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

Toshie Yoneyama; Michael Gorry; Miles A Miller; Autumn Gaither-Davis; Yan Lin; Marcia L Moss; Linda G Griffith; Douglas A Lauffenburger; Laura P Stabile; James G Herman; Nikola L Vujanovic

doi:10.7150/jca.20779

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

J Cancer. 2017 Oct 23;8(19):3916-3932. doi: 10.7150/jca.20779. eCollection 2017.

Authors

Toshie Yoneyama^{1

2}, Michael Gorry^{1

2}, Miles A Miller³, Autumn Gaither-Davis⁴, Yan Lin⁵, Marcia L Moss⁶, Linda G Griffith⁷, Douglas A Lauffenburger⁷, Laura P Stabile⁸, James G Herman⁴, Nikola L Vujanovic^{1

9

2}

Affiliations

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

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

Grants and funding

P30 CA047904/CA/NCI NIH HHS/United States