A novel function of TIMP-1

描述(申请人提供)：这项提案的长期目标是揭示金属蛋白酶组织抑制物(TIMP)-1在乳腺癌进展过程中的多效性。越来越多的临床证据表明，TIMP-1是预测乳腺癌患者预后不良的有效指标。然而，TIMP-1在乳腺上皮细胞中的作用机制尚不完全清楚。越来越多的证据表明，TIMP-1除了具有典型的金属蛋白酶(MP)活性抑制活性外，还可以促进细胞存活，而不依赖于MP的抑制。在过去的资助期间，我们的结论是TIMP-1是一种有效的内源性和外源性细胞凋亡抑制因子，不依赖于其MP抑制活性。重要的是，我们最近的研究确定了TIMP-1成员CD63是第一个与TIMP-1结合的细胞表面蛋白，它调节整合素？1的生存途径。TIMP-1激活CD63/整合素信号复合体意味着TIMP-1在许多细胞过程中可能有不同的作用。初步数据显示，TIMP-1还抑制三维基质培养中非恶性乳腺上皮MCF10A腺泡的形态发生过程中管腔的形成和细胞的凋亡。此外，TIMP-1与CD63的相互作用通过上调EMT主要转录因子Twist来诱导上皮间充质转化(EMT)，该转录因子可促进癌症进展。在结构上，TIMP-1通过其非MP抑制C末端结构域与CD63结合，就像它与潜伏的PRO-MMP9结合一样。这提示了TIMP-1在细胞表面的一种新的调控机制，在这种机制中，原-基质金属蛋白酶-9在细胞周隙的可获得性可能调节了TIMP-1/CD63的相互作用。根据我们以前和初步的研究，我们假设在肿瘤进展和转移的早期阶段，TIMP-1/CD63激活整合素1/a信号复合体激活焦点黏附(FAK)和磷脂酰肌醇3-激酶(PI3K)，导致细胞存活，增加Twist表达和EMT。为了验证我们的假设，我们建议(1)研究TIMP-1和CD63调节整合素异二聚体功能的分子机制，(2)研究TIMP-1/CD63介导的细胞内信号转导通路，以调节细胞存活和EMT，(3)建立前-基质金属蛋白酶-9在TIMP-1/CD63介导的细胞存活调节中的作用，以及(4)研究TIMP-1在乳腺癌进展中的作用。拟议研究的完成将有助于集体努力了解TIMP-1在乳腺癌进展过程中的多功能。这一信息也可能有助于开发针对TIMP-1的S信号活性与其MP抑制活性的新方法。公共卫生相关性：金属蛋白酶组织抑制因子(TIMP)-1是一种天然的基质金属蛋白酶(MPS)抑制剂，已被证明在肿瘤转移后期的动物模型中抑制肿瘤细胞的体外侵袭和肿瘤的进展。然而，新出现的临床证据表明，TIMP-1是乳腺癌患者预后不良的有效预测因子，考虑到这些先前报道的研究，这是一个意想不到的观察结果。包括我们在内的许多研究人员提供的越来越多的证据表明，TIMP-1也可以促进细胞存活，而不依赖于其对MP的抑制。有趣的是，初步数据显示，TIMP-1还可以诱导乳腺上皮细胞上皮间充质转化(EMT)表型。目前应用的目标是揭示TIMP-1调节细胞存活和EMT的分子机制，并在乳腺癌进展的早期阶段的动物模型中检查其潜在的致癌活性。拟议研究的完成将有助于集体努力了解TIMP-1在乳腺癌进展过程中的多种功能。这一信息也可能有助于开发针对TIMP-1的S信号活性与其MP抑制活性的新方法。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to unveil the pleiotropic activity of tissue inhibitor of metalloproteinases (TIMP)-1 during breast cancer progression. Emerging clinical evidence indicates that TIMP-1 is a potent predictor of poor prognosis in breast cancer patients. However, the mechanisms of action of TIMP-1 in breast epithelial cells are not completely understood. In addition to its classical activity as an inhibitor of metalloproteinase (MP) activity, accumulating evidence indicates that TIMP-1 can also promote cell survival independently of MP inhibition. During the past funding period, we conclusively showed that TIMP-1 is a potent inhibitor of both intrinsic and extrinsic apoptosis independent of its MP inhibitory activity. Importantly, our recent study identified the tetraspanin member CD63 as the first TIMP-1 binding cell surface protein which modulates the integrin ¿1 survival pathway. TIMP-1 activation of the CD63/integrin signaling complex implies potentially diverse effects of TIMP-1 on many cellular processes. Preliminary data show that TIMP-1 also inhibits lumen formation and apoptosis during morphogenesis of non-malignant breast epithelial MCF10A acini in three-dimensional matrigel cultures. Moreover, TIMP-1 interaction with CD63 induces an epithelial mesenchymal transition (EMT) via upregulation of the EMT master transcription factor Twist, known to promote cancer progression. Structurally, TIMP-1 binds to CD63 via its non-MP inhibitory C-terminal domain as it does to latent pro-MMP-9. This suggests a novel mechanism of TIMP-1 regulation at the cell surface, in which the availability of pro-MMP-9 in the pericellular space may regulate TIMP-1/CD63 interactions. Based on our previous and preliminary studies, we hypothesize that TIMP-1/CD63 activation of integrin ¿1/a signaling complex activates focal adhesion (FAK) and phosphatidylinositol 3-kinase (PI3K) leading to cell survival, increased Twist expression and EMT during the early stages of tumor progression and metastasis. To test our hypothesis, we propose (1) to investigate the molecular mechanisms by which TIMP-1 and CD63 regulate integrin heterodimer functions, (2) to investigate TIMP-1/CD63-mediated intracellular signal transduction pathways for the regulation of cell survival and EMT, (3) to establish the role of pro-MMP-9 in the regulation of TIMP-1/CD63-mediated cell survival, and (4) to examine the in vivo roles of TIMP-1 during breast cancer progression. Accomplishment of the proposed studies will contribute to the collective endeavor to understand the multi functions of TIMP-1 during breast cancer progression. This information may also be useful for the development of novel approaches for targeting TIMP-1's signaling activity versus its MP inhibitory activity. PUBLIC HEALTH RELEVANCE: Tissue inhibitor of metalloproteinases (TIMP)-1, a natural inhibitor of matrix metalloproteinases (MPs), has been shown to inhibit tumor cell invasion in vitro and tumor progression in animal models of later stages of tumor metastasis. However, emerging clinical evidence indicates that TIMP-1 is a potent predictor of poor prognosis in breast cancer patients, an unexpected observation in light of these previously reported studies. Accumulating evidence provided by many investigators including us indicates that TIMP-1 can also promote cell survival independently of its MP inhibition. Interestingly, the preliminary data show that TIMP-1 also induces epithelial mesenchymal transition (EMT) phenotype in breast epithelial cells. The goals of the current application are to unveil molecular mechanisms by which TIMP-1 regulates cell survival and EMT, and to examine its potential oncogenic activity in an animal model of the early stages of breast cancer progression. Accomplishment of the proposed studies will contribute to the collective endeavor to understand the multiple functions of TIMP-1 during breast cancer progression. This information may also be useful for the development of novel approaches for targeting TIMP-1's signaling activity versus its MP inhibitory activity.