MicroRNA-mediated loss of ADAR1 in metastatic melanoma promotes tumor growth
Yael Nemlich, … , Gideon Rechavi, Gal Markel
Yael Nemlich, … , Gideon Rechavi, Gal Markel
Published May 24, 2013
Citation Information: J Clin Invest. 2013;123(6):2703-2718. https://doi.org/10.1172/JCI62980.
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Research Article Oncology

MicroRNA-mediated loss of ADAR1 in metastatic melanoma promotes tumor growth

Abstract

Some solid tumors have reduced posttranscriptional RNA editing by adenosine deaminase acting on RNA (ADAR) enzymes, but the functional significance of this alteration has been unclear. Here, we found the primary RNA-editing enzyme ADAR1 is frequently reduced in metastatic melanomas. In situ analysis of melanoma samples using progression tissue microarrays indicated a substantial downregulation of ADAR1 during the metastatic transition. Further, ADAR1 knockdown altered cell morphology, promoted in vitro proliferation, and markedly enhanced the tumorigenicity in vivo. A comparative whole genome expression microarray analysis revealed that ADAR1 controls the expression of more than 100 microRNAs (miRNAs) that regulate many genes associated with the observed phenotypes. Importantly, we discovered that ADAR1 fundamentally regulates miRNA processing in an RNA binding–dependent, yet RNA editing–independent manner by regulating Dicer expression at the translational level via let-7. In addition, ADAR1 formed a complex with DGCR8 that was mutually exclusive with the DGCR8-Drosha complex that processes pri-miRNAs in the nucleus. We found that cancer cells silence ADAR1 by overexpressing miR-17 and miR-432, which both directly target the ADAR1 transcript. We further demonstrated that the genes encoding miR-17 and miR-432 are frequently amplified in melanoma and that aberrant hypomethylation of the imprinted DLK1-DIO3 region in chromosome 14 can also drive miR-432 overexpression.

Authors

Yael Nemlich, Eyal Greenberg, Rona Ortenberg, Michal J. Besser, Iris Barshack, Jasmine Jacob-Hirsch, Elad Jacoby, Eran Eyal, Ludmila Rivkin, Victor G. Prieto, Nitin Chakravarti, Lyn M. Duncan, David M. Kallenberg, Eitan Galun, Dorothy C. Bennett, Ninette Amariglio, Menashe Bar-Eli, Jacob Schachter, Gideon Rechavi, Gal Markel

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Figure 3

ADAR1-dependent regulation of proliferation is RNA-editing independent.

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ADAR1-dependent regulation of proliferation is RNA-editing independent. ...
(A) Ten functional clusters, determined by IPA, that were significantly affected by ADAR1 downregulation are shown. The significance was calculated by Fisher’s exact test, and is expressed as –log (P value). The number of DEGs that map to a specific pathway is indicated at the top of each column. (B) ADAR1 constructs used for functional assays. Shown are ADAR1-P110, ADAR1-P150, and His-tagged dsRBD, Z-DBD-P110, Z-DBD-P150, ΔCAT-P110, ΔCAT-P150, CAT-MUT-P110, and CAT-MUT-P150. ADAR1 fragments amplified and cloned into pCDNA3 (“truncations”). (C) The His-tagged ADAR1 domains were transfected into 624mel cells, and their expression, relative to mock-control, was detected by intracellular staining in flow cytometry; the impact of “truncation” and overexpression constructs on (D) ADAR1 expression in transductants was determined by qPCR. (E) BLCAP editing rate by ADAR1 was examined by sequenom massarray. (F) Net proliferation was monitored by standardized XTT assay. The number of cells was determined 48 hours after seeding. (G) Cells were plated and after 48 hours DNA was extracted, stained with PI, and subsequently analyzed for cell cycle by flow cytometry. Data represent the mean ± SEM of 3 independent experiments, each performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 (2-tailed t test).