Increased heparanase expression is caused by promoter hypomethylation and up-regulation of transcriptional factor early growth response-1 in human prostate cancer.
OBJECTIVE
Heparanase degrades heparan sulfate and has been implicated in tumor invasion and metastasis. The transcription factor, early growth response 1 (EGR1), is associated with the inducible transcription of the heparanase gene. We hypothesize that CpG hypomethylation in the heparanase promoter coupled with up-regulation of EGR1 levels may induce heparanase expression in human prostate cancer.


METHODS
Cultured prostate cancer cell lines (Du145, DuPro, LNCaP, and PC-3) with and without 5'-aza-2-deoxycytidine treatment, 177 prostate cancer samples, and 69 benign prostatic hyperplasia (BPH) samples were used. The frequency and level of heparanase promoter methylation were analyzed by methylation-specific primers which covered the core binding motif of EGR1 (GGCG) or SP1 (GGGCGG) or both.


RESULTS
In cultured Du145, DuPro, LNCaP, and PC-3 cell lines, mRNA transcripts of heparanase were significantly increased after 5'-aza-2-deoxycytidine treatment, suggesting that promoter methylation was involved in the regulation of heparanase mRNA transcript. Significantly higher methylation was found in BPH samples than in prostate cancer samples (P < 0.0001), whereas mRNA transcripts of the heparanase gene were inversely lower in BPH samples than in prostate cancer samples (P < 0.01). EGR1 expression in prostate cancer tissues was significantly higher than in BPH tissues (P < 0.001) and correlated with heparanase expression (P < 0.0001). Moreover, multiple regression analysis revealed that up-regulation of EGR1 contributed significantly more to heparanase expression than did promoter CpG hypomethylation in prostate cancer samples (P < 0.0001).


CONCLUSIONS
To our knowledge this is the first comprehensive study demonstrating that increased heparanase expression in prostate cancer tissues is due to promoter hypomethylation and up-regulation of transcription factor EGR1.
Ogishima
Tatsuya
T
Department of Urology, University of California-San Francisco and Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121, USA.
Shiina
Hiroaki
H
Breault
Julia E
JE
Tabatabai
Laura
L
Bassett
William W
WW
Enokida
Hideki
H
Li
Long-Cheng
LC
Kawakami
Toshifumi
T
Urakami
Shinji
S
Ribeiro-Filho
Leopoldo A
LA
Terashima
Masaharu
M
Fujime
Makoto
M
Igawa
Mikio
M
Dahiya
Rajvir
R
D000368:Aged; D000369:Aged, 80 and over; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001483:Base Sequence; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D004268:DNA-Binding Proteins / Q000235:genetics*; D000077209:Decitabine; D051766:Early Growth Response Protein 1; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005966:Glucuronidase / Q000235:genetics*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011470:Prostatic Hyperplasia / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012044:Regression Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics*; D015854:Up-Regulation / Q000235:genetics; D000099175:Heparanase
D001374:Azacitidine; D015254:DNA Modification Methylases; D004268:DNA-Binding Proteins; D000077209:Decitabine; D051766:Early Growth Response Protein 1; D004791:Enzyme Inhibitors; D005966:Glucuronidase; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D000099175:Heparanase; C497325:EGR1 protein, human