PDIA3P1 Promotes Temozolomide Resistance in Glioblastoma by Inhibiting C/EBPβ Degradation to Facilitate Proneural-to- Mesenchymal Transition

Abstract:

Background Resistance to temozolomide (TMZ) is a major obstacle to prevention of recurrence after surgery for glioblastoma (GBM). Although long noncoding RNAs (lncRNAs) play wide roles in GBM, the lncRNAs that regulate TMZ resistance have not been elucidated clearly. This study aims to identify lncRNAs that may affect TMZ treatment sensitivity and to explore novel therapeutic strategies to overcome TMZ resistance in GBM.

Methods The lncRNAs associated with TMZ resistance were explored based on Cancer Cell Line Encyclopedia (CCLE) dataset and Genomics of Drug Sensitivity in Cancer (GDSC) dataset.Quantitative real-time PCR (qRT-PRC) was used to detect the expression of PDIA3P1 in TMZ-resistant and TMZ-sensitive GBM cell lines. Both gain-of-function and loss-of-function studies were used to assess the effects of PDIA3P1 on TMZ resistance through in vitro and in vivo assays. Glioma Stem Cells (GSCs) were used to determine the effect of PDIA3P1 on GBM subtype. The hypothesis that PDIA3P1 promotes proneural-to-mesenchymal transition (PMT) was established by bioinformatics analysis and functional experiments. RNA pull-down and RNA immunoprecipitation (RIP) assays were performed to examine the interaction between PDIA3P1 and C/EBPβ. The post-translational modification mechanism of C/EBPβ was verified using ubiquitination and co-immunoprecipitation (co-IP) experiments. The CompuSyn was leveraged to calculate the combination index (CI) and the antitumor effect of TMZ with Neflamapimod (NEF) was validated both in vitro and in vivo.

Results We identified a lncRNA, PDIA3P1, which was upregulated in TMZ-resistant GBM cell lines. Overexpression of PDIA3P1 promoted the acquisition of TMZ resistance, whereas knockdown of PDIA3P1 restored TMZ sensitivity. PDIA3P1 was upregulated in MES-GBM, promoted PMT progression in GSCs, and caused GBMs to be more resistant to TMZ treatment. Mechanistically, PDIA3P1 disrupted C/EBPβ-MDM2 complex and stabilized C/EBPβ protein by preventing it from MDM2-mediated ubiquitination. The expression of PDIA3P1 was up-regulated in a time- and a concentration-dependent manner after TMZ treatment. The TMZ-induced upregulation of PDIA3P1 was mediated by p38α-MAPK signaling pathway. NEF is a small molecule drug that specifically targets p38α with excellent blood-brain barrier (BBB) permeability. NEF could block TMZ-responsive PDIA3P1 upregulation and produced synergistic effects with TMZ at specific concentrations. The combination of TMZ and NEF exhibited excellent synergistic antitumor effects in vitro and in vivo.

Conclusions PDIA3P1 promoted PMT through stabilization of C/EBPβ, thereby reducing the sensitivity of GBM cells to TMZ treatment. NEF could inhibit TMZ-responsive PDIA3P1 upregulation and NEF combined with TMZ could provide better anti-tumor effects.