Sunitinib induces PTEN expression and inhibits PDGFR signaling and migration of medulloblastoma cells

Abstract

We previously showed that inhibition of the platelet-derived growth factor receptor (PDGFR) blocks the survival and migration of medulloblastoma cells. Identification of in vitro PDGFR-targeting pharmacologic agents that are suitable for preclinical testing in medulloblastoma models in vivo will be critical for efficiently translating these agents to clinical investigation in children with medulloblastoma. In this study, we investigated whether the multi-tyrosine kinase inhibitor sunitinib, effectively inhibits PDGFR signaling required for medulloblastoma cell migration. Daoy and D556 human medulloblastoma cells pre-treated for 1 h with 0.2 μM sunitinib demonstrated induction of PTEN expression and significant inhibition of PDGFR signaling activity and transactivation of EGFR, in a RAS-independent manner, in response to PDGF-BB stimulation. Sunitinib pre-treatment markedly reduced medulloblastoma cell migration in response to both PDGF-BB and 10% serum at 4 and 24 h after treatment. Pre-treatment with sunitinib for 1 h also resulted in detachment and decreased viability of D556, but not Daoy, cells and only after 48 h following treatment. However, sunitinib did not induce apoptosis in either cell line at any time point, indicating that the anti-migratory effects of sunitinib were not due to impeding cell survival. Sunitinib similarly inhibited PDGFR signaling and migration of primary murine Smo/Smo medulloblastoma cells, suggesting that the Smo/Smo mouse is an appropriate model for preclinical testing of sunitinib. These results indicate that sunitinib may be an important pharmacologic agent for the treatment of invasive medulloblastoma, particularly given evidence of its ability to cross the blood-brain barrier to target tumor cells, and thus warrants further in vivo testing for confirmation of efficacy.

Fig. 1

Sunitinib treatment inhibits PDGFR cell signaling in a RAS-independent manner. a Representative western blot of whole cell lysates demonstrating dose-dependent inhibition of PDGFRB, PI3K, AKT and ERK phosphorylation in response to PDGF-BB (10 ng/ml) following increasing concentrations of 1 h sunitinib pre-treatment of Daoy cells. b Densitometric analysis of multiple western blots indicating significant reduction (* P < 0.05) in the phosphorylation levels of PDGFRB (upper panel) and PI3K (lower panel) in response to PDGF-BB (10 ng/ml) in Daoy and D556 cells pre-treated for 1 h with 0.2 μM sunitinib. c Representative western blot of Daoy cell lysates (upper panel) and results of multiple pull-down assays in Daoy and D556 cells (lower panel) demonstrating no change in total RAS protein or RAS-GTP activity levels, respectively, in response to PDGF-BB (10 ng/ml) following sunitinib (0.2 μM) pre-treatment for 1 h. d Representative western blot demonstrating inhibition of PDGFRB, AKT and ERK phosphorylation in response to 10% serum following 1 h sunitinib pre-treatment (0.2 μM) of Daoy cells

Fig. 2

Sunitinib treatment increases PTEN expression in PDGF-treated medulloblastoma cells. Representative western blot of Daoy cell lysates (upper panel) demonstrating a relative reduction in the level of total PTEN in response to PDGF-BB (10 ng/ml) and a relative increase in the expression of total PTEN in response to PDGF-BB when cells are pre-treated for 1 h with 0.2 μM sunitinib. Densitometry analysis of multiple western blots of Daoy cell lysates (lower panel) demonstrates a significant increase (* P < 0.05) in total PTEN expression in PDGF-stimulated Daoy cells pre-treated with sunitinib compared to untreated, PDGF-stimulated control cells

Fig. 3

Sunitinib treatment inhibits PDGF-mediated EGFR transactivation in medulloblastoma cells. a Representative western blot of Daoy whole cell lysates demonstrating that 1 h pre-treatment with 0.2 μM sunitinib abolishes PDGF-BB (10 ng/ml)-mediated transactivation (phosphorylation) of EGFR (left panel) but does not alter EGF (50 ng/ml)-mediated activation of EGFR. b Densitometric analysis of multiple western blots of Daoy (upper panel) and D556 (lower panel) whole cell lysates demonstrating that sunitinib pre-treatment completely abrogates (* P < 0.05) PDGF-BB mediated, but not EGF-mediated, phosphorylation of EGFR

Fig. 4

Sunitinib treatment inhibits medulloblastoma cell migration. a Results of multiple Boyden chamber 4 h migration assays demonstrating that the relative fold-change in PDGF-BB-mediated (20 ng/ml) migration over basal migration is significantly inhibited by 1 h sunitinib (0.2 μM) pre-treatment of Daoy and D556 cells (* P < 0.01 and * P < 0.05, respectively). b Representative photomicrographs demonstrating that in longer term 24 h wound healing migration assays, PDGF-BB (20 ng/ml)-mediated cell migration of Daoy (upper panel) and D556 (lower panel) cells is inhibited after 1 h sunitinib (0.2 μM) pre-treatment. c Results of multiple wound healing migration assays demonstrating that the calculated area (micron²) of wound closure by sunitinib-treated cells is significantly reduced (* P < 0.005) in response to both 20 ng/ml PDGF-BB (upper panel) and 10% serum (lower panel)

Fig. 5

Sunitinib induces cell detachment and inhibits viability of D556 cells at 48 h. a Representative photomicrograph demonstrating dose-dependent detachment of D556 cells at 48 h in response to increasing concentrations of 1 h sunitinib pre-treatment. b Results of multiple cell counting assays demonstrating significant inhibition (* P < 0.05) of D556, but not Daoy, cell viability in response to PDGF-BB (20 ng/ml) 48–72 h following 1 h pre-treatment with 0.2 μM sunitinib

Fig. 6

Sunitinib treatment inhibits PS125 primary murine medulloblastoma cell migration and PDGFR signaling. a Representative photomicrographs demonstrating that in a long term 24 h wound healing migration assays, PDGF-BB (20 ng/ml)-mediated cell migration of PS125 cells is inhibited after 1 h sunitinib (0.2 μM) pre-treatment. Results of multiple wound healing migration assays demonstrating that the calculated area (micron²) of wound closure by sunitinib-treated cells is significantly reduced (* P < 0.005) in response to both 20 ng/ml PDGF-BB and 10% serum. b Representative western blot of whole cell lysates demonstrating inhibition of PDGFRB, SHP2, AKT and ERK phosphorylation and increase in PTEN expression with no change in total RAS expression in response to PDGF-BB (10 ng/ml) following 1 h sunitinib pre-treatment of PS125 cells

Fig. 7

Sunitinib inhibits viability of PS125 primary murine medulloblastoma cells. Graph representing the average rate of proliferation in PS125 mouse medulloblastoma cells treated with increasing doses of sunitinib (0.1, 0.2, 0.4, and 0.8 μM, however, only 0.1 and 0.2 μM doses were plotted as there was no significant difference in response between the 0.2 μM dose and the higher doses) for 1, 2, 3, 4, 5, and 8 days compared to untreated controls. There was no significant change in cell viability as determined by the XTT colorimetric absorbance in all treatment groups at 24 and 48 h post treatment compared to untreated controls. However, by 72 h post treatment at all concentrations of drug tested led to a significant (P < 0.05) reduction in the viability of the treated cells compared to the untreated controls. Results represent the mean ± SE of mean of multiple experiments