Chemical Tools for Selective Activity Profiling of Endogenously Expressed MMP-14 in Multicellular Models

Article abstract of DOI:10.1021/acschembio.8b00562

Matrix metalloproteases (MMPs) are a large family of zinc-dependent endopeptidases involved in a diverse set of physiological and pathological processes, most notably in cancer. Current methods for imaging and quantifying MMP activity lack sufficient selectivity and spatiotemporal resolution to allow studies of specific MMP function in vivo. Previously, we reported a strategy for selective targeting of MMPs by engineering a functionally silent cysteine mutation that enables highly specific covalent modification by a designed activity-based probe. Here, we describe the translation of that technology into a mouse model of breast cancer and subsequent demonstration of the utility of the approach for studies of MMP-14 activation in the tumor microenvironment. Using this approach, we find that MMP-14 is active in late stage tumors and is predominantly associated with stromal cell populations that have been activated by specific signaling molecules (e.g., TGFβ) produced by tumor cells. Our data demonstrate the applicability of this approach for studies of MMP function in whole organisms and identify important regulatory mechanisms for MMP-14 activity in the tumor microenvironment.

Full text of DOI:10.1021/acschembio.8b00562

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Article
Chemical tools for selective activity profiling of
endogenously expressed MMP-14 in multicellular models
Neri Amara, Martina Tholen, and Matthew Bogyo
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00562 • Publication Date (Web): 30 Aug 2018
Downloaded from http://pubs.acs.org on August 31, 2018
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ACS Chemical Biology
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Chemical tools for selective activity profiling of endogenously expressed MMPꢀ14 in
multicellular models
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Neri Amara1, Martina Tholen1, Matthew Bogyo1,2,*
Departments of Pathology1, Microbiology and Immunology2 Stanford University School of
Medicine, Stanford CA 94305 USA
*Correspondence: mbogyo@stanford.edu
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Abstract
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Matrix metalloproteases (MMPs) are a large family of zincꢀdependent endopeptidases,
involved in a diverse set of physiological and pathological processes, most notably in
cancer. Current methods for imaging and quantifying MMP activity lack sufficient selectivity
and spatiotemporal resolution to allow studies of specific MMP function in vivo. Previously,
we reported a strategy for selective targeting of MMPs by engineering a functionally silent
cysteine mutation that enables highly specific covalent modification by a designed activityꢀ
based probe (ABP). Here we describe the translation of that technology into a mouse model
of breast cancer and subsequent demonstration of the utility of the approach for studies of
MMPꢀ14 activation in the tumor microenvironment. Using this approach, we find that MMPꢀ
14 is active in late stage tumors and is predominantly associated with stromal cell
populations that have been activated by specific signaling molecules (e.g. TGFβ) produced
by tumor cells. Our data demonstrates the applicability of this approach for studies of MMP
function in whole organisms and identifies important regulatory mechanisms for MMPꢀ14
activity in the tumor microenvironment.
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ACS Chemical Biology
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Introduction
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MMPs are a family of zincꢀdependent endopeptidases, consisting of more than 25 members
in humans.1 This large family of proteases is responsible for the degradation of most
extracellular proteins and has long been associated with many types and stages of cancer.2,
3 Proteolysis by MMPs is essential for a variety of physiological activities4 and signaling
events5, 6 and is tightly regulated at several levels. In addition to regulation at the
transcriptional level, all MMPs are produced as latent proꢀenzymes that require enzymatic
activation for catalytic function.1 Their activity is regulated by compartmentalization and
trafficking and is inhibited by endogenous tissue inhibitors of matrix metalloproteinases
(TIMPs).7
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MMPs promote tumor progression and regulate the tumor microenvironment through
degradation of the extracellular matrix (ECM) as well as through additional signaling
functions.6, 8 However, attempts to target MMPs for therapeutic purposes have been
hampered by a general poor understanding of the specific roles of individual MMP members
and inadequate target validation.3, 9 Contradicting roles have emerged for MMPs both as
tumor promoters and tumor suppressors.10 Depending on the circumstances, different
members of the MMPs regulate tumorigenesis during the process of microenvironment
remodeling. Despite mounting evidence of elevated MMP activity at the interface of a tumor
and its stroma,2, 8 our understanding of the specific roles, physical location and dynamics of
proteolytic activation of individual MMPs remains elusive. Current methods to visualize MMP
activity, such as fluorogenic substrates,11, 12 radiolabeled inhibitors13, 14 or fluorescent matrix
proteins15 lack the specificity and spatiotemporal precision required to map the activity of
individual MMPs in the context of a native tissue during tumor development.
Our group previously reported the design and validation of a methodology to
specifically image and selectively inhibit individual MMPs.16 This approach makes use of a
combination of protein engineering and a corresponding activityꢀbased probe (ABP) to
selectively label active forms of MMPs in intact cells (Figure 1a). A proximityꢀinduced
interaction between an engineered cysteine and an electrophilic warhead on the ABP results
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