Elucidating the role of EYA2 at centrosomes in glioblastoma stem cells

阐明 EYA2 在胶质母细胞瘤干细胞中心体的作用

• 批准号: 10751806
• 负责人: Arthur R Wolin
• 金额: $ 3.59万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751806
• 关键词: 2-tyrosine; 3' Untranslated Regions; AKAP9 gene; Adult; Antibodies; Apoptosis; Binding; Biological Assay; Biology; Brain; Cell Cycle Arrest; Cell Cycle Progression; Cells; Centrosome; Characteristics; Chromosome Segregation; Co-Immunoprecipitations; Collaborations; Cytoplasm; Data; Defect; Development; Disease; Exhibits; Eye; Foundations; G2/M Arrest; Genetic Variation; Glioblastoma; In Vitro; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mitosis; Mitotic; Mitotic spindle; Molecular; Nuclear; PLK1 gene; Peptides; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotyrosine; Population; Proliferating; Protein Tyrosine Phosphatase; Proteins; Recurrent tumor; Role; Testing; Time; Transcription Coactivator; Trimethoprim-Sulfamethoxazole; Western Blotting; Work; efficacious treatment; experimental study; immunocytochemistry; inhibitor; knock-down; mimetics; mutant; nerve stem cell; novel; overexpression; pharmacologic; progenitor; radioresistant; small hairpin RNA; stem; stem cell proliferation; stem cells; survival outcome; therapeutic target; tumorigenic

PROJECT SUMMARY/ABSTRACT Glioblastoma multiforme (GBM) is an aggressive, genetically diverse, and universally lethal malignancy of the brain. The aggressive characteristics and genetic diversity observed in GBM have been linked to a population of cells with stem-like characteristics known as glioblastoma stem cells (GSCs). In collaboration with the Rich lab, we have shown that EYA2 (eyes absent transcriptional coactivator and phosphatase 2) is highly expressed in GSCs compared to differentiated glioblastoma cells (DGCs) and neural stem cells (NSCs), where it is highly localized to centrosomes. Importantly, GSCs are uniquely reliant on EYA2’s tyrosine phosphatase (Tyr Ptase) activity for proliferation, as pharmacological inhibition of EYA2’s Tyr Ptase activity or overexpression of an EYA2 Tyr Ptase dead mutant causes cell cycle arrest, mono- and multipolar spindle abnormalities, and apoptosis when compared to DGCs and NSCs. Together, these data provide evidence that the Tyr Ptase activity of EYA2 elicits a novel function at centrosomes that is required for GSC proliferation. We have previously demonstrated that several cancers re-express EYA proteins to promote tumorigenic and metastatic phenotypes through multiple cytoplasmic and nuclear functions. The Tyr Ptase activity of EYA proteins is implicated in cell cycle progression in several cancers, but the molecular cause of these phenotypes is mostly unknown. Intriguingly, EYA proteins have not previously been shown to function at centrosomes. My data highlights that EYA2 is localized to centrosomes in GSCs and that inhibition of EYA2’s Tyr Ptase activity leads to centrosome fragmentation in mitotic GSCs, suggesting a novel, mitotic regulatory role for EYA2 in cancer. Furthermore, using EYA2 substrate-trapping experiments I have identified several centrosome- associated proteins and mitotic regulators as potential substrates of EYA2’s Tyr Ptase activity. Because the precise mechanism for EYA2’s Tyr Ptase activity at centrosomes and in mitotic spindle formation remains elusive, in this proposal I will test the hypothesis that EYA2’s tyrosine phosphatase activity is directly required at centrosomes for centrosome maturation and mitotic spindle formation, and that direct targets of the EYA2 Tyr Ptase activity can be identified that are critical for mitotic progression.

项目总结/摘要 多形性胶质母细胞瘤（GBM）是一种侵袭性，遗传多样性和普遍致命的肿瘤。 脑恶性肿瘤在GBM中观察到的攻击性特征和遗传多样性 与一群具有干细胞样特征的细胞有关，称为胶质母细胞瘤干细胞 （GSC）。在与Rich实验室的合作中，我们已经证明EYA 2（眼睛缺乏转录 与分化的胶质母细胞瘤相比， 细胞（DGC）和神经干细胞（NSC），其中它高度定位于中心体。重要的是， GSC独特地依赖于EYA 2的酪氨酸磷酸酶（Tyr Ptase）活性进行增殖， EYA 2的Tyr Ptase活性的药理学抑制或EYA 2 Tyr Ptase死亡的过表达 突变体引起细胞周期停滞，单极和多极纺锤体异常， 与DGC和NSC相比。总之，这些数据提供了证据表明， EYA 2在GSC增殖所需的中心体中具有新的功能。 我们以前已经证明，一些癌症重新表达EYA蛋白，以促进 肿瘤发生和转移表型通过多种细胞质和核功能。轮胎 EYA蛋白的Ptase活性与几种癌症的细胞周期进展有关，但EYA蛋白的Ptase活性与细胞周期进展有关。 这些表型的分子原因大多是未知的。有趣的是，EYA蛋白没有 以前被证明在中心体发挥作用。我的数据强调EYA 2被本地化为 GSC中的中心体，并且EYA 2的Tyr Ptase活性的抑制导致中心体 有丝分裂GSC中的片段，表明EYA 2在癌症中的新的有丝分裂调节作用。 此外，使用EYA 2底物捕获实验，我已经确定了几个中心体- 相关蛋白和有丝分裂调节因子作为EYA 2的Tyr Ptase活性的潜在底物。 由于EYA 2在中心体和有丝分裂纺锤体中的Tyr蛋白酶活性的精确机制 形成仍然难以捉摸，在这个建议中，我将测试的假设，EYA 2的酪氨酸 磷酸酶活性是中心体成熟所直接需要的， 有丝分裂纺锤体形成，且EYA 2 Tyr Ptase活性直接靶标可以是 对有丝分裂的进程至关重要。