Effect of ADAM28 on carcinoma cell metastasis by cleavage of von Willebrand factor.
BACKGROUND
A disintegrin and metalloproteinase 28 (ADAM28) is implicated in tumor growth and metastasis in human breast and non-small cell lung carcinomas. We explored the mechanism of ADAM28-mediated metastasis by searching for new substrates of ADAM28.


METHODS
We used a yeast two-hybrid system to screen the human lung cDNA library for ADAM28-binding proteins and identified von Willebrand factor (VWF) as a potential candidate. Binding was confirmed using yeast two-hybrid and protein binding assays, and ADAM28-mediated cleavage of VWF was analyzed by immunoblotting. Exogenous VWF-induced apoptosis in vitro was examined in human lung carcinoma (PC-9 and Calu-3), breast carcinoma (MDA-MB231 and MCF-7), renal cell carcinoma (Caki-2 and 769P), and hepatocellular carcinoma (HepG2) cells, and expression of ADAM28 was assessed by reverse transcription-polymerase chain reaction and immunoblotting. Effect on lung metastasis of PC-9 and MDA-MB231 cells was assessed by knockdown of ADAM28 expression using short hairpin RNAs (ADAM28-shRNA) and small interfering RNAs (ADAM28-siRNA), and inhibition of activity using neutralizing anti-ADAM28 antibody, in a mouse xenograft model by in vivo imaging (n = 9 mice per group). All statistical tests were two-sided.


RESULTS
ADAM28 could bind to and cleave native VWF. Cells with very low ADAM28 expression (MCF-7, 769P, and HepG2) were susceptible to VWF-induced apoptosis, whereas cells with high expression (PC-9, Calu-3, MDA-MB231, and Caki-2) were resistant. Knockdown of ADAM28 expression in PC-9 and MDA-MB231 cells by shRNA showed increased carcinoma cell apoptosis mainly in lung blood vessels and statistically significantly decreased lung metastasis at week 3 after injection (PC-9-control [n = 9 mice] vs PC-9-ADAM28-shRNA [n = 9 mice]: mean count = 198 × 10(6) vs 37 × 10(6) photons/s, difference = 161 × 10(6) photons/s, 95% confidence interval = 134 × 10(6) to 188 × 10(6) photons/s, P < .001). Similar inhibition of lung metastasis was observed with ADAM28-siRNA and anti-ADAM28 antibody.


CONCLUSIONS
ADAM28 cleaves and inactivates proapoptotic VWF in carcinoma cells and enhances lung metastasis probably by promoting carcinoma cell survival within the blood vessels.
Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-0016, Japan.
Satsuki
S
Mochizuki
Kenji
K
Soejima
Masayuki
M
Shimoda
Hitoshi
H
Abe
Aya
A
Sasaki
Hirotaka James
HJ
Okano
Hideyuki
H
Okano
Yasunori
Y
Okada
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism / Q000473:pathology; D002292:Carcinoma, Renal Cell / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007680:Kidney Neoplasms / Q000378:metabolism / Q000473:pathology; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D008163:Luminescent Measurements; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D051379:Mice; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology*; D009374:Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014183:Transplantation, Heterologous; D020798:Two-Hybrid System Techniques; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
D034741:RNA, Small Interfering; D014841:von Willebrand Factor; D051722:ADAM Proteins; C410224:ADAM28 protein, human