Inhibition of matrix proteases to sensitize medulloblastoma cells to radiation

描述（由申请人提供）：髓母细胞瘤属于一组原始神经外胚层肿瘤，是以神经元分化为主的侵袭性肿瘤。尽管神经外科、化疗和放疗的技术进步， 患有这些肿瘤的患者仍然是可变的，并且在患有转移性疾病的婴儿和成人患者中相对较差。传统的治疗方法也是有毒的，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的发病率。一个可能值得进一步探索的途径是使用抑制基质酶，有助于治疗辐射后的附带损害。这种方法还可以通过阻止神经胶质瘤生长的特征性侵袭性浸润来防止肿瘤的临床进展。尿激酶纤溶酶原激活物（uPA）和基质金属蛋白酶-9（MMP-9）的表达与生存和M期的显着关联表明，这些蛋白酶可能调节髓母细胞瘤患者的生存。照射后髓母细胞瘤细胞uPAR和MMP-9表达水平升高，细胞侵袭力增强。这些发现使我们假设通过RNAi技术抑制uPAR和MMP-9可能是一种潜在的治疗方法，以提高髓母细胞瘤患者的放疗疗效。具体目标1。在体外和体内模型中评价uPAR和MMP-9抑制和辐射单独和组合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与辐射联合对髓母细胞瘤细胞系中uPAR和MMP-9水平的影响。目标1b。在体外模型中确定puPAR、pMMP-9、pUM和辐射单独和组合对人髓母细胞瘤细胞系侵袭行为的影响。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作用。目标1d。确定puPAR、pMMP-9、pUM和辐射单独和组合对裸小鼠脑内注射的人髓母细胞瘤细胞系的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、粘附和凋亡的分子机制的影响。目标2a。研究与对照/模拟和乱序载体（pSV）对照相比，puPAR、pMMP-9、pUM和单独的辐射组合对成神经管细胞瘤细胞系中粘附和迁移的分子机制的影响。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的分子机制的影响。目标2c。与模拟和Psv对照相比，评价puPAR、pMMP-9、pUM及其组合对髓母细胞瘤细胞系中细胞凋亡的分子机制的影响。这种体外基础科学实验和体内转化研究的结合将为开发一种新的治疗方法提供基础，以治疗对传统放疗有抵抗力的髓母细胞瘤。 公众健康相关性：髓母细胞瘤是一种发现于小脑中线附近的脑肿瘤。它是一种发生在婴儿和幼儿中的脑肿瘤。手术后，建议对整个CNS进行外部放射（颅脊髓放射，或CSI），以防止肿瘤在该区域复发（复发或复发）。可以理解的是，人们对正在成长的儿童的大脑和脊柱进行放射治疗可能产生的长期并发症给予了很大的关注。这些可能包括记忆，学习和社会/情绪调节方面的缺陷以及生长问题。这些患者的主要目标是制定治疗策略，最大限度地减少对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点以带来治疗益处。本研究探讨了基因治疗抑制蛋白酶表达与辐射联合杀伤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.