ABCB1 inhibition provides a novel therapeutic target to block TWIST1-induced migration in medulloblastoma.

Aishah Nasir,Alice Cardall,Ian D Kerr,Anbarasu Lourdusamy,David Onion,Alison Ritchie,Beth Coyle,Niovi Nicolaou,Franziska Linke,Hanna Cameron,Ramadhan T Othman,Andrey Korshunov,Anna M Grabowska,Cara Valente,Stefan M Pfister,Marina Ryzhova

doi:10.1093/noajnl/vdab030

Abstract

BackgroundTherapeutic intervention in metastatic medulloblastoma is dependent on elucidating the underlying metastatic mechanism. We investigated whether an epithelial–mesenchymal transition (EMT)-like pathway could drive medulloblastoma metastasis.MethodsA 3D Basement Membrane Extract (3D-BME) model was used to investigate medulloblastoma cell migration. Cell line growth was quantified with AlamarBlue metabolic assays and the morphology assessed by time-lapse imaging. Gene expression was analyzed by qRT-PCR and protein expression by immunohistochemistry of patient tissue microarrays and mouse orthotopic xenografts. Chromatin immunoprecipitation was used to determine whether the EMT transcription factor TWIST1 bound to the promoter of the multidrug pump ABCB1. TWIST1 was overexpressed in MED6 cells by lentiviral transduction (MED6-TWIST1). Inhibition of ABCB1 was mediated by vardenafil, and TWIST1 expression was reduced by either Harmine or shRNA.ResultsMetastatic cells migrated to form large metabolically active aggregates, whereas non-tumorigenic/non-metastatic cells formed small aggregates with decreasing metabolic activity. TWIST1 expression was upregulated in the 3D-BME model. TWIST1 and ABCB1 were significantly associated with metastasis in patients (P = .041 and P = .04, respectively). High nuclear TWIST1 expression was observed in the invasive edge of the MED1 orthotopic model, and TWIST1 knockdown in cell lines was associated with reduced cell migration (P < .05). TWIST1 bound to the ABCB1 promoter (P = .03) and induced cell aggregation in metastatic and TWIST1-overexpressing, non-metastatic (MED6-TWIST1) cells, which was significantly attenuated by vardenafil (P < .05).ConclusionsIn this study, we identified a TWIST1–ABCB1 signaling axis during medulloblastoma migration, which can be therapeutically targeted with the clinically approved ABCB1 inhibitor, vardenafil.

Highlights

Therapeutic intervention in metastatic medulloblastoma is dependent on elucidating the underlying metastatic mechanism
We investigated whether previously defined nontumorigenic (C17.225 and FB8323), non-metastatic (MED620 and UW228-324), and metastatic (MED120, D283Med[27] and D458Med28) cell lines could be distinguished based on the aforementioned factors
The AlamarBlue assay and time-lapse imaging were used to assess metabolic activity (Figure 1A) and morphology (Figure 1B), respectively, of each cell line cultured in serum-free 3D Basement Membrane Extract (3D-basement membrane extract (BME))

Summary

Introduction

Therapeutic intervention in metastatic medulloblastoma is dependent on elucidating the underlying metastatic mechanism. We investigated whether an epithelial–mesenchymal transition (EMT)-like pathway could drive medulloblastoma metastasis. A 3D Basement Membrane Extract (3D-BME) model was used to investigate medulloblastoma cell migration. Cell line growth was quantified with AlamarBlue metabolic assays and the morphology assessed by timelapse imaging. Gene expression was analyzed by qRT-PCR and protein expression by immunohistochemistry of patient tissue microarrays and mouse orthotopic xenografts. Chromatin immunoprecipitation was used to determine whether the EMT transcription factor TWIST1 bound to the promoter of the multidrug pump ABCB1. TWIST1 was overexpressed in MED6 cells by lentiviral transduction (MED6-TWIST1). Inhibition of ABCB1 was mediated by vardenafil, and TWIST1 expression was reduced by either Harmine or shRNA.

Methods

Results

Conclusion