Gene expression in GBM with Cav3.2 inhibition

Glioblastoma stem cells (GSCs) have been implicated in tumor initiation, progression and resistance to therapy. We investigated the expression, function, mechanisms of action and therapeutic targeting of T-type calcium channels (Cav3.2) with the FDA approved and repurposed drug mibefradil in glioblastoma (GBM), and GSCs. We found that Cav3.2 is highly expressed in human GBM specimens and enriched in GCSs. Analyses of TCGA and REMBRANDT databases confirmed the upregulation of Cav3.2 in a subset of tumors (TCGA) and showed that overexpression is associated with worse prognosis. Mibefradil and Cav3.2 knockdown inhibited the growth, survival and stemness of GSCs and sensitized them to temozolomide (TMZ) chemotherapy. To investigate the mechanisms of action of Cav3.2 in GSC, we performed proteomic and transcriptomic screenings followed by functional rescue experiments. Inhibition of Cav3.2 altered cancer signaling pathways and gene transcription. Among other, inhibition of Cav3.2 suppressed GSC growth through inhibition of pro-survival pathways AKT/mTOR, and induction of apoptosis through upregulation of survivin, BAX and cleavage of caspase 9 and PARP. Inhibition of Cav3.2 induced a decrease in the expression of oncogenes including PDGFA, PDGFB and TGFB1 and an increase in the expression of tumor suppressors including TNFRSF14 and HSD17B14. In vivo oral administration of Cav3.2 blocker mibefradil significantly inhibited GSC-derived xenograft growth, prolonged animal survival and sensitized the tumors to TMZ. Our study represents the first comprehensive characterization of Cav3.2 in GBM tumors and GSC. The findings establish Cav3.2 inhibition with the repurposed FDA-approved drug mibefradil as a new strategy for GBM therapy.

胶质母细胞瘤干细胞（GSCs）已被证实参与肿瘤起始、进展及治疗抵抗过程。本研究针对胶质母细胞瘤（GBM）及GSCs，探究了T型钙通道（Cav3.2）的表达特征、生物学功能、作用机制与治疗靶向策略，并使用经美国食品药品监督管理局（FDA）批准且获批重用途的药物米贝拉地尔（mibefradil）开展相关实验。研究发现，Cav3.2在人类胶质母细胞瘤标本中呈高表达，并在GSCs中富集。对癌症基因组图谱（TCGA）与REMBRANDT数据库的分析证实，Cav3.2在部分肿瘤（TCGA队列）中存在上调现象，且其过表达与不良预后相关。米贝拉地尔处理与Cav3.2基因敲低均可抑制GSCs的增殖、存活与干细胞干性，并可增强其对替莫唑胺（TMZ）化疗的敏感性。为探究Cav3.2在GSCs中的作用机制，本研究开展了蛋白质组学与转录组学筛选，并随后进行了功能拯救实验。结果显示，抑制Cav3.2可改变肿瘤信号通路与基因转录模式。具体而言，Cav3.2抑制可通过阻断促存活通路AKT/mTOR抑制GSCs增殖，并通过上调生存素（survivin）、BAX的表达以及切割半胱天冬酶9（caspase 9）与多聚ADP核糖聚合酶（PARP）诱导细胞凋亡。抑制Cav3.2可降低PDGFA、PDGFB与TGFB1等癌基因的表达水平，并上调TNFRSF14与HSD17B14等抑癌基因的表达。体内实验中，口服给予Cav3.2阻滞剂米贝拉地尔可显著抑制GSCs来源的异种移植瘤生长，延长受试动物的生存期，并增强肿瘤对替莫唑胺的敏感性。本研究首次对胶质母细胞瘤与GSCs中的Cav3.2进行了全面的特征解析。本研究结果证实，利用获批重用途的FDA批准药物米贝拉地尔抑制Cav3.2，可作为胶质母细胞瘤治疗的全新策略。