Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA.
Oncogene. 2013 Jan 17;32(3):327-40. doi: 10.1038/onc.2012.52. Epub 2012 Feb 20.
Matrix metalloproteinase-2 (MMP-2) has pivotal role in the degradation of extracellular matrix, and thereby enhances the invasive, proliferative and metastatic potential in cancer. Knockdown of MMP-2 using MMP-2 small interfering RNA (pM) in human glioma xenograft cell lines 4910 and 5310 decreased cell proliferation compared with mock and pSV (scrambled vector) treatments, as determined by 5-bromo-2'-deoxyuridine incorporation, Ki-67 staining and clonogenic survival assay. Cytokine array and western blotting using tumor-conditioned media displayed modulated secretory levels of various cytokines including granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor-α, angiogenin, vascular endothelial growth factor and PDGF-BB in MMP-2 knockdown cells. Further, cDNA PCR array indicated potential negative regulation of Janus kinase/Stat3 pathway in pM-treated cells. Mechanistically, MMP-2 is involved in complex formation with α5 and β1 integrins and MMP-2 downregulation inhibited α5β1 integrin-mediated Stat3 phosphorylation and nuclear translocation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed inhibited Stat3 DNA-binding activity and recruitment at CyclinD1 and c-Myc promoters in pM-treated cells. In individual experiments, IL-6 or siRNA-insensitive MMP-2 overexpression by pM-FL-A141G counteracted and restored the pM-inhibited Stat3 DNA-binding activity, suggesting IL-6/Stat3 signaling suppression in pM-treated 4910 and 5310 cells. MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters. Activation of α5β1 signaling by Fibronectin adhesion elevated pM-inhibited Stat3 phosphorylation whereas blocking α5β1 abrogated constitutive Stat3 activation. In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments. Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels. Our data indicate the possible role of MMP-2/α5β1 interaction in the regulation of α5β1-mediated IL-6/Stat3 signaling activation and signifies the therapeutic potential of blocking MMP-2/α5β1 interaction in glioma treatment.
基质金属蛋白酶-2（MMP-2）在细胞外基质的降解中起着关键作用，从而增强了癌症的侵袭性、增殖性和转移性。在人神经胶质瘤异种细胞系 4910 和 5310 中，使用 MMP-2 小干扰 RNA（pM）敲低 MMP-2，与对照和 pSV（乱序载体）处理相比，通过 5-溴-2'-脱氧尿苷掺入、Ki-67 染色和集落形成存活测定，细胞增殖减少。使用肿瘤条件培养基进行细胞因子阵列和 Western blot 显示，MMP-2 敲低细胞中各种细胞因子的分泌水平发生了调节，包括粒细胞-巨噬细胞集落刺激因子、白细胞介素-6（IL-6）、IL-8、IL-10、肿瘤坏死因子-α、血管生成素、血管内皮生长因子和 PDGF-BB。此外，cDNA PCR 阵列表明，Janus 激酶/Stat3 通路在 pM 处理的细胞中可能受到负调控。在机制上，MMP-2 与 α5 和 β1 整合素形成复合物，MMP-2 的下调抑制了 α5β1 整合素介导的 Stat3 磷酸化和核转位。电泳迁移率变动分析和染色质免疫沉淀分析显示，在 pM 处理的细胞中，Stat3 的 DNA 结合活性和募集在 CyclinD1 和 c-Myc 启动子上受到抑制。在单独的实验中，pM-FL-A141G 通过 IL-6 或 siRNA 不敏感的 MMP-2 过表达逆转并恢复了 pM 抑制的 Stat3 DNA 结合活性，表明 pM 处理的 4910 和 5310 细胞中抑制了 IL-6/Stat3 信号转导。人重组 MMP-2 处理增强了 MMP-2/α5β1 的结合，导致 Stat3 DNA 结合活性和募集在 CyclinD1 和 c-Myc 启动子上增加。纤维连接蛋白黏附激活 α5β1 信号转导导致 pM 抑制的 Stat3 磷酸化升高，而阻断 α5β1 则消除了组成性 Stat3 激活。在原位肿瘤模型的体内实验中，与对照或 pSV 处理相比，pM 处理的肿瘤体积减小。肿瘤切片的免疫荧光研究证实了我们的体外发现，即 MMP-2/α5β1 的高表达和共定位，pM 处理后降低，同时 IL-6、磷酸化 Stat3、CyclinD1、c-Myc、Ki-67 和 PCNA 的表达水平显著降低。我们的数据表明 MMP-2/α5β1 相互作用可能在调节 α5β1 介导的 IL-6/Stat3 信号转导激活中发挥作用，并表明阻断 MMP-2/α5β1 相互作用在神经胶质瘤治疗中的治疗潜力。
