Background: Mesotrypsin is an atypical isoform of trypsin that previously has not been investigated in the context of prostate cancer. In unpublished analyses, we find that mesotrypsin expression correlates with prostate cancer progression and predicts recurrence. We further find that mesotrypsin promotes malignant growth and stimulates invasion of prostate cancer cells via a mechanism involving transcriptional upregulation of COX-2, suggesting that inhibition of mesotrypsin or its critical downstream effectors may provide new avenues for prostate cancer intervention. In crystallographic and enzyme kinetics studies, we have found that mesotrypsin possesses unique active site features that result in atypical substrate- and inhibitor-binding specificity compared with other related proteases; the distinctive nature of the mesotrypsin active site may allow development of inhibitors that can target mesotrypsin selectively. Our preliminary studies indicate that naturally occurring polypeptide inhibitors may be engineered for selectivity toward mesotrypsin, for potential use as drugs.Objectives: We propose (1) to define the role of mesotrypsin in prostate cancer progression, and (2) to develop and test mesotrypsin inhibitors with potential to block prostate cancer invasion and metastasis.Specific Aims:Specific Aim 1: To define the role of mesotrypsin in prostate cancer progression.A) Identify the specific targets of mesotrypsin cleavage in prostate cancer cells that lead to increased invasion.B) Identify the pathway by which mesotrypsin induces transcriptional upregulation of COX-2.C) Develop an animal model to define the invasion-enhancing effects of mesotrypsin.Specific Aim 2: To develop potent and selective mesotrypsin inhibitors, and evaluate mesotrypsin inhibition as a therapeutic strategy.A) Select a polypeptide inhibitor scaffold of optimal stability against mesotrypsin degradation.B) Optimize mesotrypsin inhibitor for enhanced potency and selectivity; evaluate in biological assays.Study Design: We will identify specific substrates of mesotrypsin from among a panel of candidates by Western blotting and kinetic assays, and evaluate their significance in prostate cancer cell malignant growth and invasion using RNAi techniques and three-dimensional (3D) cell culture studies. We will identify promoter elements and transcription factors involved in mesotrypsin transcriptional regulation of COX-2 using promoter-reporter studies and RNAi techniques. We will assess the effects of enhanced mesotrypsin expression on the growth, invasion, and metastasis of human prostate cancer orthotopic xenografts in nude mice, using real time in vivo tumor imaging by bioluminescence in addition to conventional endpoint analysis. For development of mesotrypsin-targeting inhibitors, we will evaluate a panel of human-derived Kunitz domain inhibitors to identify a superior template with optimal proteolytic stability, and we will identify mutations that confer improvements in mesotrypsin affinity and selectivity by screening a phage-displayed inhibitor library. The most promising inhibitors will be assessed as potential prostate cancer therapeutics in 3D cell culture morphology assays and invasion assays, and will ultimately be assessed in the orthotopic mouse model.Innovation: This study is innovative because it centers on mesotrypsin as a completely novel therapeutic target for prostate cancer. It is also innovative in integrating (a) a prostate cancer cell progression series modeling the specific changes associated with acquisition of invasiveness, (b) a sophisticated 3D culture model which provides a physiologically relevant, tractable, and cost-effective assay for initial screening of inhibitors, and (c) an orthotopic xenograft model appropriate for comprehensive evaluation of the effects of mesotrypsin and mesotrypsin inhibition on all steps involved in prostate cancer invasion and metastasis.Impact: This study is highly significant because, by targeting an enzyme that is upregulated in invasive prostate cancer and that may be involved in metastatic spread of the disease, we will potentially introduce a new treatment approach that will reduce prostate cancer mortality. We propose to engineer natural polypeptide inhibitors as potent and selective drugs targeting mesotrypsin. Protein therapeutics represent a growing segment of the drug discovery field, accounting for a quarter of recent new drug approvals, and polypeptide serine protease inhibitors possess many attributes indicative of high potential as protein therapeutics.Focus Areas: This study is highly responsive to Fiscal Year 2009 PCRP focus areas:A) Tumor Biology: We will define the extracellular targets and intracellular processes initiated by mesotrypsin that promote prostate cancer progression, potentially revealing an important role in prostate cancer for this little-known enzyme.B) Therapy: We will develop novel mesotrypsin-targeting inhibitors based on small, soluble, highly stable human proteins for assessment as new protein therapeutics in models of prostate cancer.
|Effective start/end date||1/1/09 → 8/31/13|
- U.S. Department of Defense: $688,500.00
- Congressionally Directed Medical Research Programs: $688,500.00