Molecular Pathogenesis of Neoplasia

肿瘤的分子发病机制

• 批准号: 7735339
• 负责人: Russell Lonser
• 金额: $ 103.41万
• 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/7735339
• 关键词: Acids; Address; Adult; Adverse effects; Affect; Amino Acid Sequence; Animal Model; Animals; Antibodies; Binding; Biology; Blood - brain barrier anatomy; Blood Island; Blood capillaries; Brain; Brain Neoplasms; Categories; Cell Differentiation process; Cell Line; Cell Proliferation; Cell-Mediated Cytolysis; Cells; Chemical Structure; Chemicals; Ciliary Neurotrophic Factor Receptor; Clinical; Complex; Condition; Conventional (Clear Cell) Renal Cell Carcinoma; Cooperative Research and Development Agreement; Culture Media; Cultured Cells; Cytoplasmic and Nuclear Receptors; Data; Defect; Development; Developmental Biology; Diagnostic; Disease; Drug Combinations; EPOR gene; Epigenetic Process; Erythroblasts; Erythrocytes; Erythropoietin; Gene Mutation; Gene Targeting; Genes; Genetic; Genetic Heterogeneity; Genetic Transcription; Glial Fibrillary Acidic Protein; Glioblastoma; Glioma; Growth; Heterogeneity; Histone Deacetylase; Human; In Vitro; Inherited; Investigation; Isoelectric Focusing; Lead; Liquid Chromatography; Maintenance; Mediator of activation protein; Membrane; Mesenchymal; Microdissection; Modeling; Molecular; Mus; Neoplasms; Neuraxis; Nuclear; Nuclear Receptors; Numbers; Okadaic Acid; Organ; Pathogenesis; Pathologic; Pathway interactions; Patients; Pattern; Penetration; Peptide Sequence Determination; Peptides; Pharmaceutical Preparations; Phase; Population; Primary Neoplasm; Protein Overexpression; Proteins; Proteome; Proteomics; Protocols documentation; Reagent; Retinoic Acid Receptor; Retinoids; Secondary to; Series; Staging; Stem cells; Syndrome; Techniques; Testing; Therapeutic; Therapeutic Uses; Thyroid Hormone Receptor; Time; Tissues; Tretinoin; Tumor Cell Line; Tumor Suppressor Genes; Von Hippel-Lindau Syndrome; Work; Xeno; angiogenesis; antibody-dependent cell cytotoxicity; autocrine; base; capillary; cell growth; endolymphatic sac; gel electrophoresis; gene function; genetic analysis; hemangioblastoma; histogenesis; histone deacetylase 3; inhibitor/antagonist; insight; molecular imaging; nano; neoplastic cell; new technology; novel therapeutics; outcome forecast; phosphatase inhibitor; programs; receptor; therapeutic target; tool; tumor; tumor growth; tumor progression; tumorigenesis; two-dimensional

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.

多形胶质母细胞瘤（GBM）中的差异蛋白质组学表达。 选择性组织显微解剖用于获得GBM细胞的纯群，使用二维蛋白质凝胶电泳（2-DGE）和蛋白质测序研究。 这些分子技术鉴定出在GBM中差异表达的某些靶蛋白，允许区分2个主要类别的GBM（主要和次要）。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，并产生了2种独特的蛋白质组学模式。因此，可以在蛋白质水平上识别出GBM的两种基本与次要模式，以前是由临床和遗传差异区分的，可能对预后和治疗方案有影响。我们正在使用相同的方法在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中鉴定差异表达的蛋白质。 GBM中的核受体核心压轴（N-COR）表达。 我们发现N-COR在GBM细胞中过表达。 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的细胞不表达胶质原纤维酸性蛋白（GFAP），这是星形细胞分化的标志物。 具有细胞质N-COR或N-COR Express GFAP的细胞。 N-COR与非配体的核受体（例如类维生素酸受体和甲状腺激素受体）结合。当N-Cor形成带有视网膜样和甲状腺激素受体的沉默介质（SMRT），组蛋白脱乙酰基酶3（HDAC3）和视网膜酸受体（RAR）的复合体时，RAR特异性靶基因的转录会导致细胞增殖增加。 我们发现黄质酸与视黄酸（RA）协同作用以抑制GBM细胞生长并增加细胞分化。 已知Phoshastase-1抑制剂（例如冈田酸）抑制N-COR活性。 黄氧酸不是针对N-COR的，并且可能具有明显的副作用。 我们认为，这些发现提供了关键信息，以增强对神经胶质瘤生物学的当前理解，这可能会导致患者的新治疗选择。因此，我们通过专门针对NCO-R途径而非其他化合物，而不是其他靶向，而不是其他靶向GBM的细胞生长和分化的新化学化合物，而不是其他化合物，但不是其他化合物。 最近，Lixte Biotechonolgy Inc.和Ninds之间的CRADA因研究这种新型的治疗范式和开发新的小型化合物而被执行。 目前正在研究一系列磷酸酶抑制剂和HDAC抑制剂的抑制活性。 LB1是磷酸酶抑制剂和LB 2，由我们的Crada伴侣开发并提供了HDAC抑制剂。在过去的一年中，共同计划和开发了人类GBM动物模型中的新药和药物组合的方案。该研究的结果表明，新化合物LB1和LB2对小鼠Xeno-Graft的人GBM的肿瘤生长有抑制作用。最近，已经对这两种化合物的化学结构进行了修饰，以更好地渗透到血脑屏障上。 我们计划在GBM动物肿瘤模型中测试这些化合物。 将CNTF受体在脑肿瘤中鉴定为潜在的诊断和治疗靶点。 我们使用较新开发的蛋白质组学技术通过毛细管等电聚焦（CIEF）区分GBM干细胞的蛋白质组（CIEF），该蛋白质组具有纳米逆性相液相色谱（NRPLC）肽分离和MS/MS/MS蛋白蛋白测序。 我们发现，CNTF受体蛋白始终出现在GBM蛋白质组中，但不在正常的成年大脑中。 随后的研究表明，其表达与神经胶质瘤的病理级及其细胞分化相关。 将CNTF受体作为GBM干细胞和组织中的独特表达特征及其膜定位，并具有大量蛋白质外细胞分布，我们进一步研究了GBM细胞中抗体依赖性细胞毒性（ADCC）。 该研究的初步结果表明抑制了肿瘤细胞生长和强烈的细胞毒性效应。我们计划开发一种与脑肿瘤中CNTF受体特异性结合的CNTF受体抗体，以进行治疗以及潜在的分子成像标记。 von Hippel-Lindau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相似，VHL中的肿瘤发生最常见于易感细胞中的VHL野生型缺失引发。然而，在大多数（如果不是全部）肿瘤基因综合征中，几个关键问题仍然无法解释：1）在任何器官中，只有一种特定类型的肿瘤发生。 2）肿瘤发生仅限于特定的器官集，3）肿瘤抑制基因功能与肿瘤发生之间没有明显的关联。我们最近关于血管细胞瘤组织发生的研究揭示了血管群代表发育暂停的组织的证据。我们通过发现了众多的间充质前体，这些前体在血管母细胞瘤形成之前，我们进一步确立了PVHL缺乏对中枢神经系统组织的发育效果。 与胚胎血岛分化类似，血管群中的血岛形成与红细胞生成素（EPO）受体（EPOR）的短暂表达有关。 EPOR表达与继发于VHL缺乏的EPO表达相吻合，我们最近提出，该表达是肿瘤进展的机制。这种肿瘤进展的机制似乎适用于其他与VHL疾病相关的肿瘤，包括肾透明细胞癌和内淋巴SAC肿瘤。目前关于VHL疾病相关的血管母细胞瘤的项目包括a）在血管母细胞瘤中表达与血管母细胞瘤中与血管母细胞群相关的蛋白的表达，b）血管母细胞瘤前体材料的详细表征，c）研究血管瘤细胞的血管细胞来源，通过遗传分析，通过遗传分析来探讨关键问题，以探讨tumor cellsogen的关键问题，是否直接参与tumor angi angiegen。 基于VHL相关肿瘤的EPO和EPOR的均匀表达，并表明这些肿瘤中潜在的EPO驱动的自分泌环，我们试图在培养条件下建立肿瘤细胞系。培养中肿瘤细胞的维持需要富含EPO的培养基。 通过在培养基中添加EPO或EPOR抗体，可以破坏VHL肿瘤细胞的生长。使用EPO丰富的培养基，我们培养了原发性肿瘤，并将其维持在培养物中进行体外测试。我们通过改变细胞培养条件来表征并扩展这些肿瘤细胞为核细胞和成熟的红细胞后代的成核。这些发现进一步支持，即这些肿瘤中的EPO和EPOR功能途径对于VHL肿瘤的生长和分化至关重要。此外，已建立的培养细胞系将有助于测试其他试剂。