Localization matters: nuclear-trapped Survivin sensitizes glioblastoma cells to temozolomide by elevating cellular senescence and impairing homologous recombination

Thomas R Reich,Teodora Nikolova,Fabian Mühlhäusler,Petra Beli,Maja T Tomicic,Markus Christmann,Juliana Brandstetter Vilar,Alicia Poplawski,Christian Schwarzenbach,H Irem Baymaz,Sven Unger

doi:10.1007/s00018-021-03864-0

Abstract

To clarify whether differential compartmentalization of Survivin impacts temozolomide (TMZ)-triggered end points, we established a well-defined glioblastoma cell model in vitro (LN229 and A172) and in vivo, distinguishing between its nuclear and cytoplasmic localization. Expression of nuclear export sequence (NES)-mutated Survivin (SurvNESmut-GFP) led to impaired colony formation upon TMZ. This was not due to enhanced cell death but rather due to increased senescence. Nuclear-trapped Survivin reduced homologous recombination (HR)-mediated double-strand break (DSB) repair, as evaluated by γH2AX foci formation and qPCR-based HR assay leading to pronounced induction of chromosome aberrations. Opposite, clones, expressing free-shuttling cytoplasmic but not nuclear-trapped Survivin, could repair TMZ-induced DSBs and evaded senescence. Mass spectrometry-based interactomics revealed, however, no direct interaction of Survivin with any of the repair factors. The improved TMZ-triggered HR activity in Surv-GFP was associated with enhanced mRNA and stabilized RAD51 protein expression, opposite to diminished RAD51 expression in SurvNESmut cells. Notably, cytoplasmic Survivin could significantly compensate for the viability under RAD51 knockdown. Differential Survivin localization also resulted in distinctive TMZ-triggered transcriptional pathways, associated with senescence and chromosome instability as shown by global transcriptome analysis. Orthotopic LN229 xenografts, expressing SurvNESmut exhibited diminished growth and increased DNA damage upon TMZ, as manifested by PCNA and γH2AX foci expression, respectively, in brain tissue sections. Consequently, those mice lived longer. Although tumors of high-grade glioma patients expressed majorly nuclear Survivin, they exhibited rarely NES mutations which did not correlate with survival. Based on our in vitro and xenograft data, Survivin nuclear trapping would facilitate glioma response to TMZ.

Highlights

Among the different subtypes of brain cancer, glioblastomas (WHO grade IV) account for the majority (> 50%) of malignant brain tumors [1, 2]
We addressed the question whether its differential localization alters the reproductive survival of glioblastoma cells and whether this is associated with changes in the repair of double-strand break (DSB) by homologous recombination (HR), induction of chromosome aberrations and the onset of senescence
The generated clones express either wt-Survivin fused to GFP, or a fusion protein mutated in the Survivin nuclear export sequence (LN229-SurvNESmut or A172-SurvNESmut), as shown by western-blot

Summary

Introduction

Among the different subtypes of brain cancer, glioblastomas (WHO grade IV) account for the majority (> 50%) of malignant brain tumors [1, 2]. Since TMZ exerts its cytotoxic effect by the induction of O6-methyguanine (O6MeG), and subsequent formation of DNA double-strand breaks (DSBs), success of glioblastoma therapy strongly depends on the DNA repair capacity of the tumor. Persistent O6MeG can be converted into DSBs via futile DNA mismatch repair (MMR) in the second replication cycle after TMZ exposure [4]. If these DSBs are not repaired by homologous recombination (HR), they result in chromosome aberrations and the activation of cell death via apoptosis [5, 6]. Error-prone non-homologous end-joining (NHEJ) seems not to protect against O 6-methyguanine – triggered DSBs, chromosome aberrations and cell death [8]

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