Targeting the Fn14-Rac1 signaling pathway in invasive gliomas

描述(由申请人提供)：由于不能有效地靶向侵袭细胞，用辅助疗法治疗恶性多形性胶质母细胞瘤(GB)的疗效仍然很大程度上不成功。Fn14在晚期胶质瘤中的表达水平升高。Fn14诱导的胶质瘤侵袭是由小GTPase rac1介导的；Fn14 TRAF结合区的缺失不能激活rac1蛋白。我们发现，鸟嘌呤核苷酸交换因子ECT2在GBM中高表达，ECT2和Fn14的过度表达与肿瘤分级直接相关，与患者预后呈负相关。Ect2可与Fn14胞浆复合体结合，调节rac1的激活。Rac1在许多正常组织中普遍存在，但在许多恶性状态下的信号转导病理中，rac1作为下游分子的中心位置，提示应利用靶向rac1的关键调控因子来发现侵袭胶质瘤细胞的关键易感性。我们假设Fn14通过rac1发挥作用，增强恶性胶质母细胞瘤细胞的侵袭性和存活率。具体目的如下：1)明确Fn14激活胶质瘤细胞中Rac1的近端机制(S)。我们将询问参与Fn14介导的Rac1激活的信号蛋白(S)的特定生化模型。我们将首先验证Ect2在Fn14诱导的rac1激活中的作用，并确定Ect2是Fn14下游的CDC42激活的rac1的直接交换因子还是rac1的间接交换因子。其次，我们将确定ECT2是否直接或间接结合到Fn14‘S细胞质尾部的TRAF位点。2)探讨Fn14-rac1信号轴在脑胶质瘤侵袭、转移和治疗反应中的作用。通过使用siRNAs抑制Fn14信号蛋白(Ect2，Traf)的表达水平，将进行生物学验证，以评估这些蛋白对胶质瘤细胞体外迁移、体外大鼠脑片细胞侵袭以及体外侵袭性胶质瘤细胞对细胞毒治疗的易感性的影响。此外，Fn14信号蛋白的丰度和激活的验证将使用免疫组织化学(IHC)技术来确定胶质瘤侵袭性TMA。3)确定抑制胶质母细胞瘤移植瘤中Fn14或其信号蛋白(S)是否导致体内肿瘤生长和细胞侵袭减少，并促进细胞死亡的激活。通过阻断抗人Fn14的单抗或针对Fn14信号蛋白(S)的shRNA(ECT2)对Fn14信号成分的抑制和操纵，以评估Fn14及其信号成分作为靶向治疗侵袭性原发人恶性胶质母细胞瘤异种移植瘤的适用性。这些生物疗法在细胞毒剂替莫唑胺诱导细胞死亡中的作用将被用来评估这些生物疗法的效果。中位队列生存率(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下游信号转导靶标(rac1和核因子：B的激活)的检测将被用来衡量生物治疗的有效性。 项目简介：多形性胶质母细胞瘤(GBM)的一个特征是肿瘤细胞侵袭周围正常大脑的能力。这些侵入的细胞在手术中会被遗漏，可以躲避集中的辐射，也可以躲避化疗。初步研究表明，跨膜受体Fn14随着胶质瘤的进展而上调；Fn14的表达与患者预后不良呈负相关。Fn14的激活介导了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Fn14或其信号蛋白的治疗作为手术切除的辅助手段，可能会特异性地针对侵袭性胶质瘤细胞，并改善这种毁灭性癌症的预后。

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.