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

摘要：几类内源性蛋白水解酶抑制物可调节细胞外基质的周转，与组织内稳态的丧失和肿瘤微环境的形成有关。金属蛋白酶组织抑制因子(TIMPs)是与肿瘤微环境的建立和发展相关的酶的最特异和最广泛研究的蛋白酶抑制剂。TIMP是一个由四个高度保守的小蛋白组成的家族。TIMP在从果蝇、斑马鱼、线虫到人类的各种物种中都被发现，这表明这些蛋白是古老的真核蛋白。20世纪90年代初，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，促使制药业努力开发合成的基质金属蛋白酶抑制剂，作为潜在的新的癌症治疗药物。不幸的是，这些合成的基质金属蛋白酶抑制剂在随后的所有人类临床试验中都失败了。尽管已经提出了许多假说来解释这些药物在人类癌症患者中的失败，但这种失败的真正本质仍然不清楚。哺乳动物TIMP家族有四个成员，它们在蛋白质水平上具有显著的同源性和结构相似性。作为TIMP家族的一员，TIMP-2的独特之处在于，除了抑制MMPs外，TIMP-2还选择性地与MT1-MMPs相互作用，促进原-MMP2在细胞表面的激活。因此，TIMP-2既是MMPs的抑制因子，又是激活MMP2的细胞机制所必需的。与TIMP家族的其他三个成员相比，TIMP-2也具有不同的基因结构。在TIMP和突触素基因家族之间存在着一种有趣的关系，因为TIMP家族的三个成员嵌套在突触素基因中。突触素1基因嵌套TIMP-1，突触素2嵌套TIMP-4，突触素3嵌套TIMP-3。TIMP-2是TIMP家族中唯一没有嵌套在突触素家族基因中的成员。突触素和TIMP基因的嵌套关系早在果蝇的系统发育上就开始了。最近的一份报告描述了TIMP-2基因非常大(60kb)第一内含子中的一个嵌套基因，称为差异显示克隆8(DDC8)，最初被认为编码一种睾丸特异蛋白。此外，研究还表明，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-2mRNA，这表明这两个基因之间存在选择性剪接。我们最近证实，外源性和内源性TIMP-2的表达增强促进了人微血管内皮细胞的分化。Jaworski和他的同事已经证明了TIMP-2抑制轴突增殖和促进分化。与正常组织相比，TIMP-2在肿瘤组织中的表达水平较低。这些观察结果使我们推测TIMP-2在肿瘤细胞和肿瘤移植瘤中的重新表达可能影响肿瘤的进展、肿瘤细胞的分化、上皮向间充质的转变和转移能力。我们先前已经证明TIMP-2抑制EGF诱导的人肺癌细胞系A549的生长。初步研究表明，内源性TIMP-2在A549细胞中的表达很低，强制表达TIMP-2或ALA+TIMP-2可导致肿瘤生长抑制80%以上。我们现在正在研究TIMP-2在促进肿瘤细胞分化方面的作用机制。此外，我们还试图确定TIMP-2的金属蛋白酶抑制活性、抗血管生成活性和可能的直接抗肿瘤作用对该蛋白整体强大的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