title: Specific-site methylation of tumour suppressor ANKRD11 in breast cancer.
abstract: ANKRD11 is a putative tumour suppressor gene in breast cancer, which has been shown in our laboratory to be a co-activator of p53. Our data suggest that down-regulation of ANKRD11 is associated with breast tumourigenesis. Breast cancer cell lines treated with DNA demethylating agents resulted in up-regulation of ANKRD11 expression suggesting that promoter DNA methylation may be responsible for its down-regulation. The transcriptional activity of a CpG-rich region 2kb upstream of the transcription initiation site of ANKRD11 was investigated using dual-luciferase reporter assays. The constructs carrying -661 to -571 bp promoter sequence showed significant transcriptional activity. Using the SEQUENOM Epityper Platform, the region between -770 and +399 bp was analysed in 25 breast tumours, four normal breast tissues and five normal blood samples. The region between -770 and -323 bp was shown to be frequently methylated in breast tumours. The methylation patterns of all analysed CpGs in this region were identical in the normal and tumour samples, except for a 19 bp region containing three CpG sites. These sites had significantly higher levels of methylation in tumours (40%) compared to normal samples (6%). Our findings support the role of ANKRD11 as a tumour suppressor gene and suggest that aberrant DNA methylation of three CpGs in a 19 bp region within the ANKRD11 promoter may be responsible for its down-regulation in breast cancer.
authors.affiliation: Cancer Therapeutics Laboratory, Discipline of Medicine, The University of Adelaide, South Australia, Australia. sue.lim@adelaide.edu.au
authors.forename: Sue Ping
authors.initials: SP
authors.lastname: Lim
authors.forename: Nick C
authors.initials: NC
authors.lastname: Wong
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Suetani
authors.forename: Kristen
authors.initials: K
authors.lastname: Ho
authors.forename: Jane Lee
authors.initials: JL
authors.lastname: Ng
authors.forename: Paul M
authors.initials: PM
authors.lastname: Neilsen
authors.forename: Peter G
authors.initials: PG
authors.lastname: Gill
authors.forename: Raman
authors.initials: R
authors.lastname: Kumar
authors.forename: David F
authors.initials: DF
authors.lastname: Callen
mesh_terms: D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D018899:CpG Islands; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D019175:DNA Methylation; D005260:Female; D016147:Genes, Tumor Suppressor; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics*; D000097584:DNA Methyltransferase 3B
chemical_list: D004248:DNA (Cytosine-5-)-Methyltransferases; D012097:Repressor Proteins; D000097584:DNA Methyltransferase 3B; C492515:ANKRD11 protein, human