TIMP-2-mediated Tumor Cell Differentiation and Chemo-sensitization Therapy

摘要：几种内源性蛋白酶抑制剂可以调节与组织稳态丧失和肿瘤微环境形成相关的细胞外基质的周转。组织金属蛋白酶抑制剂（TIMPs）是与肿瘤微环境的建立和进展相关的酶的最特异性和研究最充分的蛋白酶抑制剂。timp是一个由四个小的高度保守的蛋白质组成的家族。从果蝇、斑马鱼、秀丽隐杆线虫到人类等物种中都发现了TIMPs，这表明这些蛋白质是古老的真核蛋白。在20世纪90年代早期，TIMP- 1和TIMP-2在小鼠异种移植模型中抑制肿瘤细胞的侵袭和转移的证明，刺激了制药行业开发合成MMP抑制剂作为潜在的新癌症治疗药物的重大努力。不幸的是，这些合成的MMP抑制剂在随后的所有人体临床试验中都失败了。尽管已经提出了许多假设来解释这些药物在人类癌症患者中的失败，但这种失败的真正性质仍然未知。哺乳动物TIMP家族有4个成员，它们在蛋白水平上具有显著的同源性和结构同一性。TIMP-2作为TIMP家族成员的独特之处在于，除了抑制MMPs外，TIMP-2还选择性地与MT1-MMP相互作用，以促进pro-MMP-2的细胞表面活化。因此，TIMP-2既是MMPs的抑制剂，也是mmp -2激活的细胞机制所必需的。与TIMP家族的其他三个成员相比，TIMP-2也具有独特的基因结构。TIMP与突触蛋白基因家族之间存在一个有趣的关系，因为TIMP家族的三个成员嵌套在突触蛋白基因中。synapsin 1基因嵌套TIMP-1， synapsin 2基因嵌套TIMP-4， synapsin 3基因嵌套TIMP-3。TIMP-2是TIMP家族中唯一不嵌套在突触蛋白家族基因中的成员。突触素- timp基因的嵌套关系早在果蝇时代就开始了。最近的一份报告描述了一个嵌套在TIMP-2基因的非常大（60 kb）的第一个内含子内的基因，称为差异显示克隆8 (DDC8)，最初被认为是编码睾丸特异性蛋白质。此外，研究表明，由于外显子1缺失而产生的TIMP-2缺陷小鼠的大脑中含有编码DDC8序列下游外显子2-5的TIMP-2 mRNA，这表明这两个基因之间存在选择性剪接。我们最近证明了外源性和内源性TIMP-2的增强表达促进了人微血管内皮细胞的分化。Jaworski及其同事已经证明TIMP-2抑制神经突增殖并促进分化。与正常组织相比，TIMP-2在肿瘤中经常表达不足。这些观察结果使我们假设TIMP-2在肿瘤细胞和肿瘤异种移植物中的重新表达可能影响肿瘤进展、肿瘤细胞分化、上皮细胞向间质细胞的转化和转移能力。我们之前已经证明TIMP-2抑制egf诱导的人肺癌细胞系A549的生长。初步证据表明，在A549细胞系中，TIMP-2的内源性表达量很低，而强制表达TIMP-2或Ala+TIMP-2对肿瘤生长的抑制作用超过80%。我们现在正在研究TIMP-2在促进肿瘤细胞分化中的作用。此外，我们正试图描述TIMP-2的金属蛋白酶抑制剂活性、抗血管生成活性和可能的直接抗肿瘤作用对该蛋白整体有效抗肿瘤活性的相对贡献

Summary: Endogenous protease inhibitors of several classes can regulate the turnover of the extracellular matrix associated with loss of tissue homeostasis and formation of the tumor microenvironment. The tissue inhibitors of metalloproteinases (TIMPs) are the most specific and well-studied protease inhibitors for the enzymes associated with establishment and progression of the tumor microenvironment. The TIMPs are a family of four small highly conserved proteins. The TIMPs have been identified in species ranging from drosophila, zebra fish and C. elegans to humans, suggesting that these proteins are ancient eukaryotic proteins. The demonstration that TIMP 1 and TIMP-2 inhibited tumor cell invasion and metastasis in mouse xenograft models in the early 1990s, spurred a significant effort on the part of the pharmaceutical industry to develop synthetic MMP inhibitors as potential new cancer therapeutics. Unfortunately, these synthetic MMP inhibitors failed in all subsequent human clinical trials. Although many hypotheses have been put forward to explain the failure of these drugs in human cancer patients, the true nature of this failure remains unknown. The mammalian TIMP family has four members, which share significant homology and structural identity at the protein level. TIMP-2 is unique as a member of the TIMP family in that in addition to inhibiting MMPs TIMP-2 selectively interacts with MT1-MMP to facilitate the cell-surface activation of pro-MMP-2. Thus, TIMP-2 functions both as an inhibitor of MMPs, and is required for the cellular mechanism of pro-MMP-2 activation. TIMP-2 also has a distinct gene structure compared with the other three members of the TIMP family. An interesting relationship exists between the TIMPs and the synapsin gene family in that three members of the TIMP family are nested within the synapsin genes. The synapsin 1 gene nests TIMP-1, synapsin 2 nests TIMP-4 and synapsin 3 nests TIMP-3. TIMP-2 is the only member of the TIMP family that is not nested within a gene of the synapsin family. The synapsin-TIMP gene nesting relationship began phylogenetically as far back as Drosophila. A recent report describes a nested gene within the very large (60 kb) first intron of the TIMP-2 gene, known as differential display clone 8 (DDC8), which at first was thought to encode a testis specific protein. Furthermore, it has been shown that the brain of the TIMP-2-deficient mouse generated by deletion of exon 1 contains TIMP-2 mRNA encoding exons 2-5 downstream of DDC8 sequence, suggesting alternative splicing between these two genes. We have recently demonstrated that exogenous and enhanced expression of endogenous TIMP-2 promotes the differentiation of human microvascular endothelial cells. Jaworski and colleagues have demonstrated that TIMP-2 inhibits neurite proliferation and promotes differentiation. TIMP-2 is frequently under expressed in tumors compared with normal tissue. These observations led us to postulate that re-expression of TIMP-2 in tumor cells and tumor xenografts may influence tumor progression, tumor cell differentiation, epithelial-to-mesenchymal transition and metastatic capacity. We have previously demonstrated that TIMP-2 suppressed the EGF-induced growth of the human lung cancer cell line A549. Preliminary evidence shows that endogenous expression of TIMP-2 in the A549 cell line is very low and the forced expression of TIMP-2 or Ala+TIMP-2 results in over 80 % suppression of tumor growth. We are now examining the mechanism of this effect with respect to the role of TIMP-2 in promoting tumor cell differentiation. In addition we are attempting to characterize the relative contributions of the metalloproteinase inhibitor activity, anti-angiogenic activity and possible direct anti-tumor effects of TIMP-2 to the overall potent anti-tumor activity of this protein