Molecular Pathogenesis of Neoplasia

肿瘤的分子发病机制

• 批准号: 8158235
• 负责人: Russell Lonser
• 金额: $ 150.13万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8158235
• 关键词: Molecular; Neoplasms; Pathogenesis

Differential Proteomic Expression in Glioblastoma Multiforme (GBM). Selective tissue microdissection was used to obtain pure populations of GBM cells, which were studied using two-dimensional protein gel electrophoresis (2-DGE) and protein sequencing. These molecular technique identified select target proteins that were expressed differentially among GBMs that permitted distinguishing between the 2 main categories of GBMs (primary and secondary). We isolated and sequenced 11 unique proteins that were differentially expressed in the primary and secondary GBMs and that produced 2 distinctive proteomic patterns. Thus, the 2 patterns of GBMs, primary versus secondary, previously distinguished by clinical and genetic differences, can be recognized at the protein level and may have implications for prognosis and treatment options. We are using the same approach to identify differentially expressed proteins in different stages of glioma formation, as well as within gliomas with different phenotypic expression. Nuclear Receptor Corepressor (N-CoR) Expression in GBM. We have found that N-CoR is overexpressed in GBM cells. Studies indicate that the subcellular localization and presence of N-CoR is related to the differentiation state of GBM cells. Cells expressing nuclear N-CoR do not express glial fibrillary acidic protein (GFAP), a marker of astrocytic differentiation. Cells with cytoplasmic N-CoR or loss of N-CoR express GFAP. N-CoR binds to unliganded nuclear receptors such as the retinoid acid receptor and thyroid hormone receptor. When N-CoR forms a complex with silencing mediator of retinoid and thyroid hormone receptors (SMRT), histone deacetylase 3 (HDAC3) and retinoic acid receptor, (RAR), transcription of RAR specific target genes is repressed resulting in increased cell proliferation. We have found that oxadaic acid acts synergistically with retinoic acid (RA) to inhibit GBM cell growth and increase cell differentiation. Phoshastase-1 inhibitors such as okadaic acid are known to inhibit N-CoR activity. Oxadaic acid is not specifically targeted to N-CoR and is likely to have significant side-effects. We believe these findings provide critical information for enhancing current understanding of glioma biology which may lead to new therapeutic options for patients. Therefore, we seek more specific phosphatase inhibitors from new chemical compounds that affect cell growth and differentiation in GBM by specifically targeting the NCo-R pathway, but not others. Recently a CRADA between Lixte Biotechonolgy Inc. and NINDS was executed for studying this novel therapeutic paradigm and developing new small chemical compounds. Work is currently in progress studying the inhibitory activity of a series of phosphatase inhibitors and HDAC inhibitors. LB1, a phosphatase inhibitor and LB 2, an HDAC inhibitor were developed and provided by our CRADA partner. During the last year, protocols for evaluating the new drugs and combinations of drugs for activities in an animal model of human GBM were jointly planned and developed. The results of the study show an inhibitory effect on tumor growth of the mouse xeno-grafted human GBM with both new compounds, LB1 and LB2. Recently, the chemical structure of these 2 compounds have been modified for a better penetration to the blood brain barrier. We are planning to test these compounds in a GBM animal tumor model. Identification of CNTF Receptor in Brain Tumors as Potential Diagnostic and Therapeutic Target. We used a more recently developed proteomic technique to distinguish the proteome of GBM stem cells by capillary isoelectric focusing (CIEF) with nano-reversed-phase liquid chromatography (nRPLC) peptide separation and MS/MS protein sequencing. We have found that CNTF receptor protein consistently appeared in GBM proteomes but not in normal adult brain. Subsequent studies indicated that its expression correlates with the pathologic grades of the glioma and their cell differentiation. Taking CNTF receptor as an unique expression character in GBM stem cells and tissues and its membrane localization with large portion of protein extra-cellular distribution, we further studied antibody-dependent cellular cytotoxicity (ADCC) in GBM cells. The preliminary results of the study showed an inhibition of tumor cell growth and strong cellular cytotoxicity effect. We are planning to develop a CNTF receptor antibody specifically binding to CNTF receptor in brain tumor for therapeutic use, as well as potential molecular imaging marker. Developmental Biology and Tumorigenesis of von Hippel-Lindau disease (VHL). Analagous to other tumor supressor gene syndromes, tumorigenesis in VHL is most commonly initiated by a VHL wild-type deletion in susceptible cells. Several key questions, however, remain unexplained in most, if not all, tumor suppressor gene syndromes: 1) in any organ, only one specific type of tumor occurs; 2) tumorigenesis is restricted to specific sets of organs and 3) there is no obvious association between tumor suppressor gene function and tumorigenesis. Our recent studies on the histogenesis of hemangioblastomas revealed evidence that hemangioblastomas represent developmentally-arrested tissue. We have further established developmental effects of pVHL deficiency on central nervous system tissues by the discovery of numerous mesenchymal precursors that precede hemangioblastoma formation. In analogy to embryonal blood island differentiation, blood island formation in hemangioblastomas is associated with transient expression of the erythropoietin (Epo) receptor (EpoR). EpoR expression coincides with expression of Epo secondary to VHL deficiency, which we have recently proposed as a mechanism of tumor progression. This mechanism of tumor progression appears to be applicable to other VHL disease-associated tumors, including renal clear cell carcinoma and endolymphatic sac tumor. Current projects on VHL disease-associated hemangioblastomas include a) expression of developmental hemangioblast-associated proteins in hemangioblastomas, b) detailed characterization of hemangioblastoma precursor material, c) investigation of the vessel cell origin of hemangioblastoma by genetic analysis to address the critical questions whether the tumor cell participating directly to the angiogenesis. Based on the uniform expression of EPO and EPOR by VHL associated tumors and previous data indicating a potential EPO driven autocrine loop in these tumors, we attempted to establish the tumor cell line in culture conditions. The maintenance of tumor cells in culture requires an EPO-rich media. Growth of the VHL tumor cells was disrupted by addition of EPO or EPOR antibodies in the culture media. Using EPO rich culture media, we cultured the primary tumors and maintained them in culture for in vitro testing. We have characterized and expanded these tumor cells to nucleated erythrocytes and mature erythrocytic progeny by varying the cell culture conditions. These findings further support that the EPO and EPOR functional pathway in these tumors is critical in VHL tumor growth and differentiation. Further, the established culture cell lines will be useful for testing other reagents.

多形性胶质母细胞瘤 (GBM) 中的差异蛋白质组表达。 采用选择性组织显微切割获得纯 GBM 细胞群，并使用二维蛋白质凝胶电泳 (2-DGE) 和蛋白质测序对其进行研究。 这些分子技术鉴定了在 GBM 之间差异表达的特定靶蛋白，从而可以区分 GBM 的 2 个主要类别（原发性和继发性）。我们分离并测序了 11 种独特的蛋白质，这些蛋白质在初级和次级 GBM 中差异表达，并产生 2 种独特的蛋白质组模式。因此，以前通过临床和遗传差异区分的两种 GBM 模式（原发性与继发性）可以在蛋白质水平上识别，并且可能对预后和治疗选择产生影响。我们使用相同的方法来识别神经胶质瘤形成的不同阶段以及具有不同表型表达的神经胶质瘤内的差异表达蛋白。 GBM 中核受体辅阻遏物 (N-CoR) 的表达。 我们发现 N-CoR 在 GBM 细胞中过度表达。 研究表明，N-CoR 的亚细胞定位和存在与 GBM 细胞的分化状态有关。 表达核 N-CoR 的细胞不表达胶质纤维酸性蛋白 (GFAP)，这是星形胶质细胞分化的标志物。 具有细胞质 N-CoR 或 N-CoR 缺失的细胞表达 GFAP。 N-CoR 与未配体的核受体结合，例如视黄酸受体和甲状腺激素受体。当 N-CoR 与类视黄醇和甲状腺激素受体 (SMRT)、组蛋白脱乙酰酶 3 (HDAC3) 和视黄酸受体 (RAR) 的沉默介质形成复合物时，RAR 特定靶基因的转录受到抑制，导致细胞增殖增加。 我们发现草酸与视黄酸 (RA) 具有协同作用，可抑制 GBM 细胞生长并增加细胞分化。 Phoshastase-1 抑制剂（例如冈田酸）已知可抑制 N-CoR 活性。 草酸并非专门针对 N-CoR，并且可能具有显着的副作用。 我们相信这些发现为增强目前对神经胶质瘤生物学的理解提供了关键信息，这可能为患者带来新的治疗选择。因此，我们从新化合物中寻找更特异性的磷酸酶抑制剂，通过专门针对 NCo-R 途径而不是其他途径来影响 GBM 中的细胞生长和分化。 最近，Lixte Biotechonolgy Inc. 和 NINDS 之间执行了一项 CRADA，以研究这种新颖的治疗模式并开发新的小化合物。 目前正在进行研究一系列磷酸酶抑制剂和 HDAC 抑制剂的抑制活性的工作。 LB1（一种磷酸酶抑制剂）和 LB 2（一种 HDAC 抑制剂）由我们的 CRADA 合作伙伴开发和提供。去年，共同规划和制定了评估新药和药物组合在人类 GBM 动物模型中的活性的方案。研究结果表明，LB1 和 LB2 这两种新化合物对小鼠异种移植的人 GBM 的肿瘤生长具有抑制作用。最近，这两种化合物的化学结构已被修改，以更好地渗透血脑屏障。 我们计划在 GBM 动物肿瘤模型中测试这些化合物。 脑肿瘤中 CNTF 受体的鉴定作为潜在的诊断和治疗靶点。 我们使用最近开发的蛋白质组学技术，通过毛细管等电聚焦 (CIEF) 结合纳米反相液相色谱 (nRPLC) 肽分离和 MS/MS 蛋白质测序来区分 GBM 干细胞的蛋白质组。 我们发现 CNTF 受体蛋白始终出现在 GBM 蛋白质组中，但不在正常成人大脑中出现。 随后的研究表明其表达与神经胶质瘤的病理分级及其细胞分化相关。 以CNTF受体作为GBM干细胞和组织中独特的表达特征及其膜定位和大部分蛋白分布于细胞外的情况，我们进一步研究了GBM细胞中抗体依赖性细胞毒性（ADCC）。 研究初步结果显示具有抑制肿瘤细胞生长和较强的细胞毒作用。我们计划开发一种特异性结合脑肿瘤中CNTF受体的CNTF受体抗体用于治疗用途，以及潜在的分子成像标记。 希佩尔-林道病 (VHL) 的发育生物学和肿瘤发生。 与其他肿瘤抑制基因综合征类似，VHL 的肿瘤发生最常见是由易感细胞中的 VHL 野生型缺失引发的。然而，在大多数（如果不是全部）肿瘤抑制基因综合征中，有几个关键问题仍然无法解释：1）在任何器官中，只发生一种特定类型的肿瘤； 2) 肿瘤发生仅限于特定的器官组，并且 3) 抑癌基因功能与肿瘤发生之间没有明显的关联。我们最近对血管母细胞瘤的组织发生的研究揭示了血管母细胞瘤代表发育停滞组织的证据。通过发现血管母细胞瘤形成之前的许多间充质前体，我们进一步确定了 pVHL 缺陷对中枢神经系统组织的发育影响。 与胚胎血岛分化类似，血管母细胞瘤中的血岛形成与促红细胞生成素 (Epo) 受体 (EpoR) 的瞬时表达相关。 EpoR 表达与继发于 VHL 缺陷的 Epo 表达一致，我们最近提出将其作为肿瘤进展的机制。这种肿瘤进展机制似乎适用于其他 VHL 疾病相关肿瘤，包括肾透明细胞癌和内淋巴囊肿瘤。目前有关 VHL 疾病相关血管母细胞瘤的项目包括：a) 血管母细胞瘤中发育性成血管细胞相关蛋白的表达，b) 血管母细胞瘤前体物质的详细表征，c) 通过遗传分析研究血管母细胞瘤的血管细胞起源，以解决是否存在血管母细胞瘤的关键问题。肿瘤细胞直接参与血管生成。 基于 VHL 相关肿瘤 EPO 和 EPOR 的一致表达以及表明这些肿瘤中潜在的 EPO 驱动的自分泌环的先前数据，我们尝试在培养条件下建立肿瘤细胞系。肿瘤细胞在培养物中的维持需要富含 EPO 的培养基。 通过在培养基中添加 EPO 或 EPOR 抗体来破坏 VHL 肿瘤细胞的生长。使用富含 EPO 的培养基，我们培养原发性肿瘤并将其维持在培养物中以进行体外测试。我们通过改变细胞培养条件对这些肿瘤细胞进行了表征并扩增为有核红细胞和成熟红细胞后代。这些发现进一步支持这些肿瘤中的 EPO 和 EPOR 功能途径对于 VHL 肿瘤生长和分化至关重要。此外，建立的培养细胞系将可用于测试其他试剂。