CpG hypermethylation of promoter region and inactivation of E-cadherin gene in human bladder cancer.
Several studies have shown that E-cadherin expression is lost during malignant transformation. We hypothesized that CpG methylation in the promoter region may inactivate the expression of the E-cadherin gene in human bladder cancer. Normal and bladder cancer samples from 51 patients were compared in terms of E-cadherin gene expression and methylation status by immunohistochemistry, methylation-specific polymerase chain reaction (MSP), and bisulfite genome-sequencing techniques. Ten different CpG sites (nt 863, 865, 873, 879, 887, 892, 901, 918, 920, and 940) in the promoter region were studied. Thirty-five of 51 (69%) bladder cancer samples lacked E-cadherin expression, whereas only six of 51 (12%) normal bladder samples lacked E-cadherin immunoreactivity. MSP analysis of bladder cancer samples suggested that 43 of 51 (84%) showed methylation of the promoter region, whereas only 12 of 51 (24%) normal bladder samples showed hypermethylation. Sodium bisulfite genome-sequencing analysis revealed that of 10 CpG sites, two sites (nt 892 and nt 940) showed 100% methylation in all the cancer samples analyzed. Other CpG sites were partially methylated (47-91%). Normal tissue showed only 12% methylation (range, 1-33%) on various CpG sites. Also supporting these data, E-cadherin-negative bladder cancer cell lines restored expression of the E-cadherin gene after treatment with the demethylating agent 5-aza-2'-deoxycytidine. The present study showed that CpG hypermethylation was an important mechanism of E-cadherin gene inactivation in bladder cancer and also that specific CpG sites consistently presented higher methylation levels than others. These findings may provide a better strategy for the diagnosis and management of bladder cancer.
Department of Urology, San Francisco Veterans Affairs Medical Center and University of California, 94121, USA.
Leopoldo Alves
LA
Ribeiro-Filho
Joseph
J
Franks
Masahiro
M
Sasaki
Hiroaki
H
Shiina
Long-Cheng
LC
Li
Dana
D
Nojima
Sami
S
Arap
Peter
P
Carroll
Hideki
H
Enokida
Masayuki
M
Nakagawa
Suguru
S
Yonezawa
Rajvir
R
Dahiya
D000368:Aged; D015820:Cadherins / Q000235:genetics*; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D020868:Gene Silencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D012016:Reference Values; D017422:Sequence Analysis, DNA; D001749:Urinary Bladder Neoplasms / Q000235:genetics*
D015820:Cadherins