Inhibition of matrix proteases to sensitize medulloblastoma cells to radiation

描述（申请人提供）：髓母细胞瘤属于一组原始神经外胚层肿瘤，是一种以神经元分化为主的侵袭性肿瘤。尽管神经外科、化疗和放射治疗的技术取得了进步，但预后 患有这些肿瘤的患者仍然存在差异，并且在患有转移性疾病的婴儿和成人患者中相对较差。传统治疗方法也有毒，可能导致长期残疾。因此，需要新的策略来预防这些患者的治疗相关发病率。可能值得进一步探索的一个途径是使用基质酶的抑制，这些酶会在治疗性辐射后造成附带损伤。这种方法还可以通过阻止神经胶质瘤生长特征的侵袭性浸润来防止肿瘤的临床进展。尿激酶纤溶酶原激活剂 (uPA) 和基质金属蛋白酶 9 (MMP-9) 表达与生存和 M 期显着相关，表明这些蛋白酶可能调节髓母细胞瘤患者的生存。受辐射的髓母细胞瘤细胞中 uPAR 和 MMP-9 表达水平以及细胞侵袭性增加。这些发现使我们推测通过 RNAi 技术抑制 uPAR 和 MMP-9 可能是提高髓母细胞瘤患者放疗疗效的潜在治疗方法。具体目标 1. 在体外和体内模型中评估单独和联合使用 uPAR 和 MMP-9 抑制和放射对髓母细胞瘤细胞生长、侵袭和血管生成的影响。目标 1a。确定 puPAR、pMMP-9 和 pUM 与放射组合对髓母细胞瘤细胞系中 uPAR 和 MMP-9 水平的影响。目标 1b。确定 puPAR、pMMP-9、pUM 和辐射单独和组合对人髓母细胞瘤细胞系体外模型侵袭行为的影响。目标 1c。在体外和体内模型中评估 puPAR、pMMP-9、pUM 和辐射单独和组合对脑血管生成的影响。目标1d。确定 puPAR、pMMP-9、pUM 和辐射单独和组合对裸鼠脑内注射的人髓母细胞瘤细胞系预先建立的颅内肿瘤生长或侵袭性的最佳剂量。具体目标 2. 确定 puPAR、pMMP-9、pUM 和辐射单独和组合对髓母细胞瘤细胞系增殖、迁移、粘附和凋亡分子机制的影响。目标 2a。与对照/模拟和乱序载体 (pSV) 对照相比，研究 puPAR、pMMP-9、pUM 和单独放射组合对髓母细胞瘤细胞系粘附和迁移分子机制的影响。目标 2b。与模拟和 pSV 对照相比，确定 puPAR、pMMP-9、pUM 和辐射单独和组合对髓母细胞瘤细胞系增殖分子机制的影响。目标 2c。与模拟和 Psv 对照相比，评估 puPAR、pMMP-9、pUM 及其组合对髓母细胞瘤细胞系凋亡分子机制的影响。这种体外基础科学实验和体内转化研究的结合将为开发对传统放射治疗耐药的髓母细胞瘤肿瘤的新治疗方法奠定基础。 公共卫生相关性：髓母细胞瘤是一种脑肿瘤，发现于小脑中线附近。它是一种发生于婴儿和幼儿的脑肿瘤。手术后，建议对整个中枢神经系统进行外部放射（颅脊髓照射，或 CSI），以防止肿瘤在该区域复发（复发）。放射治疗对成长中的儿童的大脑和脊柱可能产生的长期并发症引起了人们的广泛关注，这是可以理解的。这些可能包括记忆力、学习力、社交/情感适应能力的缺陷，以及成长问题。这些患者的一个主要目标是制定治疗策略，最大限度地减少中枢神经系统的辐射剂量。基因治疗包括将遗传物质引入患病细胞靶标以带来治疗效果。本研究探讨了抑制蛋白酶表达的基因疗法与放射治疗相结合以较低的毒性杀死髓母细胞瘤细胞的有效性。

DESCRIPTION (provided by applicant): Medulloblastomas, which belong to a group of primitive neuroectodermal tumors, are invasive tumors with predominant neuronal differentiation. Despite technological advances in neurosurgery, chemotherapy and radiation therapy, the prognosis for patients with these tumors remains variable and is relatively poor in infants and adult patients with metastatic disease. The traditional treatments are also toxic and can lead to long-term disabilities. Therefore new strategies are needed to prevent treatment related morbidity in these patients. One avenue possibly worth exploring further is the use of inhibition of matrix enzymes that contribute to collateral damage after therapeutic irradiation. This approach may also prevent clinical progression of the tumor by thwarting the invasive infiltration that characterizes glioma growth. A significant association of urokinase plasminogen activator (uPA) and matrix metalloprotease-9 (MMP-9) expression with survival and M-stage indicates that these proteases may modulate the survival of medulloblastoma patients. The levels of uPAR and MMP-9 expression and cellular invasiveness were increased in irradiated medulloblastoma cells. These findings led us to hypothesize inhibition of uPAR and MMP-9 by RNAi technology could be a potential therapeutic approach to improve the efficacy of radiotherapy in medulloblastoma patients. Specific Aim 1. Evaluate the effect of uPAR and MMP-9 inhibition and radiation alone and in combination on medulloblastoma cell growth, invasion and angiogenesis in both in vitro and in vivo models. Aim 1a. Determine the effect of puPAR, pMMP-9 and pUM in combination with radiation on the levels of uPAR and MMP-9 in medulloblastoma cell lines. Aim 1b. Determine the effect of puPAR, pMMP-9, pUM and radiation alone and in combination on the invasive behavior of human medulloblastoma cell lines in vitro models. Aim 1c. Evaluate the effect of puPAR, pMMP-9, pUM and radiation alone and in combination on cerebral angiogenesis in both in vitro and in vivo models. Aim 1d. Determine the optimal doses of puPAR, pMMP-9, pUM and radiation alone and in combination on pre-established intracranial tumor growth or invasiveness of human medulloblastoma cell lines injected intracerebrally in nude mice. Specific Aim 2. Determine the effect of puPAR, pMMP-9, pUM and radiation alone and in combination on the molecular mechanisms of proliferation, migration, adhesion and apoptosis in medulloblastoma cell lines. Aim 2a. Investigate the effect of puPAR, pMMP-9, pUM and radiation alone in combination on the molecular mechanisms of adhesion and migration in medulloblastoma cell lines compared to control/mock and scrambled vector (pSV) controls. Aim 2b. Determine the effect of puPAR, pMMP-9, pUM and radiation alone and in combination on the molecular mechanisms of proliferation in medulloblastoma cell lines compared with mock and pSV controls. Aim 2c. Evaluate the effect of puPAR, pMMP-9, pUM and in combination on the molecular mechanisms of apoptosis in medulloblastoma cell lines compared with mock and Psv controls. This combination of in vitro basic science experiments and translational in vivo studies will provide the basis for development of a new therapeutic approach to medulloblastoma tumors which are resistant to conventional radiotherapy. PUBLIC HELATH RELEVANCE: Medulloblastomas are one type of brain tumors that are found near the midline of the cerebellum. It is a type of brain tumor that occurs in infants and young children. After surgery, external radiation to the entire CNS (craniospinal irradiation, or CSI) is recommended to prevent the tumor from coming back in this area (recurrence, or relapse). Much attention has understandably been paid to the possible long-term complications of radiation therapy to the brain and spine of a growing child. These can include deficits in memory, learning, and social/emotional adjustment, and growth problems. A major goal for these patients is to develop treatment strategies that minimize the dose of radiation to the central nervous system. Gene therapy consists of the introduction of genetic material into diseased cellular targets to bring about therapeutic benefit. The present study explores the usefulness of gene therapy to inhibit protease expression in conjunction with radiation for killing of medulloblastoma cells with lower toxicity.