Cancer biology in the zebrafish

斑马鱼的癌症生物学

• 批准号: 9012017
• 负责人: LEONARD Ira ZON
• 金额: $ 37.31万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-03 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9012017
• 关键词: Acceleration; Address; Alleles; Animal Model; BRAF gene; Binding Proteins; Biochemistry; Biology; Cancer Biology; Chemicals; Chromatin; Chromosomes, Human, Pair 1; Clinical; Clinical Trials; Combined Modality Therapy; Complex; DHODH gene; Data; Defect; Development; Disease; Dominant-Negative Mutation; Effectiveness; Embryo; Enzymes; Epigenetic Process; Family member; Gene Expression; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Growth; Health; Human; Human Chromosomes; Informatics; Laboratories; Lead; Leflunomide; Malignant Neoplasms; Melanoma Cell; Metastatic Melanoma; Modeling; Morbidity - disease rate; Mutate; Neoplasm Metastasis; Neural Crest; Neural Crest Cell; Oncogenes; Patients; Pharmaceutical Preparations; Phosphotransferases; Play; Positive Transcriptional Elongation Factor B; Proteins; Pyrimidine; RNA; Role; SETDB1 gene; Staging; Study models; System; TP53 gene; Techniques; Testing; Transcription Elongation; Transcriptional Regulation; Translating; Work; Zebrafish; chemical genetics; cofactor; epigenetic regulation; expression cloning; hepatoma-derived growth factor; in vivo; inhibitor/antagonist; knock-down; melanocyte; melanoma; mortality; mutant; novel therapeutics; overexpression; protein complex; protein expression; relapse patients; research study; response; small molecule; targeted cancer therapy; tumor; tumor initiation; tumorigenesis; vector

DESCRIPTION (provided by applicant): Cancer comprises a set of devastating diseases that cause significant morbidity and mortality. My laboratory has utilized the zebrafish as a model for studying cancer. Our zebrafish melanoma model was constructed by overexpression of the human BRAFV600 allele (mutated in about 70% of human melanomas) in combination with p53 deficiency: we developed an expression cloning strategy to examine the effect of specific genes on melanoma formation. In one example, we found the driver gene on human chromosome 1 that is amplified in 30 percent of melanoma. SETDB1, an H3K9 trimethylase, enhanced tumorigenesis in our melanoma model. SETDB1 is an epigenetic regulator that suppresses gene expression and promotes melanoma formation and invasion. We have since successfully undertaken a large-scale overexpression screen for other chromatin factors that accelerate melanoma in vivo. Here we propose to study the mechanism of action for SATB2, along with its family member SATB1 (SATB1 protein expression correlates to the stage of human melanoma), and HDGF. We will purify the SATB2 and HDGF protein complexes in human melanoma cells, and study components of the complex by gene knockdown with morpholinos and by overexpression in zebrafish. Our laboratory has also recently documented that leflunomide (LEF), known to inactivate DHODH (an enzyme required for pyrimidine synthesis), is capable of suppressing melanoma formation and, in combination with a BRAF inhibitor, lead to substantial reduction of melanoma growth. We have rapidly proceeded to a clinical trial that tests the combination therapy of a BRAF inhibitor and LEF as a new therapy for metastatic melanoma. The mechanism that underlies LEF-associated suppression of melanoma involves a block of transcriptional elongation of neural crest genes. Our study of the regulation of transcriptional pausing in melanoma cells revealed the involvement of the HEXIM complex, which is composed of RNA and proteins, and which sequesters the kinase P-TEFb that phosphorylates POLII to allow transcriptional elongation to occur. Inactivation of the HEXIM complex rescues the effects of LEF treatment of zebrafish embryos. Informatics studies have uncovered that HEXIM is substantially downregulated in a variety of melanomas. Preliminary data suggest that HEXIM overexpression can suppress melanoma formation in our model. We plan to express a constitutively active and dominant negative HEXIM mutant and evaluate the effect on melanoma formation. We have also initiated a chemical screen to find factors that alter transcriptional pausing in melanoma. Chemicals that rescue LEF could be helpful in determining the mechanism of action by which a DHODH inhibitor leads to a transcription elongation defect. We anticipate that results of the proposed experiments will have a broad impact on our understanding of the basic biology of melanoma, including epigenetic and transcriptional mechanisms, and will rapidly translate to new clinical trials on patients with melanoma.

描述（由申请人提供）：癌症包括一系列导致显着发病率和死亡率的破坏性疾病。我的实验室利用斑马鱼作为研究癌症的模型。我们的斑马鱼黑色素瘤模型是通过人类 BRAFV600 等位基因（在约 70% 的人类黑色素瘤中发生突变）的过度表达与 p53 缺陷相结合而构建的：我们开发了一种表达克隆策略来检查特定基因对黑色素瘤形成的影响。在一个例子中，我们发现人类 1 号染色体上的驱动基因在 30% 的黑色素瘤中被扩增。 SETDB1 是一种 H3K9 三甲基化酶，可增强我们的黑色素瘤模型中的肿瘤发生。 SETDB1 是一种表观遗传调节因子，可抑制基因表达并促进黑色素瘤形成和侵袭。此后，我们成功地对体内加速黑色素瘤的其他染色质因子进行了大规模的过表达筛选。在这里，我们建议研究 SATB2 及其家族成员 SATB1（SATB1 蛋白表达与人类黑色素瘤阶段相关）和 HDGF 的作用机制。我们将纯化人类黑色素瘤细胞中的 SATB2 和 HDGF 蛋白复合物，并通过吗啉代基因敲除和在斑马鱼中过度表达来研究复合物的成分。我们的实验室最近还记录了来氟米特 (LEF)，已知可以灭活 DHODH（一种嘧啶合成所需的酶），能够抑制黑色素瘤的形成，并与 BRAF 抑制剂结合使用，可大幅减少黑色素瘤的生长。我们已迅速开展一项临床试验，测试 BRAF 抑制剂和 LEF 的联合疗法作为转移性黑色素瘤的新疗法。 LEF 相关的黑色素瘤抑制机制涉及神经嵴基因转录延伸的阻断。我们对黑色素瘤细胞转录暂停调节的研究揭示了 HEXIM 复合物的参与，该复合物由 RNA 和蛋白质组成，并隔离磷酸化 POLII 的激酶 P-TEFb，以允许转录延伸发生。 HEXIM 复合物的失活可以挽救 LEF 处理斑马鱼胚胎的效果。信息学研究发现，HEXIM 在多种黑色素瘤中显着下调。初步数据表明，HEXIM 过度表达可以抑制我们模型中黑色素瘤的形成。我们计划表达组成型活性和显性失活的 HEXIM 突变体，并评估其对黑色素瘤形成的影响。我们还启动了化学筛选，以寻找改变黑色素瘤转录暂停的因素。拯救 LEF 的化学物质可能有助于确定 DHODH 抑制剂导致转录延伸缺陷的作用机制。我们预计所提出的实验结果将对我们对黑色素瘤基础生物学（包括表观遗传和转录机制）的理解产生广泛影响，并将迅速转化为针对黑色素瘤患者的新临床试验。