Persistent SIX2 expression as a first hit mechanism in Wilms tumorigenesis

SIX2 持续表达是肾母细胞瘤发生中的第一击机制

基本信息

批准号：
8812986
负责人：
Harold Newton Lovvorn
金额：
$ 4.46万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-12-01 至 2016-04-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8812986
关键词：
Address Adult Alleles Applications Grants Breeding Cell Survival Cells Childhood Cloning Codon Nucleotides Cre-LoxP Development Disease Embryo Endowment Engineering Epigenetic Process Epithelial Equilibrium Event Excision Funding Mechanisms Future Genes Genetic Genetic Recombination Goals Grant Green Fluorescent Proteins Growth Human Inbreeding Injection of therapeutic agent Kidney Laboratories Lesion Maintenance Malignant - descriptor Malignant Neoplasms Malignant childhood renal neoplasm Mesenchyme Modeling Multipotent Stem Cells Mus Nature Nephroblastoma Nephrons Pathway interactions Peptides Pharmaceutical Preparations Phenotype Plasmids Population Production Publishing Resistance Rest Rodent Model Role Scheme Signal Transduction Source Stem cells System Tamoxifen Testing Time Tissues Transgenes Transgenic Mice Transgenic Model Work analog beta catenin blastema blastocyst cancer stem cell cellular targeting clinically relevant daughter cell design embryonic stem cell implantation innovation mouse development mouse model nephrogenesis novel offspring overexpression postnatal premature prevent progenitor public health relevance pup self-renewal stem cell population stemness tool transmission process tumor tumor growth tumor initiation tumorigenesis vector

项目摘要

DESCRIPTION (provided by applicant): Wilms tumor (WT) is a pediatric renal malignancy thought to arise from aberrant differentiation and persistence of embryonic kidney stem cells. Precisely how nephron progenitors escape pathways of epithelial commitment and conversion, mechanisms that potentially represent 'first hits' in Wilms tumorigenesis, have not been clarified. WT blastema, the putative malignant analogue of these nephron progenitors, retains expression of the transcriptional regulator, Six2, which in mouse development promotes self-renewal of the cap mesenchyme (CM) and prevents its premature epithelial differentiation. Our previous work has shown that SIX2, normally absent in the adult kidney, is persistently expressed across a broad spectrum of human WT. Putting together these observations of SIX2 activity in development and disease, the fundamental question arises whether this gene provides a mechanism for the CM and its progeny to self-perpetuate in the WT sequence. The purpose of developing this mouse model is to test the hypothesis that persistent Six2 expression in the CM impairs epithelial differentiation and promotes retention of this progenitor population as a set-up to develop nephrogenic rests, the putative precursor lesion of WT. We have two aims: 1) to generate and validate a tissue-specific, Cre-activated mouse line that allows temporal control of Six2 expression in nephron progenitors, and 2) to characterize the phenotype of persistent Six2 expression in the embryonic and adult kidney. Briefly, mice will be engineered to express Six2 persistently in nephron progenitors (and daughter cells) under the temporal control of our established tamoxifen-inducible and CM-specific Cited1-Cre-ERT2 transgenic mouse line. To yield temporal control of Six2 expression, a mouse-specific ROSA26-loxP-STOP-loxP-Gfp-2a- Six2 (R26-LSL-Six2, for short) allele will be designed. Once validated to transmit this R26-LSL-Six2 allele in the germline, founders will be inbred to homozygosity for subsequent breeding with heterozygous Cited1-Cre- ERT2 mice. This breeding scheme will produce offspring that all carry the R26-LSL-Six2 allele and that half carry Cited1-Cre-ERT2, which affords control for tamoxifen effects on renal development in Cre-negative mice. Administered to all dams in this scheme, tamoxifen will induce CM-specific production of Cre and thereby excision of the STOP codon in half of the pups, and subsequent Six2 production will occur specifically in the nephron progenitors of the CM and daughter cells. Coinciding with peak Cited1 activity in the CM, tamoxifen will be administered to dams at e15.5, and the resulting phenotype on nephron progenitor differentiation will be evaluated at e19.5 and one month postnatal. In this model, we predict disruption of CM differentiation and retention of these progenitors in the form of nephrogenic rests. The impact of these studies therefore will be to develop a model for exploring persistent Six2 expression in the CM as a candidate mechanism in the initiation of the WT sequence and for evaluating in future work the interactions of Six2 with Wnt/β-catenin signaling as a targetable mechanism of cancer stem cell survival that will reveal new and more efficacious drugs.

描述（由应用提供）：Wilms肿瘤（WT）是一种小儿肾脏恶性肿瘤，认为是由异常的分化和胚胎肾脏干细胞的持续性引起的。尚未尚未澄清肾上腺祖祖细胞如何逃脱上皮承诺和转化的途径，即可能代表威尔姆斯肿瘤发生中“首次命中”的机制。 WT BLASTEMA是这些肾单位祖细胞的推定恶性类似物，保留了转录调节剂Six2的表达，在小鼠发育中促进了帽间质（CM）的自我更新，并防止其过早的上皮分化。我们以前的工作表明，通常在成年肾脏中缺乏的六个持续在人类WT中持续表达。将这些对六个活性在发育和疾病中的观察汇总在一起时，基本问题出现了该基因是否为CM及其后代提供了一种在WT序列中进行自我的机制。开发该小鼠模型的目的是检验以下假设：CM中持续的六个表达会损害上皮分化，并促进该祖先种群的保留作为设置为了形成肾原性，wt的假定前体病变。我们有两个目的：1）生成和验证组织特异性的，CRE激活的小鼠线，该系列允许对肾单位祖细胞中的Six2表达进行临时控制，而2）表征胚胎和成年肾脏中持续的SIX2表达的表型。简而言之，在我们已建立的他莫昔芬诱导的临时控制下，将在肾单位祖细胞（和子细胞）中持续地表达小鼠持续表达62，且CM特异性的CMETICETIC 1-CRET1-CRE-ERT2转基因小鼠系。为了临时控制Six2表达，将设计一个小鼠特异性Rosa26-loxp-Stop-lox-loxp-gfp-2a- six2（简称R26-LSL-SIX2）等位基因。一旦验证以在种系中传输该R26-LSL-SIX2等位基因，创始人将被赋予纯合性，以随后用杂合子引用的1-循环2小鼠繁殖。这种繁殖方案将产生所有携带R26-LSL-SIX2等位基因的后代，而一半的携带Cusited1-Cre-ERT2，该后代可以控制他莫昔佛剂对CRE阴性小鼠肾脏发育的影响。在该方案中，对所有大坝的管理，他莫昔佛剂将诱导CM特异性的CRE产生，从而在一半的幼崽中引起终止密码子的惊喜，随后的Six2产生将在CM和子女细胞的肾单位祖细胞中特别发生。与CM中的峰值峰值活性一致，他莫昔芬将在E15.5时给予大坝，并在E19.5和产后一个月的肾单位祖细胞分化的表型进行评估。在此模型中，我们预测CM分化和这些祖细胞的保留的破坏是以肾原性的形式造成的。这些研究的影响将是开发一个模型，以探索CM中持续的SIX2表达，作为启动WT序列的候选机制，并在未来的工作中评估Six2与Wnt/β-catenin信号传导的相互作用是癌症干细胞存活的一种目标机制，这些机制将揭示出新的和更有效的药物。