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: 8763694
• 负责人: William Stetler-Stevenson
• 金额: $ 62.9万
• 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/8763694
• 关键词: A549; ABCB1 gene; ABCG2 gene; AKR1C1; ATP-Binding Cassette Transporters; Active Sites; Acute Disease; Alanine; Amino Acids; Angiogenesis Inhibition; Angiogenesis Inhibitors; Apoptosis; Basement membrane; Behavior; Binding; Biological; Biological Assay; Biological Response Modifier Therapy; Breast Cancer Cell; CD29 Antigen; CD34 gene; Cancer cell line; Cell Differentiation process; Cell Line; Cell Proliferation; Cell Surface Receptors; Cell membrane; Cell model; Cell physiology; Cell surface; Cells; Chemotherapy-Oncologic Procedure; Chronic Disease; Clinical; Complex; Cultured Tumor Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cytoskeleton; Cytotoxic Chemotherapy; Cytotoxic agent; Data; Development; Diagnosis; Disintegrins; Dominant-Negative Mutation; Doxorubicin; Dyes; E-Cadherin; Elements; Endothelial Cells; Enzymes; Epidermal Growth Factor; Epithelial; Equilibrium; Extracellular Matrix; Extracellular Matrix Degradation; Extracellular Matrix Proteins; Family; Fibroblast Growth Factor; Fibroblast Growth Factor 2; Fibroblasts; Focal Adhesion Kinase 1; Future; Gelatinase A; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Genes; Glioblastoma; Goals; Growth; Growth Factor; Growth Inhibitors; Human; In Vitro; Integrins; Laboratories; Ligands; Lung; Malignant Epithelial Cell; Malignant neoplasm of lung; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Measures; Mediating; Mesenchymal; Metalloproteases; Methionine; Methods; Modeling; NOD/SCID mouse; Names; Neoplasm Metastasis; Nitric Oxide Donors; Nitric Oxide Synthase; Non-Small-Cell Lung Carcinoma; Normal tissue morphology; Pathology; Pattern; Phosphorylation; Physiology; Population; Prognostic Factor; Protein Dephosphorylation; Protein Tyrosine Phosphatase; Proteins; Proto-Oncogene Proteins c-akt; Publishing; Radiation therapy; Receptor Protein-Tyrosine Kinases; Regulation; Reporting; Research; Research Personnel; Resistance; Retroviral Vector; Role; S1-5 protein; Series; Side; Signal Pathway; Signal Transduction; Solid Neoplasm; Staging; Stimulation of Cell Proliferation; Survival Rate; Testing; Therapeutic; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinases; Tissues; Topotecan; Tumor Angiogenesis; Tumor Cell Invasion; Tumor Tissue; Tyrosine Phosphorylation; Vascular Endothelial Growth Factors; Vascular Permeabilities; Xenograft procedure; Zinc; angiogenesis; arginyllysine; base; beta catenin; cadherin 5; cancer cell; cancer stem cell; cancer therapy; cell growth; cell motility; cell type; density; fibrosarcoma; fibulin; in vivo; inhibitor/antagonist; interest; lung Carcinoma; mRNA Expression; member; migration; mutant; neoplastic cell; novel; phospholipase C gamma; prevent; receptor; research study; response; synthetic peptide; tissue procollagenase; tumor; tumor growth; tumor microenvironment; tumor progression; tumor xenograft; tumorigenic; vector control

During the past 4 years we have completed, (and published) several studies characterizing downstream signaling pathways from the TIMP-2-receptor, alpha3/beta1-integrin, involved in mediating the anti-angiogenic effects of TIMP-2, including a more detailed characterization of TIMP-2-mediated growth inhibition of endothelial cells (hMVECs) in response to fibroblast growth factor-2 (FGF-2) These studies demonstrated that inhibition of src homology protein tyrosine phosphatase-1 (SHP-1), either by pharmacologic methods or expression of dominant negative SHP-1, prevents TIMP-2-mediated inhibition of fibroblast growth factor mediated endothelial mitogenesis. These findings are consistent with previous observations that TIMP-2 activates SHP-1 resulting in inactivation (dephosphorylation) of a variety of receptor tyrosine kinases via a ligand-dependent fashion In another series of experiments, we examined the phosphorylation pattern of vascular endothelial growth factor-2 (VEGFR-2) following VEGF-A stimulation in the presence or absence of TIMP-2 (Ala+TIMP-2) Our results show that Ala+TIMP-2 selectively alters tyrosine phosphorylation of VEGFR-2 at residues implicated in endothelial cell proliferation and migration (significantly decreased phosphorylation at Y951, Y996 and Y1175). Ala+TIMP-2 disrupted downstream activation of phospholipase C-gamma, Akt and endothelial nitric oxide synthetase. TIMP-2 or Ala+TIMP-2 inhibit VEGF-A-mediated Ca+2 influx, and reduced cGMP levels normally enhanced by nitric oxide donors. The observed decrease in cGMP was sensitive to isobutylmethylxanthine inhibition. In another recent report we showed that TIMP-2 mediates the inhibition of vascular permeability via an alpha3/beta1-Shp-1-cAMP/PKA signaling pathway, which enhanced VE-cadherin association with the cytoskeleton Our results demonstrate the potential utility for TIMP-2 in cancer therapy through "normalization" of vascular permeability and function . It is this "normalization" of tumor vasculature, to which Jain and colleagues attribute much of the clinical benefit and enhanced response to radiotherapy following the use of angiogenesis inhibitors It is interesting that such "normalization" also seems to occur in vivo in our experiments utilizing forced expression of TIMP-2 in tumor xenografts (major unpublished observation, see below). We also completed our studies on the "B-C loop" synthetic peptides, identified by their alpha3/beta1-binding activity, demonstrating their anti-angiogenic and anti-tumorigenic activity in vivo. The proposed TIMP-2 mutants of the glutamic, lysine and arginine amino acid residues in the "B-C" loop region have been expressed but their activities have not yet been further characterized. As noted above these studies are incomplete because of staff departures and our focus on demonstrating in vivo MMP-independent activity of TIMP-2. As mentioned above our major focus has been to demonstrate the MMP-independent anti-angiogenic effects of TIMP-2 in contributing to the anti-tumor activity of TIMP-2 in vivo observed by a number of investigators. To this end we employed retroviral vectors to force expression of TIMP-2 and Ala+TIMP-2 in the human lung carcinoma cell line A549 and then used these cell lines in tumor xenograft experiments in both nu/nu and NOD-SCID mice. Although these cell lines showed no discernable difference in basal growth rates in vitro there was significant suppression of tumor growth in both TIMP-2 (90 %) and Ala+TIMP-2 (75%) xenografts compared to empty vector controls as late as 40 days post tumor-inoculation. The suppression of tumor growth was accompanied by a statistically significant decrease in tumor microvascular density count (CD 31+ or CD34+), a measure of antiangiogenic effects, as well as by increased tumor cell apoptosis (also possibly due to inhibition of angiogenesis). Somewhat unexpectedly, we also observed a decrease in focal adhesion kinase (FAK) in TIMP-2 expressing tumors and a significant decrease in FAK phosphorylation (Y397) in both TIMP-2 and Ala+TIMP-2 expressing tumor cells. Our observation that both FAK and/or AKT (Protein Kinase B, PKB) phosphorylation is reduced in TIMP-2 and Ala+TIMP-2 tumor tissues is significant in that: 1) FAK is upstream of AKT signaling, and both are involved in regulation of cell migration; 2) TIMP-2 and Ala+TIMP-2 expression reduced tumor cell migration in vitro. We previously reported decreased FAK phosphorylation in endothelial cells where it is involved in control of eNOS activity. In summary, these experiments using retrovirally transduced tumor cells expressing wild type (wt) TIMP-2 or metalloprotease inhibitor-deficient Ala+TIMP-2 clearly demonstrate that the MMP-independent activities of TIMP-2, including the anti-angiogenic activity, are of sufficient magnitude to significantly impact tumor growth in vivo. Our observation of the effects of TIMP-2 and Ala+TIMP-2 on A549 tumor xenografts, led us to perform transcriptional profiling of these cell lines and tumor tissues. The observed changes in gene expression are predominantly related to decreased tumor development and reduced metastasis In contrast to control A549 cells, cells expressing TIMP-2 or Ala+TIMP-2 showed increased expression of E-cadherin, and were resistant to redistribution of cell membrane associate E-cadherin and beta-catenin following epidermal growth factor (EGF) stimulation, suggestive of a mesenchymal-epithelial transition. Other genes of interest that were differentially regulated include EGF-containing fibulin-like extracellular matrix protein 1 (EGFEMP1, fibulin 3) that was up regulated in cells expressing TIMP-2 or Ala+TIMP-2. This protein is a favorable prognostic factor in gliomablastoma, and suppresses angiogenesis, cell proliferation and VEGF-A expression. However, these findings need to be confirmed (see below) and the mechanisms of the effects on downstream gene regulation remain to be identified. Additional data from our gene expression profiling also revealed changes in ATP-binding cassette (ABC) transporter gene expression. ABC proteins drive cell efflux of a variety of substrates, including cytotoxic drugs, and are known to contribute to resistance to cancer chemotherapy. The activity of ABC transporters is an important indicator of cancer stem cell (CSC) presence in various solid tumors. The Hoechst 33342 dye efflux assay identifies a tumor cell subpopulation, known as the side population (SP), that is enriched in CSCs. Based on our gene expression profiling data we posit that TIMP-2 anti-tumor activity may, in part, involve regulation of the SP in our lung cancer cell model. To this end, we determined the correlation between the SP fraction and level of endogenous TIMP-2 expression in a series of six non-small cell lung cancer (NSCLC) cell line. Interestingly, our results demonstrate a strong, highly significant inverse correlation (R2=0.073, p0.03) between the level of endogenous TIMP-2 mRNA expression and the percentage of SP determined using the Hoechst dye efflux assay. In A549 cells expressing TIMP-2, a significant decrease in the SP is observed and this decrease is associated with lower expression of ABCG2, ABCB1 and AKR1C1. Functional analysis reveals that A549 cells expressing TIMP-2 show increased sensitivity to cytotoxic drugs, including doxorubicin and topotecan. These findings suggest that TIMP-2 therapy may enhance sensitivity to cytotoxic chemotherapy, and are the first demonstration that TIMP-2 modulates SP and possibly CSC levels and function. We feel these studies demonstrate significant progress in developing a new biological activity that suppresses cancer stem cells, tumor cells, endothelial cells and tumor growth in vivo.

在过去的4年中证明通过药理方法或显性阴性SHP-1的表达抑制SRC同源蛋白酪氨酸磷酸酶-1（SHP-1），可以防止TIMP-2介导的抑制成纤维细胞生长因子介导的内皮丝质的抑制。 These findings are consistent with previous observations that TIMP-2 activates SHP-1 resulting in inactivation (dephosphorylation) of a variety of receptor tyrosine kinases via a ligand-dependent fashion In another series of experiments, we examined the phosphorylation pattern of vascular endothelial growth factor-2 (VEGFR-2) following VEGF-A stimulation in the presence or absence of TIMP-2 (Ala+TIMP-2) Our结果表明，ALA+TIMP-2在与内皮细胞增殖和迁移有关的残基上有选择地改变VEGFR-2的酪氨酸磷酸化（在Y951，Y996和Y1175时显着降低了磷酸化）。 ALA+TIMP-2破坏了磷脂酶C-Gamma，Akt和内皮一氧化氮合成酶的下游激活。 TIMP-2或ALA+TIMP-2抑制VEGF-A介导的Ca+2涌入，并且通常通过一氧化氮供体增强CGMP水平。观察到的CGMP降低对异丁基甲基黄嘌呤抑制敏感。在最近的另一份报告中，我们表明TIMP-2通过alpha3/beta1-shp-1-camp/pKA信号通路介导了血管通透性的抑制作用，从而增强了VE-Cadherin与细胞骨架的关联，我们的结果证明了TIMP-2在癌症治疗中的潜在效用，通过“正常化”的“标准化”血管渗透性和功能和功能。 It is this "normalization" of tumor vasculature, to which Jain and colleagues attribute much of the clinical benefit and enhanced response to radiotherapy following the use of angiogenesis inhibitors It is interesting that such "normalization" also seems to occur in vivo in our experiments utilizing forced expression of TIMP-2 in tumor xenografts (major unpublished observation, see below).我们还完成了关于“ B-C环”合成肽的研究，该肽通过其α3/beta1结合活性确定，证明了它们在体内的抗血管生成和抗肿瘤活性。已经表达了谷氨酸，赖氨酸和精氨酸氨基酸残基的提议的TIMP-2突变体，但尚未进一步表征它们的活性。如上所述，由于员工的出发，这些研究是不完整的，我们专注于展示TIMP-2的体内MMP独立活动。如上所述，我们的主要重点是证明TIMP-2对MMP独立的抗血管生成作用，在许多研究者观察到的TIMP-2在体内的抗肿瘤活性中有助于。为此，我们采用了逆转录病毒载体在人肺癌细胞系A549中强迫TIMP-2和ALA+TIMP-2的表达，然后在NU/NU和NOD-SCID小鼠中使用这些细胞系A549。尽管这些细胞系在体外的基础生长速率没有明显的差异，但与肿瘤接种后40天的空载体对照相比，TIMP-2（90％）和ALA+TIMP-2（75％）异种移植物的肿瘤生长显着抑制。肿瘤生长的抑制伴随着肿瘤微血管密度计数（CD 31+或CD34+）的统计学显着降低，这是一种抗血管生成作用的量度，以及肿瘤细胞凋亡的增加（也可能是由于血管生成抑制）。出乎意料的是，我们还观察到TIMP-2表达肿瘤的局灶性粘附激酶（FAK）的降低，而TIMP-2和ALA+TIMP-2表达肿瘤细胞的FAK磷酸化（Y397）显着降低。我们观察到FAK和/或AKT（蛋白激酶B，PKB）磷酸化在TIMP-2和ALA+TIMP-2肿瘤组织中均降低了：1）FAK是AKT信号的上游，并且两者都参与细胞迁移的调节； 2）TIMP-2和ALA+TIMP-2表达在体外降低了肿瘤细胞的迁移。我们先前报道了内皮细胞中FAK磷酸化的降低，其中它参与了eNOS活性的控制。总而言之，这些使用逆转录病毒转导的肿瘤细胞表达野生型（WT）TIMP-2或金属蛋白酶抑制剂缺陷型ALA+TIMP-2清楚地表明，TIMP-2的MMP独立活性，包括抗血管生成活性，具有足够的幅度，可以显着影响VMUMOR在Vivo中的生长。我们观察到TIMP-2和ALA+TIMP-2对A549肿瘤异种移植的影响，使我们对这些细胞系和肿瘤组织进行了转录分析。与对照A549的细胞相比，观察到的基因表达的变化主要与肿瘤的发展减少和转移减少有关，表达TIMP-2或ALA+TIMP-2的细胞显示出E-钙粘着蛋白的表达增加，并且耐于细胞膜的E-Cadherin和Beta-cate蛋白 - 抑制了刺激性（均具有刺激性）（均为蛋白质）（s exteremal a stresep a strope），并且 过渡。受差异调节的其他感兴趣的基因包括含有EGF的纤维蛋白样细胞外基质蛋白1（EGFEMP1，fibulin 3），该基因在表达TIMP-2或ALA+TIMP-2的细胞中受到调节。该蛋白是神经胶质瘤中的一个有利的预后因素，可抑制血管生成，细胞增殖和VEGF-A表达。但是，这些发现需要得到确认（见下文），并且对下游基因调节的影响的机制仍有待定。来自我们基因表达分析的其他数据也揭示了ATP结合盒（ABC）转运蛋白基因表达的变化。 ABC蛋白驱动各种底物（包括细胞毒性药物）的细胞外排，已知有助于抗癌化学疗法。 ABC转运蛋白的活性是各种实体瘤中癌症干细胞（CSC）存在的重要指标。 HOECHST 33342染料外排测定法鉴定了富含CSC的肿瘤细胞亚群，称为侧种群（SP）。根据我们的基因表达分析数据，我们认为TIMP-2抗肿瘤活性可能部分涉及我们的肺癌细胞模型中SP的调节。为此，我们确定了一系列六个非小细胞肺癌（NSCLC）细胞系中内源性TIMP-2表达水平的相关性。有趣的是，我们的结果表明，内源性TIMP-2 mRNA表达水平与使用HOECHST染料外排测定的SP的百分比之间存在很强的，非常显着的反相关（R2 = 0.073，P0.03）。在表达TIMP-2的A549细胞中，观察到SP显着降低，并且这种降低与ABCG2，ABCB1和AKR1C1的较低表达有关。功能分析表明，表达TIMP-2的A549细胞对包括阿霉素和拓扑素在内的细胞毒性药物的敏感性增加。这些发现表明，TIMP-2治疗可以增强对细胞毒性化疗的敏感性，并且是TIMP-2首次调节SP以及可能的CSC水平和功能的证明。我们认为这些研究表明，在开发一种新的生物学活性方面取得了重大进展，该活性抑制了癌症干细胞，肿瘤细胞，内皮细胞和体内肿瘤生长。