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

在过去的4年里，我们已经完成（并发表）了几项研究，描述了TIMP-2受体α 3/ β 1整合素的下游信号通路，参与介导TIMP-2的抗血管生成作用，包括更详细地描述了TIMP-2介导的内皮细胞（hMVECs）对成纤维细胞生长因子-2 （FGF-2）的生长抑制。这些研究表明，抑制src同源蛋白酪氨酸磷酸酶-1 (SHP-1)，无论是通过药理学方法还是显性阴性SHP-1的表达，都可以阻止timp -2介导的成纤维细胞生长因子介导的内皮细胞有丝分裂发生的抑制。这些发现与之前的观察结果一致，即TIMP-2激活SHP-1，导致多种受体酪氨酸激酶通过配体依赖的方式失活（去磷酸化）。我们研究了在存在或不存在TIMP-2 （Ala+TIMP-2）的情况下，VEGF-A刺激后血管内皮生长因子-2 （VEGFR-2）的磷酸化模式。我们的结果表明，Ala+TIMP-2选择性地改变了内皮细胞增殖和迁移相关残基上VEGFR-2的酪氨酸磷酸化（在Y951、Y996和Y1175时磷酸化显著降低）。Ala+TIMP-2破坏了磷脂酶c - γ、Akt和内皮型一氧化氮合成酶的下游活化。TIMP-2或Ala+TIMP-2抑制vegf - a介导的Ca+2内流，并降低通常由一氧化氮供体增强的cGMP水平。观察到cGMP降低对异丁基甲基黄嘌呤抑制敏感。在最近的另一篇报道中，我们发现TIMP-2通过α 3/ β 1- shp -1- camp /PKA信号通路介导血管通透性的抑制，从而增强VE-cadherin与细胞骨架的关联。我们的研究结果表明，TIMP-2通过血管通透性和功能的“正常化”，在癌症治疗中具有潜在的效用。Jain和同事将肿瘤血管的这种“正常化”归因于使用血管生成抑制剂后的临床益处和对放疗的增强反应。有趣的是，这种“正常化”似乎也发生在我们在肿瘤异种移植物中使用强制表达TIMP-2的实验中（主要未发表的观察结果，见下文）。我们还完成了“B-C环”合成肽的研究，通过其α 3/ β 1结合活性鉴定，证明其在体内具有抗血管生成和抗肿瘤活性。提出的“B-C”环区谷氨酸、赖氨酸和精氨酸残基的TIMP-2突变体已经表达，但其活性尚未进一步表征。如上所述，这些研究是不完整的，因为工作人员的离职和我们的重点是证明TIMP-2在体内不依赖于mmp的活性。如上所述，我们的主要重点是证明TIMP-2不依赖于mmp的抗血管生成作用，这有助于TIMP-2在体内的抗肿瘤活性。为此，我们利用逆转录病毒载体在人肺癌细胞系A549中强制表达TIMP-2和Ala+TIMP-2，然后将这些细胞系用于nu/nu和NOD-SCID小鼠的肿瘤异种移植实验。尽管这些细胞系在体外的基本生长速率没有明显差异，但在肿瘤接种后40天，与空载体对照相比，TIMP-2（90%）和Ala+TIMP-2（75%）异种移植物的肿瘤生长均受到显著抑制。肿瘤生长的抑制伴随着肿瘤微血管密度计数（cd31 +或CD34+）的统计学显著下降，这是一种抗血管生成作用的测量，以及肿瘤细胞凋亡的增加（也可能是由于抑制血管生成）。出乎意料的是，我们还观察到表达TIMP-2的肿瘤细胞中局灶黏附激酶（FAK）的减少，以及表达TIMP-2和Ala+TIMP-2的肿瘤细胞中FAK磷酸化（Y397）的显著减少。我们观察到FAK和/或AKT （Protein Kinase B， PKB）磷酸化在TIMP-2和Ala+TIMP-2肿瘤组织中均降低，这表明：1)FAK位于AKT信号传导的上游，并且都参与细胞迁移的调节；2)体外表达TIMP-2和Ala+TIMP-2可减少肿瘤细胞的迁移。我们之前报道了内皮细胞中FAK磷酸化降低，其中FAK磷酸化参与控制eNOS活性。总之，这些使用逆转录病毒转导的表达野生型（wt） TIMP-2或缺乏金属蛋白酶抑制剂的Ala+TIMP-2的肿瘤细胞的实验清楚地表明，TIMP-2不依赖于mmp的活性，包括抗血管生成活性，足以显著影响肿瘤在体内的生长。我们观察到TIMP-2和Ala+TIMP-2对A549肿瘤异种移植物的影响，使我们对这些细胞系和肿瘤组织进行转录谱分析。与对照A549细胞相比，表达TIMP-2或Ala+TIMP-2的细胞显示E-cadherin表达增加，并且在表皮生长因子（EGF）刺激后对细胞膜相关E-cadherin和β -catenin的再分配产生抗性，提示间质-上皮转变。其他受差异调控的基因包括含有egf的纤维蛋白样细胞外基质蛋白1 (EGFEMP1，纤维蛋白3)，在表达TIMP-2或Ala+TIMP-2的细胞中上调。该蛋白在胶质瘤中是一个有利的预后因子，并抑制血管生成、细胞增殖和VEGF-A的表达。然而，这些发现需要得到证实（见下文），对下游基因调控的影响机制仍有待确定。来自基因表达谱的其他数据也揭示了atp结合盒（ABC）转运体基因表达的变化。ABC蛋白驱动多种底物的细胞外排，包括细胞毒性药物，并且已知有助于对癌症化疗的抵抗。ABC转运蛋白的活性是各种实体肿瘤中肿瘤干细胞存在的重要指标。Hoechst 33342染料外排测定确定了肿瘤细胞亚群，称为侧群（SP），其在csc中富集。根据我们的基因表达谱数据，我们假设TIMP-2的抗肿瘤活性可能部分参与了我们肺癌细胞模型中SP的调节。为此，我们在一系列6种非小细胞肺癌（NSCLC）细胞系中测定了SP部分与内源性TIMP-2表达水平的相关性。有趣的是，我们的结果显示，内源性TIMP-2 mRNA表达水平与使用Hoechst染料外排法测定的SP百分比之间存在强烈的、高度显著的负相关（R2=0.073, p0.03）。在表达TIMP-2的A549细胞中，SP显著降低，这种降低与ABCG2、ABCB1和AKR1C1的表达降低有关。功能分析显示，表达TIMP-2的A549细胞对包括阿霉素和拓扑替康在内的细胞毒性药物的敏感性增加。这些发现表明TIMP-2治疗可能增强对细胞毒性化疗的敏感性，并且首次证明TIMP-2调节SP和可能的CSC水平和功能。我们认为这些研究在开发一种抑制肿瘤干细胞、肿瘤细胞、内皮细胞和肿瘤生长的新的生物活性方面取得了重大进展。

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.