Mi Zhiyong, Guo Hongtao, Wai Philip Y, Gao Chengjiang, Kuo Paul C
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
Carcinogenesis. 2006 Jun;27(6):1134-45. doi: 10.1093/carcin/bgi352. Epub 2006 Feb 12.
Metastasis-supporting physiological alterations are regulated by cell signaling molecules, which target signal transduction pathways and gene expression. Osteopontin (OPN) overexpression may represent a key molecular event in cancer metastasis. In this study, using metastatic 4T1 and non-metastatic 4T07 murine mammary cancer cell lines, we demonstrate that 4T1 cells exhibit significantly increased OPN, integrin-linked kinase (ILK), matrix metalloproteinase-2 (MMP-2) and urokinase-type plasminogen activator (uPA) expression in contrast to 4T07 cells. Blockade of OPN binding to 4T1 cell-surface integrins by the competitive ligand inhibitor, RGD, or a blocking antibody to alphavbeta3 integrin decreases OPN, ILK, MMP-2 and uPA expression. Conversely, exposure of 4T07 cells to exogenous OPN increases ILK, MMP-2 and uPA levels. Further experiments demonstrate that OPN-alphavbeta3 integrin binding in 4T1 with subsequent activation of ILK results in binding of AP-1 to MMP-2 and uPA promoter and increased in vitro promoter activation, as measured by transient transfection assays using MMP-2 and uPA promoter-reporter constructs. AP-1 activity is ablated by co-transfection of DN-ILK or exposure to RGD. Finally, functional correlative assays demonstrate that inhibition of ILK activity or RGD-mediated blockade of alphavbeta3 integrin binding significantly inhibits in vitro invasion, migration and invasion properties of 4T1 cells. In addition, uPA and MMP-2 have overlapping contributions to 4T1 migration and invasion characteristics. However, OPN and ILK activities contribute to 4T1 adhesion activities via mechanisms that are independent of uPA and MMP-2. Our results indicate that binding of an RGD-bearing ligand, such as OPN, to integrin receptors in metastatic 4T1 cells transcriptionally mediates MMP-2, uPA and OPN expression through ILK-dependent AP-1 activity and significantly increases in vitro functional correlates of metastasis. In 4T1 murine mammary cancer cells, we conclude that OPN mediates metastatic behavior, in part, through upregulation of MMP-2 and uPA protein expression.
支持转移的生理改变受细胞信号分子调控，这些分子作用于信号转导通路和基因表达。骨桥蛋白（OPN）过表达可能是癌症转移中的关键分子事件。在本研究中，我们使用转移性4T1和非转移性4T07小鼠乳腺癌细胞系，证明与4T07细胞相比，4T1细胞中OPN、整合素连接激酶（ILK）、基质金属蛋白酶-2（MMP-2）和尿激酶型纤溶酶原激活剂（uPA）的表达显著增加。竞争性配体抑制剂RGD或抗αvβ3整合素阻断抗体阻断OPN与4T1细胞表面整合素的结合，可降低OPN、ILK、MMP-2和uPA的表达。相反，将4T07细胞暴露于外源性OPN可增加ILK、MMP-2和uPA水平。进一步实验表明，4T1细胞中OPN与αvβ3整合素结合并随后激活ILK，导致AP-1与MMP-2和uPA启动子结合，并增加体外启动子活性，这通过使用MMP-2和uPA启动子-报告基因构建体的瞬时转染实验来测量。通过共转染DN-ILK或暴露于RGD可消除AP-1活性。最后，功能相关性分析表明，抑制ILK活性或RGD介导的αvβ3整合素结合阻断可显著抑制4T1细胞的体外侵袭、迁移和浸润特性。此外，uPA和MMP-2对4T1迁移和浸润特性有重叠作用。然而，OPN和ILK活性通过独立于uPA和MMP-2的机制促进4T1黏附活性。我们的结果表明，携带RGD的配体（如OPN）与转移性4T1细胞中的整合素受体结合，通过依赖ILK的AP-1活性转录介导MMP-2、uPA和OPN的表达，并显著增加转移的体外功能相关性。在4T1小鼠乳腺癌细胞中，我们得出结论，OPN部分通过上调MMP-2和uPA蛋白表达介导转移行为。
