Cath B and uPAR si RNA constructs regressed glioma growth

描述(申请人提供)：尽管对多形性胶质母细胞瘤采取了许多治疗策略，但患有这种侵袭性脑恶性肿瘤的患者的存活率仍然很低。即使联合使用几种治疗方法，良好的预后也是极其罕见的，因为残留的癌细胞不可避免地渗透到正常脑组织并导致肿瘤复发。局部侵袭仍然是脑肿瘤发病率和死亡率的重要原因。我的实验室的长期目标是了解人类胶质瘤肿瘤侵袭性的细胞和分子机制。放射治疗仍然是控制恶性和良性胶质母细胞瘤的主要治疗方法之一。在残留或复发的良性肿瘤患者中，人们越来越担心放射相关的副作用，即使在放射外科等高度精确的治疗下也可能发生这些副作用。尽管有一定的治疗效果，但最近的证据表明，辐射可能通过激活与肿瘤侵袭、血管生成和转移有关的几条途径，在体外和体内促进癌细胞的恶性行为。此外，胶质瘤启动细胞对放射治疗具有抵抗力。包括我们的研究在内的几项研究表明，恶性胶质瘤中组织B和uPAR水平的显著增加和辐射进一步激活了组织B和uPAR分子。我们假设Cath B和uPAR基因将被靶向，同时结合辐射，其抗癌效果将被确定。本研究的具体目的是：1.在体外模型中评价针对组织蛋白酶B和uPAR的siRNA表达载体(PCU)对胶质瘤细胞的黏附、迁移和侵袭的影响。目标1a。确定PC、PU、PCU和单独及联合照射对胶质母细胞瘤细胞系和胶质瘤启动细胞(GIC)中Cath B和uPAR水平的影响。目标1b。测定照射前后Cath B和uPAR对胶质母细胞瘤细胞系和胶质瘤起始细胞的黏附和迁移的下调作用。目标1c。评价PC、PU、PCU和辐射单独及联合对人胶质母细胞瘤细胞系和GIC侵袭行为的影响。具体目的2.研究辐射对GIC和非GIC细胞生长、增殖和凋亡影响的分子机制。目标2a。评估在非GIC和GIC中照射和不照射对细胞生长和增殖的下调作用的分子机制。目标2b。确定照射前后Cath B和uPAR下调对胶质母细胞瘤细胞系和GIC细胞凋亡的分子机制的影响。具体目的3.确定B组织和uPAR抑制非GIC和GIC裸鼠照射前后血管生成和肿瘤生长的分子机制。目标3a。评估Cath B和uPAR下调介导的信号机制改变在体外和体内有无放射治疗的脑血管生成调节中的作用。目标3b。确定PC、PU、PCU单独及联合照射对预先建立的颅内肿瘤生长或裸鼠脑内注射的人胶质母细胞瘤细胞系和GIC侵袭力的最佳剂量。建议的研究应该会对Cath B和uPAR的发病机制产生重大的洞察力，进而应该为胶质母细胞瘤的治疗干预提供新的靶点。 公共卫生相关性：尽管对多形性胶质母细胞瘤采取了许多治疗策略，但患有这种侵袭性脑恶性肿瘤的患者的存活率仍然很低。25年来，放射治疗一直是胶质母细胞瘤最有效的单一治疗方法。然而，越来越多的长期存活者有继发性副作用，以及这种治疗方法对胶质瘤启动细胞的抵抗力，突显了开发新的治疗方法的必要性。这项建议代表了一种联合治疗方法，使用针对组织蛋白酶B和uPAR的单个和双顺反子siRNA构建。这一策略可能会改善治疗胶质母细胞瘤的放射治疗结果。

DESCRIPTION (provided by applicant): Despite the many therapeutic strategies undertaken for treatment of glioblastoma multiforme, the survival rate remains low for patients afflicted with this aggressive cerebral malignancy. Even with the combined use of several therapeutic modalities, a good prognosis is extremely rare as the remaining cancer cells inevitably infiltrate the normal brain tissue and cause tumor recurrence. Local invasion remains an important cause of morbidity and mortality from brain tumors. The long-term objective of my laboratory is to understand the cellular and molecular mechanisms that underlie tumor invasiveness in human gliomas. Radiotherapy remains a major component of treatment modalities for controlling both malignant and benign glioblastomas. In patients with residual or recurring benign tumors, there is increasing concern about radiation-related side effects that may occur even with highly accurate therapies such as radiosurgery. Despite some therapeutic effect, recent evidence has shown that irradiation may promote malignant behaviors of cancer cells both in vitro and in vivo by activating several pathways involved in tumor invasiveness, angiogenesis and metastasis. Further, the glioma initiating cells are resistant to radiotherapy. Several studies, including ours, demonstrated that significantly increased levels of cath B and uPAR in malignant gliomas and radiation further activates cath B and uPAR molecules. We hypothesize that the cath B and uPAR gene will be targeted while simultaneously combined with radiation and their anti-cancer effects will be determined. The specific aims of the proposal are: Specific Aim 1. Evaluate the effect of vectors expressing siRNA targeting cathepsin B and uPAR (pCU) on glioma cell adhesion, migration and invasion in in vitro models with and without radiation. Aim 1a. Determine the effect of pC, pU, pCU and radiation alone and in combination on the levels of cath B and uPAR in glioblastoma cell lines and glioma initiating cells (GIC). Aim 1b. Determine the downregulation of cath B and uPAR on glioma cell adhesion and migration in glioblastoma cell lines and glioma initiating cells with and without radiation. Aim 1c. Evaluate the effect of pC, pU, pCU and radiation alone and in combination on the invasive behavior of human glioblastoma cell lines and GIC. Specific Aim 2. Determine the molecular mechanisms in cath B and uPAR inhibition-mediated changes on cell growth, proliferation and apoptosis in GIC and non-GIC with and without radiation. Aim 2a. Evaluate the molecular mechanisms in cath B and uPAR downregulation on cell growth and proliferation with and without radiation in non-GIC and GIC. Aim 2b. Determine the effect of cath B and uPAR downregulation on the molecular mechanisms of apoptosis in glioblastoma cell lines and GIC with and without radiation. Specific Aim 3. Determine the molecular mechanisms in cath B and uPAR inhibition-mediated changes in angiogenesis and tumor growth in non-GIC and GIC with and without radiation in nude mice. Aim 3a. Evaluate the effect of cath B and uPAR downregulation-mediated changes in the signaling mechanisms in the regulation of cerebral angiogenesis both in vitro and in vivo with and without radiation. Aim 3b. Determine the optimal doses of pC, pU, pCU and radiation alone and in combination on pre-established intracranial tumor growth or the invasiveness of human glioblastoma cell lines and GIC injected intracerebrally in nude mice. The proposed studies should generate major insight into the pathogenesis of cath B and uPAR and, in turn, should suggest novel targets for therapeutic interventions of glioblastomas. PUBLIC HEALTH RELEVANCE: Despite the many therapeutic strategies undertaken for treatment of glioblastoma multiforme, the survival rate for patients afflicted with this aggressive cerebral malignancy remains low. Radiotherapy has remained the single most effective therapy of glioblastoma for more than 25 years. However, an increasing number of long survivors with secondary side effects and the resistance of glioma initiating cells from this treatment highlighted the need for development of novel therapeutic approaches. This proposal represents a combinational therapeutic approach using a single and bicistronic siRNA construct for cathepsin B and uPAR. This strategy may improve radiotherapy outcomes for the treatment of glioblastomas.