The cell cycle checkpoint gene Rad9 is a novel oncogene activated by 11q13 amplification and DNA methylation in breast cancer.
Human Rad9 (hRad9), a structural homologue of yeast Schizosaccharomyces pombe rad9, is involved in cell cycle checkpoints and apoptosis. hRad9 can serve as a corepressor of androgen receptor in prostate cancer cells, but little is known about its role in the development of breast or other cancers. In the present study, semiquantitative reverse transcription-PCR showed that Rad9 mRNA levels were up-regulated in 52.1% (25 of 48) of breast tumors, and this up-regulation correlated with tumor size (P = 0.037) and local recurrence (P = 0.033). Overexpression of Rad9 mRNA was partly due to an increase in Rad9 gene number as measured by quantitative PCR. In other breast tumors with Rad9 mRNA overexpression but without increase in gene number, there was differential methylation of two putative Sp1/3 binding sites within the first and second introns of the Rad9 gene, which was similarly found in MCF-7 breast cancer cell line with increased Rad9 mRNA. Silencing Rad9 expression by RNA interference in MCF-7 cell line inhibited its proliferation in vitro. Promoter assays indicated that the Sp1/3 site in intron 2 may act as a silencer. In vivo binding of Sp3 to intron 2 was shown by chromatin immunoprecipitation assays. Treatment of MCF-7 cell line with 5'-aza-2'-deoxycytidine reduced Rad9 mRNA expression and also increased binding of Sp3 to the demethylated intron 2 region. Collectively, these findings suggest that Rad9 is a novel oncogene candidate activated by 11q13 amplification and DNA hypermethylation in breast cancer and may play a role in tumor proliferation and local invasion.
Cheng
Chi Keung
CK
University Department of Medicine, Queen Mary Hospital, Pokfulam, Hong Kong, China.
Chow
Louis W C
LW
Loo
Wings T Y
WT
Chan
Tai Kwong
TK
Chan
Vivian
V
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D018567:Breast Neoplasms, Male / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D048708:Cell Growth Processes / Q000235:genetics; D045744:Cell Line, Tumor; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics*; D019938:Cyclin D1 / Q000235:genetics; D019175:DNA Methylation; D000077209:Decitabine; D005260:Female; D005784:Gene Amplification; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D007438:Introns; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics
D018797:Cell Cycle Proteins; D012333:RNA, Messenger; D019938:Cyclin D1; C069930:rad9 protein; D000077209:Decitabine; D001374:Azacitidine