Urokinase type plasminogen activator receptor is involved in insulin‐like growth factor‐induced migration of rhabdomyosarcoma cells in vitro

Abstract

Urokinase-type plasminogen activator (uPA) binds to its receptor, uPAR, on the surface of cancer cells, leading to the formation of plasmin. Rhabdomyosarcoma (RMS) cell lines secrete high levels of insulin-like growth factor II (IGF-II), suggesting autocrine IGFs play a major role in the unregulated growth and metastasis of RMS. In vitro, IGF-II and IGF-I increased migration of RD cells to 124+/-9% (P<0.01) and 131+/-8% (P<0.05) of control, respectively. IGF-II-induced migration was abolished by insulin-like growth factor binding protein-6 (IGFBP-6) (P<0.01), a relatively specific inhibitor of IGF-II, and by plasminogen activator inhibitor type 1 (PAI-1) (P<0.05). Aprotinin, a plasmin inhibitor, and mannosamine, which inhibits the synthesis of glycosylphosphatidylinositol (GPI), thereby preventing anchorage of GPI-linked proteins such as uPAR to the cell membrane, also decreased IGF-II- (P<0.05 for both) but not IGF-I-induced migration. [Arg54,Arg55]IGF-II and [Leu27]IGF-II, which preferentially bind to the IGF-I and IGF-II/mannose-6-phosphate receptors (IGF-II/M6PR), respectively, both induced RD cell migration to 146+/-8% (P<0.01) and 120+/-7% (P<0.05) of control, respectively. An anti-uPAR anti-serum reduced IGF-II- and IGF-I-induced migration (P<0.05 for both). An anti-low density lipoprotein-related protein (LRP) anti-serum reduced IGF-I-induced migration (P<0.05). IGF-I and -II both increased specific 125I-single chain uPA (scuPA) binding to RD cells in a dose-dependent manner (P<0.01). These results suggest involvement of the PA/plasmin system in IGF-induced migration and indicate important roles these systems may have in RMS metastasis.

Promotion of Cancer Cell Migration

“…IGFBP-6 does not have an RGD sequence, but it contains a heparin binding domain and so can bind to glycosaminoglycans (36). Interestingly, our previous studies investigating the role of IGFBP-6 in migration using a wound assay showed that IGFBP-6 inhibited migration in an IGF-II-dependent manner (13), which conflicts with the results presented here. However, dual IGF-dependent and IGF-independent actions have previously been showed for other IGFBPs.…”

“…As shown in Fig. 5, IGF-II stimulated cancer cell migration, consistent with previous findings (13,25). Surprisingly, treatment with either wtIGFBP-6 or mIGFBP-6 (1 g/ml) also significantly increased migration in RD cells (Fig.…”

“…Because of its binding preference, IGFBP-6 inhibits IGF-II-induced cell proliferation, differentiation, and survival (12)(13)(14)(15)(16), but it has marginal or no effect on IGF-I actions. A few studies have suggested that IGFBP-6 may have IGF-independent effects, but they have not been definitively demonstrated (17)(18)(19)(20).…”

Rapamycin inhibits cell motility by suppression of mTOR-mediated S6K1 and 4E-BP1 pathways

“…As seen in many other cell lines with dysregulated IGF-I signaling, rhabdomyosarcoma cells are extremely susceptible to rapamycin in growth inhibition (IC 50 ¼ 1-3 nM) (Hosoi et al, 1998). Since IGFs (IGF-I and IGF-II) promote the motility of rhabdomyosarcoma cells (El-Badry et al, 1990;Minniti et al, 1992;Gallicchio et al, 2003), we determined whether rapamycin also inhibits motility of rhabdomyosarcoma cells. Using the wound-healing assay, we found that Rh1 and Rh30 (two rhabdomyo-sarcoma cell lines) migrated under autocrine (serum free) conditions as RD (another rhabdomyosarcoma cell line) cells did (El-Badry et al, 1990).…”

“…IGF-IR controls lung carcinoma cell motility and invasion by coordinately regulating expression and activation of matrix metalloproteinase-2 (Zhang and Brodt, 2002). IGFs are also potent stimulators of rhabdomyosarcoma cell motility (El-Badry et al, 1990;Minniti et al, 1992;Gallicchio et al, 2003). Therefore, IGF-I signaling contributes to metastatic phenotypes in various types of tumors.…”