NON-PROTEOLYTIC INTERACTIONS OF TIMP-2 AND MT1-MMP

描述（由申请人提供）：促进侵袭，致瘤性膜型1基质金属蛋白酶（MT1-MMP），一种具有细胞外催化，血凝素（PEX）和铰链结构域的跨膜蛋白酶，以及短的细胞质尾部，与其生理蛋白抑制剂，金属蛋白酶组织抑制剂-2 （TIMP-2）形成化学计量复合物。MT1-MMP的蛋白水解活性是肿瘤细胞侵袭和增殖所必需的，TIMP-2结合MT1-MMP的催化结构域并阻断其蛋白水解活性。我们最近发表的大量研究表明，除了细胞外蛋白水解外，MT1-MMP和TIMP-2还通过非蛋白水解机制控制细胞增殖和迁移。TIMP-2结合MT1-MMP诱导细胞外信号调节激酶1/2 （ERK1/2）的激活，其机制独立于MT1-MMP的催化结构域和TIMP-2的抑制结构域。这种作用涉及TIMP-2与血凝素和/或铰链结构域的结合，并由MT1-MMP的细胞质尾部介导。mt1 - mmp介导的ERK1/2激活上调细胞迁移和增殖，独立于细胞外基质蛋白水解。与这一发现一致，蛋白水解无活性的MT1-MMP在体内促进肿瘤生长，其作用与野生型MT1-MMP相当。虽然这些观察结果并没有削弱涉及TIMP-2和MT1-MMP的蛋白质水解相互作用的公认重要性，但我们的新颖，意想不到的发现强烈主张我们确定的蛋白质水解非依赖性信号机制的非常重要的作用。由于ERK1/2信号通路控制多种肿瘤细胞功能，包括增殖和迁移，因此详细了解TIMP-2 - MT1-MMP相互作用激活该信号通路的分子机制可以为设计新的抑制剂来阻断肿瘤进展提供基础信息。因此，我们建议通过制定以下具体目标来详细探索这种新的信号机制：1)确定介导TIMP-2结合和ERK1/2激活的MT1-MMP PEX和/或铰链域的区域；2)确定与MT1-MMP的PEX和/或铰链域结合所需的TIMP-2区域；3)表征MT1-MMP向Ras-ERK1/2通路信号转导的机制；4)确定TIMP-2 - MT1-MMP相互作用在体内肿瘤生长中的功能作用。为此，我们将使用最先进的分子、细胞和免疫学技术以及免疫缺陷小鼠的肿瘤异种移植模型。我们的研究结果将有助于了解MT1-MMP和TIMP-2在肿瘤生长中的非蛋白水解作用，并提供旨在阻断MT1-MMP - TIMP-2相互作用和产生促进肿瘤进展的细胞内信号的新型抑制剂的开发。公共卫生相关性：我们在实验体内模型中发现了一种意想不到的、范式转移的机制，它控制肿瘤细胞的增殖和迁移，以及肿瘤的生长。因此，我们拟详细研究这一新机制及其在肿瘤生长中的功能作用。从我们的研究中获得的知识将对设计新的药物来阻止肿瘤进展具有重要的基础意义。

DESCRIPTION (provided by applicant): The invasion-promoting, tumorigenic membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with extracellular catalytic, hemopexin (PEX) and hinge domains, and a short cytoplasmic tail, forms a stoichiometric complex with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). It is well established that the proteolytic activity of MT1-MMP is required for tumor cell invasion and proliferation, and that TIMP-2 binds to the catalytic domain of MT1-MMP and blocks its proteolytic activity. Our extensive, recently published studies have shown that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of Extracellular signal-Regulated Kinase 1/2 (ERK1/2) by a mechanism independent of the MT1-MMP catalytic domain and the inhibitory domain of TIMP- 2. This effect involves TIMP-2 binding to the hemopexin and/or hinge domain and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 upregulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Consistent with this finding, proteolytically inactive MT1-MMP promotes tumor growth in vivo with an effect comparable to that of wild- type MT1-MMP. While these observations do not diminish the well-established importance of the proteolytic interactions involving TIMP-2 and MT1-MMP, our novel, unexpected findings strongly advocate a very important role for the proteolysis-independent signaling mechanism we identified. Because the ERK1/2 signaling pathway controls a variety of tumor cell functions including proliferation and migration, a detailed understanding of the molecular mechanism by which TIMP-2 - MT1-MMP interaction activates this signaling pathway can provide fundamental information for designing novel inhibitors to block tumor progression. Therefore, we propose to explore this novel signaling mechanism in detail by developing the following Specific Aims: 1) To identify the region(s) of the MT1-MMP PEX and/or hinge domains that mediate TIMP-2 binding and ERK1/2 activation, 2) To identify the region(s) of TIMP-2 required for binding to the PEX and/or hinge domain of MT1-MMP; 3) To characterize the mechanism of signal transduction from MT1-MMP to the Ras-ERK1/2 pathway; 4) To determine the functional role of TIMP-2 - MT1-MMP interaction in tumor growth in vivo. For this purpose we will use state-of-the-art molecular, cellular and immunological techniques and a tumor xenograft model in immunodeficient mice. The results of our study will lead to the understanding of the non-proteolytic role of MT1-MMP and TIMP-2 in tumor growth, and afford the development of novel inhibitors aimed to block MT1-MMP - TIMP-2 interaction and the generation of intracellular signaling that promotes tumor progression. PUBLIC HEALTH RELEVANCE: We found an unexpected, paradigm-shifting mechanism that controls tumor cell proliferation and migration, as well as tumor growth in an experimental in vivo model. Therefore, we propose to study in detail this novel mechanism and its functional role in tumor growth. The knowledge derived from our study will be of fundamental importance for designing novel pharmacological agents to block tumor progression.