Urokinase Receptor and Fibronectin Regulate the ERK(MAPK) to p38(MAPK) Activity Ratios That Determine Carcinoma Cell Proliferation or Dormancy In Vivo

We discovered that a shift between the state of tumorigenicity and dormancy in human carcinoma (HEp3) is attained through regulation of the balance between two classical mitogen-activated protein kinase (MAPK)-signaling pathways, the mitogenic extracellular regulated kinase (ERK) and the apoptotic/growth suppressive stress-activated protein kinase 2 (p38(MAPK)), and that urokinase plasminogen activator receptor (uPAR) is an important regulator of these events. This is a novel function for uPAR whereby, when expressed at high level, it enters into frequent, activating interactions with the α5β1-integrin, which facilitates the formation of insoluble fibronectin (FN) fibrils. Activation of α5β1-integrin by uPAR generates persistently high level of active ERK necessary for tumor growth in vivo. Our results show that ERK activation is generated through a convergence of two pathways: a positive signal through uPAR-activated α5β1, which activates ERK, and a signal generated by the presence of FN fibrils that suppresses p38 activity. When fibrils are removed or their assembly is blocked, p38 activity increases. Low uPAR derivatives of HEp3 cells, which are growth arrested (dormant) in vivo, have a high p38/ERK activity ratio, but in spite of a similar level of α5β1-integrin, they do not assemble FN fibrils. However, when p38 activity is inhibited by pharmacological (SB203580) or genetic (dominant negative-p38) approaches, their ERK becomes activated, uPAR is overexpressed, α5β1-integrins are activated, and dormancy is interrupted. Restoration of these properties in dormant cells can be mimicked by a direct re-expression of uPAR through transfection with a uPAR-coding plasmid. We conclude that overexpression of uPAR and its interaction with the integrin are responsible for generating two feedback loops; one increases the ERK activity that feeds back by increasing the expression of uPAR. The second loop, through the presence of FN fibrils, suppresses p38 activity, further increasing ERK activity. Together these results indicate that uPAR and its interaction with the integrin should be considered important targets for induction of tumor dormancy.

我们发现，人癌细胞系（HEp3）的致瘤性与休眠状态之间的转变，可通过调控两类经典丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号通路的平衡实现：即促有丝分裂的细胞外调节蛋白激酶（extracellular regulated kinase, ERK）通路，与促凋亡/生长抑制的应激活化蛋白激酶2（stress-activated protein kinase 2, p38(MAPK)）通路；且尿激酶型纤溶酶原激活物受体（urokinase plasminogen activator receptor, uPAR）是上述过程的重要调控因子。
这是uPAR的一项全新功能：当其高表达时，会频繁与α5β1整合素（α5β1-integrin）发生活化性相互作用，进而促进不溶性纤连蛋白（fibronectin, FN）纤维的形成。uPAR介导的α5β1整合素活化，可持续维持高水平的活化ERK，这正是体内肿瘤生长所必需的条件。
我们的研究结果显示，ERK的活化源于两条通路的交汇：其一为uPAR活化α5β1整合素所产生的正向信号，该信号可激活ERK；其二为纤连蛋白纤维存在时所产生的信号，该信号会抑制p38的活性。当纤连蛋白纤维被移除或其组装被阻断时，p38的活性会升高。
HEp3细胞的低uPAR表达株在体内处于生长停滞（休眠）状态，其p38/ERK活性比值较高；尽管这类细胞的α5β1整合素表达水平与高uPAR表达株相似，但它们无法组装纤连蛋白纤维。不过，当通过药理学手段（SB203580）或遗传学手段（显性负性p38, dominant negative-p38）抑制p38活性后，这些细胞的ERK会被激活，uPAR表达会上调，α5β1整合素也会被活化，且细胞的休眠状态被打破。
通过转染编码uPAR的质粒（uPAR-coding plasmid）直接恢复uPAR的表达，可重现休眠细胞的上述特性恢复过程。我们据此得出结论：uPAR的过表达及其与整合素的相互作用，可形成两条反馈环路：第一条环路通过升高ERK活性，反过来进一步上调uPAR的表达；第二条环路则通过纤连蛋白纤维的存在抑制p38活性，进一步增强ERK的活性。
综上，本研究结果表明，uPAR及其与整合素的相互作用，可作为诱导肿瘤休眠的重要靶点。