Cancer biology in the zebrafish

描述（由申请人提供）：癌症包括一系列导致显著发病率和死亡率的毁灭性疾病。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过度表达人类BRAFV600等位基因（在大约70%的人类黑色素瘤中发生突变）并结合p53缺陷构建的：我们开发了一种表达克隆策略来检测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%的黑色素瘤中被放大。SETDB1，一种H3K9三甲基化酶，在我们的黑色素瘤模型中增强了肿瘤的发生。SETDB1是一种抑制基因表达并促进黑色素瘤形成和侵袭的表观遗传调控因子。我们已经成功地进行了大规模的过表达筛选其他染色质因子加速黑色素瘤在体内。在此，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的阶段相关）和HDGF的作用机制。我们将在人类黑色素瘤细胞中纯化SATB2和HDGF蛋白复合物，并通过morpholinos基因敲除和斑马鱼的过表达来研究该复合物的成分。我们的实验室最近也记录了来氟米特（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，并且与BRAF抑制剂联合使用，可以显著减少黑色素瘤的生长。我们已经迅速进行了一项临床试验，测试BRAF抑制剂和LEF联合治疗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中转录暂停调控的研究揭示了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以允许转录延长发生的激酶P-TEFb。HEXIM复合物失活可恢复LEF处理斑马鱼胚胎的效果。信息学研究发现，HEXIM在多种黑色素瘤中被下调。初步数据表明，HEXIM过表达可以抑制我们模型中黑色素瘤的形成。我们计划表达一个组成活性和显性阴性的HEXIM突变体，并评估其对黑色素瘤形成的影响。我们还启动了一项化学筛选，以发现改变黑色素瘤中转录暂停的因素。挽救LEF的化学物质可能有助于确定DHODH抑制剂导致转录伸长缺陷的作用机制。我们预计，拟议的实验结果将对我们对黑色素瘤基本生物学的理解产生广泛的影响，包括表观遗传和转录机制，并将迅速转化为黑色素瘤患者的新临床试验。

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.