The Role of TIMPs in Cell Growth, Tumor Progression and Metastasis

主要活动/具体目标。我们正在进行的研究工作的主要目标是深入了解TIMP家族成员，特别是TIMP-2的mmp独立活动的机制。我们确定了以下具体目标来实现我们的目标：1)检查TIMPs在体外改变癌症干细胞（CSCs）生长和侵袭潜力中的作用；2)研究TIMPs对体内原发性和转移性肿瘤生长的影响；3)研究TIMPs对免疫调节细胞(髓源性抑制细胞（myeleloidderived suppressor cells， MDSC）向原发肿瘤和转移小生境募集的影响。我的实验室的一个主要重点是证明mmp独立的抗血管生成作用对TIMP-2体内抗肿瘤活性的贡献，这是许多研究者观察到的。为此，我们利用逆转录病毒载体在人非小细胞肺癌（NSCLC）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的细胞显示出E-cadherin的表达增加，并且在表皮生长因子（EGF）刺激后对细胞膜相关的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。该蛋白在胶质母细胞瘤中是一个有利的预后因子，抑制血管生成、细胞增殖和VEGF-A的表达。然而，这些发现还有待证实，其对下游基因调控的作用机制仍有待确定。主要成果和成就。来自基因表达谱的其他数据也揭示了非小细胞肺癌A549中atp结合盒（ABC）转运体基因表达的变化。这为了解TIMP-2对癌症干细胞（CSC）的影响以及TIMP-2增强细胞毒性治疗的潜在应用开辟了新的研究途径。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CSC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。我们认为这些研究表明TIMP-2在抑制肿瘤干细胞、肿瘤细胞、血管生成以及体内肿瘤生长和转移的生物学活性方面取得了重大进展，并为TIMP-2联合化疗和/或放疗的潜在应用提供了生物学基础，以提高这些治疗的疗效并减少潜在的副作用。血管生成和炎症是非小细胞肺癌的重要治疗靶点。我们在TIMP-2缺失小鼠中检测了TIMP-2对非小细胞肺癌肿瘤相关血管生成和炎症的影响，并使用小鼠Lewis肺癌模型与野生型小鼠进行了比较。timp -2缺陷小鼠肿瘤生长加快，肿瘤组织中血管内皮生长因子- a （VEGF-A）水平显著升高，血管生成标志物表达增强。此外，携带肿瘤的timp -2缺陷小鼠表现出炎症介质、核因子- κ B和膜联蛋白A1的上调，血清白细胞介素-6水平升高，肿瘤浸润炎性细胞显著增加。表型分析显示，表达VEGFR-1的脾MDSC （CD11b+和Gr-1+细胞）增加，提示LL肿瘤诱导的TIMP-2缺乏具有全体性作用。相反，在使用A549细胞的人非小细胞肺癌异种移植模型中，强迫过表达TIMP-2显示MDSC向肿瘤募集的显著减少。MDSC向肿瘤募集与抑制抗肿瘤免疫和增强肿瘤血管生成有关。这些最近的研究结果表明，TIMP-2对肿瘤和宿主细胞具有多种作用，这些作用结合在一起产生了一种有效的抗肿瘤活性，可以在临床上加以利用。

Major Activities/Specific Objectives. The principal goal of our ongoing research effort is to develop an in depth mechanistic understanding of the MMP-independent activities of members of the TIMP family, in particular TIMP-2. We have identified the following specific objectives to obtain our goals: 1) examine the role of TIMPs in altering the growth and invasive potential of cancer stem cells (CSCs) in vitro; 2) study to effects of TIMPs on primary and metastatic tumor growth in vivo; 3) study the influence of TIMPs on recruitment of immune-modulatory cells (myeloid-derived suppressor cells (MDSC)) to the primary tumor and metastatic niche. A major focus in my lab has been to demonstrate the contribution of the MMP-independent anti-angiogenic effects 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 non-small cell lung carcinoma (NSCLC) 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 associated 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 glioblastoma, and suppresses angiogenesis, cell proliferation and VEGF-A expression. However, these findings need to be confirmed and the mechanisms of the effects on downstream gene regulation remain to be identified. Key outcomes and achievements. Additional data from our gene expression profiling also revealed changes in ATP-binding cassette (ABC) transporter gene expression in NSCLC A549. This has led to a new avenue of investigation directed at understanding the effects of TIMP-2 on cancer stem cells (CSC) and potential use of TIMP-2 to enhance cytotoxic therapies. 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 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 the biological activities of TIMP-2 that suppress growth of cancer stem cells, tumor cells, and angiogenesis, as well as tumor growth and metastasis in vivo, and provide a biologic basis for the potential use of TIMP-2 therapy in combination with chemo-and/or radiation therapy to enhance the efficacy of these treatments while reducing potential side effects. Angiogenesis and inflammation are important therapeutic targets in NSCLC. We examined the effects of TIMP-2 on NSCLC tumor-associated angiogenesis and inflammation were examined in TIMP-2-deficient mice and compared with wild type mice using the murine Lewis lung (LL) carcinoma model. TIMP-2-deficient mice demonstrated an increased growth of tumor, significantly elevated levels of vascular endothelial growth factor-A (VEGF-A) and enhanced expression of angiogenic markers in tumor tissues. In addition Tumor-bearing TIMP-2-deficient mice demonstrated up regulation of inflammatory mediators, nuclear factor-kappa B and Annexin A1, elevated serum levels of interleukin-6, and a significant increase of tumor infiltrating inflammatory cells. Phenotypic analysis revealed an increase of splenic MDSC (CD11b+ and Gr-1+ cells) expressing VEGFR-1 suggesting a systemic effect of TIMP-2 deficiency induced by the LL tumors. In contrast, forced overexpression of TIMP-2 in a human xenograft model of NSCLC using A549 cells demonsted a significant reduction in recruitment of MDSC to the tumors. The recruitment of MDSC to tumors has been linked to suppression of antitumor immunity and enhanced tumor angiogenesis. These recent findings suggest that TIMP-2 has a variety of effects on both tumor and host cells that combine to produce a potent anti-tumor activity that could be exploited clinically.