Effective treatment of diverse medulloblastoma models with mebendazole and its impact on tumor angiogenesis

Abstract

Background: Medulloblastoma is the most common malignant brain tumor in children. Current standard treatments cure 40%-60% of patients, while the majority of survivors suffer long-term neurological sequelae. The identification of 4 molecular groups of medulloblastoma improved the clinical management with the development of targeted therapies; however, the tumor acquires resistance quickly. Mebendazole (MBZ) has a long safety record as antiparasitic in children and has been recently implicated in inhibition of various tyrosine kinases in vitro. Here, we investigated the efficacy of MBZ in various medulloblastoma subtypes and MBZ's impact on vascular endothelial growth factor receptor 2 (VEGFR2) and tumor angiogenesis.

Methods: The inhibition of MBZ on VEGFR2 kinase was investigated in an autophosphorylation assay and a cell-free kinase assay. Mice bearing orthotopic PTCH1-mutant medulloblastoma allografts, a group 3 medulloblastoma xenograft, and a PTCH1-mutant medulloblastoma with acquired resistance to the smoothened inhibitor vismodegib were treated with MBZ. The survival benefit and the impact on tumor angiogenesis and VEGFR2 kinase function were analyzed.

Results: We determined that MBZ interferes with VEGFR2 kinase by competing with ATP. MBZ selectively inhibited tumor angiogenesis but not the normal brain vasculatures in orthotopic medulloblastoma models and suppressed VEGFR2 kinase in vivo. MBZ significantly extended the survival of medulloblastoma models derived from different molecular backgrounds.

Conclusion: Our findings support testing of MBZ as a possible low-toxicity therapy for medulloblastomas of various molecular subtypes, including tumors with acquired vismodegib resistance. Its antitumor mechanism may be partially explained by inhibition of tumor angiogenesis.

Keywords: VEGF; VEGFR2; angiogenesis; mebendazole; medulloblastoma.

Fig. 1.

MBZ inhibits VEGFR2 kinase activity and competes with ATP. (A) MBZ inhibited the autophosphorylation of VEGFR2-Y1175 in HUVECs. HUVECs were starved and treated with MBZ 30 min prior to the stimulation of 50 ng/mL VEGF. Western blot of anti-pVEGFR2-Y1175 antibody indicated the autophosphorylation of tyrosine 1175, and the levels of VEGFR2 were reflected by the anti-VEGFR2 western blot. (B) Cell-free kinase assay with a purified VEGFR2 (aa789-1356) revealed an IC₅₀ of MBZ at 4.3 μM. Three measurements were performed. KD, kinase domain; Y1054/Y1059/Y1175: 3 of the major autophosphorylation sites of VEGFR2. (C, D) Effect of MBZ on the kinetics of VEGFR2 kinase activity with the ATP gradient. Kinase assays were performed with 1 μg poly (Glu₄,Tyr₁) peptide and 0, 2, 3, 5, 10, 50, 500, 2000, or 4000 μM ATP. The reactions with 0 μM ATP served as background. On the V⁻¹ and ATP⁻¹ plot (D), the linear regressions of the reactions with Con, 3, 10, and 30 μM MBZ converge closely within the statistic deviations at the intersection with the y-axis at 0.013, 0.024, 0.045, and 0.028 (×10⁻⁷), respectively. (E, F) Effect of MBZ on the kinetics of VEGFR2 kinase activity with the poly (Glu₄,Tyr₁) peptide gradient. Kinase assays were performed with 50 μM ATP and 0, 0.0025, 0.005, 0.01, 0.04, or 0.4 μg/μL poly (Glu₄,Tyr₁) peptide. The activities at 0 μg/μL poly (Glu₄,Tyr₁) peptide presumably reflected the autophosphorylation of VEGFR2. The reactions without VEGFR2 kinase with 50 μM ATP, peptide, and MBZ (0, 3, 10, or 30 μM) were used as the background controls.

Fig. 2.

MBZ inhibited the orthotopic PTCH+/−, p53−/− allograft and the vismodegib-resistant SMO-D477G medulloblastoma in mice. (A) The PTCH+/−, p53−/− allograft tumor was implanted in the right vermis of the cerebellum of nude mice. An H&E slide shows a fully grown medulloblastoma (MB) that broke into the fourth ventricle (black arrowhead). (B) Treatment with 50 mg/kg MBZ via oral gavage starting from day 5 after tumor implantation improved the median (m) survival by 150% vs the control group. (C) Xenogen scan demonstrated that the tumor growth was significantly inhibited by MBZ treatment. The tumor load in a set of representative mice after 6 days of MBZ treatment is displayed in the left picture. The right graph shows the average xenogen counts before and after the MBZ treatment. (D) D477G mutant allograft derived from a PTCH+/−, p53−/− medulloblastoma-bearing mouse treated with the Hh inhibitor vismodegib was implanted in the cerebellum of nude mice. The H&E slide shows the tumor growth in the right vermis of the cerebellum. (E) Daily treatment of mice with MBZ extended the median (m) survival from 12 days to 30 days. (F) Xenogen scan shows the inhibition of tumor growth following 6 days of MBZ treatment. The right graph shows the average xenogen counts without (Con) and with MBZ treatment.

Fig. 3.

MBZ markedly extended the survival of D425 xenograft medulloblastoma of group 3. (A) D425 medulloblastoma (MB) cells belong to group 3 of molecular classification and carry c-MYC and OTX2 genomic amplification. The cells were implanted into the right vermis of the cerebellum of nude mice. H&E staining demonstrated the fully grown cerebellar tumor. (B) Treatment by MBZ starting from day 5 of tumor implantation improved the median (m) survival from 21 to 48 days by 129%. (C) Xenogen scan demonstrated the inhibition of tumor growth after 12 days of MBZ treatment. The right graph shows the average xenogen counts without (Con) and with MBZ treatment.

Fig. 4.

MBZ reduced the formation of tumor vessels in the PTCH mutant allograft and D425 xenograft medulloblastoma. (A) The sections of PTCH mutant allograft (PTCH+/−, p53−/−) medulloblastoma (MB) were stained with anti-CD31 antibody and green fluorescent secondary antibody. Nuclei were stained with DAPI (blue). Microvessel density was counted on 200× fields (“hotspot”) in 5 independent slides in each group, graphed in (B). The same frames of green fluorescence pictures were observed with the Texas Red fluorescence filter, in order to rule out possible autofluorescence. All pictures were taken with the setting of 800 msec exposure to green fluorescence and 200 msec to DAPI channel. All scale bars are 30 μm. (C, D) Similar staining and measurement were performed with D425 medulloblastoma xenografts.

Fig. 5.

MBZ did not affect the normal vasculature in the brain tissues. The sections of mouse cerebellum bearing the PTCH mutant allograft (A) or D425 xenograft tumor (C) were stained with anti-CD31 antibody and green fluorescent secondary antibody, with DAPI staining the nuclei. All scale bars are 30 μm. The microvessels in the normal brain tissues on anatomically comparable sites distant from the respective tumors are counted and graphed in (B) and (D). The microvessel density was not affected by MBZ treatment. Five independent sections were used for each group.

Fig. 6.

MBZ inhibits VEGFR2 kinase activity in vivo. (A) Anti-VEGF immunohistochemistry staining of PTCH+/−, p53−/− medulloblastoma allografts treated or untreated with MBZ. VEGF was visualized by diaminobenzidine (brown), and the tissues were counterstained by hematoxylin (blue). All scale bars are 30 μm. (B, C) Anti-VEGFR2 (left panels, green) and anti-pVEGFR2-Y1175 (B) or anti-pVEGFR2-Y1054/1059 (C) (middle panels, red) staining of PTCH+/−, p53−/− medulloblastoma allografts untreated or treated with MBZ. The nuclei were visualized by DAPI staining (blue). MBZ treatment markedly reduced the autophosphorylation of VEGFR2-Y1175 and -Y1054/1059. The yellow color in the merged picture indicates the strong costaining of both antibodies in the control allografts. All pictures were taken with the setting of 800 msec exposure to green fluorescence, 1500 msec to Texas Red, and 200 msec to DAPI channel. All scale bars are 30 μm.