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VEGF-A或FGF-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。本项目的目的是鉴定和描述这些途径，目的是开发Ala+TIMP-2作为一种新的癌症治疗方法，并寻找潜在的新治疗靶点。金属蛋白酶抑制因子-2(TIMP-2)通过抑制基质金属蛋白酶(MMPs)或直接结合内皮细胞来抑制血管生成。本研究的主要目的是确定TIMP-2区域参与与先前确定的整合素受体α3beta1结合，并确定从该区域衍生的合成肽是否具有血管抑制和肿瘤抑制活性。我们证实TIMP-2的N-末端结构域(N-TIMP-2)与α3β1结合并在体外抑制血管内皮生长因子刺激的内皮细胞生长，提示TIMP-2的α3β1结合域和生长抑制活性均定位于N-末端结构域。利用肽阵列方法，我们鉴定了TIMP-2初级序列的24个氨基酸区域，由残基Ile43-Ala66组成，显示出与α3β1结合的活性。随后，我们证明了来自该区域的合成肽竞争TIMP-2与α3beta1结合，并在体外抑制内皮生长。我们定义了一个最小的肽序列(肽8-9)，它既具有血管抑制作用，又具有体内抗肿瘤活性，使用的是Kapsis肉瘤的小鼠移植模型。因此，α3β1结合活性和血管抑制活性都共定位于TIMP-2初级序列中暴露于溶剂的灵活区域，与TIMP家族的其他成员相比，该区域在氨基酸序列上是独一无二的。此外，对该区域TIMP-2和TIMP-1蛋白三维结构的比较也发现了独特的结构差异。我们的发现表明，TIMP-2的整合素结合、肿瘤生长抑制和体内血管抑制活性在一个独特的序列/结构环(B-C环)中密切相关。2012年的进展表明，TIMP-2具有直接的抗肿瘤特性，独立于其体内的基质金属蛋白酶抑制活性(见Bourboulia等人，AMER)。J.Pathol 179：2589-2600,2011)。这表明，在A549人肺癌细胞中强制表达TIMP-2和Ala+TIMP-2(缺乏基质金属蛋白酶抑制活性)会直接导致抑制这些途径的肿瘤细胞中Fak和AKT的磷酸化水平降低。我们还使用TIMP-2缺陷的小鼠模型以及表达TIMP-2的A549异种移植瘤，证明了TIMP-2具有抑制肿瘤相关髓系抑制细胞的渗透的功能，而髓系抑制细胞对肿瘤血管生成至关重要。这些发现支持TIMP-2作为癌症治疗的一种新的生物疗法的临床前持续开发。

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.Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits angiogenesis by several mechanisms involving either MMP inhibition or direct endothelial cell binding. The primary aim of this study was to identify the TIMP-2 region involved in binding to the previously identified receptor integrin alpha3beta1, and to determine whether synthetic peptides derived from this region retained angio-inhibitory and tumor suppressor activity. We demonstrated that the N-terminal domain of TIMP-2 (N-TIMP-2) binds to alpha3beta1 and inhibits vascular endothelial growth factor-stimulated endothelial cell growth in vitro, suggesting that both the alpha3beta1-binding domain and growth suppressor activity of TIMP-2 localize to the N-terminal domain. Using a peptide array approach we identify a 24 amino acid region of TIMP-2 primary sequence, consisting of residues Ile43-Ala66, which shows alpha3beta1-binding activity. Subsequently we demonstrate that synthetic peptides from this region compete for TIMP-2 binding to alpha3beta1 and suppress endothelial growth in vitro. We define a minimal peptide sequence (peptide 8-9) that posses both angio-inhibitory and, using a murine xenograft model of Kaposis sarcoma, anti-tumorigenic activity in vivo. Thus, both the alpha3beta1-binding and angio-inhibitory activities co-localize to a solvent exposed, flexible region in the TIMP-2 primary sequence that is unique in amino acid sequence compared with other members of the TIMP family. Furthermore, comparison of the TIMP-2 and TIMP-1 protein 3-D structures in this region also identified unique structural differences. Our findings demonstrate that the integrin binding, tumor growth suppressor and in vivo angio-inhibitory activities of TIMP-2 are intimately associated within a unique sequence/structural loop (B-C loop). Advances in 2012 are the demonstration that TIMP-2 has direct antitumoral properties independent of its MMP inhibitory activity in vivo (See Bourboulia et al., Amer. J. Pathol 179:2589-2600, 2011). This worked demonstrated that forced expression of TIMP-2 and Ala+TIMP-2 (lacking MMP inhibitory activity) in A549 human lung cancer cells results in decreased phosphorylation of Fak and AKT directly in the tumor cells inhibiting these pathways. We also demonstrated using a TIMP-2-deficient mouse model, as well as A549 expressing TIMP-2 xenografts, that TIMP-2 functions to suppress the infiltration of tumor-associated myeloid suppressor cells that are critical for tumor angiogenesis. These findings support the continued preclinical development of TIMP-2 as a novel biological therapy for cancer treatment.