Prostate cancer, the most commonly diagnosed malignancy and the second leading cause of cancer mortality in American men, has a particularly high propensity to metastasize to bone. Unfortunately, PC bone metastasis has no cure. Our long-term goal is to investigate the therapeutic potential of maspin in treating PC bone metastasis. Maspin is a novel epithelial-specific serine protease inhibitor (serpin). Clinical studies indicate that maspin expression is down regulated in invasive and metastatic carcinomas. Experimental evidence from our laboratory and others demonstrates a tumor suppressive role of maspin at the steps of tumor invasion and metastasis. In particular, we have shown that maspin inhibits PC bone tumor growth, PC-induced osteolysis, and PC bone tumor angiogenesis. To explore the therapeutic potential of maspin, we need to have a better understanding of its underlying molecular mechanism. We have shown that maspin dramatically reduces the presentation as well as the activity of PC cell surface-associated urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR). uPA and its zymogen pro-uPA can both bind to uPAR to regulate extracellular proteolysis, cell adhesion and signal transduction. Interestingly, maspin exhibits a novel affinity for pro-uPA. Our preliminary evidence suggests that maspin may trigger receptor-mediated endocytosis of the pro-uPA/uPAR complex before pro-uPA becomes activated. We also showed that maspin specifically binds to type I collagen, the most abundant protein in bone matrix. While maspin stabilized PC cell attachment to type I collagen, type I collagen further enhanced the maspin-mediated pro-uPA endocytosis. Since uPA promotes both osteoblastic and osteolytic tumor bone metastasis, our data collectively support a novel hypothesis: Extracellular maspin may block PC/bone interaction by quenching the cell surface-associated pro-uPA/uPAR complex. Furthermore, the maspin effect on PC/bone interaction is directly regulated by type I collagen. To address this new hypothesis, we will focus on three specific aims.
Specific Aim 1 is to investigate the regulation of pro-uPA by maspin.
Specific Aim 2 is to investigate the maspin/Col I Interaction.
Specific Aim 3 is to investigate the regulation of uPA/uPAR by maspin in PC/bone interaction. Despite the consensus that uPA and uPAR are potential therapeutic targets in PC metastasis, there is no uPA- or uPAR-targeting drug for cancer treatments. To this end, results from this study may provide novel insights regarding how maspin may block the uPA/uPAR complex in the vicious cycle of PC/bone interaction. We will characterize rational maspin mutants in clinically relevant models for PC/bone metastasis to delineate the functional determinants of maspin. The expected results are likely to shed new lights on how to improve the therapeutic intervention of PC bone metastasis.
The current application addresses a novel hypothesis that extracellular maspin, a noninhibitory serine protease inhibitor, may block prostate cancer (PC)/bone interaction by quenching the cell surface-associated pro-urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) complex. Furthermore, the maspin effect on PC/bone interaction is directly regulated by type I collagen. This hypothesis not only is conceptually paradigm-shifting but also has high clinical significance. At center is the novel serine protease-like property of maspin to antagonize multifaceted activities of the uPA/uPAR complex. A valuable lesson from the past effort to inhibit the uPA/uPAR system by strong enzymatic antagonists such as PAI-1 and synthetic peptides is that inhibition of an active enzyme without inhibiting its activation may not be sufficient. Furthermore, the enzymatically inert or less active zymogens such as pro-uPA may have very important biological functions that can not be neutralized by specific enzymatic inhibitors. In light of the opposite effects of maspin and the uPA/uPAR complex in tumor invasion and metastasis, maspin offers a unique opportunity to quench cell surface associated pro-uPA before it become proteolytically active. To our knowledge, no other naturally occurring or synthetic uPA inhibitors have been shown to do the same. The current study is highly relevant to human PC bone metastasis. Maspin, uPA, and uPAR are important regulators of tumor progression and metastasis. Pertinent to the current application, the uPA/uPAR complex has been shown to play a key role in facilitating the vicious cycle of tumor/bone interaction and the tumor bone metastasis, thus are promising therapeutic targets. In contrast, maspin has been shown to act as a tumor suppressor at the step of tumor invasion and metastasis. Furthermore, the maspin-binding protein, type I collagen (Col I), is not only a key component in tumor host tissue remodeling, but also the most abundant protein in bone matrix. We are aware that these molecules, including maspin, may be just as important in the bone metastasis of other types of cancer such as breast cancer. However, bone metastasis of different types of cancer may involve different regulating mechanisms. To focus the current application on human PC bone metastasis and PC/bone interaction, we will use appropriate cell lines and clinically relevant tumor models. To prepare for the translation of our bench study to eventual development of maspin-based anti-metastasis drugs, we put an emphasis on the structural-functional relationship of maspin. From an evolution point of view, the activity of maspin to target serine protease-like molecules may not be substituted by other serpins that have evolved to acquire higher target specificities. The current literature, understandably based mostly on classical inhibitory serpins, offers little guideline for maspin research. Yet, to our knowledge, no other serpins including ov-serpins have been investigated as serine protease-like inhibitors against serine protease-like targets. To this end, we were given a timely and valuable gift, the X-ray crystal structure of maspin (10), which confirms many of the novel features of maspin that took us a decade to uncover. This structure will be used as the blue print for our rational designs of maspin mutagenesis. The expected results are likely to shed new lights on how to convert maspin into a drug.
