Heparanase gene silencing, tumor invasiveness, angiogenesis, and metastasis.
BACKGROUND
Heparanase is an endoglycosidase that degrades heparan sulfate, the main polysaccharide constituent of the extracellular matrix and basement membrane. Expression of the heparanase gene is associated with the invasive, angiogenic, and metastatic potential of diverse malignant tumors and cell lines. We used gene-silencing strategies to evaluate the role of heparanase in malignancy and to explore the therapeutic potential of its specific targeting.


METHODS
We designed plasmid vectors to express hammerhead ribozymes or small interfering RNAs (siRNAs) directed against the human or mouse heparanase mRNAs. Human breast carcinoma (MDA-MB-435) and mouse lymphoma (Eb) and melanoma (B16-BL6) tumor cell lines, which have naturally high levels of endogenous heparanase or have been genetically engineered to overexpress heparanase, were transfected with anti-heparanase ribozyme or siRNA. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and measurements of enzymatic activity were used to confirm the efficient silencing of heparanase gene expression. Cells transfected with the anti-heparanase ribozyme and siRNA vectors were tested for invasiveness in vitro and metastatic dissemination in animal models of experimental and spontaneous metastasis.


RESULTS
Compared with cells transfected with control constructs, cells transfected with the anti-heparanase ribozyme or siRNA vectors had profoundly reduced invasion and adhesion in vitro, regardless of cell type, and expressed less heparanase. In vivo, tumors produced by cells transfected with the anti-heparanase ribozyme and siRNA vectors were less vascularized and less metastatic than tumors produced by cells transfected with the control vectors. Mice injected with cells transfected with the anti-heparanase ribozyme and siRNA vectors lived longer than mice injected with control cells.


CONCLUSIONS
The association of reduced levels of heparanase and altered tumorigenic properties in cells with anti-heparanase ribozyme- or siRNA-mediated gene-silencing vectors suggests that heparanase is important in cancer progression. Heparanase gene silencing has potential use as a target for anticancer drug development.
Department of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Evgeny
E
Edovitsky
Michael
M
Elkin
Eyal
E
Zcharia
Tamar
T
Peretz
Israel
I
Vlodavsky
D043925:Angiogenesis Inducing Agents / Q000378:metabolism; D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D001485:Basement Membrane / Q000201:enzymology / Q000473:pathology; D018450:Disease Progression; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D005822:Genetic Vectors; D005966:Glucuronidase / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D006133:Growth Substances / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008175:Lung Neoplasms / Q000201:enzymology / Q000235:genetics / Q000556:secondary; D008297:Male; D008546:Melanoma, Experimental / Q000201:enzymology / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D009361:Neoplasm Invasiveness / Q000235:genetics; D009362:Neoplasm Metastasis / Q000188:drug therapy / Q000235:genetics; D009389:Neovascularization, Pathologic / Q000188:drug therapy / Q000235:genetics; D010957:Plasmids; D016337:RNA, Catalytic / Q000494:pharmacology*; D012334:RNA, Neoplasm / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D000099175:Heparanase
D043925:Angiogenesis Inducing Agents; D020533:Angiogenesis Inhibitors; D004791:Enzyme Inhibitors; D005966:Glucuronidase; D006133:Growth Substances; D006497:Heparan Sulfate; D016337:RNA, Catalytic; D012334:RNA, Neoplasm; D034741:RNA, Small Interfering; D000099175:Heparanase