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Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin

Abstract

Several lines of evidence suggest that aberrant Notch signaling contributes to the development of several types of cancer. Activation of Notch receptor is executed through intramembrane proteolysis by γ-secretase, which is a multimeric membrane-embedded protease comprised of presenilin, nicastrin (NCT), anterior pharynx defective 1 and PEN-2. In this study, we report the neutralization of the γ-secretase activity by a novel monoclonal antibody A5226A against the extracellular domain of NCT, generated by using a recombinant budded baculovirus as an immunogen. This antibody recognized fully glycosylated mature NCT in the active γ-secretase complex on the cell surface, and inhibited the γ-secretase activity by competing with the substrate binding in vitro. Moreover, A5226A abolished the γ-secretase activity-dependent growth of cancer cells in a xenograft model. Our data provide compelling evidence that NCT is a molecular target for the mechanism-based inhibition of γ-secretase, and that targeting NCT might be a novel therapeutic strategy against cancer caused by aberrant γ-secretase activity and Notch signaling.

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Acknowledgements

We are grateful to Drs T Fukuyama, S Yokoshima (The University of Tokyo), C Haass (Ludwig-Maximilians-University München), H Natsugari (Teikyo University), R Kopan (Washington University in St Louis), G Thinakaran (The University of Chicago), M Vooijs (University Medical Center Utrecht Cancer Center) and G Yu (The University of Texas Southwestern Medical Center) for valuable reagents and our current and previous laboratory members for helpful discussions. We also would like to thank Keiko Tamura-Kawakami and Maiko Nampo for their excellent technical support. This work is supported in part by Grants-in-Aid for Young Scientists (S) (for TT) from Japan Society for the Promotion of Science (JSPS), by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (for TT, TI), Scientific Research on Priority Areas ‘Research on Pathomechanisms of Brain Disorders’ from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (for TT, TI), by Targeted Proteins Research Program grant from the MEXT (for TT, TI, JT), by Core Research for Evolutional Science and Technology grant from the MEXT (for TT, TI), Japan. IH and ST were research fellows of JSPS.

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Hayashi, I., Takatori, S., Urano, Y. et al. Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin. Oncogene 31, 787–798 (2012). https://doi.org/10.1038/onc.2011.265

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