Plexin-B2 function in glioma invasion and glioma stem cell maintenance

 描述(申请人提供)：多形性胶质母细胞瘤(GBM)仍然是最致命的脑癌形式。目前迫切需要更好地确定调控GBM恶性特征的分子途径，特别是胶质瘤干细胞(GSC)的侵袭性和持久性，GSC也被称为肿瘤增殖细胞，由于其致瘤潜力和治疗耐药性，被认为是肿瘤复发的主要来源。Plexin-B2是一种轴突导向受体，最初被认为是在胶质瘤中高度上调的基因。我们最近的患者数据库分析发现，丛状蛋白-B2的上调与胶质瘤的分级和低存活率有关。这促使我们研究这一重要联系的机制基础。我们对人ATCC胶质瘤细胞系的研究表明，Plexin-B2的激活改变了肌动蛋白的细胞骨架，并通过激活Rho GTP酶促进了细胞的迁移。在颅内移植中，Plexin-B2 shRNA敲除阻碍了肿瘤的侵袭并破坏了胶质瘤的血管形成。我们从患者来源的GSC系获得的新的初步数据表明，除了前迁移作用外，Plexin-B2还可能调节胶质瘤干细胞的表型，包括干细胞标志物的表达、自我更新能力、增殖和分化潜力。在这些令人兴奋的结果的基础上，我们建议检验中心假设，即丛状蛋白-B2在促进胶质瘤侵袭和干细胞特性方面发挥多方面的作用。为了方便我们的研究，我们采用了CRISPR基因编辑技术，在患者来源的GSC系中产生了Plexin-B2的双等位突变。在目标1中，我们专注于胶质瘤的侵袭，并通过进行培养试验、脑片培养和颅内移植来探讨Plexin-B2信号是否影响GBM细胞的细胞骨架动力学和细胞运动性。通过比较GSCs及其分化后代的细胞迁移，这组研究还探讨了迁移潜力是否取决于分化等级。在目标2中，我们关注胶质瘤干细胞的特性，并询问丛状蛋白-B2是否有助于干细胞标记物的表达、自我更新能力、增殖和分化潜力以及治疗耐药。我们将在GSC颅内移植的存活队列中进行研究，以确定Plexin-B2缺失是否带来生存益处，以及它是否降低了辐射抵抗。在目标3中，我们专注于丛状蛋白-B2信号在介导迁移和干细胞特性中的功能靶点和下游效应器。我们询问Plexin-B2的哪个胞内域是主要的信号臂，如果细胞迁移是通过调节Rho GTP酶来介导的，STAT3是否作为核心信号枢纽发挥作用，以及干细胞因子SOX2或OLIG2是否在STAT3下游发挥作用以维持茎的完整性。最后，我们将研究黏附分子L1CAM和钙粘蛋白/原钙粘蛋白，这是我们最近对Plexin-B2基因敲除的GSC进行的RNA-Seq分析中发现的最受调控的一组基因。了解Plexin-B2如何调控GBM的恶性特征将为抑制胶质瘤生长和侵袭的新靶点开辟可能性。

 DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) remains the most deadly form of brain cancer. There is a dire need to better define the molecular pathways that regulate the malignant features of GBM, in particular, its invasiveness and the persistence of glioma stem cells (GSC), also known as tumor propagating cells, which are thought to be the main source of tumor recurrence due to their tumorigenic potential and therapy resistance. Plexin-B2 is an axon guidance receptor originally identified as a gene highly upregulated in glioma. Our recent patient database analyses found that upregulation of Plexin-B2 correlates with glioma grade and poor survival. This prompted us to investigate the mechanistic basis of this important link. Our studies in human ATCC glioma cell lines demonstrated that Plexin-B2 activation alters actin cytoskeleton and promotes cell migration through activation of Rho GTPases. In intracranial transplants, Plexin-B2 shRNA knockdown hindered invasion and disrupted glioma vascularization. Our new preliminary data from patient-derived GSC lines suggest that besides a promigratory role, Plexin-B2 may also regulate glioma stem cell phenotypes, including stem cell marker expression, self-renewal capacity, proliferation and differentiation potential. Building on these exciting results, we propose to test the central hypothesis that Plexin-B2 plays a multifaceted role in promoting glioma invasion and stem cell characteristics. To facilitate our studies, we have adopted CRISPR gene editing technology to generate bi-allelic mutations of Plexin-B2 in patient-derived GSC lines. In Aim 1, we focus on glioma invasion and ask if Plexin-B2 signaling influences cytoskeletal dynamics and cellular motility of GBM cells by performing culture assays, brain slice cultures, and intracranial transplants. By comparing cell migration of GSCs and their differentiated progeny, this set of studies also addresses whether migratory potential is contingent on differentiation hierarchy. In Aim 2, we focus on glioma stem cell characteristics and ask if Plexin-B2 contributes to stem cell marker expression, self-renewal capacity, proliferation and differentiation potential, and treatment resistance. We will investigate in survival cohorts with GSC intracranial transplants if Plexin-B2 deletion confers survival benefit and if it reduces radiation resistance. In Aim 3, we focus on functional targets and downstream effectors of Plexin-B2 signaling in mediating migration and stem cell properties. We ask which intracellular domain of Plexin-B2 is the main signaling arm, if cellular migration is mediated through modulation of Rho GTPases, whether STAT3 functions as a core signaling hub, and if stem cell factors SOX2 or OLIG2 function downstream of STAT3 for stemness maintenance. Lastly, we will study the adhesion molecules L1CAM and cadherins/protocadherins, the most significantly regulated group of genes that were identified in our recent RNA-Seq analysis of Plexin-B2 knockout GSC. Understanding how Plexin-B2 regulates the malignant features of GBM will open up possibilities for new targets to inhibit glioma growth and invasion.