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.

Targeting c-IAP1, c-IAP2, and Bcl-2 Eliminates Senescent Glioblastoma Cells Following Temozolomide Treatment

“…Of note, also for A172, the amount of senescent cells increased up to 75% at later post-exposure times. In all cell lines, senescence was accompanied by arrest in the G2-phase of the cell cycle, which confirms previous reports [ 8 , 48 ]. All cell lines showed the emergence of polyploid cells (20–30% of the population) upon treatment with 50 µM TMZ and a post-exposure time of 120 h. Very recently, we reported that glioma cells showing a strong accumulation of nuclear Survivin also exhibit an increased polyploid cell population after exposure to TMZ [ 48 ].…”

“…Overall, our data presented here and previously published indicate that in glioblastoma cells, TMZ induces senescence to a significantly higher level than cell death through apoptosis and necrosis [ 7 , 8 , 43 , 48 ]. Senescence is associated with activation of several anti-apoptotic factors, such as Bcl-2, c-IAP1, and c-IAP2.…”

Regulation of temozolomide resistance via lncRNAs: Clinical and biological properties of lncRNAs in gliomas (Review)

“…Song et al ( 115 ) previously indicated that the knockdown of survivin promoted glioma cell sensitivity to TMZ treatment by inducing the apoptosis of senescent cells. Furthermore, survivin nuclear trapping has been found to facilitate GBM response to TMZ ( 116 ).…”

Sensitive Imaging of Cellular RNA via Cascaded Proximity-Induced Fluorogenic Reactions

“…The first well-studied type of RNA as a biomarker is messenger RNA (mRNA), which is a single-stranded RNA transcribed from a specific DNA to guide protein synthesis. A number of mRNAs have been employed in disease diagnosis , or therapy. , For instance, survivin mRNA is known to be upregulated in most cancers, and its expression level is important for early diagnosis and medical prognosis. , To be noted, mRNAs exhibit complex dynamics in cells, including the expression, degradation, and translocation; thus, in situ live-cell imaging of target mRNAs is highly desirable to obtain their spatial–temporal information for better understanding the functions, identifying diseases, and evaluating therapy efficacy …”

“…3,4 For instance, survivin mRNA is known to be upregulated in most cancers, and its expression level is important for early diagnosis and medical prognosis. 5,6 To be noted, mRNAs exhibit complex dynamics in cells, including the expression, 5 degradation, 7 and translocation; 8 thus, in situ live-cell imaging of target mRNAs is highly desirable to obtain their spatial−temporal information for better understanding the functions, identifying diseases, and evaluating therapy efficacy. 9 Most available non-polymerase chain reaction (PCR) detection methods rely on probes without signal amplification [e.g., using classic molecular beacons (MBs) 10 or linear templated reactions 11−13 ], where the signal and the target mRNA are in an equivalent ratio (1:1), hindering the detection of low-expression mRNA.…”