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

改变的 miRNA 模式是众所周知的癌症特征，但直到最近鉴定出肾母细胞瘤中 miRNA 加工基因的反复突变，才确定 miRNA 生物合成失调是肿瘤形成的驱动因素。肾母细胞瘤是源自肾前体细胞的胚胎肿瘤。特别是在胚芽型肿瘤中，胚胎前体细胞显然无法终末分化并保持高度增殖状态。通过外显子组测序，我们发现胚芽亚组中微处理器基因 DROSHA 和 DGCR8 存在反复突变，且预后较差。大多数突变预计要么使 DROSHA 的催化中心失活，要么改变 DGCR8 的 RNA 结合域，这两者都会导致 RNA 代谢的改变。再加上 DICER1、DIS3L2 和 LIN28B 在（遗传性）肾母细胞瘤罕见病例中发挥作用的证据，这清楚地表明 miRNA 及其加工机制对于调节肾前体细胞的细胞命运和恶性转化至关重要，即使目前完全未知其潜在机制。我们计划描述此类突变对临床肿瘤特征的影响。我们工作的重点将是体外分析突变体 DROSHA/DGCR8 对培养肿瘤细胞以及作为遗传上更容易处理的模型的小鼠 ES 细胞中细胞 RNA 表达和功能的影响。这些还将用于测试本次筛选中确定的关键靶标 RNA 的功能相关性，以评估是否只是单个基因或对 miRNA 甚至 mRNA/lncRNA 的全局影响可能很重要。为了生成微处理器依赖性肾母细胞瘤形成的模型，我们将建立具有可诱导突变体 DROSHA 和 DGCR8 cDNA 的小鼠品系，这将使我们能够将功能研究扩展到体内情况。这些实验应该为我们提供关于 miRNA 驱动肿瘤形成的新途径的独特见解，该途径应该在更广泛的背景下相关。

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.