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

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.

SUBMITTER: Reich TR

PROVIDER: S-EPMC8257519 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHigh-grade glioma (HGG) is a fatal human cancer. Bortezomib, a proteasome inhibitor, has been approved for the treatment of multiple myeloma but its use in glioma awaits further investigation. This study aimed to explore the chemotherapeutic effect and the underlying mechanism of bortezomib on gliomas.MethodsU251 and U87 cell viability and proliferation were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, tumor cell spheroid growth, and colony formation assay. Cell apoptosis and cell cycle were detected by flow cytometry. Temozolomide (TMZ)-insensitive cell lines were induced by long-term TMZ treatment, and cells with stem cell characteristics were enriched with stem cell culture medium. The mRNA levels of interested genes were measured via reverse transcription-quantitative polymerase chain reaction, and protein levels were determined via Western blotting/immunofluorescent staining in cell lines and immunohistochemical staining in paraffin-embedded sections. Via inoculating U87 cells subcutaneously, glioma xenograft models in nude mice were established for drug experiments. Patient survival data were analyzed using the Kaplan-Meier method.ResultsBortezomib inhibited the viability and proliferation of U251 and U87 cells in a dose- and time-dependent manner by inducing apoptosis and cell cycle arrest. Bortezomib also significantly inhibited the spheroid growth, colony formation, and stem-like cell proliferation of U251 and U87 cells. When administrated in combination, bortezomib showed synergistic effect with TMZ in vitro and sensitized glioma to TMZ treatment both in vitro and in vivo. Bortezomib reduced both the mRNA and protein levels of Forkhead Box M1 (FOXM1) and its target gene Survivin. The FOXM1-Survivin axis was markedly up-regulated in established TMZ-insensitive glioma cell lines and HGG patients. Expression levels of FOXM1 and Survivin were positively correlated with each other and both related to poor prognosis in glioma patients.ConclusionsBortezomib was found to inhibit glioma growth and improved TMZ chemotherapy efficacy, probably via down-regulating the FOXM1-Survivin axis. Bortezomib might be a promising agent for treating malignant glioma, alone or in combination with TMZ.

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

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