MicroRNA-mediated loss of ADAR1 in metastatic melanoma promotes tumor growth
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
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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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 1

Reduced ADAR1 expression is a common event in melanoma.

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Reduced ADAR1 expression is a common event in melanoma. (A) Analysis of ...
(A) Analysis of the immunohistochemical expression levels of ADAR1. Lines in the boxes denote the mean. (B) ADAR1 expression in low-passage metastatic melanoma cultures, as determined by qPCR. Results are expressed as fold above the average values in all normal melanocyte specimens. Cutoffs for overexpression and downregulation were determined as greater than 2 and less than 0.5, respectively. Data represent the mean ± SEM of 2 experiments on independent RNA purifications, each performed in triplicate. (C) ADAR1 expression at the protein level of selected low-passage melanoma cultures (samples A–G in part B), as determined by Western blot. A representative blot is shown. (D) ADAR1 expression at the protein level as determined by Western blot and (E) A-to-I editing rate of BLCAP by ADAR1 as determined by Sequenom MassArray in ADAR1-manipulated 624mel cell system: stable KD of ADAR1 (ADAR1-KD), rescue of ADAR1-P150 or ADAR1-P110 (rescue-P150, rescue-P110, respectively), and transfection with scrambled sequence and empty pcDNA3 (control). Data represent the mean ± SEM of 3 independent experiments, each performed in triplicate. **P < 0.01 (2-tailed t test, ANOVA); ***P < 0.0001 (ANOVA).