Abstract
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.
Highlights
The complex process of tumor invasion and metastasis requires a network of different proteases
In the present paper, we provide evidence that Src induces urokinase plasminogen activator (u-PA) receptor (u-PAR) promoter activity via an activator protein-1 (AP-1) consensus motif (À190/ À171) bound especially with phospho – c-Jun, in cultured colon cancer cells, this being paralleled by specific JNK activation and phosphorylation of c-Jun at Ser73
This adds new knowledge on molecular mechanisms by which Src regulates this important invasion-related gene because in previous studies, we implicated Sp1 bound to the u-PAR promoter region À152/À135 as a transcription factor mediating Srcinduced u-PAR regulation
Summary
The complex process of tumor invasion and metastasis requires a network of different proteases. U-PA leads to an activation of plasminogen and further proteases such as matrix metalloproteinases-2 (MMP-2) and MMP-9, and to the degradation of main components of the extracellular matrix such as fibrin, fibronectin, and laminin. This effect of u-PA is potentiated by binding to its specific cell receptor u-PAR, which has been shown to be overexpressed in diverse human tumors, such as gastric, colorectal, breast, and other cancers [3, 4]. It has been shown that u-PAR gene expression can be induced by diverse transcription factors [e.g., Sp1, activator protein-1 (AP-1), AP-2, or nuclear factor-nB (NFnB)] bound to different promoter motifs, following stimulation with different growth factors or cytokines [6]. We and others have characterized two main cis-elements of the u-PAR
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