The overall aim of this project is to understand the biochemistry, biology, and pathology of cell surface-associated serine proteases; in particular, their relation to the development, regeneration, and malignant transformation of oral tissues. Novel cell surface serine proteases in epidermal development, repair, and malignancy Identification and characterization of novel type II transmembrane serine proteases Bioinformatic analysis predicts the existence of a large family of type II transmembrane serine proteases (TTSPs). Working with Dr. Toni Antalis, University of Maryland, we performed a detailed molecular and biochemical characterization of one of these novel TTSPs, Downregulated in Epidermal Squamous Cell Carcinoma (DESC)1, identified as a transcript that is frequently extinguish in oral cancer. The novel gene resides in a cluster of five DESC1-like genes on human chromosome 4q13.3, syntenic to the mouse chromosome 5E1 locus containing seven DESC1-like genes. cDNA cloning of the DESC1 mRNA identified a multi-domain serine protease with an N-terminal signal anchor, a SEA domain, and a C-terminal serine protease domain that is expressed in epidermal, oral, and male reproductive tissues. The DESC1 mRNA directed the translation of a 60 kDa N-glycosylated trypsin-activatable transmembrane serine protease zymogen. Activated DESC1 cleaved synthetic peptides with arginine in the P1 position and proteolytic activity was abolished by generic inhibitors of serine proteases, but not by other classes of protease inhibitors. Unexpectedly, DESC1 formed inhibitory complexes with both plasminogen activator inhibitor-1 and protein C inhibitor that are co-expressed with DESC1, suggesting that TTSPs may be novel targets for serpin inhibition. Together, these data show that DESC1 encodes a functional cell surface serine protease that may have important functions in the epidermis, oral, and reproductive epithelium. Matriptase in squamous cell carcinogenesis The recently identified TTSP, matriptase, is expressed with remarkable consistency in human epithelial tumors, including oral squamous cell carcinoma. In collaboration with the MCU, we found that matriptase differs from other tumor-associated proteases by possessing a strong oncogenic potential when unopposed by its endogenous inhibitor HAI-1. Modest orthotopic overexpression of matriptase in the epidermis of transgenic mice caused spontaneous squamous cell carcinoma and dramatically potentiated squamous cell carcinogenesis after genotoxic exposure. Matriptase-induced malignancies were preceded by progressive interfollicular hyperplasia, dysplasia, fibrosis, and dermal inflammation. Molecular analysis uncovered activating mutations in either Ha-ras or K-ras in carcinogen-induced tumors, while matriptase-induced spontaneous carcinoma formation did not require ras activation. Increasing epidermal HAI-1 expression completely negated the oncogenic effects of matriptase. The data implicate dysregulated matriptase proteolysis in malignant epithelial transformation, and predict a critical role of the matriptase/HAI-1 balance in regulating malignant conversion of carcinogen-exposed tissues, including oral epithelium. Plasminogen activation in malignant tissue remodeling The uPAR-Associated Protein, uPARAP The uPAR-associated protein (uPARAP) is highly overexpressed at sites of active tissue remodeling, including the reactive stroma of human oral cancer. Unexpectedly, we found that the novel transmembrane glycoprotein targets collagen for endocytic uptake, lysosomal delivery, and proteolytic degradation. In uPARAP-sufficient cells, fluorescent collagen is first internalized into vesicular structures a subset of which were identified as lysosomes by staining for LAMP-1. In contrast, collagen remains extracellular and associated with fiber-like structttttures in uPARAP-deficient cells. Blocking lysosomal cysteine proteases with the inhibitor E64d resulted in strong accumulation of collagen in lysosomes in uPARAP-sufficient cells, showing that endocytosed collagen is degraded by lysosomal cathepsins. Taken together, these findings predict a central role of uPARAP in collagen catabolism during physiological and pathological tissue remodeling, including cancer. Initiation of the urokinase-mediated plasminogen activation cascade Attempts to identify the initiation routes and the sites of plasminogen (Plg) activation during tissue remodeling in vivo are hampered by the absence of reagents capable of detecting the activation state of the minute quantities of Plg activators that are present in living tissues. This absence of tools to image cell surface proteolytic activity is generic to the protease field and significantly impairs research progress and the use of protease inhibitors in the clinic for cancer treatment. We are working along three separate lines to image uPA activity. With the MCU, we have shown that laser capture microdissection combined with Plg-casein zymography can be used as a novel and sensitive way to detect uPA in human oral squamous cell carcinomas, using as little as 0.5 microgram protein lysate from microdissected tumors. In complementary studies, we have worked with Ching H. Tung, Harvard Medical School, on the development of selective uPA-activatable near-infrared fluorescent imaging probes for specific detection of uPA activity in biological systems. Finally, with Steve Leppla, MPS, NIAID, we are developing modified uPA-cleaved anthrax toxin-beta-lactamase fusion proteins combined with beta-lactamase-activated fluorescence resonance energy transfer probes to detect uPA activity in living cells. Unexpected roles of the plasminogen activation system in brain disease We have continued our collaboration with Dr. Daniel Lawrence, University of Maryland, on the pleiotropic functions of tissue plasminogen activator (tPA) in brain pathology. Our most recent studies have documented that tPA mediates blood brain barrier breakdown via a Plg-independent process that involves tPA binding to the low density lipoprotein receptor (LRP). Injection of tPA into the cerebrospinal fluid in the absence of ischemia results in a rapid dose-dependent increase in vascular permeability. This activity is not seen with uPA and can be induced in Plg-/- mice, demonstrating that the effect is Plg-independent. However, the activity is blocked by antibodies to the LRP and by the LRP antagonist, RAP, suggesting a receptor-mediated process. Together these studies demonstrate that tPA is both necessary and sufficient to directly increase vascular permeability in the early stages of blood brain barrier opening, and suggest that tPA is critical to the detrimental vasogenic edema frequently associated with stroke and head trauma. Urokinase-activated bacterial cytotoxins for the treatment of tumors In collaboration with the MPS, NIAID, we recently showed that engineered anthrax toxins that are activated by cell surface uPA display potent antitumor activity and reduced toxicity to normal tissues. We have continued our preclinical development of uPA activated anthrax toxins, working via a CRADA with the biopharmaceutical company, OncoTac Pharmaceuticals. These studies have shown that uPA-activated anthrax toxins provide potent antitumor activity when administered systemically via several different routes, and that the therapeutic index can be substantially increased by co-administration of dexamethasone to specifically suppress uPA production by normal tissues. In a collaboration with Dr. Arthur Frankel, Wake Forest University, we also have found that our novel strategy for redirecting toxins to tumors can also be used to improve the therapeutic index of bacterial toxins already in experimental clinical use.
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