p53基因突变促进Wilms 肿瘤发展转移的小鼠动物模型研究

Wilms tumor as a pediatric renal cancer raises serious health risk to children. The mechanism of WT metastasis is unclear, no effective therapies are available for these patients. We recently discovered that somatic Wt1 knockout induced WT tumorigenesis ，and constitutive Igf2 upregulation accelerates tumorigenesis. It is the first WT mouse model for therapuetic study (JCI 2011). Preliminary data indicated that other mechanisms contribute to WT tumorigenesis and metastasis. A p53R172H mouse model corresponding to human mutation is demonstrated to often cause metastatic tumors. Thus, we propose to (1) cross the our previuosly generated WT mice with the mice carrying p53R172H (equivalent to human p53R175H) mutation to investigate the role of p53 mutation in WT genesis and metastasis. (2) identify gene/protein expression alterations between the non-metastaic and metastaic tumors via high-throughput RNA-seq, miRNA-seq and Reverse Phase Protein Array analyses. (3) develop a molecular signature for metastasis after validating the top prioritized molecular changes in mouse xenograft model and human metastatic WT cell line WiT49 with various assays. This study will reveal the mechanism of WT metastasis, and help to develop more effective and personalized therapy.

Wilms 肿瘤（WT）是严重危害儿童健康的肾脏癌，其远位转移的原因不明，且缺乏有效的治疗方法。申请人前期发现Wt1基因剔除会诱导WT生成，而上调Igf2具促进作用，这首次为WT的治疗研究提供了小鼠模型（JCI, 2011）。预实验表明存在其他机制加速WT生成，转移；而模拟人p53突变的小鼠p53R172H模型多生成转移性肿瘤。据此本项目提出（1）通过杂交Wt1和 p53R172H的小鼠模型，研究p53 突变对肿瘤发展转移的影响，建立WT转移的模型。（2）通过RNA-seq, miRNA-seq 和 Reverse Phase Protein Array分析，比较转移与非转移肿瘤之间差异表达基因和蛋白。（3）在小鼠转移瘤及人WT转移肿瘤细胞WiT49中核实差异表达分子功能，确定转移标志分子。本项目将揭示WT肿瘤发生转移的机制，并提出针对性治疗方法。

Wilms 肿瘤（WT）是最常见的儿童肾脏恶性瘤。大约25%患者在治疗后没有好的效果，仅有约50%的患者会存活。4成在治疗后无效果的患者具有p53突变，并伴有转移情况发生。我们对于WT发生转移的生物学机制了解很少，部分是由于缺少模型动物。.本研究中建立了具有突变p53表达的肾母细胞瘤发生的Wt1/Igf2/p53+/515A模型老鼠，在这个模型老鼠中p53515A等位基因被引入。p53515A等位基因等同于人的p53R175H热点突变，并且实验证明在具有骨肉瘤和其他肿瘤的老鼠中具有这个突变会发生肿瘤转移。Wt1/Igf2/p53+/515A模型老鼠与Wt1/Igf2模型老鼠相比，它会提早两个月产生Wilms肿瘤。Wilms肿瘤发生在Wt1/Igf2/p53+/515A模型老鼠要比Wt1/Igf2模型老鼠提早大约两个月。但Wt1/Igf2/p53+/515A模型老鼠并没有自发产生转移瘤。通过NOD/SCID老鼠移植瘤（腹腔注射和尾静脉注射自发产生的Wilms肿瘤细胞）实验发现，Wt1/Igf2/p53+/515A模型老鼠肿瘤细胞比Wt1/Igf2模型老鼠肿瘤细胞具有更高的转移能力。在Wilms肿瘤发生转移时，p53稳定性增强与之相伴随。通过细胞体外实验，敲除突变p53后WiT49细胞侵袭能力降低。同时通过RNAseq测序发现了一些差异表达基因。