Regulation of the LIN28/let-7 pathway in cancer The RNA-binding proteins LIN28A/B control the biogenesis of let-7 tumor suppressor miRNAs. Reactivation of this embryonic pathway is sufficient to promote cellular transformation and drive tumorigenesis in mouse models. Notably, LIN28B functions as an oncogene in neuroblastoma, Wilms tumors (nephroblastoma), hepatoblastoma, and others. At the molecular level, LIN28A recruits the Terminal Uridyl Transferases (TUTases), ZCCHC6/11, to let-7 precursor RNAs targeting them for destruction by the DIS3L2 exonuclease. Germline DIS3L2 mutations cause Perlman syndrome - a rare developmental and cancer predisposition disorder with ~6,000-fold elevated risk of Wilms tumorigenesis. Somatic mutations in DIS3L2 have also been found in ~30% sporadic Wilms tumors. Despite this genetic evidence that DIS3L2 functions as a tumor suppressor its molecular and cellular function remains unaddressed. Similarly, the Beckwith-Wiedemann syndrome (BWS) of fetal overgrowth is associated with ~600-fold elevated risk of developing Wilms tumors, yet the molecular and cellular function of the H19 long-non-coding RNA, a likely tumor suppressor gene in BWS, remains unknown. Considering the strong links between miRNA, LIN28, DIS3L2, and Wilms as well as preliminary data linking H19 to the LIN28/let-7 pathway, leads us to hypothesize that dysregulation of let-7 biogenesis is a unifying mechanism in Wilms tumorigenesis. Loss-of-function studies will be performed to address the possible redundant role of ZCCHC6/11 in LIN28B regulation of let-7, and biochemical approaches will be employed to elucidate the mechanism by which LIN28B suppresses let-7 in human cancer cells. Relative levels of let-7, H19, LIN28A/B, and DIS3L2 will be measured in a panel of cell lines and primary human Wilms tumors and RNA sequencing analysis will be performed on a subset. H19- and DIS3L2 loss- and gain-of-function experiments will be performed in human cancer cells and the effects on gene expression, cell growth, and tumorigenicity will be measured. Novel approaches will be applied to identify new DIS3L2 target RNAs. This will provide important mechanistic insight into the LIN28/let-7 pathway in human cancer cells, uncover the tumor suppressive role of H19 in pediatric cancer, and highlight the relevance of RNA decay mechanisms in cancer by elucidating DIS3L2 tumor suppressive function.
This study will investigate uncharacterized tumor suppressor genes and uncover their role in RNA regulation in human cancer cells and may lead to novel therapeutic strategies to restore gene expression in neuroblastoma, hepatoblastoma, Wilms, and other childhood tumors
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