Inhibiting serine protease-induced prostate cancer progression

描述（由申请人提供）：我们已经确定丝氨酸蛋白酶PRSS3/中间胰蛋白酶是前列腺切除术后前列腺癌全身进展的预后分子，并且在转移性前列腺癌组织中上调。我们还发现，在原位小鼠模型中，沉默前列腺癌细胞中间胰蛋白酶的表达可抑制培养和转移的侵袭性，而用重组间胰蛋白酶治疗前列腺癌细胞可刺激侵袭行为。我们的初步数据表明，中胰蛋白酶通过切割细胞外基质（ECM）中的一种或多种特定底物来促进前列腺癌的进展，从而导致COX-2的上调和侵袭行为的刺激。我们假设中胰蛋白酶可能既是转移性前列腺癌的潜在治疗靶点，也是系统性进展的有用的预后组织生物标志物。在这里，我们提出了以下实验：(1)进一步确定间胰蛋白酶促进前列腺癌进展的生物学机制；(2)开发有效和选择性的间胰蛋白酶抑制剂，以促进培养和动物模型的机制研究，并为间胰蛋白酶靶向治疗提供候选药物；(3)评估间胰蛋白酶作为预后组织生物标志物的作用。在目标1中，我们将使用蛋白质组学方法识别ECM中中间胰蛋白酶的蛋白水解底物，并使用3D培养模型测试它们对入侵的影响。我们还将使用启动子报告子测定和RNA干扰方法确定中胰蛋白酶调节COX-2转录的机制。在Aim 2中，我们将采用结构引导蛋白工程优化一种中胰蛋白酶多肽抑制剂，并在培养试验中评估该抑制剂对侵袭性的影响，以及对原位小鼠模型中肿瘤生长和转移的影响。在Aim 3中，我们将使用新开发的选择性间胰蛋白酶抗体，评估间胰蛋白酶蛋白染色作为高风险根治性前列腺切除术队列中潜在的预后组织生物标志物。我们还将确定中胰蛋白酶表达是否与转移到特定器官部位有关，使用不同的转移组织小组。这些目标的成功完成将极大地提高我们对前列腺癌进展的新分子机制的理解。

DESCRIPTION (provided by applicant): We have identified the serine protease PRSS3/mesotrypsin as a molecule that is prognostic for prostate cancer systemic progression following prostatectomy, and that is upregulated in metastatic prostate cancer tissue. We have also found that silencing of mesotrypsin expression in prostate cancer cells inhibits invasiveness in culture and metastasis in an orthotopic mouse model, while treatment of prostate cancer cells with recombinant mesotrypsin stimulates invasive behavior. Our preliminary data suggest that mesotrypsin promotes prostate cancer progression through cleavage of one or more specific substrates in the extracellular matrix (ECM), leading to upregulation of COX-2 and stimulation of invasive behavior. We hypothesize that mesotrypsin may represent both a potential therapeutic target for metastatic prostate cancer and a useful prognostic tissue biomarker of systemic progression. Here, we propose experiments (1) to further define the biological mechanisms by which mesotrypsin promotes prostate cancer progression, (2) to develop potent and selective mesotrypsin inhibitors that will facilitate mechanistic studies in culture and animal models and offer candidates for mesotrypsin-targeted therapeutics, and (3) to evaluate mesotrypsin as a prognostic tissue biomarker. In Aim 1, we will identify proteolytic substrates of mesotrypsin in ECM using a proteomic approach, and test their impact on invasion using 3D culture models. We will also determine the mechanisms by which mesotrypsin regulates COX-2 transcription using promoter-reporter assays and RNA interference approaches. In Aim 2, we will employ structure-guided protein engineering to optimize a polypeptide inhibitor of mesotrypsin, and evaluate the impact of this inhibitor on invasion in culture assays and on tumor growth and metastasis in an orthotopic mouse model. In Aim 3, we will assess mesotrypsin protein staining as a potential prognostic tissue biomarker in a high-risk radical prostatectomy cohort, using a newly developed selective mesotrypsin antibody. We will also determine whether mesotrypsin expression is associated with metastasis to specific organ sites, using a diverse panel of metastatic tissues. The successful completion of these aims will critically improve our understanding of novel molecular mechanisms that underlie prostate cancer progression.