The Role of PDGFR in Medulloblastoma Progression

描述(申请人提供)：髓母细胞瘤是儿童最常见的恶性脑肿瘤。为了探讨有效治疗的问题，必须首先了解人类髓母细胞瘤的进展情况。我们假设血小板衍生生长因子(PDGFR)介导的信号转导可增强肿瘤细胞的反应，从而促进髓母细胞瘤的生长和转移扩散，并已表明(I)转移性髓母细胞瘤显著过度表达PDGFR；(Ii)抑制髓母细胞瘤细胞PDGFR活性的药物显著减少了下游信号转导靶点MAPK的磷酸化，改变了基因表达，降低了细胞迁移和存活；(Iii)在独立的髓母细胞瘤微阵列基因表达数据库中，仅有三个基因，包括DDGFR，在至少三分之一的转移性肿瘤中显示了可检测到的mRNA表达，并降低了细胞的迁移和存活。这些数据结合初步数据表明，该受体的α(PDGFRA)和β(PDGFRb)亚型均可在髓母细胞瘤和细胞的RNA和蛋白水平上表达，2)在正常脑组织中少量表达，3)在髓母细胞瘤细胞中以自分泌和旁分泌方式激活，4)在PDGFR酪氨酸激酶活性选择性抑制剂治疗后，以一种剂量依赖的方式诱导髓母细胞瘤细胞凋亡，提示PDGFR在髓母细胞瘤的生长和进展中可能是至关重要的机制。因此，PDGFR是一种潜在的治疗干预新靶点。为了验证这一假设，我们将使用人类髓母细胞瘤细胞系。我们将(I)进行全面的体外研究，以确定pdgfr信号级联及其对生存、增殖、黏附、迁移和侵袭的影响，(Ii)确定pdgfr信号在这些细胞中诱导的关键基因表达的变化，(Iii)通过可诱导的针对每种pdgfr的siRNA转染法建立pdgfr表达缺陷的髓母细胞瘤细胞，并确定抑制的表达和活性对体外存活、增殖和迁移以及体内生长和转移的影响，以及(Iv)确定磷酸化pdgfr蛋白的表达模式及其与转移和预后的相关性，以此作为评估pdgfr抑制剂治疗该疾病的潜在临床有效性的方法。

DESCRIPTION (provided by applicant): Medulloblastoma is the most common malignant brain tumor in children. To approach the problem of effective therapy, the progression of human medulloblastoma must first be understood. We hypothesize that platelet-derived growth factor (PDGFR)-mediated signal transduction enhances tumor cell responses that promote the growth and metastatic spread of medulloblastoma, and have shown that (i) PDGFR is significantly overexpressed by metastatic medulloblastomas, (ii) inhibitors of medulloblastoma cell PDGFR activity decrease phosphorylation of the downstream signaling target, MAPK, alter gene expression, and decrease cell migration and survival and (iii) in silico analysis of 135 known pro-metastatic genes within independent datasets of microarray gene expression of medulloblastomas, only three genes, including DDGFR, demonstrated detectable mRNA expression in at least one third of all tumors analyzed and significant overexpression by metastatic tumors in each dataset. These data, combined with the preliminary data showing that both alpha (PDGFRA) and beta (PDGFRB) subtypes of the receptor are 1) expressed on the RNA and protein level by medulloblastoma tumors and cells, 2) sparsely expressed by normal brain, 3) activated in an autocrine and paracrine fashion in medulloblastoma cells, and 4) capable of inducing apoptosis of medulloblastoma cells in a dose-dependent manner following treatment with a selective inhibitor of PDGFR tyrosine kinase activity, suggest a mechanism by which PDGFR may be vital for medulloblastoma growth and progression. Thus, PDGFR is a potential novel target for therapeutic intervention. To test this hypothesis we will employ human medulloblastoma cell lines. We will (i) conduct comprehensive in vitro studies to determine the PDGFR signaling cascade and its effects on survival, proliferation, adhesion, migration and invasion, (ii) determine the key gene expression changes induced by PDGFR signaling in these cells, (iii) develop medulloblastoma cells with deficient PDGFR expression by inducible siRNA transfection specific for each PDGFR and determine the effect of inhibited expression and activity on survival, proliferation and migration in vitro and growth and metastasis in vivo, and (iv) determine the expression pattern of phosphorylated PDGFR protein and its correlation with metastasis and outcome in human medulloblastomas as a way to assess the potential clinical utility of PDGFR inhibitors for this disease