Cysteine Protease Network in Tumor Progression and Therapy

描述（由申请人提供）：我们首次报道了豆科蛋白，一种溶酶体半胱氨酸蛋白酶，在大多数啮齿动物和人类实体瘤中通过肿瘤和基质细胞高度表达。我们发现豆科蛋白的表达是由缺氧诱导的，并在肿瘤发展的早期发生。我们证明了豆类蛋白通过复杂而精确的调节组织蛋白酶和半胱天蛋白酶网络，增强肿瘤细胞的侵袭/转移和保护细胞免于凋亡。豆类是哺乳动物中唯一的天冬酰胺内肽酶，具有类似半胱天冬酶的催化位点。半胱天冬酶在植物中是不存在的，据报道豆类蛋白酶是植物细胞凋亡的效应蛋白酶。豆科蛋白通过与细胞表面整合素结合，激活MMP-2和组织蛋白酶l，促进肿瘤细胞的侵袭和转移。我们在哺乳动物中证明，豆科蛋白进化获得抗凋亡活性。豆科蛋白通过催化caspase 9失活和通过结合和调节组织蛋白酶B活性阻止组织蛋白酶B激活Bid，从而保护细胞免于程序性细胞死亡。我们已经报道了靶向肿瘤微环境（TME）中仅由豆科蛋白激活的细胞不渗透前药的策略。在这里，我们证明了高亲和力，细胞不渗透的天冬酰胺内肽酶抑制剂（AEPI-1）对TME中豆类素的抑制作用，抑制血管生成和肿瘤细胞的侵袭/转移。AEPI-1治疗的肿瘤表现出细胞外基质的严重破坏和胶原含量的显著降低，并导致药物渗透和保留增强。鉴于豆科蛋白的高度限制性特异性及其在肿瘤发展中的功能，我们正在寻求一种假设驱动的方法来推进癌症治疗的后续策略。1. 靶向肿瘤微环境（TME）的细胞不渗透性豆类激活前药及基于紫杉醇的前药开发。2. 用细胞不渗透的AEPI抑制TME中的豆科蛋白抑制肿瘤侵袭/转移和血管生成。3. 提高AEPI的细胞通透性，进一步抑制细胞内豆科蛋白，使耐药肿瘤细胞对凋亡敏感。给予细胞渗透性AEPI可能会延长AEPI的疗效，但可能导致毒性。我们将评估细胞不渗透性和渗透性AEPIs与现有癌症治疗方法的协同作用。

DESCRIPTION (provided by applicant): We first reported that legumain, a lysosomal cysteine protease, is highly expressed in majority of rodent and human solid tumors by tumor as well as stromal cells. We found legumain expression is induced by hypoxia and occurs early during tumor development. We demonstrated that legumain enhances tumor cell invasion/ metastasis and protects cells from apoptosis through complex and precise regulation of cathepsin and caspase network. Legumain is the only asparaginyl endopeptidase in mammals and it has a caspase-like catalytic site. The caspases are absent in plants, and legumain was reported to be the effector protease for plant cell apoptosis. Legumain contributes to tumor cell invasion and metastasis through binding to cell surface integrins and activate both MMP-2 and cathepsin L. We demonstrated in mammals legumain evolved to obtain an anti-apoptotic activity. Legumain protects cells from programmed cell death by catalytic inactivation of caspase 9 and by preventing Bid activation by cathepsin B through binding and modulating cathepsin B activity. We have reported the strategy of targeting cell-impermeable prodrug activated only by legumain in the tumor microenvironment (TME). Here, we demonstrated inhibition of legumain in TME by a high affinity, cell impermeable asparaginyl endopeptidase inhibitor (AEPI-1) suppress angiogenesis and tumor cell invasion/metastasis. AEPI-1 treated tumors demonstrated a profound disorganization of extracellular matrix and a significant reduction of collagen content and resulted in enhanced drug penetration and retention. Given the highly restrictive specificity of legumain and its functions in tumor development, we are pursuing a hypothesis-driven approach to advance following strategies as cancer therapies. 1. Targeting cell-Impermeable legumain-activated prodrug to the tumor microenvironment (TME) and developing a paclitaxel based prodrug. 2. Inhibiting legumain in the TME with a cell-impermeable AEPI to suppress tumor invasion/metastasis and angiogenesis. 3. Improving cell permeability of AEPI to further inhibit intracellular legumain and sensitize resistant tumor cells to apoptosis. Administration of cell permeable AEPI will likely extend AEPI efficacy, but may lead to toxicity. We will evaluate both cell- impermeable and permeable AEPIs for synergy with established cancer therapies.