NF-κB 和 FOSL1 的相互作用驱动神经胶质瘤干细胞特性。

Interaction of NF-κB and FOSL1 drives glioma stemness.

机构信息

Department of Microbiology, Biochemistry & Immunology, Morehouse School of Medicine, Atlanta, USA.

Department of Chemistry, Xavier University, 1 Drexel Dr, New Orleans, LA, USA.

出版信息

Cell Mol Life Sci. 2024 Jun 10;81(1):255. doi: 10.1007/s00018-024-05293-1.

DOI:10.1007/s00018-024-05293-1

PMID:38856747

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11335291/

Abstract

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor; GBM's inevitable recurrence suggests that glioblastoma stem cells (GSC) allow these tumors to persist. Our previous work showed that FOSL1, transactivated by the STAT3 gene, functions as a tumorigenic gene in glioma pathogenesis and acts as a diagnostic marker and potential drug target in glioma patients. Accumulating evidence shows that STAT3 and NF-κB cooperate to promote the development and progression of various cancers. The link between STAT3 and NF-κB suggests that NF-κB can also transcriptionally regulate FOSL1 and contribute to gliomagenesis. To investigate downstream molecules of FOSL1, we analyzed the transcriptome after overexpressing FOSL1 in a PDX-L14 line characterized by deficient FOSL1 expression. We then conducted immunohistochemical staining for FOSL1 and NF-κB p65 using rabbit polyclonal anti-FOSL1 and NF-κB p65 in glioma tissue microarrays (TMA) derived from 141 glioma patients and 15 healthy individuals. Next, mutants of the human FOSL1 promoter, featuring mutations in essential binding sites for NF-κB were generated using a Q5 site-directed mutagenesis kit. Subsequently, we examined luciferase activity in glioma cells and compared it to the wild-type FOSL1 promoter. Then, we explored the mutual regulation between NF-κB signaling and FOSL1 by modulating the expression of NF-κB or FOSL1. Subsequently, we assessed the activity of FOSL1 and NF-κB. To understand the role of FOSL1 in cell growth and stemness, we conducted a CCK-8 assay and cell cycle analysis, assessing apoptosis and GSC markers, ALDH1, and CD133 under varying FOSL1 expression conditions. Transcriptome analyses of downstream molecules of FOSL1 show that NF-κB signaling pathway is regulated by FOSL1. NF-κB p65 protein expression correlates to the expression of FOSL1 in glioma patients, and both are associated with glioma grades. NF-κB is a crucial transcription factor activating the FOSL1 promoter in glioma cells. Mutual regulation between NF-κB and FOSL1 contributes to glioma tumorigenesis and stemness through promoting G1/S transition and inhibiting apoptosis. Therefore, the FOSL1 molecular pathway is functionally connected to NF-κB activation, enhances stemness, and is indicative that FOSL1 may potentially be a novel GBM drug target.

摘要

多形性胶质母细胞瘤（GBM）是最常见和最恶性的原发性脑肿瘤；GBM 的不可避免的复发表明胶质母细胞瘤干细胞（GSC）允许这些肿瘤持续存在。我们之前的工作表明，STAT3 基因转录激活的 FOSL1 在神经胶质瘤发病机制中起致癌基因的作用，并作为神经胶质瘤患者的诊断标志物和潜在的药物靶点。越来越多的证据表明 STAT3 和 NF-κB 合作促进各种癌症的发展和进展。STAT3 和 NF-κB 之间的联系表明 NF-κB 也可以转录调节 FOSL1 并有助于神经胶质瘤的发生。为了研究 FOSL1 的下游分子，我们在 PDX-L14 系中过表达 FOSL1 后分析了转录组，该系特征是 FOSL1 表达不足。然后，我们使用兔多克隆抗 FOSL1 和 NF-κB p65 在源自 141 名神经胶质瘤患者和 15 名健康个体的神经胶质瘤组织微阵列（TMA）中进行了 FOSL1 和 NF-κB p65 的免疫组织化学染色。接下来，使用 Q5 定点诱变试剂盒生成了人类 FOSL1 启动子的突变体，该突变体中的 NF-κB 必需结合位点发生突变。随后，我们检查了神经胶质瘤细胞中的荧光素酶活性，并将其与野生型 FOSL1 启动子进行了比较。然后，我们通过调节 NF-κB 或 FOSL1 的表达来探索 NF-κB 信号与 FOSL1 之间的相互调节。随后，我们评估了 FOSL1 和 NF-κB 的活性。为了了解 FOSL1 在细胞生长和干细胞特性中的作用，我们在不同的 FOSL1 表达条件下进行了 CCK-8 测定和细胞周期分析，评估了细胞凋亡和 GSC 标志物 ALDH1 和 CD133。FOSL1 下游分子的转录组分析表明，NF-κB 信号通路受 FOSL1 调节。NF-κB p65 蛋白表达与神经胶质瘤患者中 FOSL1 的表达相关，并且两者均与神经胶质瘤分级相关。NF-κB 是一种关键的转录因子，可激活神经胶质瘤细胞中的 FOSL1 启动子。NF-κB 和 FOSL1 之间的相互调节通过促进 G1/S 过渡和抑制细胞凋亡有助于神经胶质瘤的发生和干细胞特性。因此，FOSL1 分子途径与 NF-κB 激活功能相关，增强了干细胞特性，并表明 FOSL1 可能是一种新的 GBM 药物靶点。