title: Epigenetic regulation of cancer stem cell genes in triple-negative breast cancer.
abstract: Expression of specific breast cancer stem cells (BCSCs) is seen in aggressive tumors, but their regulation is unclear. Epigenetic changes influence gene expression and are implicated in breast cancer progression. We hypothesized that promoter methylation regulates specific BCSC-related genes [CD44, CD133, CD24, MSH1 (alias, Musashi-1), and ALDH1] and that this epigenetic profile can identify aggressive subtypes, such as triple-negative breast cancer (TNBC). Methylation analysis was performed using MassARRAY EpiTYPER sequencing; CpG-rich sites were identified in the promoter regions of BCSC genes, except ALDH1. These sites were screened by treatment with 5-aza-2'-deoxycytidine in four TN and five non-TNBC cell lines. The specific regulatory CpG site demonstrating the most significant inverse correlation between CpG site methylation and mRNA expression was identified for CD44, CD133, and Musashi-1, but not for CD24. Methylation of CD44, CD133, and Musashi-1 was evaluated in 91 American Joint Committee on Cancer stage I to III primary breast cancer tumors, and these sites were significantly hypomethylated in TNBC versus non-TNBC. The IHC staining of primary tumors with the highest and lowest methylation levels revealed the strongest staining in hypomethylated specimens, suggesting that hypomethylation leads to gene activation. We demonstrate that methylation is a significant mechanism regulating CD44, CD133, and Musashi-1, and that gene hypomethylation correlates with TNBC. Assessment of epigenetic changes in BCSC genes may provide a more accurate classification of TNBC and could be developed as potential therapeutic targets.
authors.affiliation: Department of Molecular Oncology, John Wayne Cancer Institute, Saint John's Health Center, Santa Monica, CA 90404, USA.
authors.forename: Naofumi
authors.initials: N
authors.lastname: Kagara
authors.forename: Kelly T
authors.initials: KT
authors.lastname: Huynh
authors.forename: Christine
authors.initials: C
authors.lastname: Kuo
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Okano
authors.forename: Myung Shin
authors.initials: MS
authors.lastname: Sim
authors.forename: David
authors.initials: D
authors.lastname: Elashoff
authors.forename: Kelly
authors.initials: K
authors.lastname: Chong
authors.forename: Armando E
authors.initials: AE
authors.lastname: Giuliano
authors.forename: Dave S B
authors.initials: DS
authors.lastname: Hoon
mesh_terms: D000071916:AC133 Antigen; D000328:Adult; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051927:CD24 Antigen / Q000235:genetics / Q000378:metabolism; D018270:Carcinoma, Ductal, Breast / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics; D044127:Epigenesis, Genetic / Q000235:genetics*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D052138:Genes, Neoplasm; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D014408:Biomarkers, Tumor; D051927:CD24 Antigen; C497889:CD24 protein, human; C497483:CD44 protein, human; D006023:Glycoproteins; D018960:Hyaluronan Receptors; C491297:MSI1 protein, human; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; C000606023:PROM1 protein, human; D010455:Peptides; D016601:RNA-Binding Proteins