School of Biomedicine, Far Eastern Federal University, Vladivostok 690091, Russia.
N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia, Moscow 115478, Russia.
Oncol Rep. 2019 May;41(5):3080-3088. doi: 10.3892/or.2019.7043. Epub 2019 Mar 5.
Glioblastoma multiforme (GBM) is one of the most aggressive types of brain tumor and is highly resistant to therapy. The median survival time for patients with GBM is 15 months. GBM resistance to treatment is associated with cancer stem cells (CSCs). CD133 membrane glycoprotein is the best‑known marker of GBM CSCs. The Wnt signaling pathway plays an important role in the proliferation of all stem cells. To the best of our knowledge, the present study was the first to examine the expression levels of proteins associated with the Wnt signaling pathway in СD133+ CSCs of human GBM. Furthermore, potential targets that may regulate СD133+ CSCs in human GBM were investigated. The human GBM U‑87MG cell line was cultured in neurobasal medium supplemented with B27, fibroblast growth factor, epidermal growth factor and no serum. Immunohistochemical characteristics of glioma spheres were investigated based on the expression of key markers of CSCs. CD133+ cells were extracted from glioma spheres by cell sorting and then lysed. High‑performance liquid chromatography‑mass spectrometry was used for proteome analysis. Lysates of CD133‑ cells in GBM were used for comparison. The present study was the first to describe the conceptual proteome differences between GBM and CD133+ CSCs of the common pool. Major differences were identified in the glycolysis/gluconeogenesis, focal adhesion, tight junction and Wnt signaling pathways. This study aimed to analyze the crucial role that proteins of the Wnt signaling pathway play in stem cell proliferation. The identified proteins were analyzed for their association with the Wnt signaling pathway using the international open databases PubMed, Protein Analysis Through Evolutionary Relationships, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and Search Tool for the Retrieval of Interacting Genes/Proteins. An increased expression of 12 proteins associated with the Wnt signaling pathway were identified in GBM CD133+ CSCs, which included catenin β‑1, disheveled associated activator of morphogenesis 1, RAC family small GTPase 2 and RAS homolog gene family member A, a number of which are also associated with adherens junctions. The Wnt signaling pathway is not upregulated in CSCs; however, the high expression levels of adenomatous polyposis coli, β‑catenin, C‑terminal binding protein (CtBP) and RuvB‑like AAA ATPase 1 (RUVBL1 or Pontin52) proteins suggest the possibility of alternative activation of specific genes in the nuclei of these cells. Calcyclin‑binding protein, casein kinase II α, casein kinase II β, CtBP1, CtBP2, CUL1 and RUVBL1 proteins may be used as targets for the pharmaceutical regulation of CSCs in complex GBM treatment.
多形性胶质母细胞瘤（GBM）是最具侵袭性的脑肿瘤之一，对治疗具有高度抗性。GBM 患者的中位生存时间为 15 个月。GBM 对治疗的抗性与癌症干细胞（CSC）有关。CD133 膜糖蛋白是 GBM CSC 的最佳标志物。Wnt 信号通路在所有干细胞的增殖中起着重要作用。据我们所知，本研究首次研究了与人类 GBM 的 CD133+CSC 相关的 Wnt 信号通路蛋白的表达水平。此外，还研究了可能调节人类 GBM 中 CD133+CSC 的潜在靶标。将人 GBM U-87MG 细胞系在含有 B27、成纤维细胞生长因子、表皮生长因子和无血清的神经基底培养基中培养。根据 CSC 关键标志物的表达，研究了神经胶质瘤球体的免疫组织化学特征。通过细胞分选从神经胶质瘤球体中提取 CD133+细胞，然后裂解。采用高效液相色谱-质谱法进行蛋白质组分析。将 GBM 中 CD133-细胞的裂解物用于比较。本研究首次描述了 GBM 和常见池中的 CD133+CSC 之间概念性蛋白质组的差异。在糖酵解/糖异生、焦点粘连、紧密连接和 Wnt 信号通路中发现了主要差异。本研究旨在分析 Wnt 信号通路蛋白在干细胞增殖中的关键作用。使用国际开放数据库 PubMed、通过进化关系进行蛋白质分析、基因本体论、京都基因与基因组百科全书和检索相互作用基因/蛋白质的工具，对与 Wnt 信号通路相关的鉴定蛋白进行分析。在 GBM CD133+CSC 中鉴定出与 Wnt 信号通路相关的 12 种蛋白表达增加，其中包括连环蛋白β-1、形态发生的卷曲相关激活物 1、RAC 家族小 GTP 酶 2 和 RAS 同源基因家族成员 A，其中一些也与黏着斑有关。Wnt 信号通路在 CSC 中并未上调；然而，腺瘤性结肠息肉病蛋白、β-连环蛋白、C 端结合蛋白（CtBP）和 RuvB 样 AAA ATPase 1（RUVBL1 或 Pontin52）蛋白的高表达表明，这些细胞的核中可能存在特定基因的替代激活。钙调蛋白结合蛋白、酪蛋白激酶 IIα、酪蛋白激酶 IIβ、CtBP1、CtBP2、CUL1 和 RUVBL1 蛋白可用作复杂 GBM 治疗中 CSC 药物调节的靶标。
