MPS1 kinase as a potential therapeutic target in medulloblastoma

Abstract

Medulloblastoma is the most common type of malignant brain tumor that affects children. Although recent advances in chemotherapy and radiation have improved outcomes, high-risk patients perform poorly with significant morbidity. Gene expression profiling has revealed that monopolar spindle 1 (MPS1) (TTK1) is highly expressed in medulloblastoma patient samples compared to that noted in normal cerebellum. MPS1 is a key regulator of the spindle assembly checkpoint (SAC), a mitotic mechanism specifically required for proper chromosomal alignment and segregation. The SAC can be activated in aneuploid cancer cells and MPS1 is overexpressed in many types of cancers. A previous study has demonstrated the effectiveness of inhibiting MPS1 with small-molecule inhibitors, but the role of MPS1 in medulloblastoma is unknown. In the present study, we demonstrated that MPS1 inhibition by shRNA or with a small-molecule drug, NMS-P715, resulted in decreased cell growth, inhibition of clonogenic potential and induction of apoptosis in cells belonging to both the Shh and group 3 medulloblastoma genomic signature. These findings highlight MPS1 as a rational therapeutic target for medulloblastoma.

Figure 1

Overexpression of MPS1 in pediatric brain tumors. (A) Microarray expression of MPS1 mRNA in 90 pediatric brain tumors showed significant overexpression of MPS1 in high-grade pediatric brain tumors when compared to normal brain. Error bars represent standard error of the mean (SEM). (B) Microarray expression of MPS1 mRNA in 120 patients with medulloblastoma demonstrated that MPS1 mRNA is significantly elevated in medulloblas-toma compared to adult and fetal cerebellum but not significantly different among the genomic subgroups (WNT, SHH, groups 3 and 4). Error bars represent SEM. (C) MPS1 protein expression in patient tumor samples including two group 3 and two group 4 samples compared to normal pediatric cerebellum (CB). (D) MPS1 protein expression in commonly used medulloblastoma cell lines (D283, D458 and D425, group 3 genomic subgroup; Daoy, ONS-76 and UW228, Shh genomic subgroup). ATRT, atypical teratoid/rhabdoid tumor: GMB, glioblastoma multiforme; MED, medulloblastoma; PA, pilocytic astrocytoma.

Figure 2

Inhibition of MPS1 suppresses medulloblastoma cell growth in vitro. Knockdown of MPS1 mRNA with shTTK significantly decreased the number of viable cells in both (A) Daoy and (B) ONS-76 cell lines as measured by MTS assay. Inhibition of medulloblastoma cell growth by MPS1 kinase inhibitor, NMS-P715 in a concentration-dependent manner in the (C) Daoy and (D) ONS-76 cells. (E) Activity of NMS-P715 (2 µM) against a panel of Myc-driven medulloblastoma cell lines. All cells were treated for 72 h and each study was repeated 3 times.

Figure 3

NMS-P715 inhibits clonogenic potential of (A) Daoy, (B) ONS-76 and (C) UW228 medulloblastoma cells. Quantification of colony formation with representative wells in control and NMS-P715-treated cells (*P<0.01, **P<0.001). Cells were treated for 10 days. Error bars represent SEM.

Figure 4

(A) Western blotting of NMS-P715-treated medulloblastoma cells, showing that NMS-P715 strongly decreased autophosphorylation of serine 821 on MPS1 in the Daoy and UW228 medulloblastoma cells. (B) The suppression of phosphorylation was dose-dependent for both cell lines as quantified by densitometry (pMPS1 levels relative to total MPS1 protein).

Figure 5

Chemical inhibition of MPS1 in medulloblastoma induces cell cycle arrest. (A) NMS-P715 induced G2/M arrest in medulloblastoma cells. Representative flow plots with quantification of cell cycle fractions. (B) Western blot analysis of cell cycle-related proteins upon MPS1 inhibition with NMS-P715. MPS1 inhibition resulted in decreased cyclin B2 as well as decreased CDC2 protein levels in the Daoy and D425 cells, but not in the D458 cells.

Figure 6

Inhibition of MPS1 induces apoptosis in medulloblastoma cells. Representative flow plots of Annexin V expression and quantification of apoptosis in the control and NMS-P715-treated cells.