Members of the platelet-derived growth factor family have long been known as potent stimulators of cell proliferation, migration, and transformation. PDGF ligands are typically secreted by tumor cells or inflammatory cells, which then activate the PDGF receptors on the cell surfaces of surrounding mesenchymal cells, mediating cell-cell communication. PDGFR correlation to poor prognosis in prostate cancer and the therapeutic value of PDGFR-antagonistic chemotherapeutic drugs are now being established. Both α- and β-PDGFR expression levels are upregulated in prostatic intraepithelial neoplasia and adenocarcinoma. Interestingly, even though increased expression of especially the β-PDGFR protein is often found in advanced stages of prostate cancer with phenotypic changes typical to β-PDGFR activation, its cognate ligand PDGF B expression has not been reported in prostate cancer clinical samples (10
). This raises the possibility that PDGF D, a newly discovered ligand for β-PDGFR, may be responsible for β-PDGFR-mediated signal transduction during prostate cancer progression. In phase I and phase II clinical trials, the PDGFR antagonistic drugs ST1571 (also known as Gleevac or imatinib mesylate) and SU101 have shown promise in reducing prostate-specific antigen levels, tumor progression, and resultant bony pain (8
). Similar to the clinical studies, a mouse model of prostate cancer bone metastasis exhibits increased β-PDGFR phosphorylation in prostate tumor xenographs as well as in the surrounding mouse bone endothelial cells and vascular smooth muscle cells (36
). Accordingly, administration of ST1571 either alone or in combination with other cancer therapy drugs results in a decrease of PDGFR phosphorylation, significant reduction of tumor growth and angiogenesis, and reduced destruction of host bone in prostate cancer murine models (16
To better understand the roles of PDGF/β-PDGFR signaling in prostate cancer progression, we have investigated the effects of PDGF D expression, a newly discovered ligand for β-PDGFR, in prostate carcinoma cells. Whereas PDGF AA, PDGF AB, and PDGF BB are processed intracellularly and secreted as active dimers that can readily activate PDGFRs, PDGF CC and PDGF DD are secreted as latent dimers. Thus, the proteolytic cleavage of PDGF DD is an essential step in regulating its activity for β-PDGFR-mediated signal transduction. PDGF D is produced in many secretory organs, including the adrenal and salivary glands, and its expression is often upregulated in many human tumors (19
). Yet, a protease responsible for PDGF D processing in the circulation or at the tumor site was unknown. This present study identifies uPA as an activator of PDGF D in human prostate carcinoma cells. Our data are in good agreement with the recent paper by Fredriksson et al., which focuses on the tPA activation of PDGF CC but also reports that tPA does not activate PDGF DD (9
). The levels of active uPA expressed by PC3 correlate with the efficiency of PDGF D processing, suggesting uPA as a critical regulator of PDGF-D/β-PDGFR signaling for prostate cancer progression.
uPA has been localized to the sites of cell-cell contact, as well as the leading edge of migrating cells, and is well correlated with cancer metastasis (reviewed in references 2
). Increased serum levels of uPA are also associated with prostate cancer development and are elevated even more in patients with metastasis to the bone (24
). The most well-known substrate of uPA is plasminogen, and until recently, it was believed that its pathogenic effects involved the aberrant activation of plasminogen into the active protease plasmin. Plasmin degrades the extracellular matrix of the cell, allowing for the release of growth factors and other matrix degrading enzymes, such as the metalloproteases. However, recent studies suggest that uPA can induce cell migration and proliferation independent of plasmin generation. The plasmin-independent uPA activity is thought to induce cell proliferation/migration by proteolytically activating growth factors (hepatocyte growth factor, vascular endothelial growth factor) or by interacting with an as-yet-unidentified cell surface receptor other than uPAR (25
Our finding further supports the direct role of uPA in the regulation of growth factor signaling critical for cancer progression. Considering potent roles for β-PDGFR signaling in cell growth, migration, transformation, and angiogenesis, uPA activation of PDGF D has a significant implication in coordinating the oncogenic activities of uPA and PDGF D at the tumor site. In fact, our previous study showed that PDGF DD stimulates prostate cancer cell proliferation in an autocrine manner and also induces the cell proliferation and migration of prostate fibroblast cells in a paracrine manner. More importantly, PDGF D drastically enhances tumor cell interactions with the surrounding stroma in a mouse tumor model (38
), demonstrating a potential oncogenic activity of PDGF D in prostate cancer progression. Now, in this study, we show that PDGF D and its activator, uPA, can colocalize in human prostate carcinoma; therefore, uPA and PDGF D interaction can be biologically significant in a pathological context. Interestingly, recent studies showed a synergistic effect of uPA with PDGF BB signaling in inducing human smooth muscle cell migration and proliferation that is reliant on the uPA catalytic domain but independent of plasmin generation (5
). It would be of particular importance to examine whether PDGF DD expressed in smooth muscle cells is also involved in the synergistic signaling loop between uPA and PDGF BB signaling.
Active uPA protein is found in both CM and cell lysate collected from PC3 cells, which correlates with our observations that PDGF D is activated when incubated with either PC3 cells or CM. In contrast, very low levels of active uPA are detected only in LNCaP cell lysate, which correlates with our observation that PDGF D is inefficiently activated in the presence of LNCaP cells, but not in CM collected from LNCaP. Interestingly, when PDGF D signaling is constitutively activated by PC3 cells, active tc-uPA levels significantly increase. Although we do not understand the molecular mechanisms of this feedback signaling loop at present, this may be critical both for uPA localization to the leading edge of migrating tumor cells which are often found in vivo and for uPA activation of PDGF D at or near the cell surface. Recent studies report that localized PDGF BB expression in tumor cells, compared to diffuse PDGF BB expression, has a profound impact on the recruitment of pericytes to the new vasculature developing within the tumor (1
). Our preliminary histological data reveal that PDGF D in normal prostate is preferentially expressed in the mesenchyme of the gland and that the expression of PDGF D is increased and more profuse in prostate carcinoma. Interestingly, colocalization of uPA and PDGF D appears to be preferentially localized to the border of the carcinoma. Therefore, localized activation of PDGF DD by uPA at prostate tumor-stromal cell contacts could contribute to vascularization of the tumor and the migration and invasion of cancer cells.