J Cancer 2017; 8(19):3916-3932. doi:10.7150/jca.20779

Research Paper

Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates

Toshie Yoneyama1,6, Michael Gorry1,6, Miles A Miller9, Autumn Gaither-Davis5, Yan Lin3, Marcia L. Moss7, Linda G. Griffith8, Douglas A. Lauffenburger8, Laura P. Stabile4, James G. Herman5, Nikola L. Vujanovic1,2,6✉

1. Department of Pathology, University of Pittsburgh Cancer Institute, Pittsburgh, PA;
2. Department of Immunology, University of Pittsburgh Cancer Institute, Pittsburgh, PA;
3. Department of Biostatistics, University of Pittsburgh Cancer Institute, Pittsburgh, PA;
4. Department of Pharmacology and Chemical Biology, University of Pittsburgh Cancer Institute, Pittsburgh, PA;
5. Department of Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, PA;
6. VAPHS, Pittsburgh, PA;
7. BioZyme Inc, Apex, NC;
8. Department of Biologic Engineering, Massachusetts Institute of Technology;
9. Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA

Abstract

Increases in expression of ADAM10 and ADAM17 genes and proteins have been evaluated, but not validated as cancer biomarkers. Specific enzyme activities better reflect enzyme cellular functions, and might be better biomarkers than enzyme genes or proteins. However, no high throughput assay is available to test this possibility. Recent studies have developed the high throughput real-time proteolytic activity matrix analysis (PrAMA) that integrates the enzymatic processing of multiple enzyme substrates with mathematical-modeling computation. The original PrAMA measures with significant accuracy the activities of individual metalloproteinases expressed on live cells. To make the biomarker assay usable in clinical practice, we modified PrAMA by testing enzymatic activities in cell and tissue lysates supplemented with broad-spectrum non-MP enzyme inhibitors, and by maximizing the assay specificity using systematic mathematical-modeling analyses. The modified PrAMA accurately measured the absence and decreases of ADAM10 sheddase activity (ADAM10sa) and ADAM17sa in ADAM10-/- and ADAM17-/- mouse embryonic fibroblasts (MEFs), and ADAM10- and ADAM17-siRNA transfected human cancer cells, respectively. It also measured the restoration and inhibition of ADAM10sa in ADAM10-cDNA-transfected ADAM10-/- MEFs and GI254023X-treated human cancer cell and tissue lysates, respectively. Additionally, the modified PrAMA simultaneously quantified with significant accuracy ADAM10sa and ADAM17sa in multiple human tumor specimens, and showed the essential characteristics of a robust high throughput multiplex assay that could be broadly used in biomarker studies. Selectively measuring specific enzyme activities, this new clinically applicable assay is potentially superior to the standard protein- and gene-expression assays that do not distinguish active and inactive enzyme forms.

Keywords: Cancer biomarker, Sheddase activity, ADAM10, ADAM17, Fluorogenic peptide substrates, Proteolytic activity matrix analysis, Cell lysate, Tissues, lysate, Gene knockout, Gene silence, Gene restoration, Protease inhibitors

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How to cite this article:
Yoneyama T, Gorry M, Miller MA, Gaither-Davis A, Lin Y, Moss ML, Griffith LG, Lauffenburger DA, Stabile LP, Herman JG, Vujanovic NL. Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates. J Cancer 2017; 8(19):3916-3932. doi:10.7150/jca.20779. Available from http://www.jcancer.org/v08p3916.htm