title: Invasion suppressor cystatin E/M (CST6): high-level cell type-specific expression in normal brain and epigenetic silencing in gliomas.
abstract: DNA hypermethylation-mediated gene silencing is a frequent and early contributor to aberrant cell growth and invasion in cancer. Malignant gliomas are the most common primary brain tumors in adults and the second most common tumor in children. Morbidity and mortality are high in glioma patients because tumors are resistant to treatment and are highly invasive into surrounding brain tissue rendering complete surgical resection impossible. Invasiveness is regulated by the interplay between secreted proteases (eg, cathepsins) and their endogenous inhibitors (cystatins). In our previous studies we identified cystatin E/M (CST6) as a frequent target of epigenetic silencing in glioma. Cystatin E/M is a potent inhibitor of cathepsin B, which is frequently overexpressed in glioma. Here, we study the expression of cystatin E/M in normal brain and show that it is highly and moderately expressed in oligodendrocytes and astrocytes, respectively, but not in neurons. Consistent with this, the CST6 promoter is hypomethylated in all normal samples using methylation-specific PCR, bisulfite genomic sequencing, and pyrosequencing. In contrast, 78% of 28 primary brain tumors demonstrated reduced/absent cystatin E/M expression using a tissue microarray and this reduced expression correlated with CST6 promoter hypermethylation. Interestingly, CST6 was expressed in neural stem cells (NSC) and markedly induced upon differentiation, whereas a glioma tumor initiating cell (TIC) line was completely blocked for CST6 expression by promoter methylation. Analysis of primary pediatric brain tumor-derived lines also showed CST6 downregulation and methylation in nearly 100% of 12 cases. Finally, ectopic expression of cystatin E/M in glioma lines reduced cell motility and invasion. These results demonstrate that epigenetic silencing of CST6 is frequent in adult and pediatric brain tumors and occurs in TICs, which are thought to give rise to the tumor. CST6 methylation may therefore represent a novel prognostic marker and therapeutic target specifically altered in TICs.
authors.affiliation: Department of Pathology, UF-Shands Cancer Center Program in Cancer Genetics, Epigenetics, and Tumor Virology, University of Florida College of Medicine, Gainesville, FL, USA.
authors.forename: Jingxin
authors.initials: J
authors.lastname: Qiu
authors.forename: Lingbao
authors.initials: L
authors.lastname: Ai
authors.forename: Cheppail
authors.initials: C
authors.lastname: Ramachandran
authors.forename: Bing
authors.initials: B
authors.lastname: Yao
authors.forename: Suhasni
authors.initials: S
authors.lastname: Gopalakrishnan
authors.forename: C Robert
authors.initials: CR
authors.lastname: Fields
authors.forename: Amber L
authors.initials: AL
authors.lastname: Delmas
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Dyer
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Melnick
authors.forename: Anthony T
authors.initials: AT
authors.lastname: Yachnis
authors.forename: Philip H
authors.initials: PH
authors.lastname: Schwartz
authors.forename: Howard A
authors.initials: HA
authors.lastname: Fine
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Brown
authors.forename: Keith D
authors.initials: KD
authors.lastname: Robertson
mesh_terms: D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D001932:Brain Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D055332:Cystatin M; D015891:Cystatins / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D017931:DNA Primers; D044127:Epigenesis, Genetic; D020868:Gene Silencing; D005910:Glioma / Q000235:genetics* / Q000473:pathology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009361:Neoplasm Invasiveness; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D046888:Tissue Array Analysis
chemical_list: C525104:CST6 protein, human; D055332:Cystatin M; D015891:Cystatins; D017931:DNA Primers