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

癌症进展取决于蛋白水解系统的作用，该系统促进肿瘤细胞的生长和侵袭。 肿瘤细胞膜型基质金属蛋白酶（MT 1-MMP）赋予肿瘤细胞 在胶原基质中侵入和生长，因此是癌症进展中的关键蛋白酶。作为 膜系蛋白酶MT 1-MMP受多种调节机制的动态相互作用调节， 共同控制肿瘤细胞表面和细胞周围空间中活性酶的水平。的 本申请的长期目标是揭示调节肿瘤中MT 1-MMP活性的机制 细胞表面，并将这些知识应用于开发旨在抑制 MT 1-MMP在癌症中的作用 我们以前的工作一直集中在阐明结构特征和生化过程 其定义了MT 1-MMP在细胞上进行自催化加工和胞外域脱落的能力 膜，酶调节的两个基本过程。活性MT 1-MMP的加工产生一种 一种44 kDa的无活性膜系片段，维持关键酶结构域，但其在 MT 1-MMP的调控机制尚不清楚。MT 1-MMP的胞外结构域脱落产生50-kDa的可溶性 存在于肿瘤中，是一种完全胜任的蛋白酶。然而，它对肿瘤的贡献 蛋白水解是未知的。在此，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，并鉴定了这一点。 片段作为肿瘤细胞中酶功能的复杂调节剂。自然脱落的胞外域 MT 1-MMP已被表征并发现是催化活性蛋白酶，对TIMP-2敏感， 其具有将MT 1-MMP的蛋白水解谱从细胞的范围扩大的潜力 通过调节膜锚定的MT 1-MMP的活性， 总的来说，这些观察结果在MT 1-MMP活性的调节中提出了一个新的范例，并表明 MT 1-MMP的加工形式和可溶形式在肿瘤恶性程度中发挥关键作用的假设。到 为了验证这一假设，我们提出：（1）研究加工和主动之间的动态相互作用 MT 1-MMP在酶功能中的形式，（2）定义44-kDa物质作用的结构基础 探讨可溶性MT 1-MMP在MT 1-MMP调节中的作用 活性和（4）研究MT 1-MMP的加工和可溶形式在功能测定中的作用， 体外和体内肿瘤细胞侵袭和生长。这一应用的结果将有助于我们 了解MT 1-MMP在肿瘤细胞中的功能，并有助于共同努力，旨在抑制 它在癌组织中的活性 绩效S\“TE（S）（组织、城市、州） 韦恩州立大学底特律密歇根州 PHS 398（2004年9月修订版）第2页表格第2页 主要研究者/项目负责人（最后，第一，中间）：FridmaD，Rafael A 关键人员。参见说明。根据需要使用续页，以下列格式提供所需信息。 从首席研究员开始。按字母顺序列出所有其他关键人员，姓在前。 名称eRA Commons用户名组织在项目中的角色 韦恩州立大学PI 其他重要贡献者 名称组织。在项目中的作用 韦恩，露西亚·舒格州立大学顾问 希舍娃，阿西娅韦恩州立大学顾问 人胚胎干细胞（HES） 如果拟开展的项目涉及人类胚胎干细胞，请在下面列出以下列表中特定细胞系的注册号： http://stemcells.nih.gov/reqistrv/index.asp.根据需要使用续页。 如果此时无法引用特定的行，请包括一个声明，说明将使用注册表中的一行。 细胞系 披露许可声明。仅适用于SBIR/STTR。参见SBIR/STTR说明。FJ是FJ否 PHS 398（2004年9月修订版）第3页表格第2页-续 将下列各页依次编号 应用程序.不要使用后缀，如4a，4 b。 主要研究者/项目负责人（最后，第一，中间）：Fridman，Rafael 主要研究者/项目负责人的姓名必须在打印页和续页的顶部提供。 研究资助 目录 页码 首页1_ 说明，

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. PERFORMANCE S\"TE(S)(organization, city, state) Wayne State University Detroit Ml PHS 398 (Rev. 09/04) Page _2 Form Page 2 Principal Investigator/ProgramDirector (Last, First, Middle): FridmaD, Rafael A KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Fridman, Rafael RFRIDMAN Wayne State University PI OTHER SIGNIFICANT CONTRIBUTORS Name Organization . Role on Project Schuger, Lucia Wayne State University Consultant Shisheva, Assia Wayne State University Consultant Human Embryonic Stem Cells ^ No FJ yes If the proposed project Involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://stemcells.nih.gov/reqistrv/index.asp. Use continuation pages as needed. If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used. Cell Line Disclosure Permission Statement. Applicable to SBIR/STTR Only. SeeSBIR/STTR instructions. FJ Yes FJ No PHS 398 (Rev.09/04) Page 3 Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Fridman, Rafael The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1_ Description,