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

TIMP 在细胞生长和分化中的作用：肿瘤血管生成

• 批准号: 8350064
• 负责人: William Stetler-Stevenson
• 金额: $ 64.29万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8350064
• 关键词: 3-Dimensional; A549; Amino Acid Sequence; Amino Acids; Antibodies; Apoptotic; Basement membrane; Behavior; Binding; Biochemical; Biological; Biology; Breast Cancer Cell; Cancer cell line; Cell Differentiation process; Cell Line; Cell Surface Receptors; Cells; Chemicals; Chronic Disease; Complex; Cues; Development; EGF gene; Elements; Endothelial Cells; Epidermal Growth Factor Receptor; Evolution; Excision; Extracellular Matrix; Family; Fibroblast Growth Factor; Fibroblast Growth Factor 2; Fibroblasts; Future; Gene Expression; Genes; Goals; Growth; Growth Factor; Growth Factor Receptors; Homeostasis; Human; Immune response; In Vitro; Inflammation; Inhibition of Matrix Metalloproteinases Pathway; Injection of therapeutic agent; Integrin Binding; Integrin alpha3beta1; Integrins; Kaposi Sarcoma; Laboratories; Lead; Malignant - descriptor; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of lung; Matrix Metalloproteinases; Mediating; Mus; Mutation; N-terminal; Neoplasm Metastasis; Non-Malignant; Normal Cell; Normal tissue morphology; Oncogenes; Organ; Organism; Pathway interactions; Peptides; Phenotype; Phosphorylation; Play; Primary Neoplasm; Process; Protein Tyrosine Phosphatase; Proteins; Reagent; Receptor Activation; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Recombinants; Regulation; Relative (related person); Role; Signaling Molecule; Site; Solvents; Stimulation of Cell Proliferation; Structure; Teratocarcinoma; Therapeutic; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinases; Tissues; Tumor Angiogenesis; Tumor Cell Invasion; Tumor Suppression; Tumor Suppressor Genes; Tumor Suppressor Proteins; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; Work; Xenograft Model; angiogenesis; base; blastocyst; cancer therapy; cell behavior; cell growth; cell type; cytokine; epithelial to mesenchymal transition; extracellular; fibrosarcoma; flexibility; human tissue; in vivo; in vivo Model; lung Carcinoma; member; metastatic process; neoplastic cell; new therapeutic target; novel; novel therapeutics; overexpression; precursor cell; pressure; protein aminoacid sequence; receptor; research study; response; synthetic peptide; tumor; tumor growth; tumor progression; tumorigenic

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).

摘要：A549人肺癌细胞系的EGF刺激表明，细胞生长反应的抑制是通过蛋白酪氨酸磷酸酶活性的激活介导的，并导致EGF受体磷酸化降低。使用抗整合蛋白抗体的竞争结合实验将整联蛋白alpha3beta1鉴定为人类微血管内皮细胞（HMVECS）的TIMP-2的假定细胞表面受体。 ALA+TIMP-2还抑制了VEGF-A或FGF-2在体内刺激的有丝分裂发生和血管生成，因此表明TIMP-2的血管抑制活性与MMP抑制作用可分离。这种作用的机制涉及生长因子受体信号传导的整合素受体失活，称为异源受体失活。这是首次证明整联蛋白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，与细胞表面受体并与这些受体相互作用来定义了TIMP生物学的新范式，它们可以直接影响细胞行为。使用体外和体内模型，我们的当前和将来的工作都集中在TIMP-2中识别alpha3beta1整合蛋白结合域（S），并在正常和肿瘤细胞中与alpha3beta1相互作用后，进一步了解了我们对TIMP-2相互作用后的细胞效应的理解，以及随后的Micro Enronmerm Micro Enronmment中的变化。我们的目标是进一步表征TIMP在肿瘤微环境中及其对肿瘤抑制和/或进展的相对贡献的TIMP的依赖性和MMP依赖性作用。这些研究应确定正常组织中细胞外基质和肿瘤微环境调节细胞行为的关键机制，并可能导致新的治疗策略用于癌症治疗。这些发现表明，定义负责TIMP-2结合到alpha3beta1的结构域对于进一步剖析该复合物分子的多种生物学活性至关重要，并定义了这种活性对正常组织和恶性组织中微环境的功能贡献。该项目的重点是确定ALA+TIMP-2的抗血管生成和抗肿瘤效应的机制。与人类微血管内皮细胞的初步工作表明了一种称为异源受体失活的机制。在这种效果中，TIMP-2受体α3Beta1降低受体酪氨酸激酶的磷酸化和激活，例如血管内皮生长因子受体受体（VEGFR）-2，成纤维细胞生长因子recetpor（FGFR）-1和表皮生长因子受体受体（EGFR）通过激活磷酸酶磷酸酶磷酸酶磷酸酶（1）。然而，最近在肿瘤细胞和内皮细胞中进行的实验表明，ALA+TIMP-2的生长抑制活性更为复杂，似乎涉及凋亡途径以及上皮细胞对间质转变的基因表达的变化，这对于肿瘤侵袭和转移至关重要。该项目的目的是识别和描述这些途径，目的是将ALA+TIMP-2开发为一种新型的癌症治疗，并确定潜在的新治疗靶标。金属蛋白酶-2（TIMP-2）的组织抑制剂通过涉及MMP抑制或直接内皮细胞结合的几种机制抑制血管生成。这项研究的主要目的是确定与先前鉴定的受体整合素α3BETA1结合涉及的TIMP-2区域，并确定从该区域衍生的合成肽是否保留了血管抑制性和肿瘤抑制剂活性。我们证明了TIMP-2（N-TIMP-2）的N末端结构域与alpha3beta1结合，并抑制体外的血管内皮生长因子刺激的内皮细胞生长，这表明alpha3beta1结合域和TIMP-2型TIMP-2的生长抑制剂均在N-末端末端域上的生长抑制活性。使用肽阵列方法，我们鉴定了TIMP-2主要序列的24个氨基酸区域，该序列由残基ILE43-ALA66组成，该残基显示了Alpha3beta1结合活性。随后，我们证明了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。我们定义了最小的肽序列（肽8-9），该序列同时具有血管抑制性，并使用kaposis肉瘤的鼠异种移植模型，体内抗肿瘤活性。因此，与TIMP家族的其他成员相比，在TIMP-2主要序列中，Alpha3beta1结合和血管抑制活性都将其在TIMP-2主要序列中共定位在TIMP-2主要序列中是独一无二的。此外，该区域中TIMP-2和TIMP-1蛋白3-D结构的比较也确定了独特的结构差异。我们的发现表明，TIMP-2的整联蛋白结合，肿瘤生长抑制和体内抑制性活性与独特的序列/结构环（B-C环）密切相关。