Functional analysis of DROSHA and DGCR8 microprocessor mutations as a cause of Wilms tumors

miRNA模式的改变是众所周知的癌症特征，但直到最近才在Wilms肿瘤中发现miRNA加工基因的复发性突变，现在才确定miRNA生物发生的失调是肿瘤形成的驱动因素。肾母细胞瘤是来源于肾前体细胞的胚胎性肿瘤。特别是在胚型肿瘤中，胚胎前体细胞显然不能最终分化并保持高度增殖状态。通过外显子组测序，我们发现微处理器基因DROSHA和DGCR8在胚亚群中反复突变，结果不佳。据预测，大多数突变要么使DROSHA的催化中心失活，要么改变DGCR8的RNA结合结构域，这两种突变都会导致RNA代谢的改变。再加上DICER1、DIS3L2和LIN28B在罕见的（遗传性）肾母细胞瘤中发挥作用的证据，这清楚地表明，mirna及其加工机制对于调节肾前细胞的细胞命运和恶性转化至关重要，即使其潜在机制目前还完全未知。我们计划描述这些突变对临床肿瘤特征的影响。我们的工作重点将是在体外分析突变体DROSHA/DGCR8对培养肿瘤细胞和小鼠胚胎干细胞中细胞RNA表达和功能的影响，因为它们在遗传上更容易处理。这些也将用于测试在该筛选中鉴定的关键靶rna的功能相关性，以评估是否只有单个基因或对miRNA甚至mRNA/lncRNA的整体影响可能是重要的。为了建立微处理器依赖的Wilms肿瘤形成模型，我们将建立具有诱导突变DROSHA和DGCR8 cdna的小鼠系，这将使我们能够将我们的功能研究扩展到体内情况。这些实验应该为我们提供对miRNA驱动肿瘤形成的新途径的独特见解，这应该与更广泛的背景相关。

Altered miRNA patterns are well known features of cancer, but only the recent identification of recurrent mutations in miRNA processing genes in Wilms tumors has now established deregulation of miRNA biogenesis as a driver of tumor formation. Wilms tumors are embryonal neoplasms that are derived from renal precursor cells. Especially in blastemal-type tumors, embryonic precursor cells apparently fail to terminally differentiate and remain in a highly proliferative state. By exome sequencing, we have identified recurrent mutations in the microprocessor genes DROSHA and DGCR8 in the blastemal subgroup with poor outcome. Most mutations are predicted either to inactivate the catalytic center of DROSHA or to change the RNA binding domain of DGCR8, both of which should lead to altered RNA metabolism. Together with evidence for a role of DICER1, DIS3L2 and LIN28B in rare cases of (inherited) Wilms tumors, this clearly suggests that miRNAs and their processing machinery must be critical for regulating cell fate and malignant transformation in kidney precursor cells, even if the underlying mechanism is completely unknown at this point.We plan to characterize the consequences of such mutations on clinical tumor characteristics. The focus of our work will be on in vitro analyses of the effects of mutant DROSHA/DGCR8 on cellular RNA representations and functions in cultured tumor cells as well as in mouse ES cells as genetically more tractable models. These will also be used to test the functional relevance of critical target RNAs identified in this screen to evaluate if just single genes or global effects on miRNA or even on mRNA/lncRNA may be important. To generate a model of microprocessor dependent Wilms tumor formation, we will establish mouse lines with inducible mutant DROSHA and DGCR8 cDNAs that will allow us to extend our functional studies to the in vivo situation. These experiments should provide us with unique insights into this novel pathway of miRNA driven tumor formation that should be relevant in a much broader context.