SOXF Transcription Factors / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: C410024:BRMS1 protein, human; C525104:CST6 protein, human; D055332:Cystatin M; D053539:Keratin-19; D009363:Neoplasm Proteins; D012097:Repressor Proteins; C109188:SOX17 protein, human; D055760:SOXF Transcription Factors