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

癌症的进展依赖于促进肿瘤生长和侵袭的蛋白分解系统的作用 肿瘤细胞。膜型基质金属蛋白酶-1(MT1-MMPs)使肿瘤细胞具有 侵袭和生长在胶原蛋白基质中，因此是癌症进展过程中的关键蛋白酶。作为一名 膜拴系蛋白水解酶，MT1-基质金属蛋白酶是由一种动态相互作用的调节机制来调节的， 共同控制肿瘤细胞表面和细胞周隙中的活性酶水平。这个 该应用的长期目标是揭示在肿瘤中调节MT1-MMP活性的机制 并将这一知识应用于开发旨在抑制 MT1-MMPs在肿瘤中的表达 我们之前的工作一直集中在阐明结构特征和生化过程 它们定义了MT1-基质金属蛋白酶在细胞上经历自我催化处理和胞外结构域脱落的能力 膜是酶调节的两个基本过程。活性MT1-基质金属蛋白酶的加工产生 44 kDa的非活性膜拴系片段，维持关键酶结构域，但其功能 目前对MT1-MMPs的调控知之甚少。MT1-MMP胞外区脱除可产生50 kDa的可溶性 存在于肿瘤中的一种形式，是一种具有完全活性的蛋白酶。然而，它对肿瘤的贡献 蛋白水解酶是未知的。在此，体外和异种肿瘤移植的新证据表明 膜连接的44 kDa物种，与活性的MT1-MMP呈现动态相互作用，并鉴定这一点 片段作为肿瘤细胞中酶功能的复杂调节器。自然脱落的外域 MT1-MMP已被鉴定并发现是一种催化活性的蛋白酶，对TIMP-2敏感， 它有可能将MT1-基质金属蛋白酶的蛋白水解谱从细胞的限制范围扩大 膜结合到细胞周隙，调节膜锚定的MT1-基质金属蛋白酶的活性。 总而言之，这些观察为MT1-MMPs活性的调节提供了一个新的范式，并提示 认为MT1-基质金属蛋白酶的加工和溶解形式在肿瘤恶性中起关键作用的假说。至 检验我们提出的这一假设：(1)研究被加工者和被激活者之间的动态相互作用 MT1-MMP在酶功能中的形式，(2)确定了44-kDa物种作用的结构基础 关于MT1-MMPs的调节，(3)探讨可溶性MT1-MMPs在MT1-MMPs调节中的作用 活性和(4)研究MT1-基质金属蛋白酶的加工和溶解形式在功能分析中的作用。 肿瘤细胞在体外和体内的侵袭和生长。这项申请的结果将有助于我们的 了解MT1-基质金属蛋白酶在肿瘤细胞中的功能，并为旨在抑制的集体努力做出贡献 它在癌组织中的活性。 演出S\“特(S)(组织、市、州) 韦恩州立大学底特律分校 小灵通398(09/04版)第_2页表格第2页 首席调查员/项目主任(最后、第一、中间)：Fridmad，Rafael A 关键人员。请参阅说明。根据需要使用续页，以如下所示的格式提供所需信息。 从首席调查员开始。按字母顺序列出所有其他关键人员，姓氏在前。 名称时代共享用户名项目上的组织角色 弗里德曼，拉斐尔·弗里德曼·韦恩州立大学 其他重要贡献者 名称组织。在项目中的角色 露西娅·韦恩州立大学顾问舒格 Shisheva，Assia Wayne州立大学顾问 人类胚胎干细胞^否FJ是 如果建议的项目涉及人类胚胎干细胞，请从下面的列表中列出特定细胞系(S)的注册号： Http://stemcells.nih.gov/reqistrv/index.asp.根据需要使用续页。 如果此时不能引用某一特定行，请附上一条声明，说明将使用注册处的一行。 细胞系 披露许可声明。仅适用于SBIR/STTR。请参阅SBIR/STTR说明。FJ是FJ否 小灵通398(09/04版)第3页表格第2页-续 从头到尾连续地给下面的页面编号 应用程序。不要使用诸如4a、4b之类的后缀。 首席调查员/项目主任(最后、第一、中间)：拉斐尔·弗里德曼 必须在每张打印页和每张续页的顶部提供首席调查员/项目主任的姓名。 研究补助金 目录 页码 正面第1页_ 描述，

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,