Inhibiting serine protease-induced prostate cancer progression

描述（由申请人提供）：我们已经鉴定了丝氨酸蛋白酶PRSS 3/中胰蛋白酶作为一种分子，其是前列腺切除术后前列腺癌系统性进展的预后，并且在转移性前列腺癌组织中上调。我们还发现，前列腺癌细胞中的中胰蛋白酶表达的沉默抑制了原位小鼠模型中培养物的侵袭性和转移，而用重组中胰蛋白酶处理前列腺癌细胞刺激了侵袭行为。我们的初步数据表明，中胰蛋白酶通过切割细胞外基质（ECM）中的一种或多种特异性底物促进前列腺癌进展，导致考克斯-2上调和刺激侵袭行为。我们假设中胰蛋白酶可能是转移性前列腺癌的潜在治疗靶点，也是系统进展的有用预后组织生物标志物。在这里，我们提出了实验（1），以进一步确定中胰蛋白酶促进前列腺癌进展的生物学机制，（2）开发有效的和选择性的中胰蛋白酶抑制剂，这将有助于在培养和动物模型中的机制研究，并提供中胰蛋白酶靶向治疗的候选人，和（3）评估中胰蛋白酶作为预后组织生物标志物。在目标1中，我们将使用蛋白质组学方法鉴定ECM中胰蛋白酶的蛋白水解底物，并使用3D培养模型测试其对侵袭的影响。我们还将确定中胰蛋白酶调节考克斯-2转录的机制，使用启动子-报告分析和RNA干扰方法。在目标2中，我们将采用结构导向的蛋白质工程来优化中胰蛋白酶的多肽抑制剂，并评估该抑制剂对培养测定中的侵袭和原位小鼠模型中的肿瘤生长和转移的影响。在目标3中，我们将使用新开发的选择性中胰蛋白酶抗体评估中胰蛋白酶蛋白染色作为高危根治性直肠癌切除术队列中的潜在预后组织生物标志物。我们还将使用多种转移组织来确定中胰蛋白酶表达是否与转移到特定器官部位相关。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.