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

TIMP 在细胞生长、肿瘤进展和转移中的作用

• 批准号: 8938403
• 负责人: William Stetler-Stevenson
• 金额: $ 58.38万
• 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/8938403
• 关键词: A549; ABCB1 gene; ABCG2 gene; AKR1C1; ATP-Binding Cassette Transporters; Achievement; Acute Disease; Adverse effects; Alanine; Angiogenesis Inhibition; Angiogenesis Inhibitors; Annexin A1; Apoptosis; Basement membrane; Behavior; Binding; Biological; Biological Assay; Breast Cancer Cell; CD34 gene; Cancer cell line; Cell Line; Cell Proliferation; Cell Surface Receptors; Cell membrane; Cell model; Cell physiology; Cell surface; Cells; Chemotherapy-Oncologic Procedure; Chronic Disease; Complex; Cultured Tumor Cells; Cytotoxic Chemotherapy; Cytotoxic agent; Data; Development; Disintegrins; Doxorubicin; Dyes; E-Cadherin; Elements; Endothelial Cells; Epidermal Growth Factor; Epithelial; Equilibrium; Extracellular Matrix; Extracellular Matrix Proteins; Family; Fibroblasts; Focal Adhesion Kinase 1; Future; Gelatinase A; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Genes; Glioblastoma; Goals; Growth; Growth Factor; Human; ITGAM gene; Immune; Immunity; In Vitro; Inflammation; Inflammation Mediators; Inflammatory Infiltrate; Interleukin-6; Investigation; Laboratories; Lewis Lung Carcinoma; Link; Lung; Lung Neoplasms; Malignant neoplasm of lung; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Measures; Mediating; Mesenchymal; Metalloproteases; Methods; Modeling; Mus; Myelogenous; Myeloid Cells; NF-kappa B; NOD/SCID mouse; Neoplasm Metastasis; Non-Small-Cell Lung Carcinoma; Normal tissue morphology; Outcome; Pathology; Phosphorylation; Physiology; Population; Primary Neoplasm; Prognostic Factor; Protease Inhibitor; Proteins; Proto-Oncogene Proteins c-akt; Radiation therapy; Receptor Protein-Tyrosine Kinases; Regulation; Reporting; Research; Research Personnel; Resistance; Retroviral Vector; Role; S1-5 protein; Series; Serum; Side; Signal Pathway; Signal Transduction; Solid Neoplasm; Suppressor-Effector T-Lymphocytes; Therapeutic; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinases; Tissues; Topotecan; Treatment Efficacy; Tumor Angiogenesis; Tumor Tissue; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Wild Type Mouse; Xenograft Model; Xenograft procedure; Zinc; angiogenesis; base; beta catenin; cancer cell; cancer stem cell; cell growth; cell motility; cell type; density; fibrosarcoma; fibulin; in vivo; inhibitor/antagonist; interest; mRNA Expression; member; migration; neoplastic cell; novel; overexpression; research study; therapeutic target; tissue procollagenase; tumor; tumor growth; tumor microenvironment; tumor progression; tumor xenograft; tumorigenic; vector control

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.

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）独立于 MMP 的活动。为了实现我们的目标，我们确定了以下具体目标：1）检查 TIMP 在体外改变癌症干细胞（CSC）生长和侵袭潜力方面的作用； 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（骨髓源性抑制细胞（MDSC））募集至原发肿瘤和转移灶的影响。我实验室的一个主要重点是证明许多研究人员观察到的不依赖 MMP 的抗血管生成作用对 TIMP-2 体内抗肿瘤活性的贡献。为此，我们采用逆转录病毒载体强制在人非小细胞肺癌 (NSCLC) 系 A549 中表达 TIMP-2 和 Ala+TIMP-2，然后将这些细胞系用于 nu/nu 和 nu/nu 的肿瘤异种移植实验中。 NOD-SCID 小鼠。尽管这些细胞系在体外基础生长率上没有明显差异，但与空载体对照相比，TIMP-2 (90%) 和 Ala+TIMP-2 (75%) 异种移植物中的肿瘤生长在晚至 40 年后仍显着受到抑制。肿瘤接种后几天。肿瘤生长的抑制伴随着肿瘤微血管密度计数（CD 31+ 或 CD34+）（抗血管生成作用的衡量标准）的统计显着下降，以及肿瘤细胞凋亡的增加（也可能是由于血管生成的抑制）。有点出乎意料的是，我们还观察到表达 TIMP-2 的肿瘤中粘着斑激酶 (FAK) 减少，并且表达 TIMP-2 和 Ala+TIMP-2 的肿瘤细胞中 FAK 磷酸化 (Y397) 显着减少。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，其意义在于：1) FAK 是 AKT 信号传导的上游，并且两者都参与细胞迁移的调节； 2)TIMP-2和Ala+TIMP-2表达减少体外肿瘤细胞迁移。我们之前报道过内皮细胞中 FAK 磷酸化降低，FAK 参与 eNOS 活性的控制。总之，这些实验使用表达野生型（wt）TIMP-2 或金属蛋白酶抑制剂缺陷型 Ala+TIMP-2 的逆转录病毒转导肿瘤细胞，清楚地证明 TIMP-2 的 MMP 独立活性（包括抗血管生成活性）是足以显着影响体内肿瘤生长。我们观察了 TIMP-2 和 Ala+TIMP-2 对 A549 肿瘤异种移植物的影响，从而对这些细胞系和肿瘤组织进行了转录分析。观察到的基因表达变化主要与肿瘤发展减少和转移减少有关。与对照 A549 细胞相比，表达 TIMP-2 或 Ala+TIMP-2 的细胞显示 E-钙粘蛋白表达增加，并且对细胞膜重新分布具有抵抗力表皮生长因子（EGF）刺激后相关的E-钙粘蛋白和β-连环蛋白，提示间充质-上皮转变。其他受到差异调节的感兴趣基因包括含有 EGF 的 fibulin 样细胞外基质蛋白 1（EGFEMP1、fibulin 3），该蛋白在表达 TIMP-2 或 Ala+TIMP-2 的细胞中上调。该蛋白是胶质母细胞瘤的有利预后因素，可抑制血管生成、细胞增殖和 VEGF-A 表达。然而，这些发现需要得到证实，并且对下游基因调控的影响机制仍有待确定。主要成果和成就。我们的基因表达谱的其他数据还揭示了 NSCLC A549 中 ATP 结合盒 (ABC) 转运蛋白基因表达的变化。这带来了一条新的研究途径，旨在了解 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用途。 ABC 蛋白驱动多种底物（包括细胞毒性药物）的细胞流出，并且已知有助于抵抗癌症化疗。 ABC 转运蛋白的活性是各种实体瘤中 CSC 存在的重要指标。 Hoechst 33342 染料流出检测可识别富含 CSC 的肿瘤细胞亚群，称为侧群 (SP)。根据我们的基因表达谱数据，我们推测 TIMP-2 抗肿瘤活性可能部分涉及肺癌细胞模型中 SP 的调节。为此，我们确定了一系列六种非小细胞肺癌 (NSCLC) 细胞系中 SP 分数与内源 TIMP-2 表达水平之间的相关性。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 染料流出测定法测定的 SP 百分比之间存在强烈且高度显着的负相关（R2=0.073，p0.03）。在表达 TIMP-2 的 A549 细胞中，观察到 SP 显着降低，这种降低与 ABCG2、ABCB1 和 AKR1C1 的表达降低有关。功能分析表明，表达 TIMP-2 的 A549 细胞对细胞毒性药物（包括阿霉素和拓扑替康）表现出更高的敏感性。这些发现表明 TIMP-2 疗法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 以及可能的 CSC 水平和功能。我们认为这些研究表明TIMP-2在抑制癌症干细胞、肿瘤细胞和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的潜在用途提供了生物学基础-2 疗法与化疗和/或放射疗法相结合，以增强这些治疗的功效，同时减少潜在的副作用。血管生成和炎症是非小细胞肺癌的重要治疗靶点。我们检查了 TIMP-2 对 NSCLC 肿瘤相关血管生成和炎症的影响，在 TIMP-2 缺陷小鼠中进行了检查，并使用鼠 Lewis 肺癌 (LL) 癌模型与野生型小鼠进行了比较。 TIMP-2缺陷小鼠表现出肿瘤生长加快、血管内皮生长因子-A (VEGF-A)水平显着升高以及肿瘤组织中血管生成标记物表达增强。此外，携带肿瘤的TIMP-2缺陷小鼠表现出炎症介质、核因子-κB和膜联蛋白A1的上调、白细胞介素6的血清水平升高以及肿瘤浸润炎症细胞的显着增加。表型分析显示，表达 VEGFR-1 的脾 MDSC（CD11b+ 和 Gr-1+ 细胞）增加，表明 LL 肿瘤诱导的 TIMP-2 缺陷具有全身效应。相比之下，在使用 A549 细胞的人类 NSCLC 异种移植模型中强制过度表达 TIMP-2 表明 MDSC 向肿瘤的募集显着减少。 MDSC 向肿瘤的募集与抗肿瘤免疫的抑制和肿瘤血管生成的增强有关。这些最新研究结果表明，TIMP-2 对肿瘤和宿主细胞具有多种作用，这些作用结合起来产生可在临床上利用的有效抗肿瘤活性。