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

描述（由申请人提供）：癌症进展取决于促进肿瘤细胞生长和侵袭的蛋白水解系统的作用。膜型基质金属蛋白酶（MT 1-MMP）赋予肿瘤细胞在胶原基质中侵袭和生长的能力，因此是癌症进展中的关键蛋白酶。作为一种膜系蛋白酶，MT 1-MMP受到多种调节机制的动态相互作用的调节，这些机制共同控制肿瘤细胞表面和细胞周围空间中活性酶的水平。本申请的长期目标是揭示调节肿瘤细胞表面MT 1-MMP活性的机制，并将这些知识应用于开发旨在抑制癌症中MT 1-MMP的新方法。我们以前的努力一直集中在阐明的结构特征和生化过程，定义的能力MT 1-MMP进行自催化加工和胞外域脱落的细胞膜上，酶的调节的两个基本过程。活性MT 1-MMP的加工产生44 kDa的非活性膜栓系片段，其维持关键酶结构域，但其在MT 1-MMP调节中的功能知之甚少。MT 1-MMP的胞外结构域脱落产生存在于肿瘤中的50-kDa可溶形式，并且是完全胜任的蛋白酶。然而，其对肿瘤蛋白水解的贡献是未知的。在此，在体外和肿瘤异种移植物中的新证据表明，膜栓系的44-kDa物质显示与活性MT 1-MMP的动态相互作用，并将该片段鉴定为肿瘤细胞中酶功能的复杂调节剂。MT 1-MMP的天然脱落的胞外域已被表征并被发现是一种催化活性蛋白酶，对TIMP-2敏感，其具有将MT 1-MMP的蛋白水解谱从细胞膜的范围扩展到细胞周围空间并调节膜锚定的MT 1-MMP的活性的潜力。总的来说，这些观察提出了一个新的范例，在MT 1-MMP活性的调节，并建议的假设，加工和可溶性形式的MT 1-MMP在肿瘤恶性发挥关键作用。为了检验这一假设，我们建议：（1）研究在酶功能中MT 1-MMP的加工形式和活性形式之间的动态相互作用，（2）确定44-kDa物质对MT 1-MMP调节作用的结构基础，（3）研究可溶性MT 1-MMP在调节MT 1-MMP活性中的作用，和（4）研究加工的和可溶性形式的MT 1-MMP在调节MT 1-MMP活性中的作用。MMP在体内外肿瘤细胞侵袭和生长的功能测定中的应用。该应用的结果将有助于我们理解MT 1-MMP在肿瘤细胞中的功能，并有助于旨在抑制其在癌组织中的活性的集体努力。

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.