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A Cellular Based Assay to Monitor the Cleavage of the Extracellular Matrix by Matrix Metalloproteinases as a Tool for Drug Discovery Against Metastasis
Author(s) -
Doyle Ryan,
Canter Ben,
Nader Joy,
Wolkowicz Roland,
Tandon Ruby
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.03612
Subject(s) - extracellular matrix , proteases , cancer cell , matrix metalloproteinase , chemistry , microbiology and biotechnology , protease , drug discovery , biology , biochemistry , enzyme , cancer , genetics
One of the most important genes up‐regulated in cancer is known as Matrix Metalloproteinase (MMPs). MMP’s are enzymes critical for remodeling the protein matrix, called extracellular matrix (ECM), that surrounds and supports cells. This protein can be secreted or membrane‐bound as it remodels the extracellular environments in healthy individuals. However, in cancerous cells, its overexpression leads to the metastasis of cancer cells. To investigate novel ways to block metastasis, our lab engineered a new cellular Two‐Tag Assay as a tool for drug discovery, enabling the search for inhibitors. The assay, based on a system we developed for HIV and Dengue Virus, was adapted to MMP‐14. It relies on a scaffold composed of two antibody epitopes, HA and FLAG, flanking a protein substrate. Because the FLAG epitope is encoded upstream of the substrate and HA, it is cleaved while HA remains attached to the cell membrane. We then use flow cytometry to monitor the presence of HA and FLAG. Based on the presence of one or two tags, we can determine the robustness of cleavage. Previously, we inserted viral proteases between the tags and showed autocatalytic cleavage or lack of while the protein travels through the classical secretory pathway. The MMP‐14 assay has a consensus substrate of MMP‐14 instead of a viral protease, as well as a cell line obtained through retroviral technology. In addition, we expressed MMP‐14 enzyme in cells expressing substrate, as well as naïve cells. Comparing substrate‐expressing cells with substrate and enzyme expressing cells, we showed that MMP‐14 cleaves the substrate. We performed mixing experiments with substrate‐expressing and enzyme‐expressing cells to specifically corroborate cleavage at the cell surface. Results were corroborated with confocal microscopy and western blotting. Decrease in FLAG expression in the presence of MMP‐14 proved the assay’s ability to discover new inhibitors of MMP‐14. Support or Funding Information This research is supported by fellow researchers in the Wolkowicz Lab and the National Cancer Institute of the National Institutes of Health under award numbers: U54CA132384 & U54CA132379