Leupold Jörg H, Asangani Irfan, Maurer Gabriele D, Lengyel Ernst, Post Stefan, Allgayer Heike
Department of Experimental Surgery Mannheim Faculty, University of Heidelberg, Heidelberg, Germany.
Mol Cancer Res. 2007 May;5(5):485-96. doi: 10.1158/1541-7786.MCR-06-0211.
The urokinase receptor [urokinase plasminogen activator receptor (u-PAR)] promotes invasion and metastasis and is associated with poor patient survival. Recently, it was shown that Src induces u-PAR gene expression via Sp1 bound to the u-PAR promoter region -152/-135. However, u-PAR is regulated by diverse promoter motifs, among them being an essential activator protein-1 (AP-1) motif at -190/-171. Moreover, an in vivo relevance of Src-induced transcriptional regulators of u-PAR-mediated invasion, in particular intravasation, and a relevance in resected patient tumors have not sufficiently been shown. The present study was conducted (a) to investigate if, in particular, AP-1-related transcriptional mediators are required for Src-induced u-PAR-gene expression, (b) to show in vivo relevance of AP-1-mediated Src-induced u-PAR gene expression for invasion/intravasation and for resected tissues from colorectal cancer patients. Src stimulation of the u-PAR promoter deleted for AP-1 region -190/-171 was reduced as compared with the wild-type promoter in cultured colon cancer cells. In gelshifts/chromatin immunoprecipitation, Src-transfected SW480 cells showed an increase of phospho-c-Jun, in addition to JunD and Fra-1, bound to region -190/-171. Src-transfected cells showed a significant increase in c-Jun phosphorylated at Ser(73) and also Ser(63), which was paralleled by increased phospho-c-jun-NH(2)-kinase. Significant decreases of invasion/in vivo intravasation (chorionallantoic membrane model) were observed in Src-overexpressing cells treated with Src inhibitors, u-PAR-small interfering RNA, and dominant negative c-Jun (TAM67). In resected tissues of 20 colorectal cancer patients, a significant correlation between Src activity, AP-1 complexes bound to u-PAR region -190/-171, and advanced pN stage were observed. These data suggest that Src-induced u-PAR gene expression and invasion/intravasation in vivo is also mediated via AP-1 region -190/-171, especially bound with c-Jun phosphorylated at Ser(73/63), and that this pathway is biologically relevant for colorectal cancer patients, suggesting therapeutic potential.
尿激酶受体[尿激酶型纤溶酶原激活物受体（u-PAR）]可促进侵袭和转移，且与患者的不良预后相关。最近有研究表明，Src通过与u-PAR启动子区域-152/-135结合的Sp1诱导u-PAR基因表达。然而，u-PAR受多种启动子基序调控，其中位于-190/-171的激活蛋白-1（AP-1）基序至关重要。此外，Src诱导的u-PAR介导的侵袭（尤其是内渗）转录调节因子在体内的相关性以及在切除的患者肿瘤中的相关性尚未得到充分证实。本研究旨在：（a）调查Src诱导的u-PAR基因表达是否特别需要AP-1相关转录介质；（b）证明AP-1介导的Src诱导的u-PAR基因表达在侵袭/内渗以及结直肠癌患者切除组织中的体内相关性。与野生型启动子相比，在培养的结肠癌细胞中，缺失AP-1区域-190/-171的u-PAR启动子受Src刺激的程度降低。在凝胶迁移/染色质免疫沉淀实验中，转染Src的SW480细胞显示，除了JunD和Fra-1外，与区域-190/-171结合的磷酸化c-Jun增加。转染Src的细胞中，Ser(73)以及Ser(63)位点磷酸化的c-Jun显著增加，同时磷酸化c-jun-NH(2)-激酶也增加。在用Src抑制剂、u-PAR小干扰RNA和显性负性c-Jun（TAM67）处理的过表达Src的细胞中，观察到侵袭/体内内渗（绒毛尿囊膜模型）显著降低。在20例结直肠癌患者的切除组织中，观察到Src活性、与u-PAR区域-190/-171结合的AP-1复合物以及晚期pN分期之间存在显著相关性。这些数据表明，Src诱导的u-PAR基因表达和体内侵袭/内渗也通过AP-1区域-190/-171介导，尤其是与Ser(73/63)位点磷酸化的c-Jun结合，并且该途径对结直肠癌患者具有生物学相关性，提示其具有治疗潜力。
