Elucidating the role of EYA2 at centrosomes in glioblastoma stem cells

项目摘要/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性的肿瘤。 脑部恶性肿瘤。在GBM观察到的攻击性特征和遗传多样性 与一组具有干细胞样特征的细胞群相关联，称为胶质母细胞瘤干细胞 (GSC)。在与Rich实验室的合作中，我们已经证明了EYA2(眼睛缺席转录 与分化的胶质母细胞瘤相比，共激活因子和磷酸酶2在胶质母细胞瘤中高表达 细胞(DGC)和神经干细胞(NSCs)，在那里它高度定位于中心体。重要的是 GSC的增殖唯一依赖于EYA2‘S酪氨酸磷酸酶(Tyr Ptase)的活性，AS 药物抑制EYA2‘S酪氨酸酶活性或过表达EYA2酪氨酸酶 突变会导致细胞周期停滞、单极和多极纺锤体异常以及以下情况下的细胞凋亡 与DGC和NSCs相比。综上所述，这些数据提供了证据，证明了酪氨酸酶活性 EYA2在中心体诱导了一种新的功能，这是GSC增殖所必需的。 我们之前已经证明，几种癌症重新表达EYA蛋白可以促进 多种细胞质和核功能的致瘤和转移表型。轮胎 EYA蛋白的Ptase活性与几种癌症的细胞周期进展有关，但 这些表型的分子原因大多尚不清楚。有趣的是，EYA蛋白并没有 以前被证明在中心体起作用。我的数据显示，EYA2已本地化到 星形胶质细胞的中心体及抑制EYA2‘S酪氨酸蛋白酶活性导致中心体的形成 有丝分裂的GSCs中的碎裂，表明EYA2在癌症中具有新的有丝分裂调节作用。 此外，通过EYA2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剂作为EYA2‘S酪氨酸酶活性的潜在底物。 因为EYA2‘S酪氨酸磷酸酶在中心体和有丝分裂纺锤体中活性的确切机制 形成仍然难以捉摸，在这个提议中，我将检验EYA2‘S酪氨酸的假设 中心体成熟和中心体成熟直接需要中心体的磷酸酶活性。 有丝分裂纺锤体形成，且EYA2 Tyr Ptase活性直接靶点可以是 确定了对有丝分裂进程至关重要的基因。

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.