The Role of TIMPs in Cell Growth and Differentiation: Tumor Angiogenesis

摘要：EGF对A549人肺癌细胞株的刺激表明，抑制细胞生长反应是通过激活蛋白酪氨酸磷酸酶活性和导致EGF受体磷酸化降低而实现的。使用抗整合素抗体的竞争结合实验证实，整合素α3beta1可能是人微血管内皮细胞(HMVECs)上TIMP-2的细胞表面受体。ALA-TIMP-2在体外也能抑制血管内皮生长因子-A或成纤维细胞生长因子-2刺激的有丝分裂和体内血管生成，从而证明TIMP-2的血管抑制活性与对基质金属蛋白酶的抑制作用是分离的。这种作用的机制包括整合素受体失活生长因子受体信号，称为异源受体失活。这是第一次证明整合素可以负向调节受体酪氨酸激酶的激活。这项工作通过证明TIMP是多功能蛋白质，与细胞表面受体结合，并通过与这些受体的相互作用直接影响细胞行为，为TIMP生物学定义了一个新的范式。利用体外和体内模型，我们目前和未来的工作集中在确定TIMP-2中的α3β1整合素结合域(S)，并进一步了解TIMP-2与α3β1在正常细胞和肿瘤细胞中相互作用后的细胞效应，以及随后肿瘤微环境的变化。我们的目标是进一步表征TIMPs在肿瘤微环境中的非依赖于和依赖于基质金属的作用，以及它们对肿瘤抑制和/或进展的相对贡献。这些研究将确定正常组织和肿瘤微环境中细胞外基质调节细胞行为的关键机制，并可能为癌症治疗带来新的治疗策略。这些发现表明，确定TIMP-2与α3β1结合的结构域(S)对于进一步剖析这种复杂分子的多种生物学活性以及确定这种活性对正常组织和恶性肿瘤组织微环境的功能贡献至关重要。本项目的重点是确定ALA TIMP-2的抗血管生成和抗肿瘤作用的机制。对人类微血管内皮细胞的初步研究证明了一种称为异源受体失活的机制。在这种作用中，TIMP-2受体α3beta1通过激活一种名为SHP-1的磷酸酪氨酸磷酸酶来减少受体酪氨酸激酶的磷酸化和激活，这些受体包括血管内皮生长因子受体(VEGFR)-2、成纤维细胞生长因子受体(FGFR)-1和表皮生长因子受体(EGFR)。然而，最近在肿瘤细胞和内皮细胞中的实验表明，ALA TIMP-2的生长抑制活性更加复杂，似乎涉及细胞凋亡途径和上皮向间充质转化的基因表达变化，这是肿瘤侵袭和转移所必需的。本项目的目的是确定和描绘这些途径，目的是开发ALA TIMP-2作为一种新的癌症治疗方法，并确定潜在的新治疗靶点。

Summary: EGF-stimulation of the A549 human lung carcinoma cell line demonstrated that the suppression of cell growth response was mediated by the activation of protein tyrosine phosphatase activity and resulted in reduced EGF receptor phosphorylation. Competition binding experiments using anti-integrin antibodies identified integrin alpha3beta1 as a putative cell surface receptor for TIMP-2 on human microvascular endothelial cells (hMVECs). Ala+TIMP-2 also inhibited VEGF-A or FGF-2 stimulated mitogenesis in vitro and angiogenesis in vivo a, thus demonstrating that the angio-inhibitory activity of TIMP-2 is dissociable from MMP-inhibition. The mechanism of this effect involves an integrin receptor inactivation of growth factor receptor signaling, known as heterologous receptor inactivation. This was the first demonstration that integrins could negatively regulate activation of a receptor tyrosine kinase This work has defined a new paradigm for TIMP biology by demonstrating that TIMPs are multifunctional proteins, with cell surface receptors and through interaction with these receptors they can directly influence cellular behavior. Using both in vitro and in vivo models our current and future work is focused on identifying the alpha3beta1 integrin binding domain(s) in TIMP-2 and furthering our understanding of the cellular effects following TIMP-2 interaction with alpha3beta1 in both normal and neoplastic cells, as well as the subsequent alterations in the tumor microenvironment. It is our goal to further characterize the MMP-independent and MMP-dependent effects of TIMPs in the tumor microenvironment and their relative contribution to tumor suppression and/or progression. These studies should identify crucial mechanisms in the regulation of cell behavior by the extracellular matrix in normal tissues and the tumor microenvironment, and possibly lead to new therapeutic strategies for cancer treatment. These findings suggest that defining the domain(s) responsible for TIMP-2-binding to alpha3beta1 will be critical to further dissecting the multiple biological activities of this complex molecule, as well as defining the functional contributions of this activity to the microenvironment in both normal and malignant tissues. The focus of this project is to determine the mechanisms of the anti-angiogenic and anti-tumorigenic effects of Ala+TIMP-2. Preliminary work with human microvascular endothelial cells has demonstrated a mechanism known as heterologous receptor inactivation. In this effect the TIMP-2 receptor alpha3beta1 decreases phosphorylation and activation of receptor tyrosine kinases such as the vascular endothelial growth factor receptor (VEGFR)-2, fibroblast growth factor recetpor (FGFR)-1 and epidermal growth factor receptor (EGFR) by activation a phosphotyrosine phosphatase known as Shp-1. However, recent experiments in tumor cells and endothelial cells have revealed that the growth suppressor activity of Ala+TIMP-2 is more complex and appears to involve apoptotic pathways and changes in gene expression of the epithelial to mesenchymal transition that is essential to tumor invasion and metastasis. It is the purpose of this project to identify and delineate these pathways with the aim of developing Ala+TIMP-2 as a novel cancer therapeutic and identifying potential new therapeutic targets.