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Medulloblastomas are the most frequent malignant brain tumors in children. Sorafenib (Nexavar, BAY43-9006), a multi-kinase inhibitor, blocks cell proliferation and induces apoptosis in a variety of tumor cells. Sorafenib inhibited proliferation and induced apoptosis in two established cell lines (Daoy and D283) and a primary culture (VC312) of human medulloblastomas. In addition, sorafenib inhibited phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) in both cell lines and the primary tumor cells. The inhibition of phosphorylated STAT3 (Tyr705) occurs in a dose- and time-dependent manner. In contrast, AKT (protein kinase B) was only decreased in D283 and VC312 medulloblastoma cells and MAPKs (ERK1/2) were not inhibited by sorafenib in these cells. Both D-type cyclins (D1, D2, D3) and E-type cyclin were down-regulated by sorafenib. Also, expression of the anti-apoptotic protein Mcl-1, a member of the Bcl-2 family, was decreased and correlated with apoptosis induced by sorafenib. Finally, sorafenib suppressed the growth of human medulloblastoma cells in a mouse xenograft model. Together, our data demonstrate that sorafenib blocks STAT3 signaling as well as expression of cell cycle and apoptosis regulatory proteins, associated with inhibition of cell proliferation and induction of apoptosis in medulloblastomas. These findings provide a rationale for treatment of pediatric medulloblastomas with sorafenib.
Primitive Neuro-ectodermal Tumors (PNETs) are a highly heterogeneous group of tumors arising mainly in children up to 9 years of age (1). Cerebellar PNETs, known as medulloblastoma (MB), are the most prevalent malignancy of the central nervous system in childhood. The etiology of medulloblastomas is still unclear, although several signaling pathways that control cell proliferation are thought to be involved in disease progression. The Sonic hedgehog (SHH) and the Wingless (WNT) pathways have been linked to the development of medulloblastomas (2-6). In addition, AKT (protein kinase B) and MAPK/ERK (extracellular signal-regulated kinase) may contribute to the progression of the tumor in certain cases (7).
The activity of STAT (signal transducer and activator of transcription) proteins, particularly STAT3, is frequently elevated in a variety of solid tumors and hematological malignancies (8). STAT3 proteins have dual roles as cytoplasmic signaling proteins and nuclear transcription factors that activate a diverse set of genes, including some that implicated in malignant progression (9, 10). STAT3 is found to be constitutively activated in medulloblastomas (11) and the level of STAT3 activation in medulloblastomas exceeds that of all other brain tumors examined, including glioblastomas, ependymomas and astrocytomas (12). Therefore, the formation and maintenance of medulloblastomas may be regulated in part by STAT3.
Sorafenib (BAY43-9006, Nexavar) is an oral multi-kinase inhibitor that was originally developed based on its inhibitory effect on Raf and receptor tyrosine kinase (RTK) signaling (13). Deregulation of the Raf-MEK-MAPK signaling pathway is associated with development of solid tumors (14-16). Recent findings showed that sorafenib inhibited tumor growth and angiogenesis, and induced apoptosis, through either Raf-MEK-MAPK dependent or independent pathways, depending on the type of tumors being investigated (17-19). Evaluation of sorafenib from Phase I and II clinical trials on several types of advanced solid tumors showed favorable tolerability and promising clinical antitumor activity (20-22).
Our present results show that sorafenib inhibits proliferation and induces apoptosis in two established human cell lines (D283, Daoy) and a primary culture (VC312) of human medulloblastomas. Sorafenib also inhibits the in vivo growth of human medulloblastoma cells in nude mice. The biological effects of sorafenib on medulloblastomas are associated with inhibition of STAT3 signaling as well as down-regulation of cyclins D/E and Mcl-1 proteins. These findings suggest that sorafenib may be effective for the treatment of pediatric medulloblastoma tumors through inhibition of STAT3 signaling.
Sorafenib was kindly provided by Onyx and Bayer Pharmaceuticals. Anti-cyclin D1 and D3 were obtained from Calbiochem. Anti-cyclin E was obtained from BD Biosciences. Anti-cyclin D2 and anti-Mcl-1 were obtained from Santa Cruz. Horseradish peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies were from GE Healthcare. All other antibodies were obtained from Cell Signaling.
Two human meduloblastoma cell lines, D283 (D283med) and Daoy, were from American Type Culture Collection (ATCC). All cells were maintained in MEM (Eagle) with L-glutamine supplemented with 10% fetal bovine serum (FBS), and 1% Antibiotic-Antimycotic (AA). The primary culture (VC312) of medulloblastoma was derived from a tumor of a 4-year old male patient treated at the Virginia Commonwealth University Health System’s Medical College of Virginia Hospital under an IRB approved protocol. Briefly, samples of the tumor were first obtained to allow full neuropathologic evaluation and diagnosis, as required for the clinical management of the patient’s disease. The site of origin of all the tumor samples was cerebellum. The sterile dissection of tumor biopsy was dissociated and plated in 6-well tissue culture plates and expanded in DMEM/F12 medium supplemented with 1% N-2 supplement (Invitrogen), 5% FBS, 20 ng/ml recombinant human EGF and 10 ng/ml recombinant human bFGF (Beckton Dickenson). VC312 cells were subsequently maintained in DMEM (with L-glutamine) supplemented with 10% FBS and utilized at low passage number (below passage 20 for all studies).
Cell proliferation assays were performed with CellTiter 96 Aqueous One Solution Cell proliferation Assay (Promega) which contains 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). Each well of 96-well plates was seeded with 5000 cells in culture medium with 1% FBS. After overnight culture the cells were treated with different concentrations of sorafenib and controls were treated with vehicle (DMSO). After 24 h or 48 h treatments, MTS was added to the cells according to the supplier’s protocol and absorbance was measured at 490 nm using an automated ELISA plate reader.
Daoy, D283 or VC312 cells (2 × 105) were seeded in 60 mm culture dishes in culture medium with 1% FBS. The following day the cells were treated with indicated concentrations of sorafenib for a 24 h or 48 h period. After treatment, all cells including both floating and attached cells were collected, and the apoptotic cells were detected by Annexin V-FITC Apoptosis Detection Kit (BD Biosciences). The cells were stained with Annexin V-FITC and propidium iodide (PI) according to the supplier’s instructions. Viable and dead cells were detected by flow cytometry in the Analytical Cytometry Core at City of Hope National Medical Center.
Twenty μg total proteins were resolved in 4-15% gradient Tris-HCl gels (BIO-RAD). After gel electrophoresis, the proteins were transferred to Hybond-C membranes (Amersham). The membranes were blocked for 1 h at room temperature (RT) in 10% non-fat dry milk in PBST (1 × PBS with 0.1% Tween-20), followed by an overnight incubation at 4 ° C with primary antibodies in PBST with 2% non-fat dry milk. The membranes were then incubated with horseradish peroxidase labeled anti-mouse or anti-rabbit secondary antibodies for 1 h at RT. Immunoreactivity was detected with SuperSignal West Pico substrate (Pierce).
Daoy cells were plated in 100 mm culture dishes at 5 × 105 per dish. After 24 h treatment with sorafenib, cells were harvested by trypsinization and washed with PBS. Cells were fixed in ice-cold 75% ethanol, washed and resuspended in 1 ml PBS with 50 μg PI and 0.25 mg RNase A. Samples were assayed by Flow Cytometry.
The 27mer dicer substrate siRNA against STAT3 was synthesized by IDT (Coralville, IA). Cells were transfected with 50 nM STAT3 siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the supplier’s protocol. Control cells were transfected with negative control siRNA #1 (Ambion). After 24 h transfection, cells were seeded in 96-well plates for proliferation assays.
Mouse xenograft studies were performed in 4- to 6-week-old nude mice obtained from National Cancer Institute-Charles Rivers Laboratories. The mice were held in a pathogen-free animal facility at City of Hope Medical Center and were fed standard rodent chow and water ad libitum. All procedures followed the NIH guidelines for the care and use of laboratory animals. Tumors were generated by harvesting Daoy cells from mid-log phase cultures. Cells were pelleted and resuspended in a 50% mixture of Matrigel (BD Biosciences) in MEM (Eagle) medium to 2.5 × 107 cells/mL. This cell suspension (0.2 mL) was injected s.c. in the right flank of each mouse. Sorafenib treatment was started at 10 days after injection of Daoy cells. Animals with palpably established tumors of at least 65 mm3 were designated to treatment groups. Treatment with 100 μl of 10 μM sorafenib in 1 × PBS was administered by intratumoral injection three times per week for five weeks. Mice in the control group were injected with the same amount of PBS with vehicle. Tumors were measured every 3-4 days with Vernier calipers, and tumor volumes were calculated by the formula π/6 × (larger diameter) × (small diameter)2.
Student’s t test was used to evaluate statistical significance of differences between two groups and p < 0.05 was considered statistically significant.
To characterize the effects of sorafenib on cell proliferation in medulloblastomas, we performed dose-response and time-course studies in D283 and Daoy cells (Fig. 1A and 1B). Cells were treated with increasing concentrations of sorafenib (2.5, 5, 10 μM) for 24 h and 48 h, or with the vehicle (DMSO) only as control. Because previous studies suggest that sorafenib binds to serum proteins (13), these assays were performed in 1% serum to reduce the effect of serum. Sorafenib markedly inhibited proliferation of both D283 and Daoy cells in a dose- and time-dependent manner. We next investigated whether sorafenib could induce apoptosis in D283 and Daoy cells. After treatment with increasing concentrations of sorafenib (2.5, 5, 10, 20 μM) for 48 h, cells were analyzed by Annexin V/propidium iodide staining and flow cytometry. Apoptotic cells shown in Fig. 1C and 1D were Annexin V and propidium iodide double-positive. Results show that D283 cells were more sensitive to sorafenib than Daoy cells.
We investigated the levels of total and phosphorylated STAT3, AKT and MAPK (p42/44) proteins in D283 and Daoy cells after sorafenib treatment. Total protein levels of STAT3, AKT, and MAPK were not significantly changed after 4 h or 24 h sorafenib treatment (Fig. 2A and B). By contrast, phosphorylation of STAT3 at Tyr705 was substantially reduced at both an early time point (4 h) and a late time point (24 h) in D283 and Daoy cells following sorafenib treatment (Fig. 2A and 2B). The phosphorylation of STAT3 at Tyr705 was inhibited in a dose- and time-dependent manner. Phosphorylated AKT was only decreased in D283 cells after 24 h treatment, and MAPK (p42/44) was not reduced by sorafenib treatment. Phosphorylation of STAT3 at Tyr705 is essential for STAT3 activation (23). These results indicate that inhibition of STAT3 signaling is an early response to sorafenib treatment, and that STAT3 inhibition is a common response to sorafenib in both D283 and Daoy cells.
Since sorafenib significantly inhibits proliferation of D283 and Daoy cells (Fig. 1), we investigated the effect of sorafenib on key cell-cycle regulators, including D-type and E-type cyclins. Immunoblot analyses were performed to determine the expression of cyclin D1/D2/D3 and cyclin E in D283 and Daoy cells after 24 h sorafenib treatment. Figure 2C showed that sorafenib decreased the expression of cyclin D1/D2/D3 and cyclin E in a dose-dependent manner. Lower levels of cyclin D1 protein expression in D283 cells correlated with lower levels of cyclin D1 mRNA in these cells as confirmed by real-time RT-PCR (data not shown). We also analyzed the effect of sorafenib on cell-cycle distribution in Daoy cells by FACS with PI staining. Figure 2D shows a decrease of cells in G1 phase and an increase of cells arrested in S phase with increasing concentrations of sorafenib. Reducing the expression of cyclin D and E is consistent with the S-phase arrest. The S phase arrest of Daoy cells by sorafenib is similar to an earlier report in hepatocellular carcinoma (17).
Bcl-2 family proteins have a key role in survival of normal and tumor cells (24). The expression of three anti-apoptotic proteins in this family, Mcl-1, Bcl-xL and Bcl-2, was investigated after sorafenib treatment. Though Bcl-xL and Bcl-2 were not decreased, Mcl-1 was decreased in both D283 and Daoy cells after 24 h of sorafenib treatment (Fig. 2C). These results are consistent with the induction of apoptosis by sorafenib (Fig. 1C and 1D), and implicate Mcl-1 in this response.
Since STAT3 proteins have an important role in the progression and maintenance of many malignant tumors, we investigated the expression of STAT3 proteins in pediatric medulloblastomas. Six human medulloblastoma biopsies (MB1-6) were examined by Western blot analysis with specific STAT3 antibodies. Normal rat brains including embryonic brain day 15 (E15), postnatal day 4 cerebellum (P4), and 8 week adult cerebellum (8wk) were used as controls. As shown in Figure 3A, STAT3 protein was expressed in both rat normal brains and human tumor samples. STAT3 phosphorylation at Ser727 was only detected in tumor samples. Importantly, STAT3 phosphorylation at Tyr705 was observed in all six tumor biopsies and early developmental time points in rat brains, but not in adult rat brains.
To further assess the role of STAT3 proteins in tumor cells from medulloblastoma, we employed STAT3 siRNA to inhibit the expression of STAT3 in a primary culture (VC312), which was derived from the MB3 biopsy (Fig. 3A). After transfection with STAT3 siRNA, expression of STAT3 proteins in VC312 cells was partially inhibited (Fig. 3B), and the proliferation of VC312 cells was partially decreased (Fig. 3C).
To evaluate whether sorafenib has the same effect on primary cultures as on established cell lines, adherent VC312 cells were treated with sorafenib in the same manner as D283 and Daoy cells. Proliferation of VC312 cells was inhibited by sorafenib in a dose- and time-dependent manner (Fig. 4A). Sorafenib also induced apoptosis of VC312 cells in a dose-dependent manner after 48 h treatment (Fig. 4B). A lower concentration (2.5 μM) of sorafenib effectively inhibited the proliferation of VC312 cells, but it could not induce apoptosis. These results indicate that relatively higher concentrations are required to induce cell death than to inhibit proliferation. This inhibitory effect on the proliferation and survival of VC312 cells is similar to that observed for D283 and Daoy cells (Fig. 1).
We next examined signaling by STAT3, AKT, and MAPK (p42/44) proteins after sorafenib treatment in VC312 cells. Total protein levels of STAT3, AKT, and MAPK were not changed (Fig. 4C). The levels of phosphorylated STAT3 at Tyr705 proteins were significantly decreased at both an early time point (4 h) and a late time point (24 h) in a dose and time-dependent manner (Fig. 4C). Phosphorylated AKT was found to be diminished by sorafenib treatment, but inhibition of phosphorylated STAT3 was greater than that of phosphorylated AKT, especially at 4 h. Interestingly, although high levels of phosphorylated MAPK (p44/42) were present in VC312 cells, phosphorylation of MAPK (p42/44) was not decreased by sorafenib (Fig. 4C). These results indicate that the effect of sorafenib in VC312 cells is independent of the MAPK pathway.
We also investigated the effects of sorafenib on cell-cycle regulators, cyclin D and E, and anti-apoptotic genes, Mcl-1, Bcl-2 and Bcl-xL in VC312 cells. Sorafenib inhibited expression of cyclins D1/D2/D3, cyclin E and Mcl-1 in VC312 cells (Fig. 4D) in a similar manner to D283 and Daoy cells (Fig. 2).
To determine whether inhibition of STAT3 phosphorylation at Tyr705 is an early event, we treated Daoy and VC312 cells with 10 μM sorafenib for 5, 15 and 30 min. Western blot analysis (Fig. 5A) showed that the inhibition of p-STAT3 Tyr705 by sorafenib was detected as early as 5 min after treatment. We also investigated whether sorafenib inhibited phosphorylation of STAT3 at Ser727 in tumor cells from medulloblastomas. Our results (Fig. 5B) show that sorafenib did not inhibit the phosphorylation of STAT3 at Ser727 in Daoy and VC312 cells.
To examine the effect of sorafenib on growth of medulloblastoma cells in vivo, nude mice were inoculated with Daoy cells subcutaneously and were administered sorafenib by intratumoral injection (100 μl of 10 μM, three times/week). At this dose, no lethal toxicity or weight loss (greater than 10% body weight) was observed among treated animals. Sorafenib significantly inhibited the growth of medullobastomas in mice as shown in Fig. 6A (52% growth inhibition, p < 0.001, n = 8). Even though sorafenib was only administered three times per week, the inhibitory effect of sorafenib on growth of tumor xenografts was obvious compared to control after one week treatment (day 18, Fig. 6A). The expression of total and phosphorylated STAT3 was analyzed in the tumors treated with sorafenib and control tumors. Phosphorylation of STAT3 at Tyr705 in two treated tumors was decreased compared with two controls (Fig. 6B), while the total STAT3 protein in treated tumors was not decreased. These results provide proof-of-concept that local delivery of sorafenib inhibits the growth of human medulloblastoma tumors associated with decreased tyrosine phosphorylation of STAT3.
Here we provide evidence that sorafenib inhibits cell proliferation and survival of two established cell lines (D283 and Daoy) as well as a primary culture (VC312) from human medulloblastomas. Our results suggest that sorafenib inhibits growth of these medulloblastoma cells at least in part through blockade of the STAT3 signaling pathway. Although these tumor cells exhibit different growth properties, inhibition STAT3 phosphorylation at Tyr705 in response to sorafenib is common among all of them. Furthermore, inhibition of STAT3 phosphorylation at Tyr705 by sorafenib is detectable five minutes after treatment, indicating that the effect of sorafenib on STAT3 phosphorylation is an early and relatively direct event.
STAT3 activation requires Tyr705 phosphorylation, resulting in dimerization, nuclear translocation, DNA binding and transcriptional activation of target genes (23). Phoshorylation of STAT3 at Ser727 further enhances transcriptional activation of genes (23). Our data show that total STAT3 proteins as well as phosphorylation on both Tyr705 and Ser727 were constitutively expressed in biopsies from human medulloblastomas. STAT3 regulates basic biologic processes important in tumorigenesis including cell-cycle progression, apoptosis, tumor angiogenesis, and tumor-cell evasion of the immune system (8, 25). Key genes in cell-cycle control, such as cyclin D1, are regulated by STAT3 (8). The expression of cyclin D (D1/D2/D3) and cyclin E are decreased by sorafenib in medulloblastoma cells, consistent with the observed inhibition of cell-cycle progression.
Expression of Mcl-1, an anti-apoptotic protein, is also regulated by STAT3 signaling (8). Mcl-1 has been shown to have a critical role in the survival of malignant cells, especially in leukemia and myeloma (26). Sorafenib has been reported to down-regulate the expression of Mcl-1 in diverse types of tumor cells (27, 28). Here, we show that sorafenib inhibited Mcl-1 expression in both established cell lines and a primary culture of medulloblastomas. Blocking STAT3 protein in human tumor cells has been shown to down-regulate Mcl-1 expression and induce tumor-cell apoptosis (8). STAT3 and Mcl-1 are the only proteins inhibited in common among the D283, Daoy and VC312 cells. Therefore, down-regulation of Mcl-1 by inhibition of phosphorylated STAT3 may be an important mechanism of action of sorafenib in medulloblastomas.
In addition to STAT3 signaling, both Raf-MEK-MAPK and PI3K/AKT have important roles in the proliferation and survival of tumor cells (7, 14). Our data suggest that active AKT might also be suppressed by sorafenib, although MAPK signaling does not appear to be affected under the conditions examined in this study. Because sorafenib inhibits vascular endothelial growth factor receptors (VEGFRs) (13), key regulators of tumor neoangiogenesis in medulloblastoma (29), it is possible that sorafenib could inhibit angiogenesis in medulloblastoma.
Sorafenib shows good tolerability and promising antitumor activity from clinical trials in several types of solid tumors (20-22). An insidious feature of medullobastomas is their ability to metastasize and disseminate through the cerebrospinal fluid (30). Treatment of medulloblastoma is complicated by the blood-brain barrier, which acts as a physiologic barrier for delivery of drugs to the central nervous system. Various approaches have been developed for local delivery of drugs to brain tumors, including convection-enhanced delivery (31). Thus, local delivery of sorafenib to the cerebrospinal fluid by convection-enhanced delivery may result in more effective antitumor activity with reduced systemic toxicity. In summary, sorafenib is potentially a promising drug for the treatment of pediatric medulloblastomas.
Supported by the Sunshine Project of the Pediatric Cancer Foundation and NCI grant CA1155674 (to R.J.).