Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
Institute of Radiation Oncology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
J Exp Clin Cancer Res. 2024 Jan 23;43(1):28. doi: 10.1186/s13046-024-02954-8.
Glioblastoma (GBM) is the most common malignant tumor of the central nervous system. It is an aggressive tumor characterized by rapid proliferation, diffuse tumor morphology, and poor prognosis. Unfortunately, current treatments, such as surgery, radiotherapy, and chemotherapy, are unable to achieve good outcomes. Therefore, there is an urgent need to explore new treatment targets. A detailed mechanistic exploration of the role of the nuclear pore transporter KPNB1 in GBM is lacking. This study demonstrated that KPNB1 regulated GBM progression through a transcription factor YBX1 to promote the expression of post-protrusion membrane protein NLGN3. This regulation was mediated by the deubiquitinating enzyme USP7.
A tissue microarray was used to measure the expression of KPNB1 and USP7 in glioma tissues. The effects of KPNB1 knockdown on the tumorigenic properties of glioma cells were characterized by colony formation assays, Transwell migration assay, EdU proliferation assays, CCK-8 viability assays, and apoptosis analysis using flow cytometry. Transcriptome sequencing identified NLGN3 as a downstream molecule that is regulated by KPNB1. Mass spectrometry and immunoprecipitation were performed to analyze the potential interaction between KPNB1 and YBX1. Moreover, the nuclear translocation of YBX1 was determined with nuclear-cytoplasmic fractionation and immunofluorescence staining, and chromatin immunoprecipitation assays were conducted to study DNA binding with YBX1. Ubiquitination assays were performed to determine the effects of USP7 on KPNB1 stability. The intracranial orthotopic tumor model was used to detect the efficacy in vivo.
In this study, we found that the nuclear receptor KPNB1 was highly expressed in GBM and could mediate the nuclear translocation of macromolecules to promote GBM progression. Knockdown of KPNB1 inhibited the progression of GBM, both in vitro and in vivo. In addition, we found that KPNB1 could regulate the downstream expression of Neuroligin-3 (NLGN3) by mediating the nuclear import of transcription factor YBX1, which could bind to the NLGN3 promoter. NLGN3 was necessary and sufficient to promote glioma cell growth. Furthermore, we found that deubiquitinase USP7 played a critical role in stabilizing KPNB1 through deubiquitination. Knockdown of USP7 expression or inhibition of its activity could effectively impair GBM progression. In vivo experiments also demonstrated the promoting effects of USP7, KPNB1, and NLGN3 on GBM progression. Overall, our results suggested that KPNB1 stability was enhanced by USP7-mediated deubiquitination, and the overexpression of KPNB1 could promote GBM progression via the nuclear translocation of YBX1 and the subsequent increase in NLGN3 expression.
This study identified a novel and targetable USP7/KPNB1/YBX1/NLGN3 signaling axis in GBM cells.
胶质母细胞瘤（GBM）是中枢神经系统最常见的恶性肿瘤。它是一种侵袭性肿瘤，其特征为快速增殖、弥漫性肿瘤形态和预后不良。不幸的是，目前的治疗方法，如手术、放疗和化疗，无法取得良好的效果。因此，迫切需要探索新的治疗靶点。核孔转运蛋白 KPNB1 在 GBM 中的作用机制尚不清楚。本研究表明，KPNB1 通过转录因子 YBX1 调节 GBM 进展，从而促进突起后膜蛋白 NLGN3 的表达。这种调节是由去泛素化酶 USP7 介导的。
使用组织微阵列测量Glioma 组织中 KPNB1 和 USP7 的表达。通过集落形成实验、Transwell 迁移实验、EdU 增殖实验、CCK-8 活力实验和流式细胞术分析细胞凋亡来研究 KPNB1 敲低对Glioma 细胞致瘤特性的影响。转录组测序确定 NLGN3 是受 KPNB1 调节的下游分子。通过质谱和免疫沉淀分析 KPNB1 与 YBX1 之间的潜在相互作用。此外，通过核质分离和免疫荧光染色来确定 YBX1 的核转位，并通过染色质免疫沉淀实验研究 YBX1 与 DNA 的结合。进行泛素化实验以确定 USP7 对 KPNB1 稳定性的影响。使用颅内原位肿瘤模型在体内检测疗效。
在这项研究中，我们发现核受体 KPNB1 在 GBM 中高度表达，能够介导大分子的核内转运，从而促进 GBM 的进展。KPNB1 敲低抑制了 GBM 的进展，无论是在体外还是体内。此外，我们发现 KPNB1 可以通过介导转录因子 YBX1 的核内输入来调节下游 Neuroligin-3（NLGN3）的表达，YBX1 可以与 NLGN3 启动子结合。NLGN3 是促进Glioma 细胞生长所必需的。此外，我们发现去泛素酶 USP7 通过去泛素化在稳定 KPNB1 中起关键作用。敲低 USP7 的表达或抑制其活性可有效损害 GBM 的进展。体内实验也证明了 USP7、KPNB1 和 NLGN3 对 GBM 进展的促进作用。总的来说，我们的研究结果表明，USP7 介导的去泛素化增强了 KPNB1 的稳定性，KPNB1 的过表达可通过核内 YBX1 的转位和随后 NLGN3 表达的增加来促进 GBM 的进展。
本研究在 GBM 细胞中鉴定了一个新的可靶向的 USP7/KPNB1/YBX1/NLGN3 信号轴。
