Laminin-332 cleavage by matriptase alters motility parameters of prostate cancer cells.
ABSTRACT Matriptase, a type II transmembrane serine protease, has been linked to initiation and promotion of epidermal carcinogenesis in a murine model, suggesting that deregulation of its role in epithelia contributes to transformation. In human prostate cancer, matriptase expression correlates with progression. It is therefore of interest to determine how matriptase may contribute to epithelial neoplastic progression. One approach for studying this is to identify potential matriptase substrates involved in epithelial integrity and/or transformation like the extracellular matrix macromolecule, laminin-332 (Ln-332), which is found in the basement membrane of many epithelia, including prostate. Proteolytic processing of Ln-332 regulates cell motility of both normal and transformed cells, which has implications in cancer progression.
In vitro cleavage experiments were performed with purified Ln-332 protein and matriptase. Western blotting, enzyme inhibition assays, and mass spectrometry were used to confirm cleavage events. Matriptase overexpressing LNCaP prostate cancer cells were generated and included in Transwell migration assays and single cell motility assays, along with other prostate cells.
We report that matriptase proteolytically cleaves Ln-332 in the β3 chain. Substrate specificity was confirmed by blocking cleavage with the matriptase inhibitor, Kunitz domain-1. Transwell migration assays showed that DU145 cell motility was significantly enhanced when plated on matriptase-cleaved Ln-332. Similarly, Transwell migration of matriptase-overexpressing LNCaP cells was significantly increased on Ln-332 and, as determined by live single-cell microscopy, two motility parameters of this cell line, speed and directional persistence, were also higher.
Proteolytic processing of Ln-332 by matriptase enhances speed and directional persistence of prostate cancer cells.
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ABSTRACT: This paper reports a sensitive method with electrochemical technique to detect various proteases, which can be used for the diagnosis of prostate cancer. For the proposed assay method, the working electrode is modified with the peptide probes for the target proteases. These probes contain the substrate sequence of target proteases, as well as the seed peptide sequence that can accelerate the misfolding of amyloid-beta. If there are proteases in the test solution, after protease cleavage of the substrate peptides, the distal seed peptide will be removed from the electrode surface. So, in the absence of proteases, the seed peptides can initiate and accelerate amyloid-beta misfolding on the electrode surface. Consequently, the formed aggregates strongly block the electron transfer of the in-solution electroactive species with the electrode, resulting in suppressed signal readout. Nevertheless, in the presence of proteases, enzyme cleavage may lead to greatly mitigated protein misfolding and evident signal enhancement. Since the contrast in signal readout between the two cases can be amplified by using the protein misfolding step, high sensitivity suitable for direct detection of proteases in serum can be achieved. These results may suggest the feasibility of our new method for the detection of a panel of proteases in offering detailed diagnosis of prostate cancer and a better treatment of the cancer.Theranostics 01/2014; 4(7):701-7. · 7.81 Impact Factor
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ABSTRACT: Matriptase is a type-II transmembrane serine protease involved in epithelial homeostasis in both health and disease, and is implicated in the development and progression of a variety of cancers. Matriptase mediates its biological effects both via as yet undefined substrates and pathways, and also by proteolytic cleavage of a variety of well-defined protein substrates, several of which it shares with the closely-related protease hepsin. Development of targeted therapeutic strategies will require discrimination between these proteases. Here we have investigated cyclic microproteins of the squash Momordica cochinchinensis trypsin-inhibitor family (generated by total chemical synthesis) and found MCoTI-II to be a high-affinity (Ki 9 nM) and highly selective (> 1,000-fold) inhibitor of matriptase. MCoTI-II efficiently inhibited the proteolytic activation of pro-hepatocyte growth factor (HGF) by matriptase but not by hepsin, in both purified and cell-based systems, and inhibited HGF-dependent cell scattering. MCoTI-II also selectively inhibited the invasion of matriptase-expressing prostate cancer cells. Using a model of epithelial cell tight junction assembly, we also found that MCoTI-II could effectively inhibit the re-establishment of tight junctions and epithelial barrier function in MDCK-I cells after disruption, consistent with the role of matriptase in regulating epithelial integrity. Surprisingly, MCoTI-II was unable to inhibit matriptase-dependent proteolytic activation of prostasin, a GPI-anchored serine protease also implicated in epithelial homeostasis. These observations suggest that the unusually high selectivity afforded by MCoTI-II and its biological effectiveness might represent a useful starting point for the development of therapeutic inhibitors, and further highlight the role of matriptase in epithelial maintenance.Thrombosis and Haemostasis 04/2014; 112(2). · 5.76 Impact Factor
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ABSTRACT: Matriptase is a serine protease implicated in cancer invasion and metastasis. Expression of matriptase is frequently dysregulated in human cancers and matriptase has been reported to activate latent growth factors such as hepatocyte growth factor/scatter factor, and proteases such as urokinase plasminogen activator suggesting that matriptase inhibitors could have therapeutic potential in treatment of cancer. Here we report a structure-based approach which led to the discovery of selective and potent matriptase inhibitors with benzene as central core having 1,3,5 tri-substitution pattern. X-ray crystallography of one of the potent analogs in complex with matriptase revealed strong hydrogen bonding and salt-bridge interactions in the S1 pocket, as well as strong CH-π contacts between the P2/P4 cyclohexyl and Trp215 side-chain. An additional interaction of the pendant amine at cyclohexyl with Gln175 side-chain results in substantial improvement in matriptase inhibition and selectivity against other related serine proteases. Compounds 15 and 26 showed tumor growth inhibition in a subcutaneous DU-145 prostate cancer mouse model. These compounds could be useful as tools to further explore the biology of matriptase as a drug target.Bioorganic & medicinal chemistry 04/2014; · 2.82 Impact Factor