Department of Chemistry, Xavier University, 1 Drexel Dr, New Orleans, LA, USA.
Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, USA.
Cell Mol Life Sci. 2023 Aug 29;80(9):270. doi: 10.1007/s00018-023-04921-6.
We previously reported that TRPM7 regulates glioma cells' stemness through STAT3. In addition, we demonstrated that FOSL1 is a response gene for TRPM7, and the FOSL1 gene serves as an oncogene to promote glioma proliferation and invasion.
In the present study, we determined the effects of FOSL1 on glioma stem cell (GSC) markers CD133 and ALDH1 by flow cytometry, and the maintenance of stem cell activity by extreme limiting dilution assays (ELDA). To further gain insight into the mechanism by which TRPM7 activates transcription of the FOSL1 gene to contribute to glioma stemness, we constructed a FOSL1 promoter and its GAS mutants followed by luciferase reporter assays and ChIP-qPCR in a glioma cell line and glioma patient-derived xenoline. We further examined GSC markers ALDH1 and TRPM7 as well as FOSL1 by immunohistochemistry staining (IHC) in brain tissue microarray (TMA) of glioma patients.
We revealed that FOSL1 knockdown reduces the expression of GSC markers CD133 and ALDH1, and FOSL1 is required to maintain stem cell activity in glioma cells. The experiments also showed that mutations of - 328 to - 336 and - 378 to - 386 GAS elements markedly reduced FOSL1 promoter activity. Constitutively active STAT3 increased while dominant-negative STAT3 decreased FOSL1 promoter activity. Furthermore, overexpression of TRPM7 enhanced while silencing of TRPM7 reduced FOSL1 promoter activity. ChIP-qPCR assays revealed that STAT3, present in nuclear lysates of glioma cells stimulated by constitutively activated STAT3, can bind to two GAS elements, respectively. We demonstrated that deacetylation of FOSL1 at the Lys-116 residue located within its DNA binding domain led to an increase in FOSL1 transcriptional activity. We found that the expression of TRPM7, ALDH1, and FOSL1 protein is associated with grades of malignant glioma, and TRPM7 protein expression correlates to the expression of ALDH1 and FOSL1 in glioma patients.
These combined results demonstrated that TRPM7 induced FOSL1 transcriptional activation, which is mediated by the action of STAT3, a mechanism shown to be important in glioma stemness. These results indicated that FOSL1, similar to GSC markers ALDH1 and TRPM7, is a diagnostic marker and potential drug target for glioma patients.
我们之前的研究表明，TRPM7 通过 STAT3 调节神经胶质瘤细胞的干性。此外，我们还证实 FOSL1 是 TRPM7 的反应基因，并且该基因作为癌基因促进神经胶质瘤的增殖和侵袭。
在本研究中，我们通过流式细胞术确定了 FOSL1 对神经胶质瘤干细胞（GSC）标志物 CD133 和 ALDH1 的影响，并通过极端有限稀释测定（ELDA）维持干细胞活性。为了进一步深入了解 TRPM7 激活 FOSL1 基因转录以促进神经胶质瘤干性的机制，我们构建了 FOSL1 启动子及其 GAS 突变体，然后进行荧光素酶报告基因检测和 ChIP-qPCR 在神经胶质瘤细胞系和神经胶质瘤患者来源的异种系中。我们还通过免疫组织化学染色（IHC）在神经胶质瘤患者的组织微阵列（TMA）中进一步检查了 GSC 标志物 ALDH1、TRPM7 和 FOSL1。
我们揭示了 FOSL1 敲低降低了 GSC 标志物 CD133 和 ALDH1 的表达，并且 FOSL1 是维持神经胶质瘤细胞中干细胞活性所必需的。实验还表明，-328 到-336 和-378 到-386 GAS 元件的突变显着降低了 FOSL1 启动子活性。组成性激活的 STAT3 增加而显性失活的 STAT3 减少 FOSL1 启动子活性。此外，TRPM7 的过表达增强了 FOSL1 启动子活性，而 TRPM7 的沉默则降低了 FOSL1 启动子活性。ChIP-qPCR 检测显示，存在于由组成性激活的 STAT3 刺激的神经胶质瘤细胞核裂解物中的 STAT3 可以分别结合两个 GAS 元件。我们证明了位于其 DNA 结合结构域内的 Lys-116 残基上的 FOSL1 的去乙酰化导致 FOSL1 转录活性增加。我们发现 TRPM7、ALDH1 和 FOSL1 蛋白的表达与恶性神经胶质瘤的分级有关，并且 TRPM7 蛋白的表达与神经胶质瘤患者中 ALDH1 和 FOSL1 的表达相关。
这些综合结果表明，TRPM7 通过 STAT3 介导的作用诱导 FOSL1 转录激活，这一机制在神经胶质瘤干性中很重要。这些结果表明，FOSL1 与 GSC 标志物 ALDH1 和 TRPM7 一样，是神经胶质瘤患者的诊断标志物和潜在药物靶点。
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