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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：
8976148
负责人：
Harold Newton Lovvorn
金额：
$ 7.9万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
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 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8976148
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 Health Human Inbreeding Injection of therapeutic agent Kidney Laboratories Lesion LoxP-flanked allele 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 pup self-renewal stem cell population stemness therapy resistant 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（由申请人提供）：肾母细胞瘤（WT）是一种小儿肾脏恶性肿瘤，被认为是由胚胎肾干细胞的异常分化和持续存在引起的。肾单位祖细胞究竟如何逃离上皮定型和转化途径（可能代表肾母细胞瘤发生中“第一击”的机制）尚未阐明。 WT 胚基是这些肾单位祖细胞的假定恶性类似物，保留了转录调节因子 Six2 的表达，该调节因子在小鼠发育中促进帽间充质 (CM) 的自我更新并防止其过早上皮分化。我们之前的工作表明，通常在成人肾脏中不存在的 SIX2 在广泛的人类 WT 中持续表达。将发育和疾病中 SIX2 活性的这些观察结果放在一起，出现了一个根本问题：该基因是否为 CM 及其后代在 WT 序列中自我延续提供了一种机制。开发该小鼠模型的目的是测试以下假设：CM 中的持续 Six2 表达会损害上皮分化并促进该祖细胞群体的保留发展为肾源性休息，即 WT 的假定前体病变。我们有两个目标：1) 生成并验证组织特异性、Cre 激活的小鼠系，该小鼠系允许对肾单位祖细胞中 Six2 的表达进行时间控制；2) 表征胚胎和成年肾脏中持续 Six2 表达的表型。简而言之，小鼠将被改造为在我们建立的他莫昔芬诱导型和 CM 特异性 Cited1-Cre-ERT2 转基因小鼠系的时间控制下在肾单位祖细胞（和子细胞）中持续表达 Six2。为了产生对 Six2 表达的时间控制，将设计小鼠特异性 ROSA26-loxP-STOP-loxP-Gfp-2a- Six2 （简称 R26-LSL-Six2）等位基因。一旦验证在种系中传递此 R26-LSL-Six2 等位基因，创始人将进行近交以达到纯合性，以便随后与杂合 Cited1-Cre-ERT2 小鼠进行繁殖。该育种计划将产生全部携带 R26-LSL-Six2 等位基因的后代，一半携带 Cited1-Cre-ERT2，这可以控制他莫昔芬对 Cre 阴性小鼠肾脏发育的影响。在该方案中，对所有母鼠施用他莫昔芬将诱导 CM 特异性产生 Cre，从而切除一半幼仔中的终止密码子，随后的 Six2 产生将特异性发生在 CM 和子细胞的肾单位祖细胞中。与 CM 中 Cited1 活性峰值一致，将在 e15.5 时对母鼠施用他莫昔芬，并将在 e19.5 和出生后一个月评估由此产生的肾单位祖细胞分化表型。在这个模型中，我们预测 CM 分化的破坏和这些祖细胞以肾源性休息的形式保留。因此，这些研究的影响将是开发一个模型，用于探索 CM 中持续 Six2 表达作为 WT 序列启动的候选机制，并在未来的工作中评估 Six2 与 Wnt/β-catenin 信号传导的相互作用，作为癌症干细胞存活的靶向机制，从而揭示新的、更有效的药物。