Imatinib blocks migration and invasion of medulloblastoma cells by concurrently inhibiting activation of platelet-derived growth factor receptor and transactivation of epidermal growth factor receptor

Abstract

Platelet-derived growth factor (PDGF) receptor (PDGFR) expression correlates with metastatic medulloblastoma. PDGF stimulation of medulloblastoma cells phosphorylates extracellular signal-regulated kinase (ERK) and promotes migration. We sought to determine whether blocking PDGFR activity effectively inhibits signaling required for medulloblastoma cell migration and invasion. DAOY and D556 human medulloblastoma cells were treated with imatinib mesylate (Gleevec), a PDGFR tyrosine kinase inhibitor, or transfected with small interfering RNA (siRNA) to PDGFRB to test the effects of blocking PDGFR phosphorylation and expression, respectively. PDGFR cell signaling, migration, invasion, survival, and proliferation following PDGF-BB stimulation, with and without PDGFR inhibition, were measured. PDGF-BB treatment of cells increased PDGFRB, Akt and ERK phosphorylation, and transactivated epidermal growth factor receptor (EGFR), which correlated with enhanced migration, survival, and proliferation. Imatinib (1 μmol/L) treatment of DAOY and D556 cells inhibited PDGF-BB- and serum-mediated migration and invasion at 24 and 48 h, respectively, and concomitantly inhibited PDGF-BB activation of PDGFRB, Akt, and ERK but increased PTEN expression and activity. Imatinib treatment also induced DAOY cell apoptosis at 72 h and inhibited DAOY and D556 cell proliferation at 48 h. siRNA silencing of PDGFRB similarly inhibited signaling, migration, and survival and both siRNA and imatinib treatment inhibited PDGF-BB-mediated EGFR transactivation, indicating that the effects of imatinib treatment are specific to PDGFRB target inhibition. These results indicate that PDGFRB tyrosine kinase activity is critical for migration and invasion of medulloblastoma cells possibly by transactivating EGFR; thus, imatinib may represent an important novel therapeutic agent for the treatment of medulloblastoma.

Figure 1. Imatinib inhibits medulloblastoma cell Akt and Erk1/2 activation in a Ras-independent manner

Serum-starved medulloblastoma cells were first treated with increasing concentrations of imatinib or vehicle control (‘0’) for 1 h before stimulation with PDFG-BB (10 ng/ml). After an optimal dose of imatinib was identified, cells were treated with (‘+’) or without (‘−’) imatinib and PDGF-BB. Changes in target proteins and phosphorylation were detected by Western blot. A, Representative Western blot of imatinib-treated DAOY cells shows reduced activation (phosphorylation) of PDGFRB and the downstream signal transduction effectors Akt and Erk in a dose-dependent manner compared to control cells stimulated with PDGF-BB. B, Densitometry of multiple corresponding Western blots confirms significant inhibition (P < 0.005) of Akt and Erk phosphorylation in PDGF-BB stimulated DAOY cells treated with 1 uM imatinib. C, Representative Western blot of total Ras in DAOY cells treated with increasing concentrations of imatinib and D, Densitometry of multiple corresponding Western blots for GTP-Ras (DAOY and D556) following 1 uM imatinib treatment shows that Ras expression and activity is not significantly changed following imatinib treatment and PDGF-BB stimulation. *, Indicates statistically significant decrease in PDGF-BB-mediated Akt and Erk1/2 phosphorylation after imatinib treatment compared to untreated control cells. Results represent the mean ± the standard error of the mean of multiple experiments.

Figure 2. Imatinib treatment increases PTEN expression and activity in DAOY medulloblastoma cells stimulated with PDGF-BB

Serum-deprived DAOY cells were treated with (‘+’) 1 uM imatinib or vehicle control (‘−’) for 1 h before stimulation with (‘+’) or without (‘−’) PDFG-BB (10 ng/ml). Changes in PTEN protein and phosphorylation were detected by Western blot. Representative Western blots show A, PTEN expression and B, PTEN phosphorylation is increased in imatinib-treated cells stimulated with PDGF-BB compared to untreated control cells. C and D, Densitometric analysis of multiple corresponding Western blots confirms significantly (indicated by *) higher levels (P < 0.05) of total and phosphorylated PTEN, respectively, in imatinib-treated cells stimulated with PDGF-BB compared to untreated control cells. Results represent the mean ± the standard error of the mean of multiple experiments.

Figure 3. Imatinib abolishes PDGF-BB-mediated EGFR transactivation in medulloblastoima cells in a dose-dependent manner

Serum-starved DAOY cells were treated with increasing doses of imatinib or vehicle control (‘0’) for 1 h before stimulation with (‘+’) or without (‘−’) PDFG-BB (10 ng/ml) or EGF (50 ng/ml). Changes in target proteins and phosphorylation were detected by Western blot and co-immunpreciptation was used to detect PDGFR-EGFR heterodimers. Representative Western blots show that A, Imatinib treatment inhibits PDGF-mediated transactivation of EGFR in a dose-dependent manner, but has no effect on EGF-mediated activation of EGFR and B, Imatinib (1 uM) treatment of PDGF-stimulated DAOY cells abolished the PDGFRB-phospho(p)-EGFR association compared to untreated (‘−’) control cells as detected by co-immunoprecipitation (blot was cropped to improve clarity; full-length blot is provided in supplemental Fig. 3A). C and D, Densitometry of multiple corresponding Western blots confirms that DAOY and D556 cells, respectively, have significantly reduced levels of PDGF-BB-mediated EGFR transactivation (P < 0.0005) following 1 uM imatinib treatment compared to untreated (‘−’) control cells. #, Indicates statistically significant increase in EGFR activation in cells stimulated with PDGF-BB compared to unstimulated cells; * indicates statistically significant decrease in PDGF-mediated EGFR transactivation in imatinib-treated cells compared to untreated control cells. Results represent the mean ± the standard error of the mean of multiple experiments.

Figure 4. PDGFRB silencing by siRNA significantly reduces Akt and Erk1/2 activation in a Ras-independent manner, maintains PTEN and abolishes PDGF-BB-mediated EGFR trans-activation in medulloblastoma cells

Medulloblastoma cells transfected with either PDGFRB siRNA (designated by ‘+’) or negative control siRNA (designated by ‘−’) were stimulated with (‘+’) or without (‘−’) PDGF-BB (10 ng/ml) or EGF (50 ng/ml). Changes in target proteins and phosphorylation were detected by Western blot. DAOY and D556 cells transfected with PDGFRB siRNA showed significant knock-down of PDGFRB protein expression and activation (phosphorylation) (supplemental Fig. 4); Representative Western blots (upper panels) and densitometry of multiple corresponding Western blots (lower panels) show that A, DAOY cells with PDGFRB siRNA knock-down have significantly reduced levels of PDGF-activated Erk (P < 0.0005) and Akt (P < 0.005); B, Total Ras and GTP-Ras (active) expression levels were not significantly altered by PDGFRB siRNA knock-down (P > 0.05) in DAOY and D556 cells stimulated with PDGF-BB compared to control cells (‘−’); C, DAOY cells with PDGFRB siRNA knock-down maintained basal levels of total and phosphorylated PTEN, which were significantly higher compared to control siRNA transfected cells stimulated with PDGF-BB (P < 0.05) and D, DAOY (upper and lower panels) and D556 (lower panel) cells have significantly lower levels of PDGF-BB-mediated EGFR transactivation (P < 0.01) compared to negative control cells (‘−’). Results represent the mean ± the standard error of the mean of multiple experiments. *, Indicates statistically significant lower levels of PDGF-BB-mediated activation of Akt, Erk, and EGFR trans-activation, but higher levels of total and activated PTEN, in PDGFRB siRNA knock-down cells compared to negative control cells. #, Indicates statistically significant increase in PDGF-mediated EGFR transactivation in cells stimulated with PDGF-BB compared to unstimulated controls. Results represent the mean ± the standard error of the mean of multiple experiments.

Figure 4. PDGFRB silencing by siRNA significantly reduces Akt and Erk1/2 activation in a Ras-independent manner, maintains PTEN and abolishes PDGF-BB-mediated EGFR trans-activation in medulloblastoma cells

Medulloblastoma cells transfected with either PDGFRB siRNA (designated by ‘+’) or negative control siRNA (designated by ‘−’) were stimulated with (‘+’) or without (‘−’) PDGF-BB (10 ng/ml) or EGF (50 ng/ml). Changes in target proteins and phosphorylation were detected by Western blot. DAOY and D556 cells transfected with PDGFRB siRNA showed significant knock-down of PDGFRB protein expression and activation (phosphorylation) (supplemental Fig. 4); Representative Western blots (upper panels) and densitometry of multiple corresponding Western blots (lower panels) show that A, DAOY cells with PDGFRB siRNA knock-down have significantly reduced levels of PDGF-activated Erk (P < 0.0005) and Akt (P < 0.005); B, Total Ras and GTP-Ras (active) expression levels were not significantly altered by PDGFRB siRNA knock-down (P > 0.05) in DAOY and D556 cells stimulated with PDGF-BB compared to control cells (‘−’); C, DAOY cells with PDGFRB siRNA knock-down maintained basal levels of total and phosphorylated PTEN, which were significantly higher compared to control siRNA transfected cells stimulated with PDGF-BB (P < 0.05) and D, DAOY (upper and lower panels) and D556 (lower panel) cells have significantly lower levels of PDGF-BB-mediated EGFR transactivation (P < 0.01) compared to negative control cells (‘−’). Results represent the mean ± the standard error of the mean of multiple experiments. *, Indicates statistically significant lower levels of PDGF-BB-mediated activation of Akt, Erk, and EGFR trans-activation, but higher levels of total and activated PTEN, in PDGFRB siRNA knock-down cells compared to negative control cells. #, Indicates statistically significant increase in PDGF-mediated EGFR transactivation in cells stimulated with PDGF-BB compared to unstimulated controls. Results represent the mean ± the standard error of the mean of multiple experiments.

Figure 5. Imatinib inhibits medulloblastoma cell migration and invasion

For short-term assessment of migration, serum-starved medulloblastoma cells were treated with either 1 uM imatinib (‘+’) or vehicle control (‘−’) for 1 h and then seeded into the upper well of a Transwell chamber with (‘+’) or without (‘−’) PDGF-BB (10 ng/ml)-containing media in the lower chamber as chemo-attractant and cell migration quantified after 4 h incubation. For longer-term assessment of migration, cells grown in culture dish were scraped, washed and imatinib-containing media replenished with PDGF-BB (10 ng/ml) or 10% FCS and then cells were allowed to fill in the wound over 24 h. Photomicrographs of the wound were taken at time of scraping and 24 h after. Invasion over 48 h was measured in a similar fashion to Transwell migration except ECMatrix chambers were used. A, Imatinib significantly inhibited PDGF-BB-mediated short-term cell migration of DAOY cells, and nearly significant for D556 (P = 0.05; P = 0.06, respectively), compared to control cells. B, Representative photomicrographs (10X, upper panel) and sum of calculated areas of corresponding wound closure from photomicrographs (lower panel) show that imatinib significantly inhibited PDGF-BB-mediated longer-term migration of DAOY and D556 cells (P < 0.01, each; lower-left panel) and significantly inhibited serum-mediated migration of DAOY and D556 cells compared to control cells (P < 0.005, each; lower-right panel). C, DAOY and D556 cells transfected with PDGFRB siRNA (+) showed significantly inhibited longer-term PDGF-mediated migration compared to cells transfected with negative control siRNA (−) (P < 0.05, each). D, Treatment with imatinib significantly inhibited the invasion of DAOY and D556 cells compared to negative control treated cells (P < 0.001, each). Results represent the mean ± the standard error of the mean of multiple experiments. *, Indicates a statistically significant decrease in cell migration or invasion of imatinib-treated cells compared to untreated control cells.

Figure 5. Imatinib inhibits medulloblastoma cell migration and invasion

For short-term assessment of migration, serum-starved medulloblastoma cells were treated with either 1 uM imatinib (‘+’) or vehicle control (‘−’) for 1 h and then seeded into the upper well of a Transwell chamber with (‘+’) or without (‘−’) PDGF-BB (10 ng/ml)-containing media in the lower chamber as chemo-attractant and cell migration quantified after 4 h incubation. For longer-term assessment of migration, cells grown in culture dish were scraped, washed and imatinib-containing media replenished with PDGF-BB (10 ng/ml) or 10% FCS and then cells were allowed to fill in the wound over 24 h. Photomicrographs of the wound were taken at time of scraping and 24 h after. Invasion over 48 h was measured in a similar fashion to Transwell migration except ECMatrix chambers were used. A, Imatinib significantly inhibited PDGF-BB-mediated short-term cell migration of DAOY cells, and nearly significant for D556 (P = 0.05; P = 0.06, respectively), compared to control cells. B, Representative photomicrographs (10X, upper panel) and sum of calculated areas of corresponding wound closure from photomicrographs (lower panel) show that imatinib significantly inhibited PDGF-BB-mediated longer-term migration of DAOY and D556 cells (P < 0.01, each; lower-left panel) and significantly inhibited serum-mediated migration of DAOY and D556 cells compared to control cells (P < 0.005, each; lower-right panel). C, DAOY and D556 cells transfected with PDGFRB siRNA (+) showed significantly inhibited longer-term PDGF-mediated migration compared to cells transfected with negative control siRNA (−) (P < 0.05, each). D, Treatment with imatinib significantly inhibited the invasion of DAOY and D556 cells compared to negative control treated cells (P < 0.001, each). Results represent the mean ± the standard error of the mean of multiple experiments. *, Indicates a statistically significant decrease in cell migration or invasion of imatinib-treated cells compared to untreated control cells.

Figure 6. Imatinib induces delayed apoptosis and inhibits medulloblastoma cell proliferation

Serum-starved medulloblastoma cells were treated with 1 uM imatinib (‘+’) or vehicle control (‘−’). Apoptosis was determined by 7-AAD/AnnexinV immunostaining and detection by FACS at 24, 48, and 72 h after a single dose of imatinib or 48 h after transfection with PDGFRB siRNA (‘+’) or negative control siRNA (‘−’). Cell proliferation was determined by the number of viable cells counted at 0, 24, 48 and 72 h after a single dose of imatinib. A, Imatinib treatment induced a significant increase in apoptosis only at 72 h after treatment in DAOY (P = 0.006, indicated by *), but not D556 cells, compared to untreated control cells. B, PDGFRB siRNA transfection resulted in a significant increase in apoptosis of DAOY (P = 0.002, indicated by *), but not D556 cells, compared to negative control transfected cells. C, Imatinib treatment of DAOY and D556 cells significantly reduced cell proliferation (P < 0.005, each, indicated by *) only at 72 h after treatment compared to untreated control cells. Results represent the mean ± the standard error of the mean of multiple experiments.