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的神经外胚层肿瘤，是一种以神经元分化为主的侵袭性肿瘤。尽管在神经外科、化疗和放射治疗方面取得了技术进步，但 这些肿瘤的患者仍然多种多样，在婴儿和成人转移性疾病患者中相对较差。传统的治疗方法也是有毒的，可能会导致长期残疾。因此，需要新的策略来预防这些患者与治疗相关的发病率。一种可能值得进一步探索的方法是使用抑制基质酶，这种酶会导致治疗性照射后的附带损害。这种方法还可以通过抑制以胶质瘤生长为特征的侵袭性浸润性病变来防止肿瘤的临床进展。尿激酶型纤溶酶原激活物(UPA)和基质金属蛋白酶-9(MMP9)的表达与患者的生存期和M分期显著相关，提示这些蛋白可能调节髓母细胞瘤患者的生存期。照射后髓母细胞瘤细胞uPAR和MMP9表达水平增加，细胞侵袭力增加。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射治疗疗效的潜在治疗方法。具体目的1.评价uPAR和基质金属蛋白酶-9的抑制作用以及单独和联合应用对髓母细胞瘤细胞生长、侵袭和血管生成的影响。目标1a。检测puPAR、PMMP-9和PUM联合放射治疗对髓母细胞瘤细胞株uPAR和MMP9水平的影响。目标1b。确定puPAR、PMMP-9、PUM和辐射单独及联合作用对人髓母细胞瘤细胞系体外侵袭行为的影响。目标1c。评价puPAR、PMMP-9、PUM和辐射单独及联合应用对体外和体内脑血管生成的影响。目标1D。确定puPAR、PMMP-9、PUM和单独及联合照射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力的最佳剂量。具体目的2.研究puPAR、PMMP-9、PUM和放射治疗对髓母细胞瘤细胞系增殖、迁移、黏附和凋亡分子机制的影响。目标2a。研究puPAR、PMMP-9、PUM和放射单独作用对髓母细胞瘤细胞系黏附和迁移的分子机制的影响，并与对照/模拟和干扰载体(PSV)对照进行比较。目标2b。确定puPAR、PMMP-9、PUM和辐射单独及联合作用对髓母细胞瘤细胞系增殖的分子机制的影响，并与假手术和PSV对照进行比较。目标2c。评价puPAR、PMMP-9、PUM及其联合作用对髓母细胞瘤细胞系凋亡分子机制的影响，并与假手术对照组和PSV对照组进行比较。这种体外基础科学实验和体内翻译研究的结合，将为开发对常规放射治疗耐药的髓母细胞瘤的新治疗方法提供基础。公共卫生相关性：髓母细胞瘤是一种发现在小脑中线附近的脑肿瘤。这是一种发生在婴幼儿身上的脑瘤。手术后，建议对整个中枢神经系统进行外照射(颅脑脊髓照射，或CSI)，以防止肿瘤在该区域复发(复发或复发)。可以理解的是，人们对放射治疗可能对成长中儿童的大脑和脊柱产生的长期并发症给予了极大的关注。这些问题可能包括记忆、学习、社交/情绪适应方面的缺陷，以及成长问题。这些患者的一个主要目标是制定治疗策略，将对中枢神经系统的辐射剂量降至最低。基因治疗包括将遗传物质引入疾病的细胞靶点，以产生治疗效益。本研究探讨了基因治疗抑制蛋白水解酶表达联合放射治疗对低毒的髓母细胞瘤细胞的杀伤作用。

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.