T-type calcium channels regulate medulloblastoma and can be targeted for therapy

Abstract

Purpose: The goal of this study was to investigate the role and therapeutic targeting of T-type calcium channels in medulloblastoma, a common and deadly pediatric brain tumor that arises in the cerebellum.

Methods: T-type calcium channel expression was assessed in publicly available bulk and single cell RNA-seq datasets. The effects of T-type calcium channel blocker mibefradil on cell growth, death and invasion were assessed with cell counting, alamar blue, trypan blue and transwell assays. Proteomic-based drug target and signaling pathway mapping was performed with Reverse Phase Protein Arrays (RPPA). Co-expression modules of single cell RNA-seq data were generated using high dimensional weighted gene co-expression network analysis (hdWGCNA). Orthotopic xenografts were used for therapeutic studies with the T-Type calcium channel blocker mibefradil.

Results: T-type calcium channels were upregulated in more than 30% of medulloblastoma tumors and patients with high expression associated with a worse prognosis. T-type calcium channels had variable expression across all the subgroups of medulloblastoma at the bulk RNA-seq and single-cell RNA-seq level. Mibefradil treatment or siRNA mediated silencing of T-type calcium channels inhibited tumor cell growth, viability and invasion. RPPA-based protein/phosphoprotein signal pathway activation mapping of T-type calcium channel inhibition and single cell hdWGCNA identified altered cancer signaling pathways. Oral administration of mibefradil inhibited medulloblastoma xenograft growth and prolonged animal survival.

Conclusion: Our results represent a first comprehensive multi-omic characterization of T-type calcium channels in medulloblastoma and provide preclinical data for repurposing mibefradil as a treatment strategy for these relatively common pediatric brain tumors.

Keywords: Medulloblastoma; Mibefradil; T-type calcium channels.

Fig. 1

T-type calcium channels are dysregulated in medulloblastoma. (A) Genetic dysregulation of T-type calcium channels in medulloblastoma samples of the Pediatric Brain Tumor Atlas (PBTA). (B) Correlation of T-type calcium channel dysregulation with patient survival based on PBTA analysis. (C) Analysis of T-type calcium channel expression in subgroups from the Pediatric Brain Tumor Atlas (PBTA). (D) Relative expression Cav3.1 and Cav3.2 expression in medulloblastoma cell lines via RT-qPCR. (E & F) Violin plot showing expression of Cav3.1 and Cav3.2 in the different medulloblastoma subtypes in scRNA-seq data

Fig. 2

Cav3.1 and Cav3.2 are co-expressed with different pathways. (A) Network plot of top differentially expressed module for Cav3.1 positive cells in Group 3 tumors and associated biological processes. (B) Network plot of the top differentially expressed module for Cav3.2 positive cells in Group 3 tumors and associated biological processes. (C) Network plot of the top differentially expressed module for Cav3.1 positive cells in SHH tumors and associated biological processes. (D) Network plot of the top differentially expressed module for Cav3.2 positive cells in SHH tumors and associated biological processes

Fig. 3

T-type calcium channel blocker mibefradil inhibits cell growth and induces cell death in medulloblastoma. (A) ONS-76 and DAOY cells were treated with vehicle or mibefradil (1–10 µM) and assessed cell viability changes by alamar blue 72 h later. (B) ONS-76 and DAOY cells were treated with vehicle or mibefradil and cell viability was assessed 48 h later by trypan blue cell counting. (C) Cell growth assay of vehicle and mibefradil treated cells (ONS-76. DAOY). (D) Representative images of invaded cells and quantification of invasive cell number in vehicle and mibefradil treated cells. N = 3 independent experiments for all assays. *p < 0.05

Fig. 4

Mibefradil downregulates important apoptosis and growth regulating proteins. (A) Reverse phase protein array of mibefradil treated cells showed upregulation of BAK and BAD in medulloblastoma cells. (B) Reverse phase protein array of mibefradil treated cells showed downregulation of cell growth and cell cycle proteins such as CDK6, CDK2 and Ki67. (C) Western blot confirmation showing downregulation of Ki67 in mibefradil treated ONS-76 cells

Fig. 5

Mibefradil inhibits DAOY xenograft growth and prolongs survival. (A) Schematic of in vivo experimental design. (B) Representative MRIs of the tumor with quantification of tumor volume at the pretreatment time point as well as the post treatment timepoint for both Vehicle and Mibefradil-treated mice. (C) Kaplan-Meier survival curves of vehicle and mibefradil-treated mice. N = 7 mice per group. ***p < 0.001