Targeting the Fn14-Rac1 signaling pathway in invasive gliomas

描述（由申请人提供）：由于无法有效靶向入侵细胞，使用辅助疗法治疗恶性多形性胶质母细胞瘤（GB）的疗效在很大程度上仍然不成功。Fn 14表达水平在晚期胶质瘤中升高。Fn 14诱导的胶质瘤侵袭是由小的GTF受体Rac 1介导的; Fn 14的TRAF结合区的缺失未能激活Rac 1蛋白。我们发现Ect 2，一种鸟嘌呤核苷酸交换因子（GEF），在GBM中过表达，Ect 2和Fn 14的过表达与肿瘤分级直接相关，与患者预后呈负相关。Ect 2可以与Fn 14胞质复合物结合并调节Rac 1的活化。Rac 1在许多正常组织中是普遍存在的，然而Rac 1作为在恶性状态下操作的许多信号传导病理学中的下游参与者的中心位置表明，应利用靶向Rac 1的关键调节子来发现入侵胶质瘤细胞的关键漏洞。我们假设Fn 14通过Rac 1起作用，以增强恶性胶质母细胞瘤细胞的侵袭和存活。具体目的如下：1）确定Fn 14在胶质瘤细胞中激活Rac 1的近端机制。我们将查询参与Fn 14介导的Rac 1激活的信号蛋白的特定生化模型。我们将首先验证Ect 2在Fn 14诱导的Rac 1激活中的作用，并确定Ect 2是否是Rac 1的直接交换因子或通过Fn 14下游的Cdc 42激活的Rac 1的间接交换因子。其次，我们将确定Ect 2是否直接或间接结合到Fn 14的胞质尾区的TRAF位点。2)以证明Fn 14-Rac 1信号传导轴对胶质瘤迁移、侵袭和治疗反应的影响。将进行使用siRNA抑制Fn 14信号传导蛋白（Ect 2，TRAFs）表达水平的生物学验证，以评估这些蛋白对体外胶质瘤细胞迁移、使用离体大鼠脑切片的细胞侵袭以及体外侵袭性胶质瘤细胞对细胞毒性治疗的脆弱性的影响。此外，Fn 14信号蛋白的丰度和活化的验证将使用免疫组织化学（IHC）技术对胶质瘤侵袭TMA进行测定。3)以确定在胶质母细胞瘤异种移植物中Fn 14或其信号传导蛋白的抑制是否导致肿瘤生长和细胞侵袭减少以及促进体内细胞死亡的激活。使用针对人Fn 14的阻断性单克隆抗体或针对Fn 14信号传导蛋白（Ect 2）的shRNA抑制和操纵Fn 14信号传导组分，将用于评估Fn 14及其信号传导组分作为侵袭性原发性人恶性胶质母细胞瘤异种移植物靶向治疗的适用性。GBM的原位模型将用于评估这些生物疗法对细胞毒性剂替莫唑胺（Temador）诱导细胞死亡的影响。将采用中位队列生存期（Kaplan-Meier）以及肿瘤大小、侵袭模式和细胞死亡的组织学评估，以及通过ELISA和IHC技术检查下游信号传导靶点（Rac 1和NF：B活化）来测量生物疗法的有效性。 项目叙述：多形性胶质母细胞瘤（GBM）的一个标志是肿瘤细胞侵入周围正常大脑的能力。这些入侵的细胞被手术错过，逃避聚焦辐射，并躲避化疗。初步研究表明，跨膜受体，Fn 14，上调与胶质瘤进展; Fn 14表达与不良患者的结果呈负相关。Fn 14的活化介导这些恶性细胞的细胞侵袭和存活表型。因此，使用针对跨膜受体Fn 14或其信号传导蛋白的疗法作为手术摘除的辅助剂可以特异性地靶向侵袭性神经胶质瘤细胞并改善这种毁灭性癌症的结果。

DESCRIPTION (provided by applicant): The efficacy of treating malignant glioblastoma multiforme (GB) with adjuvant therapies remains largely unsuccessful due to the inability to effectively target invading cells. Fn14 expression level is elevated in advanced glial tumors. Fn14-induced glioma invasion is mediated by the small GTPase Rac1; deletion of the TRAF binding region of Fn14 failed to activate Rac1 protein. We found that Ect2, a guanine nucleotide exchange factor (GEF), is overexpressed in GBM, and that overexpression of both Ect2 and Fn14 is correlated directly with tumor grade and inversely with patient outcome. Ect2 can associate with the Fn14 cytoplasmic complex and regulate Rac1 activation. Rac1 is a ubiquitous in many normal tissues, however the central position of Rac1 as a downstream player in many signaling pathologies operating in malignant states, suggest that targeting key regulators of Rac1 should be exploited to discover key vulnerabilities for invading glioma cells. We HYPOTHESIZE that Fn14 operates via Rac1 to enhance malignant glioblastoma cell invasion and survival. The specific aims are as follow: 1) to define the proximal mechanism(s) by which the Fn14 activates Rac1 in glioma cells. We will query a specific biochemical model of the signaling protein(s) involved in Fn14- mediated Rac1 activation. We will first validate the role of Ect2 in this Fn14 induced Rac1 activation and determine whether Ect2 is a direct exchange factor for Rac1 or indirect exchange factor for Rac1 via Cdc42 activation downstream of Fn14. Secondly, we will determine whether Ect2 binds directly or indirectly to the TRAF site of the Fn14's cytoplasmic tail. 2) to demonstrate the impact of the Fn14-Rac1 signaling axis on glioma migration, invasion and therapy response. Biological validation using siRNAs directed to suppress the expression levels of the Fn14 signaling proteins (Ect2, TRAFs) will be performed to assess the effects of these proteins on glioma cell migration in vitro, cell invasion using ex vivo rat brain slices, and vulnerability of invasive glioma cells to cytotoxic therapy in vitro. Additionally, validation of the abundance and activation of Fn14 signaling proteins will be determined using immunohistochemical (IHC) techniques on glioma invasion TMAs. 3) to determine whether inhibition of Fn14 or its signaling protein(s) in glioblastoma xenografts leads to reduced tumor growth and cell invasion and facilitated activation of cell death in vivo. Inhibition and manipulation of Fn14 signaling components using blocking monoclonal antibodies to human Fn14 or shRNA directed against Fn14 signaling protein(s) (Ect2) will be used to assess the suitability of Fn14 and its signaling components as targeted therapies for invasive primary human malignant glioblastoma xenografts. Orthotopic models of GBMs will be used to assess effects of these biological therapies on induction of cell death by the cytotoxic agent temozolomide (Temador). Median cohort survival (Kaplan-Meier) as well as histological assessment of tumor size, invasion pattern and cell death in addition to examination of downstream signaling targets (Rac1 and NF:B activation) by ELISA and IHC techniques will be employed to measure the effectiveness of the biological therapies. PROJECT NARRATIVE: A hallmark of glioblastoma multiforme (GBM) is the ability of tumor cells to invade into surrounding normal brain. These invading cells are missed by surgery, escape focused radiation, and hide from chemotherapy. Preliminary studies demonstrate that the transmembrane receptor, Fn14, is upregulated with glioma progression; Fn14 expression correlates inversely with poor patient outcome. The activation of Fn14 mediates cell invasion and survival phenotypes of these malignant cells. Thus, the use of therapy against the transmembrane receptor Fn14 or its signaling proteins as an adjuvant to surgical extirpation may specifically target invasive glioma cells and improve the outcome of this devastating cancer.