Promoter CpG hypomethylation and transcription factor EGR1 hyperactivate heparanase expression in bladder cancer.
Heparanase plays a critical role in the degradation of extracellular matrix and cell membrane and is frequently upregulated in malignant tumors. Transcription factor, early growth response 1 (EGR1), is closely associated with inducible transcription of the heparanase gene. We hypothesized that promoter CpG hypomethylation with increased EGR1 expression could determine heparanase expression during the pathogenesis of bladder cancer. Bladder cancer cell lines (J82, T24 and transitional cell carcinoma) significantly restored heparanase expression after 5-Aza-dC treatment. Transfection of EGR1 siRNA with T24 bladder cancer cell line significantly downregulated heparanase expression compared to the control siRNA transfection. In 54 bladder cancer and paired normal bladder samples, heparanase expression was significantly higher in bladder cancer than in normal bladder (P<0.01). We performed methylation-specific PCR targeting the CpG sites within the core-binding consensus motifs of EGR1 (GGCG) and Sp1 (GGGCGG). Methylation prevalence was significantly higher in normal bladder than in bladder cancer (P<0.05) and inversely correlated with heparanase expression (P=0.055). In the total series of bladder cancer and normal bladder samples, the combination of promoter CpG methylation and EGR1 expression regulated heparanase expression in a stepwise manner, where heparanase expression was the lowest in methylation-positive and EGR1-negative samples and the highest in methylation-negative and EGR1-positive samples. To our knowledge, this is the first study demonstrating that increased heparanase expression during the pathogenesis of bladder cancer is due to promoter hypomethylation and transcription factor EGR1.
Ogishima
Tatsuya
T
Department of Urology, Veterans Affairs Medical Center, San Francisco and University of California, San Francisco, 4150 Clement Street, CA 94121, USA.
Shiina
Hiroaki
H
Breault
Julia E
JE
Terashima
Masaharu
M
Honda
Satoshi
S
Enokida
Hideki
H
Urakami
Shinji
S
Tokizane
Takashi
T
Kawakami
Toshifumi
T
Ribeiro-Filho
Leopoldo A
LA
Fujime
Makoto
M
Kane
Christopher J
CJ
Carroll
Peter R
PR
Igawa
Mikio
M
Dahiya
Rajvir
R
D001483:Base Sequence; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D004273:DNA, Neoplasm; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000502:physiology*; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D001743:Urinary Bladder / Q000201:enzymology; D000099175:Heparanase
D004273:DNA, Neoplasm; D051766:Early Growth Response Protein 1; D005966:Glucuronidase; D034741:RNA, Small Interfering; D000099175:Heparanase; C497325:EGR1 protein, human