Biochemistry of MT1-MMP activation in malignancy

描述(由申请人提供)：本申请是基于我们之前资助的五年项目，该项目产生了40多份同行评议的出版物。促侵袭、促肿瘤发生的MT1-MMPs被认为是肿瘤生物学中最重要的MMPs，并且作为细胞功能的调节剂发挥着特殊的作用。科学家们一致认为MT1-MMPs在细胞表面蛋白降解事件中起关键作用。我们现在知道，在多个模型系统中，MT1-MMP作为癌基因，刺激肿瘤细胞的侵袭和转移，并接管肿瘤的生长控制。一致认为，在人类中，MT1-基质金属蛋白酶活性与转移、临床分期和肿瘤大小显著相关。然而，我们对MT1-MMPs生物学的了解是有限的，尤其是与该酶在恶性肿瘤中所起的作用相比。到目前为止，MT1-基质金属蛋白酶的激活机制还不完全清楚。作为我们研究的结果，我们现在可以认为，在任何类型的细胞中，促进侵袭的MT1-MMP似乎具有激活机制层次，而不是呈现出统一的激活途径。为了增加他们的蛋白水解库，肿瘤细胞利用导致MT1-基质金属蛋白酶的自抑制原结构域完全失活的机制，而在正常细胞中，MT1-基质金属蛋白酶的活性受完整的原结构域控制，该原结构域由Furin单独切割释放。因此，完整的原结构域阻止了在正常微环境中亲侵袭、促肿瘤形成的MT1-MMPs潜在的破坏性功能。自催化激活的Furin在多种原蛋白的加工和激活过程中起着重要作用，其中包括MT1-MMPs和MT1-MMPs下游靶点MMP2。由于其在癌症中的重要作用，MT1-MMPs是一种很有前景的治疗疾病的药物靶点，其选择性生物传感器和抑制剂是迫切需要的。总体而言，很明显，需要额外的工作来更好地了解MT1-基质金属蛋白酶的结构-功能关系。我们设计的三个具体目标是：(1)确定MT1-MMP激活的两步前结构域处理机制的功能意义；(2)设计和测试高效和选择性的、切割激活的MT1-MMP和Furin(MT1-MMP的激活剂)的生物传感器；以及(3)设计和评估高选择性和强大的、活性位点靶向、抑制性Fab/scFv人源性抗MT1-MMP抗体的抗肿瘤活性。我们提出的研究的结果将是彻底了解MT1-MMP及其激活剂Furin所扮演的许多角色。我们的计划将使用一种集成的系统方法，包括生化、分子、细胞和计算机模拟研究。我们还将使用小鼠的肿瘤异种移植进行研究，以验证我们在体外获得的结果。我们的计划将导致对恶性细胞如何从其原始位置运输并在全身扩散的精细理解。这些有价值的信息将直接导致开发新的和有效的策略来检测、预测和治疗广泛的恶性肿瘤。

DESCRIPTION (provided by applicant): This application is a based on our previously funded 5-year project that generated over 40 peer-reviewed publications. Invasion-promoting, pro-tumorigenic MT1-MMP is implicated as the most important MMP in cancer biology and is known to play specialized roles as a modifier of cell function. There is a consensus among scientists that MT1-MMP is a key player in cell surface proteolytic events. We now know that in multiple model systems MT1-MMP acts as an oncogene, stimulates tumor cell invasion and metastasis, and takes over tumor growth control. In agreement, in humans MT1-MMP activity correlates significantly with metastases, clinical stage, and tumor size. Our knowledge of the MT1-MMP biology, however, is limited as yet especially when compared with the role this proteinase plays in malignancy. As of now, the mechanisms of MT1-MMP activation are not completely understood. As a result of our study we can now suggest that rather than exhibiting a uniform activation pathway in any cell type, invasion-promoting MT1-MMP appears to have an activation mechanism hierarchy. To increase their proteolytic arsenal, tumor cells exploit the mechanism leading to the complete inactivation of the MT1-MMP's autoinhibitory prodomain while in normal cells MT1- MMP activity is controlled by the intact prodomain that is released by furin cleavage alone. As a result, the intact prodomain prevents the potentially damaging function of pro-invasive, pro-tumorigenic MT1-MMP in the normal microenvironment. Autocatalytically activated furin plays an important role in the processing and activation of multiple proproteins including MT1-MMP and MMP-2, the downstream target of MT1-MMP. Because of its importance in cancer, MT1-MMP is a promising drug target in disease and its selective biosensor and inhibitors are urgently needed. Overall, it is obvious that additional work is needed to gain a better understanding of the MT1-MMP structural-functional relationships. Our three Specific Aims, which we have designed to achieve a better understanding of MT1-MMP are: (1) Determine the functional significance of a two-step prodomain processing mechanism of MT1-MMP activation in normal and cancer cells, (2) Design and test efficient and selective, cleavage-activated biosensors of MT1-MMP and furin (an activator of MT1-MMP), and (3) Design and evaluate the anti-tumor activity of the highly selective and potent, active site- targeting, inhibitory Fab/scFv human antibodies to MT1-MMP. The result of our proposed studies will be a thorough understanding of the many roles played by MT1-MMP and its activator, furin. Our program will use an integrated systems approach involving biochemical, molecular, cellular and computer modeling studies. We will also conduct studies using tumor xenografts in mice to validate our results gained in vitro. Our program will result in a refined understanding of how malignant cells traffic from their original location and spread throughout the body. This valuable information will lead directly to the development of novel and effective strategies for the detection, prognosis, and treatment of a broad range of malignancies.