PDIA3P1 通过抑制 C/EBPβ 降解促进 Temozolomide 耐药，从而促进神经前体细胞向间充质转化。

PDIA3P1 promotes Temozolomide resistance in glioblastoma by inhibiting C/EBPβ degradation to facilitate proneural-to-mesenchymal transition.

机构信息

Department of Neurosurgery, Qilu Hospital of Shandong University, Cheeloo College of Medicine and Institute of Brain and Brain-Inspired Science, Shandong University, 107 Wenhua Western Road, Jinan, 250012, Shandong, China.

Shandong Key Laboratory of Brain Function Remodeling, Jinan, 250012, Shandong, China.

出版信息

J Exp Clin Cancer Res. 2022 Jul 15;41(1):223. doi: 10.1186/s13046-022-02431-0.

DOI:10.1186/s13046-022-02431-0

PMID:35836243

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9284800/

Abstract

BACKGROUND

Resistance to temozolomide (TMZ) is a major obstacle to preventing glioblastoma (GBM) recurrence after surgery. Although long noncoding RNAs (lncRNAs) play a variety of roles in GBM, the lncRNAs that regulate TMZ resistance have not yet been clearly elucidated. 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

LncRNAs associated with TMZ resistance were identified using the Cancer Cell Line Encyclopedia (CCLE) and Genomics of Drug Sensitivity in Cancer (GDSC) datasets. Quantitative real-time PCR (qRT-PCR) was used to determine 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 using in vitro and in vivo assays. Glioma stem cells (GSCs) were used to determine the effect of PDIA3P1 on the GBM subtype. The hypothesis that PDIA3P1 promotes proneural-to-mesenchymal transition (PMT) was established using 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 posttranslational modification mechanism of C/EBPβ was verified using ubiquitination and coimmunoprecipitation (co-IP) experiments. CompuSyn was leveraged to calculate the combination index (CI), and the antitumor effect of TMZ combined with nefllamapimod (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 the C/EBPβ-MDM2 complex and stabilized the C/EBPβ protein by preventing MDM2-mediated ubiquitination. Expression of PDIA3P1 was upregulated in a time- and concentration-dependent manner in response to TMZ treatment, and TMZ-induced upregulation of PDIA3P1 was mediated by the p38α-MAPK signaling pathway. NEF is a small molecule drug that specifically targets p38α with excellent blood-brain barrier (BBB) permeability. NEF blocked TMZ-responsive PDIA3P1 upregulation and produced synergistic effects when combined with TMZ at specific concentrations. The combination of TMZ and NEF exhibited excellent synergistic antitumor effects both in vitro and in vivo.

CONCLUSION

PDIA3P1 promotes PMT by stabilizing C/EBPβ, reducing the sensitivity of GBM cells to TMZ treatment. NEF inhibits TMZ-responsive PDIA3P1 upregulation, and NEF combined with TMZ provides better antitumor effects.

摘要

背景

替莫唑胺（TMZ）耐药是手术后防止胶质母细胞瘤（GBM）复发的主要障碍。虽然长链非编码 RNA（lncRNA）在 GBM 中发挥多种作用，但调节 TMZ 耐药的 lncRNA 尚未得到明确阐明。本研究旨在鉴定可能影响 TMZ 治疗敏感性的 lncRNA，并探索克服 GBM 中 TMZ 耐药的新治疗策略。

方法

使用癌症细胞系百科全书（CCLE）和癌症药物敏感性基因组学（GDSC）数据集鉴定与 TMZ 耐药相关的 lncRNA。使用定量实时 PCR（qRT-PCR）测定 TMZ 耐药和 TMZ 敏感 GBM 细胞系中 PDIA3P1 的表达。使用体外和体内测定，通过增益功能和丧失功能研究来评估 PDIA3P1 对 TMZ 耐药的影响。使用神经胶质瘤干细胞（GSCs）来确定 PDIA3P1 对 GBM 亚型的影响。通过生物信息学分析和功能实验建立了 PDIA3P1 促进神经前体细胞向间充质转化（PMT）的假说。使用 RNA 下拉和 RNA 免疫沉淀（RIP）测定来检查 PDIA3P1 与 C/EBPβ 之间的相互作用。使用泛素化和免疫共沉淀（co-IP）实验验证了 C/EBPβ 的翻译后修饰机制。利用 CompuSyn 计算组合指数（CI），并在体外和体内验证 TMZ 联合 nefllamapimod（NEF）的抗肿瘤作用。

结果

我们鉴定出一种在 TMZ 耐药 GBM 细胞系中上调的 lncRNA，PDIA3P1。PDIA3P1 的过表达促进了 TMZ 耐药的获得，而 PDIA3P1 的敲低则恢复了 TMZ 敏感性。PDIA3P1 在 MES-GBM 中上调，促进 GSCs 中的 PMT 进展，并使 GBM 对 TMZ 治疗更具耐药性。在机制上，PDIA3P1 通过阻止 MDM2 介导的泛素化破坏了 C/EBPβ-MDM2 复合物并稳定了 C/EBPβ 蛋白。PDIA3P1 的表达在 TMZ 处理时呈时间和浓度依赖性上调，并且 TMZ 诱导的 PDIA3P1 上调是由 p38α-MAPK 信号通路介导的。NEF 是一种小分子药物，对 p38α 具有特异性，血脑屏障（BBB）通透性良好。NEF 阻断 TMZ 反应性 PDIA3P1 的上调，并在特定浓度下与 TMZ 联合使用时产生协同作用。TMZ 和 NEF 的联合在体外和体内均表现出优异的协同抗肿瘤作用。