Dynamic regulation of MT1-MMP at the tumor cell surface and malignancy

描述(由申请人提供)：癌症的进展取决于促进肿瘤细胞生长和侵袭的蛋白分解系统的作用。膜型基质金属蛋白酶(MT1-MMPs)赋予肿瘤细胞侵袭和生长在胶原基质中的能力，因此是肿瘤进展过程中的关键蛋白酶。作为一种膜连接的蛋白水解酶，MT1-MMPs受到多种调控机制的动态相互作用，这些调控机制共同控制着肿瘤细胞表面和细胞周隙中活性酶的水平。这项应用的长期目标是揭示调节肿瘤细胞表面MT1-MMP活性的机制，并将这一知识应用于开发旨在抑制癌症中MT1-MMP的新方法。我们之前的工作集中在阐明MT1-MMP的结构特征和生化过程，这些过程定义了MT1-MMP经历自催化加工和细胞膜上的胞外结构域脱落的能力，这是酶调节的两个基本过程。对活性MT1-MMPs的处理产生一个44 kDa的非活性膜系留片段，该片段维持关键的酶结构域，但其在MT1-MMPs调节中的功能尚不清楚。MT1-MMP胞外域的脱落产生了一种50 kDa的可溶性形式，它存在于肿瘤中，是一种完全有活性的蛋白酶。然而，它对肿瘤蛋白分解的作用尚不清楚。在此，在体外和在肿瘤移植瘤中的新证据表明，膜拴系的44 kDa物种，表现出与活性MT1-MMP的动态相互作用，并确认该片段是肿瘤细胞中酶功能的复杂调节因子。MT1-MMP胞外域是一种具有催化活性的酶，对TIMP-2敏感，它有可能将MT1-MMP膜上的蛋白水解谱从细胞膜扩展到细胞周隙，并调节膜锚定的MT1-MMPs的活性。综上所述，这些观察结果为MT1-MMPs活性的调控提供了一个新的范式，并提出了加工和溶解形式的MT1-MMPs在肿瘤恶性中起关键作用的假说。为了验证这一假说，我们建议：(1)研究加工形式和活性形式之间的酶功能的动态相互作用，(2)确定44 kDa物种对MT1-MMP调控的结构基础，(3)研究可溶性MT1-MMP在调节MT1-MMP活性中的作用，以及(4)研究加工形式和可溶性形式的MT1-MMP在体外和体内肿瘤细胞侵袭和生长的功能分析中的作用。这一应用的结果将有助于我们了解MT1-MMPs在肿瘤细胞中的功能，并有助于旨在抑制其在肿瘤组织中活性的集体努力。

DESCRIPTION (provided by applicant): Cancer progression depends on the action of proteolytic systems that facilitate the growth and invasion of tumor cells. The membrane type-1 matrix metalloproteinase (MT1-MMP) endows tumor cells with the ability to invade and grow within collagenous matrices and thus is a key protease in cancer progression. As a membrane-tethered protease, MT1-MMP is regulated by a dynamic interplay of regulatory mechanisms that collectively control the level of active enzyme on the tumor cell surface and in the pericellular space. The long term objective of this application is to unveil the mechanisms regulating MT1-MMP activity at the tumor cell surface and apply this knowledge towards the development of new approaches aimed at inhibiting MT1-MMP in cancer. Our previous effort has been focused on elucidating the structural features and biochemical processes that define the ability of MT1-MMP to undergo autocatalytic processing and ectodomain shedding on the cell membrane, two fundamental processes of enzyme regulation. Processing of active MT1-MMP yields an inactive membrane-tethered fragment of 44 kDa that maintains key enzyme domains but its function in MT1-MMP regulation is poorly understood. Ectodomain shedding of MT1-MMP yields a 50-kDa soluble form that is present in tumors and is a fully competent protease. However its contribution to tumor proteolysis is unknown. Herein, new evidence in vitro and in tumor xenografts shows that the membrane-tethered 44-kDa species, displays a dynamic interaction with active MT1-MMP and identifies this fragment as a complex regulator of enzyme function in tumor cells. The naturally shed ectodomain of MT1-MMP has been characterized and found to be a catalytically competent protease, sensitive to TIMP-2, which has the potential to expand the proteolytic repertoire of MT1-MMP from the confines of the cell membrane to the pericellular space and regulate the activity of membrane-anchored MT1-MMP. Collectively, these observations pose a new paradigm in the regulation of MT1-MMP activity and suggest the hypothesis that processed and soluble forms of MT1-MMP play critical roles in tumor malignancy. To test this hypothesis we propose to: (1) investigate the dynamic interplay between the processed and active forms of MT1-MMP in enzyme function, (2) define the structural basis for the effects of the 44-kDa species on MT1-MMP regulation, (3) investigate the role of the soluble MT1-MMP in the regulation of MT1-MMP activity and (4) investigate the role of processed and soluble forms of MT1-MMP in functional assays of tumor cell invasion and growth in vitro and in vivo. The results of this application will contribute to our understanding of MT1-MMP function in tumor cells and contribute to the collective effort aimed at inhibiting its activity in cancerous tissues.