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

总结：EGF刺激A549人肺癌细胞系表明，细胞生长反应的抑制是由蛋白质酪氨酸磷酸酶活性的激活介导的，并导致EGF受体磷酸化减少。使用抗整联蛋白抗体的竞争结合实验将整联蛋白α 3 β 1鉴定为人微血管内皮细胞（hMVEC）上TIMP-2的推定细胞表面受体。Ala+TIMP-2还抑制VEGF-A或FGF-2刺激的体外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MMP抑制无关。这种效应的机制涉及生长因子受体信号传导的整联蛋白受体失活，称为异源受体失活。这是第一次证明整合素可以负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，具有细胞表面受体，并通过与这些受体的相互作用，它们可以直接影响细胞行为，为TIMP生物学定义了一个新的范例。使用体外和体内模型，我们目前和未来的工作重点是确定TIMP-2中的α 3 β 1整合素结合结构域，并进一步了解TIMP-2与正常和肿瘤细胞中的α 3 β 1相互作用后的细胞效应，以及随后的肿瘤微环境改变。我们的目标是进一步表征TIMP在肿瘤微环境中的MMP非依赖性和MMP依赖性作用及其对肿瘤抑制和/或进展的相对贡献。这些研究应该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，并可能导致癌症治疗的新治疗策略。这些发现表明，确定负责TIMP-2与α 3 β 1结合的结构域对于进一步剖析这种复杂分子的多种生物活性以及确定这种活性对正常和恶性组织中微环境的功能贡献至关重要。本课题的重点是研究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已经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α 3 β 1通过激活称为Shp-1的磷酸酪氨酸磷酸酶来降低受体酪氨酸激酶如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）的磷酸化和激活。然而，最近在肿瘤细胞和内皮细胞中的实验已经揭示，Ala+TIMP-2的生长抑制活性更复杂，并且似乎涉及凋亡途径和上皮向间充质转化的基因表达的变化，上皮向间充质转化是肿瘤侵袭和转移所必需的。本项目的目的是鉴定和描述这些通路，旨在开发Ala+TIMP-2作为一种新型癌症治疗剂，并鉴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.