TY - JOUR
T1 - Effect of ADAM28 on carcinoma cell metastasis by cleavage of von willebrand factor
AU - Mochizuki, Satsuki
AU - Soejima, Kenji
AU - Shimoda, Masayuki
AU - Abe, Hitoshi
AU - Sasaki, Aya
AU - Okano, Hirotaka James
AU - Okano, Hideyuki
AU - Okada, Yasunori
N1 - Funding Information:
This work was supported by Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) (to SM), Grant-in-Aid for Scientific Research (S) ( 19109004 ) and Grant-in-Aid for Scientific Research (A) (24249022) from MEXT, Third Term 10-year Strategy for Cancer Control from the Ministry of Health and Welfare (to YO), and Grant-in-Aid for the Global COE program from MEXT (to Keio University).
PY - 2012/6/20
Y1 - 2012/6/20
N2 - 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.MethodsWe 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. Conclusion ADAM28 cleaves and inactivates proapoptotic VWF in carcinoma cells and enhances lung metastasis probably by promoting carcinoma cell survival within the blood vessels.
AB - 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.MethodsWe 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. Conclusion ADAM28 cleaves and inactivates proapoptotic VWF in carcinoma cells and enhances lung metastasis probably by promoting carcinoma cell survival within the blood vessels.
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U2 - 10.1093/jnci/djs232
DO - 10.1093/jnci/djs232
M3 - Article
C2 - 22636800
AN - SCOPUS:84864130581
SN - 0027-8874
VL - 104
SP - 906
EP - 922
JO - Journal of the National Cancer Institute
JF - Journal of the National Cancer Institute
IS - 12
ER -
