RESUMO
'Gain' of supernumerary copies of the 8q24.21 chromosomal region has been shown to be common in many human cancers and is associated with poor prognosis. The well-characterized myelocytomatosis (MYC) oncogene resides in the 8q24.21 region and is consistently co-gained with an adjacent 'gene desert' of approximately 2 megabases that contains the long non-coding RNA gene PVT1, the CCDC26 gene candidate and the GSDMC gene. Whether low copy-number gain of one or more of these genes drives neoplasia is not known. Here we use chromosome engineering in mice to show that a single extra copy of either the Myc gene or the region encompassing Pvt1, Ccdc26 and Gsdmc fails to advance cancer measurably, whereas a single supernumerary segment encompassing all four genes successfully promotes cancer. Gain of PVT1 long non-coding RNA expression was required for high MYC protein levels in 8q24-amplified human cancer cells. PVT1 RNA and MYC protein expression correlated in primary human tumours, and copy number of PVT1 was co-increased in more than 98% of MYC-copy-increase cancers. Ablation of PVT1 from MYC-driven colon cancer line HCT116 diminished its tumorigenic potency. As MYC protein has been refractory to small-molecule inhibition, the dependence of high MYC protein levels on PVT1 long non-coding RNA provides a much needed therapeutic target.
Assuntos
Variações do Número de Cópias de DNA/genética , Amplificação de Genes/genética , Dosagem de Genes/genética , Genes myc/genética , Proteína Oncogênica p55(v-myc)/genética , RNA Longo não Codificante/genética , Animais , Transformação Celular Neoplásica , Cromossomos Humanos Par 8/genética , Modelos Animais de Doenças , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína Oncogênica p55(v-myc)/metabolismo , FenótipoRESUMO
RATIONALE: The lung extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) mediates progression of fibrosis by decreasing fibroblast expression of miR-29 (microRNA-29), a master negative regulator of ECM production. The molecular mechanism is undefined. IPF-ECM is stiffer than normal. Stiffness drives fibroblast ECM production in a YAP (yes-associated protein)-dependent manner, and YAP is a known regulator of miR-29. Therefore, we tested the hypothesis that negative regulation of miR-29 by IPF-ECM was mediated by mechanotransduction of stiffness. OBJECTIVES: To determine how IPF-ECM negatively regulates miR-29. METHODS: We decellularized lung ECM using detergents and prepared polyacrylamide hydrogels of defined stiffness by varying acrylamide concentrations. Mechanistic studies were guided by immunohistochemistry of IPF lung and used cell culture, RNA-binding protein assays, and xenograft models. MEASUREMENTS AND MAIN RESULTS: Contrary to our hypothesis, we excluded fibroblast mechanotransduction of ECM stiffness as the primary mechanism deregulating miR-29. Instead, systematic examination of miR-29 biogenesis revealed a microRNA processing defect that impeded processing of miR-29 into its mature bioactive forms. Immunohistochemical analysis of the microRNA processing machinery in IPF lung specimens revealed decreased Dicer1 expression in the procollagen-rich myofibroblastic core of fibroblastic foci compared with the focus perimeter and adjacent alveolar walls. Mechanistically, IPF-ECM increased association of the Dicer1 transcript with RNA binding protein AUF1 (AU-binding factor 1), and Dicer1 knockdown conferred primary human lung fibroblasts with cell-autonomous fibrogenicity in zebrafish and mouse lung xenograft models. CONCLUSIONS: Our data identify suppression of fibroblast Dicer1 expression in the myofibroblast-rich IPF fibroblastic focus core as a central step in the mechanism by which the ECM sustains fibrosis progression in IPF.
Assuntos
RNA Helicases DEAD-box/genética , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , MicroRNAs/metabolismo , Ribonuclease III/genética , Animais , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibrose/genética , Fibrose/patologia , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Peixe-ZebraRESUMO
A causal link between hematopoietic stem/progenitor cell (HSPC) dysfunction and DNA damage accrual has been proposed. Clinically relevant strategies to maintain genome integrity in these cells are needed. Here we report that eltrombopag, a small molecule agonist of the thrombopoietin (TPO) receptor used in the clinic, promotes DNA double-strand break (DSB) repair in human HSPCs. We found that eltrombopag specifically activates the classic nonhomologous end-joining (C-NHEJ) DNA repair mechanism, a pathway known to support genome integrity. Eltrombopag-mediated DNA repair results in enhanced genome stability, survival, and function of primary human HSPCs, as demonstrated in karyotyping analyses, colony-forming unit assays and after transplantation in immunodeficient NSG mice. Eltrombopag may offer a new therapeutic modality to protect human HSPCs against genome insults.