Exploring the Therapeutic Potential of Stem Cell Biology in Gliomas

探索干细胞生物学在神经胶质瘤中的治疗潜力

基本信息

批准号：
8937868
负责人：
Ronald Gress
金额：
$ 17.76万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/8937868
关键词：
Adult Bioinformatics Biological Biological Assay Biological Models Biology Bone Morphogenetic Proteins Brain Brain Neoplasms Cancer cell line Cell Culture Techniques Cell Line Cells Cellular biology ChIP-seq Characteristics Ciliary Neurotrophic Factor Clinic Collaborations Cues Cytostatics Data Development Embryo Equilibrium Gene Expression Genes Genetic Genomics Genotype Glioblastoma Glioma Homeostasis Human Hypermethylation In Vitro Janus kinase Laboratories Lead Mediating Messenger RNA MicroRNAs Modeling Molecular Molecular Profiling Mus Normal tissue morphology Oncogenes Pathway interactions Patients Phenotype Primary Neoplasm Proliferating Radiation STAT protein STAT3 gene Secondary to Serum Signal Pathway Signal Transduction Staging Stem cells Study models Surveys System Technical Expertise Therapeutic Therapeutic Agents Transcriptional Activation Tretinoin Tumor Suppressor Proteins Tumor-Derived Tumorigenicity Ursidae Family Xenograft procedure base bone morphogenetic protein receptors chemotherapeutic agent demethylation gene function genome-wide glioma cell line human EZH2 protein in vivo neoplastic cell nerve stem cell new therapeutic target novel therapeutics pre-clinical promoter response screening self-renewal stem cell biology stem cell differentiation tool transcription factor transgene expression tumor tumor xenograft tumorigenesis tumorigenic

项目摘要

Molecular understanding of deregulated differentiation pathways in TSCs: The delicate balance between stem cell self-renewal and differentiation is controlled by various cell intrinsic and extrinsic factors that are critical for normal tissue homeostasis. Despite extensive phenotypic and functional similarities between TSCs and normal stem cells, the differentiation potentials of TSCs are not entirely normal. Elucidation of the differentiation pathways operative in both normal stem cells and TSCs will be critical for fully understanding tumorigenesis and will likely lead to novel therapeutic targets. We have identified a set of deregulated differentiation pathways in TSCs derived from human primary glioblastoma. We demonstrated that both bone morphogenetic protein (BMP)-mediated and ciliary neurotrophic factor (CNTF)-mediated Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathways elicit distinct biological consequences in adult brain derived TSCs compared to normal neural stem cells (NSCs). Like very early embryonic NSCs, 0308 TSCs proliferate in response to BMP and elicit marginal STAT3 activation after CNTF challenge. However, unlike normal NSCs in later developmental stages that acquire responsiveness to CNTF-triggered STAT3 activation in association with increased expression of BMP receptor 1B (BMPR1B), 0308 cells do not express BMPR1B secondary to Enhancer of Zeste homolog 2 (EZH2)-dependent BMPR1B promoter hypermethylation. Forced expression of BMPR1B in 0308 TICs either by transgene expression or demethylation of the promoter fully restores their differentiation capabilities and induces loss of their tumorigenicity not only via a BMP-mediated pathway but also by CNTF-mediated Jak/STAT activation. A survey of 54 primary human glioblastomas reveals that approximately 20% have suppressed expression of BMPR1B associated with promoter hypermethylation. Taken together, these data implicate that deregulation of the BMP developmental differentiation pathway in a subset of glioblastoma TSCs contributes to their tumorigenic phenotype by not only desensitizing TIC to normal differentiation cues, but also by converting otherwise cytostatic signals to pro-proliferative signals. Extensive in vitro and in vivo characterization of GBM TSCs by using differentiation-inducing agents such as retinoic acid demonstrated that these TSCs differentiate efficiently and stop proliferation. We have demonstrated that retinoic acid treatment achieve cyctostatic effect by decreasing the proportion of CD133 positive cells, a putative marker for brain TSCs, from tumors and by inducing differentiation into astroglial lineage. Interestingly, a subset of GBM TICs pretreated with radiation and chemotherapeutic agents in vivo, do not reveal significant retinoic acid-mediated differentiation. Elucidation of underlying molecular mechanism will provide important clue for predicting sensitivity of differentiation therapeutic approach. Characterization of TSCs in aspect of differentiation-inducing agents further revealed the limitation of traditional glioma cell lines grown in serum. For example, retinoic acid treatment and CNTF exposure potently induce differentiation in most GBM TICs but not of traditional cell lines. This prompted us to question whether many of potential tumor suppressors and/or cytostatic genes previously studied in cell lines, were not recognized. Given the ever increasing number of potential TSGs and oncogenes in glioblastoma TSCs identified from bioinformatics approach and technical expertise of stem cell culture accumulated in the laboratories, we have set up screening systems to study the function of these genes in stem cell cultures. We have also made significant progress on one of keystone projects that is to understand the genomic and molecular signaling similarities and differences between our glioma TSCs and normal neural stem cells (NSC). We have performed a very large scale study of 7 different GBM-derived TSC and normal embryonic NSC lines under both proliferative and differentiating conditions and derived high-throughput mRNA and microRNAs profiling. We are in the midst of performing the computational analyses of the signaling pathways similar and different between NSC and TSC and are about to compliment the array data by performing ChIP-seq analyses of certain transcription factors we have found to be pivotal in TSC biology.

分子对TSC中不受管制的分化途径的理解：干细胞自我更新和分化之间的微妙平衡受到各种细胞内在和外在因子的控制，这对于正常组织稳态至关重要。尽管TSC和正常干细胞之间存在广泛的表型和功能相似性，但TSC的分化势并不完全正常。在正常干细胞和TSC中阐明分化途径的作用对于完全理解肿瘤发生至关重要，并且可能导致新的治疗靶标。我们已经确定了来自人类原发性胶质母细胞瘤的TSC中的一组不受管制的分化途径。我们证明了骨形态发生蛋白（BMP）介导的和睫状神经营养因子（CNTF）介导的JANUS激酶/信号传感器和转录激活因子（JAK/STAT）途径与正常的神经干细胞相比，在成人脑衍生的TSC中引起了不同的生物学后果（NSC）。像非常早期的胚胎NSC一样，0308 TSC响应BMP而增殖，并引起CNTF挑战后的边际STAT3激活。但是，与正常的NSC在后期发育阶段不同，这些阶段获得了对CNTF触发的STAT3激活的响应能力，随着BMP受体1b（BMPR1B）的表达增加，0308细胞不表达继发于增强ZESTE同源性2（EZH2）依赖性BMPR1B促进剂的BMPR1B。通过转基因表达或启动子的脱甲基化，BMPR1B强迫表达BMPR1B完全恢复了它们的分化能力，并不仅通过BMP介导的途径，而且还通过CNTF介导的JAK/STAT激活引起其肿瘤性丧失。对54个原发性胶质母细胞瘤的调查表明，大约20％抑制了与启动子高甲基化相关的BMPR1B的表达。综上所述，这些数据表明，在胶质母细胞瘤TSC的子集中，BMP发育分化途径的放松管制导致了其肿瘤性表型，这不仅是通过将TIC脱敏到正常分化提示，还通过将其他细胞抑制性信号转换为促培养地信号。通过使用分化诱导剂（例如视黄酸），体外和体内表征GBM TSC的体外表征表明，这些TSC有效地区分并停止增殖。我们已经证明，视黄酸治疗通过降低CD133阳性细胞的比例，这是脑TSC的推定标记，从肿瘤和诱导分化为星形胶质细胞谱系，从而实现了细胞静态效应。有趣的是，在体内用辐射和化学治疗剂预处理的GBM TICs的子集未显示出明显的视黄酸介导的分化。阐明潜在的分子机制将为预测分化治疗方法的敏感性提供重要的线索。 TSC在分化诱导剂方面的表征进一步揭示了血清中生长的传统神经胶质瘤细胞系的局限性。例如，视黄酸治疗和CNTF暴露在大多数GBM TICS中有效地诱导分化，而不是传统细胞系的分化。这促使我们质疑是否尚未认识到先前在细胞系中研究的许多潜在抑制肿瘤和/或细胞抑制基因。鉴于从生物信息学方法和实验室中积累的干细胞培养的技术专长中确定的胶质母细胞瘤TSC中潜在的TSG和致癌基因的数量越来越多，我们已经建立了筛选系统来研究这些基因在干细胞培养中的功能。我们还在基石项目之一上取得了重大进展，该项目是了解我们的神经胶质瘤TSC和正常神经干细胞（NSC）之间的基因组和分子信号传导相似性和差异。我们对7种不同的GBM衍生的TSC和正常的胚胎NSC系进行了非常大规模的研究，并在增殖和分化条件下以及衍生的高通量mRNA和microRNAS分析。我们正在进行NSC和TSC之间相似和不同的信号通路的计算分析，并通过对某些转录因子进行chip-seq分析来补充阵列数据，我们发现在TSC生物学中是关键的。