Matrix metalloproteinases (MMPs) have been implicated in diverse roles in breast cancer development and progression. While many of the different MMPs expressed in breast cancer are produced by stromal cells MMP-9 is produced mainly by the tumor cells themselves. To date, the functional role of tumor cell-produced MMP-9 has remained unclear. Here, we show that human breast cancer cell-produced MMP-9 is specifically required for invasion in cell culture and for pulmonary metastasis in a mouse orthotopic model of basal-like breast cancer. We also find that tumor cell-produced MMP-9 promotes tumor vascularization with only modest impact on primary tumor growth, and that silencing of MMP-9 expression in tumor cells leads to an altered transcriptional program consistent with reversion to a less malignant phenotype. MMP-9 is most highly expressed in human basal-like and triple negative tumors, where our data suggest that it contributes to metastatic progression. Our results suggest that MMP9 may offer a target for anti-metastatic therapies for basal-like triple negative breast cancers, a poor prognosis subtype with few available molecularly targeted therapeutic options.

Human mesotrypsin is an isoform of trypsin that displays unusual resistance to polypeptide trypsin inhibitors and has been observed to cleave several such inhibitors as substrates. Whereas substitution of arginine for the highly conserved glycine 193 in the trypsin active site has been implicated as a critical factor in the inhibitor resistance of mesotrypsin, how this substitution leads to accelerated inhibitor cleavage is not clear. Bovine pancreatic trypsin inhibitor (BPTI) forms an extremely stable and cleavage-resistant complex with trypsin, and thus provides a rigorous challenge of mesotrypsin catalytic activity toward polypeptide inhibitors. Here, we report kinetic constants for mesotrypsin and the highly homologous (but inhibitor sensitive) human cationic trypsin, describing inhibition by, and cleavage of BPTI, as well as crystal structures of the mesotrypsin-BPTI and human cationic trypsin-BPTI complexes. We find that mesotrypsin cleaves BPTI with a rate constant accelerated 350-fold over that of human cationic trypsin and 150,000-fold over that of bovine trypsin. From the crystal structures, we see that small conformational adjustments limited to several side chains enable mesotrypsin-BPTI complex formation, surmounting the predicted steric clash introduced by Arg-193. Our results show that the mesotrypsin-BPTI interface favors catalysis through (a) electrostatic repulsion between the closely spaced mesotrypsin Arg-193 and BPTI Arg-17, and (b) elimination of two hydrogen bonds between the enzyme and the amine leaving group portion of BPTI. Our model predicts that these deleterious interactions accelerate leaving group dissociation and deacylation.There are three human trypsins encoded by different genes; cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2) are located at proximal loci on chromosome 7q35, while mesotrypsinogen (PRSS3) is found on chromosome 9p13 (1). All three isoforms are secreted as digestive zymogens in the pancreas and activated by enteropeptidase in the duodenum (2). A differentially spliced form of mesotrypsinogen termed trypsinogen 4, transcribed from an alternative promoter (3) and utilizing an unconventional CUG translation initiation codon (4), is highly expressed in brain tissue (5) and in some epithelial cell lines (6) and tumors (7). The two zymogen forms differ only at the N terminus, and processing of either form by removal of the prodomain results in active mesotrypsin of identical amino acid sequence (3). Trypsinogen 4 lacks a recognizable signal sequence and it is not known whether or how the enzyme might be secreted, though there is some evidence for processing of the prodomain and deposition of activated mesotrypsin in the extracellular neuronal matrix (8).The most striking characteristic of mesotrypsin is its unique resistance to polypeptide trypsin inhibitors (9, 10). Canonical trypsin inhibitors feature characteristic binding loops that bind to the trypsin active site extremely tightly, mimicking a substrate, yet are cleaved extremely slowly (11-13). Mesot...

Engineered protein therapeutics offer advantages, including strong target affinity, selectivity, and low toxicity, but like natural proteins can be susceptible to proteolytic degradation, thereby limiting their effectiveness. A compelling therapeutic target is mesotrypsin, a protease upregulated with tumor progression, associated with poor prognosis, and implicated in tumor growth and progression of many cancers. However, with its unique capability for cleavage and inactivation of proteinaceous inhibitors, mesotrypsin presents a formidable challenge to the development of biologic inhibitors. We used a powerful yeast display platform for directed evolution, employing a novel multi-modal library screening strategy, to engineer the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) simultaneously for increased proteolytic stability, stronger binding affinity, and improved selectivity for mesotrypsin inhibition. We identified a triple mutant APPIM17G/I18F/F34V, with a mesotrypsin inhibition constant (Ki) of 89 pM, as the strongest mesotrypsin inhibitor yet reported; this variant displays 1459-fold improved affinity, up to 350,000-fold greater specificity, and 83-fold improved proteolytic stability vs wild-type APPI. We demonstrated that APPIM17G/I18F/F34V acts as a functional inhibitor in cell-based models of mesotrypsin-dependent prostate cancer cellular invasiveness. Additionally, by solving the crystal structure of the APPIM17G/I18F/F34V/mesotrypsin complex, we obtained new insights into the structural and mechanistic basis for improved binding and proteolytic resistance. Our study identifies a promising mesotrypsin inhibitor as a starting point for development of anticancer protein therapeutics and establishes proof-of-principle for a novel library screening approach that will be widely applicable for simultaneously evolving proteolytic stability in tandem with desired functionality for diverse protein scaffolds.

PRSS3/mesotrypsin is an atypical isoform of trypsin that has been associated with breast, lung, and pancreatic cancer cell malignancy. In analyses of open source transcriptional microarray data, we find that PRSS3 expression is upregulated in metastatic prostate cancer tissue, and that expression of PRSS3 in primary prostate tumors is prognostic of systemic progression following prostatectomy. Using a mouse orthotopic model with bioluminescent imaging, we show that PRSS3/mesotrypsin is critical for prostate cancer metastasis. Silencing of PRSS3 inhibits anchorage independent growth of prostate cancer cells in soft agar assays, and suppresses invasiveness in Matrigel transwell assays and three-dimensional (3D) cell culture models. We further show that treatment with recombinant mesotrypsin directly promotes an invasive cellular phenotype in prostate cancer cells, and find that these effects are specific and require the proteolytic activity of mesotrypsin, because neither cationic trypsin nor a mesotrypsin mutant lacking activity can drive the invasive phenotype. Finally, we demonstrate that a newly developed, potent inhibitor of mesotrypsin activity can suppress prostate cancer cell invasion to a similar extent as PRSS3 gene silencing. This study defines mesotrypsin as an important mediator of prostate cancer progression and metastasis, and suggests that inhibition of mesotrypsin activity may provide a novel modality for prostate cancer treatment.