Biochemistry of MT1-MMP activation in malignancy

描述（由申请人提供）：此申请是基于我们之前资助的5年项目，该项目产生了40多篇同行评审的出版物。促进侵袭，促肿瘤发生的MT1-MMP被认为是癌症生物学中最重要的MMP，并被认为是细胞功能调节剂的特殊作用。科学家们一致认为MT1-MMP是细胞表面蛋白水解事件的关键参与者。我们现在知道，在多个模型系统中，MT1-MMP作为癌基因，刺激肿瘤细胞侵袭和转移，并接管肿瘤生长控制。在人类中，MT1-MMP活性与转移、临床分期和肿瘤大小显著相关。然而，我们对MT1-MMP生物学的了解是有限的，特别是当与这种蛋白酶在恶性肿瘤中的作用相比时。到目前为止，MT1-MMP激活的机制尚未完全了解。作为我们研究的结果，我们现在可以提出，而不是在任何细胞类型中表现出统一的激活途径，促进侵袭的MT1-MMP似乎有一个激活机制层次。为了增加它们的蛋白水解库，肿瘤细胞利用导致MT1-MMP自身抑制前域完全失活的机制，而在正常细胞中，MT1-MMP活性仅由furin切割释放的完整前域控制。因此，完整的前结构域在正常微环境中阻止了促侵袭性、促致瘤性MT1-MMP的潜在损伤功能。自催化活化的furin在包括MT1-MMP和MT1-MMP的下游靶标MMP-2在内的多种原蛋白的加工和激活中发挥重要作用。由于其在癌症中的重要作用，MT1-MMP是一种很有前景的疾病药物靶点，迫切需要其选择性生物传感器和抑制剂。总的来说，很明显需要做更多的工作来更好地理解MT1-MMP的结构-功能关系。我们为更好地了解MT1-MMP而设计的三个具体目标是：(1)确定正常细胞和癌细胞中MT1-MMP激活的两步前域加工机制的功能意义；(2)设计和测试高效、选择性、切割激活的MT1-MMP和furin （MT1-MMP的激活剂）生物传感器；(3)设计和评估高选择性、强效、活性位点靶向、抑制性Fab/scFv人MT1-MMP抗体的抗肿瘤活性。我们提出的研究结果将是对MT1-MMP及其激活剂furin所起的许多作用的透彻理解。我们的课程将采用综合系统方法，包括生化、分子、细胞和计算机建模研究。我们还将在小鼠身上进行肿瘤异种移植的研究，以验证我们在体外获得的结果。我们的程序将使我们对恶性细胞如何从它们的原始位置转移到全身扩散有了更精确的了解。这一有价值的信息将直接导致新的和有效的策略发展的检测，预后和治疗范围广泛的恶性肿瘤。

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.