Promoter CpG Hypomethylation and Transcription Factor EGR1 Hyperactivate Heparanase Expression in Bladder Cancer

Heparanase plays a critical role in the degradation ofextracellular matrix and cell membrane and is frequentlyupregulated in malignant tumors. Transcription factor,earlygrowth response 1 (EGR1),is closely associated withinducible transcription of the heparanase gene. We hypothe-sized that promoter CpG hypomethylation with increasedEGR1 expression could determine heparanase expressionduring the pathogenesis of bladder cancer. Bladder cancercell lines (J82,T24 and transitional cell carcinoma)significantly restored heparanase expression after 5-Aza-dC treatment. Transfection of EGR1 siRNA with T24bladder cancer cell line significantly downregulated hepar-anase expression compared to the control siRNA transfec-tion. In 54 bladder cancer and paired normal bladdersamples,heparanase expression was significantly higher inbladder cancer than in normal bladder ( Po0.01). Weperformed methylation-specific PCR targeting the CpGsites within the core-binding consensus motifs of EGR1(GGCG) and Sp1 (GGGCGG). Methylation prevalence wassignificantly higher in normal bladder than in bladder cancer(Po0.05) and inversely correlated with heparanase expres-sion (P¼0.055). In the total series of bladder cancer andnormal bladder samples,the combination of promoter CpGmethylation and EGR1 expression regulated heparanaseexpression in a stepwise manner,where heparanase expres-sion was the lowest in methylation-positive and EGR1-negative samples and the highest in methylation-negativeand EGR1-positive samples. To our knowledge,this is thefirst study demonstrating that increased heparanase expres-sion during the pathogenesis of bladder cancer is due topromoter hypomethylation and transcription factor EGR1.Oncogene (2005) 24, 6765–6772.doi:10.1038/sj.onc.1208811;published online 20 June 2005Keywords: heparanase; methylation; epigenetics; blad-der cancerIntroductionDegradation ofbasement membrane as well as extra-cellular matrix (ECM) comprises an initial and essentialstep for cancer cells to invade surrounding tissue andmetastasize to distant organs (Vlodavsky and Fried-mann, 2001). Both the basement membrane and ECMcontain heparan sulfite (HS) and heparan sulfateproteoglycans (HSPGs) as major structural compo-nents, which in turn serve as good substrates forheparanase (Hulett et al., 1999). Therefore, heparanase,an endo-beta-