改进基质金属蛋白酶组织抑制因子TIMPs对癌细胞表面锚定金属蛋白酶的抑制功能

MT1-MMP (a.k.a. MMP-14) is the most important membrane-associated Matrix MetalloProteinase (MMP) that degrades extracellular matrix components and sheds a great variety of cell surface-anchored bioactive molecules. Unregulated activities of the enzyme lead to rampant cell metastasis and cancer invasion. As with other MMPs, the physiological activities of MT1-MMP are modulated by the TIMPs. There are four human TIMPs (1 to 4) but only TIMP-2, -3 and -4 are potent inhibitors against MT1-MMP. TIMP-1 is inactive against the enzyme. TIMPs exert their inhibitory action against MT1-MMP by forming tight non-covalent 1:1 binary complex with the catalytic domain of the MMP. Given the pivotal role of MT1-MMP in cancer development, the principal aim of this proposal is to develop the TIMPs into an effective therapeutic agent for the treatment of MT1-MMP related diseases in particular cancers. Native MT1-MMP forms homophilic dimer on the cell surface via hemopexin domain in order to activate pro-MMP-2 and degrade collagen. Here, we propose a novel strategy for MT1-MMP inhibition by employing a tactic that would allow the TIMPs to be specifically targeted to the enzyme. Our plan is to fuse the TIMPs to a "carrier" which, in effect, is consist of a truncated form of MT1-MMP that encompasses the hemopexin and the transmembrane domains (P316 to R563; named "TIMP:MT1-PEX" hereafter). In MT1-MMP producing cancer cells, we believe TIMP:MT1-PEX recombinant protein will be attracted to native MT1-MMP through the hemopexin domain and by so doing, inhibit the target enzyme. Two panels of TIMP:MT1-PEX constructs will be studied in this proposal: (i) the first panel will consist of seven full-length and N-terminal TIMPs fused directly to the MT1-MMP hemopexin/transmembrane domains whereas (ii) the second panel will consist of constructs of similar design but with a MT1-MMP cleavable linker "PLGLAR" inserted in between the TIMPs and the hemopexin domain carrier. The inclusion of a cleavable linker would permit the TIMPs to be released from its carrier upon encountering an active MT1-MMP and thus, increase the effectiveness of inhibition. The TIMP:MT1-PEX chimera will be studied in two MT1-MMP producing cancer cell lines HT1080 and HTC75. Independent assessment of the TIMPs' potency will be performed in cell-based settings using three established assays specific for MT1-MMP, namely cell migration, gelatin degradation and CD44 shedding assays. Both transiently and stably transfected cells (by lentivirus) are to be examined in parallel. Localisation of the TIMPs will be tracked by confocal microscopy with a C-terminal histidine tag. TIMP:MT1-PEX constructs of outstanding potency are to be evaluated in vivo by subcutaneous transplantation of HT1080/HTC75 cells stably expressing the TIMPs into NOD SCID mice and the tumour development monitored over 3 months. In essence, we believe this is a novel and viable project tailored towards intellectual property development.

MT1-MMP是重要的膜型基质金属蛋白酶。非受控酶活动会导致猖獗的细胞转移和癌症入侵。MT1-MMP的生理活动受TIMP调节。本文主要目的是要将TIMP开发成一种有效的治疗剂。天然的MT1-MMP经由血红素结合蛋白样结构域在细胞表面形成同种二聚体。在此，我们提出一种新的抑制策略以改良TIMPs对MT1-MMP的抑制及选择功能。我们计划将TIMP融合于一个由MT1-MMP血红素结合蛋白和跨膜两个结构域组成的"载体"上（称为TIMP：MT1-PEX）。在表达MT1-MMP的癌细胞中，TIMP：MT1-PEX将通过血红素结合蛋白样结构域被吸引至天然MT1-MMP而抑制目标酶。我们将对表达MT1-MMP的癌细胞株进行细胞迁移, 明胶降解和CD44脱落化验研究。表现突出的TIMP：MT1-PEX将会被移植入老鼠体内以进行体内观察。这是一个专为知识产权发展而定制的既新奇而又可行的项目。

背景：.膜型1基质金属蛋白酶（MT1-MMP）是参与癌细胞转移和肿瘤增殖的膜锚定的锌依赖性基质金属蛋白酶（MMP）。 MT1-MMP 的生理活性由内源抑制剂，金属蛋白酶组织抑制剂（TIMP）-2调节。..研究目的：.该 NSFC 课题的目的是通过使用一组不同的跨膜锚定物将 TIMP 移位到细胞表面来增强 TIMP-2 对 MT1-MMP 的作用，所述跨膜锚定物包括 MT1-MMP 的血红素蛋白+/-跨膜结构域和糖基磷脂酰肌醇（GPI）锚定的其他跨膜蛋白如 RECK 和朊蛋白。..重要结果：.我们已经成功地设计出一组改造体 TIMP-2，其可以与细胞内以及细胞表面上的 MT1-MMP 形成复合物。我们通过融合 TIMP-2 到目标酶 MT1-MMP 的血红素+ +/-跨膜结构域来实现这一点。到目前为止，我们已经创建了能够与 MT1-MMP 缔合并且与野生型 TIMP-2 相比能显著降低 MMP 的酶活性的两个改造体 TIMP-2（命名为 “T2：PEX+TM”和 “T2：PEX”）。..关键数据：.表达改造体 TIMP 的宫颈癌（HeLa）细胞显示出比对照细胞显著降低的明胶溶解活性（<90％）和迁移率（<50％）。在小鼠异种移植中，与对照相比，TIMP 组的宫颈肿瘤组织体积和重量减少多达85％。..发现的意义：.这些发现在以下几个方面非常有意义。.1. 融合方法是创新的。这是 TIMP 以这种方式设计专一用于抑制 MMP 的第一个实例。.2. MT1-MMP 抑制的模式是高度特异性的。由于血红素结合蛋白结构域对 MT1-MMP 的固有亲和力，我们的改造体 TIMP “T2：PEX+TM” 和 “T2：PEX” 可以选择性地识别 MT1-MMP 而不是其他 MMP。.3. 到目前为止，我们已经表明我们的改造体 TIMP 在小鼠异种移植物中的肿瘤抑制中是高度有效的。我们相信 TIMP 可以进一步发展为人类癌症治疗中的生物制剂。