Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Sep 19;10(497):eaah3941.
doi: 10.1126/scisignal.aah3941.

The RNA-editing enzyme ADAR promotes lung adenocarcinoma migration and invasion by stabilizing FAK

Elianna M Amin  1 , Yuan Liu  1 , Su Deng  1 , Kay See Tan  2 , Neel Chudgar  1 , Marty W Mayo  3 , Francisco Sanchez-Vega  2 , Prasad S Adusumilli  1 , Nikolaus Schultz  2 , David R Jones  4
Affiliations

The RNA-editing enzyme ADAR promotes lung adenocarcinoma migration and invasion by stabilizing FAK

Elianna M Amin et al. Sci Signal. .

Abstract

Large-scale, genome-wide studies report that RNA binding proteins are altered in cancers, but it is unclear how these proteins control tumor progression. We found that the RNA-editing protein ADAR (adenosine deaminase acting on double-stranded RNA) acted as a facilitator of lung adenocarcinoma (LUAD) progression through its ability to stabilize transcripts encoding focal adhesion kinase (FAK). In samples from 802 stage I LUAD patients, increased abundance of ADAR at both the mRNA and protein level correlated with tumor recurrence. Knocking down ADAR in LUAD cells suppressed their mesenchymal properties, migration, and invasion in culture. Analysis of gene expression patterns in LUAD cells identified ADAR-associated enrichment of a subset of genes involved in cell migration pathways; among these, FAK is the most notable gene whose expression was increased in the presence of ADAR. Molecular analyses revealed that ADAR posttranscriptionally increased FAK protein abundance by binding to the FAK transcript and editing a specific intronic site that resulted in the increased stabilization of FAK mRNA. Pharmacological inhibition of FAK blocked ADAR-induced invasiveness of LUAD cells, suggesting a potential therapeutic application for LUAD that has a high abundance of ADAR.

PubMed Disclaimer

Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. ADAR is overexpressed in lung adenocarcinoma (LUAD) and correlates with tumor recurrence
(A) ADAR DNA copy numbers were determined by droplet digital PCR in human bronchial epithelial cells (HBECs) and the indicated LUAD cells. Data are in triplicate from three experiments. (B) ADAR mRNA expression in HBEC and the indicated LUAD cells were assessed by qRT-PCR. HPRT was amplified as a reference. Data are means ± SEM and in triplicate from three experiments. (C) Western blot of ADAR protein expression in HBEC and LUAD cells. N = 3 experiments. (D) Kaplan-Meier curve of progression-free survival based on ADAR mRNA expression in 162 stage I LUAD patients in the NCCRI cohort (log-rank test: p<0.0001). (E) Immunohistochemical analysis showing low and high ADAR expression in two representative stage I LUAD tumors. Scale bars: 100μm (Upper), 50μm (Lower) (F) Cumulative incidence of recurrence based on ADAR protein expression in 802 patients with stage I LUAD (Gray’s test: p=0.016).
Figure 2
Figure 2. ADAR KD inhibits cell migration and invasion
(A) qRT-PCR for ADAR mRNA relative to 18s in H358 and H1975 scramble and ADAR KD, cells. *p<0.05 and **p<0.01, compared with scramble (Mann-Whitney test). Data are means ± SEM from three independent experiments. (B) Western blot of ADAR expression in H358 and H1975 scramble and ADAR KD cells. N = 3 independent experiments. (C) Immunofluorescent staining with phalloidin (Red: F-actin) in H358 and H1975 scramble and ADAR KD cells. Scale bar: 25μm. N = 3 independent experiments. (D) Cell migration of H358 and H1975 scramble control and ADAR KD cells. cells. *p<0.05, compared with scramble (Mann-Whitney test). Data are means ± SEM from three independent experiments. Scale bar: 100μm. (E) Cell invasion of H358 and H1975 scramble and ADAR KD cells. cells. **p<0.01, compared with scramble (Mann-Whitney test). Data are means ± SEM from three experiments. Scale bar: 100μm. (F) Soft agar colony formation of H358 and H1975 scramble and ADAR KD cells. *p<0.05 and **p<0.01, compared with scramble (Mann-Whitney test). Data are means ± SEM from three independent experiments. Scale bar: 200μm.
Figure 3
Figure 3. ADAR expression increases FAK expression
(A) Heat map of genes related to cellular movement differentially expressed in H358 scramble and ADAR KD cells (p = 0.0009). N = 2 biological replicates. (B) qRT-PCR of FAK mRNA relative to 18s in the indicated cells. *p<0.05, **p<0.01, and ***p<0.001 compared with scramble (Mann-Whitney test). Data are means ± SEM from three independent experiments. (C) Western blot of FAK in the indicated cells. N = 3 independent experiments. (D) Flag-tagged ADAR was stably transfected into H1975 ADAR KD cells. Western blot for the indicated proteins. N = 3 independent experiments. (E) The correlation of ADAR and FAK mRNA in advanced stages LUAD (TCGA cohort, N = 57, p=0.031). (F) Western blot of the indicated proteins in selected patient LUAD samples. N = 3 independent experiments. (G) Immunofluorescence staining cortactin in the indicated cells. Scale bar: 25μm. N = 3 independent experiments. (H) Phospho-cortactin in the indicated cells was detected by coimmunoprecipitation using an antibody against pan-phospho-tyrosine followed by immunoblotting with an antibody against cortactin. N = 3 independent experiments. (I) Western blot for phospho-paxillin Tyr118 and paxillin in the indicated cells. N = 3 independent experiments.
Figure 4
Figure 4. ADAR stabilizes FAK transcript
(A) Percentage of remaining FAK mRNA in the indicated cells following treatment with Actinomycin D. **p<0.01 compared with scramble (Wilcoxon rank sum test of area under the curve). Data are means ± SEM from three independent experiments. (B) Diagram of ADAR protein. Blue: Zα and Zβ domains, Purple: dsRBDs Red: deaminase domain. (C) qRT-PCR for FAK mRNA relative to 18s in HCC827 and H1299 cells transfected with ADAR wt, mutants or empty vector as control. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 compared with controls (Mann-Whitney test). Data are means ± SEM from three independent experiments. (D) Western blot for the indicated proteins in the indicated HCC827 and H1299 cells. N = 3 independent experiments. (E) Phosphocortactin in the indicated HCC827 and H1299 cells was detected by coimmunoprecipitation using an antibody against pan-phospho-tyrosine followed by immunoblotting with cortactin. N = 3 independent experiments. (F) Percentage of remaining FAK mRNA in the indicated HCC827 and H1299 cells following treatment with Actinomycin D. **p<0.01 compared with control. ##p < 0.01 compared with the deaminase mutant (Wilcoxon rank sum test of area under the curve). Data are means ± SEM from three independent experiments.
Figure 5
Figure 5. ADAR stabilizes FAK through RNA-binding and editing
(A) FAK mRNA in tumors with (edit, N=41) or without (non-edit, N=189) A-to-I editing in the TCGA cohort. ***p<0.001, compared to non-edited tumors (Mann-Whitney test). (B) Chromatograms of FAK transcripts in the indicated cells. Arrow: the site chr8:141,702,274. The percentage of edited FAK detected by Sanger sequencing. N=3 biological replicates. (C) RIPs for region A in H1975 parental cells. Data are means ± SEM from three independent experiments. (D) RIPs for region A on the indicated H1299 stable cells. *p<0.05, comparing groups (Mann-Whitney test). Data are means ± SEM from three independent experiments. (E) RNA-protein interaction of in vitro dsRNA and ADARs. n = 3 independent experiments. (F) Percentage of remaining pcDNA-FAK mRNA in indicated H1975 cells following Actinomycin D treatment. *p<0.05 compared with FAKE+I WT in scramble (Blue); #p<0.05 compared with FAKE+I edited in ADAR KD (Purple); ϕp<0.05 compared with FAKE+I edited in scramble (Red) (Wilcoxon rank sum test of area under the curve). Data are means ± SEM from three independent experiments. (G) Schematic ADAR binding and editing FAK in the intron 26. Red lines: regions A and B used for RIP assays. Arrow: the editing site.
Figure 6
Figure 6. ADAR-induced cell migration and invasion is FAK dependent
(A) Invasion assays of H358 and H1975 scramble, ADAR KD, and ADAR KD with Flag-FAK cells. #p<0.05, compared with corresponding ADAR KD cells. *p<0.05 and **p<0.01, compared with scramble (Mann-Whitney test). Data are means ± SEM from three independent experiments. Scale bar: 100μm (B) Western blot for the indicated proteins in H358 and H1975 scramble control, ADAR KD, and ADAR KD with Flag-FAK cells. N = 3 independent experiments. (C) Coimmunoprecipitation using antibody against pan-phospho-tyrosine in HCC827 and H1299 control or ADAR expressed cells treated with PND1186 or VS-6063 (2.5 μM) or DMSO for 72 h. Western blot for phospho-FAK and phospho-paxillin with antibody against FAK or paxillin. N = 3 independent experiments. (D) Invasion assays of HCC827 and H1299 control and ADAR expressing cells treated with FAK inhibitors PND1186 or VS-6063 (2.5 μM) or DMSO for 72 h. #p<0.05, compared with DMSO treated control cells. *p<0.05 and **p<0.01, compared with DMSO with the same vector (Mann-Whitney test). Data are means ± SEM from three independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin. 2015;65:5–29. - PubMed
    1. Pao W, Hutchinson KE. Chipping away at the lung cancer genome. Nat Med. 2012;18:349–351. - PubMed
    1. Cooper TA, Wan L, Dreyfuss G. RNA and disease. Cell. 2009;136:777–793. - PMC - PubMed
    1. Fumagalli D, Gacquer D, Rothe F, Lefort A, Libert F, Brown D, Kheddoumi N, Shlien A, Konopka T, Salgado R, Larsimont D, Polyak K, Willard-Gallo K, Desmedt C, Piccart M, Abramowicz M, Campbell PJ, Sotiriou C, Detours V. Principles Governing A-to-I RNA Editing in the Breast Cancer Transcriptome. Cell Rep. 2015;13:277–289. - PMC - PubMed
    1. Han L, Diao L, Yu S, Xu X, Li J, Zhang R, Yang Y, Werner HM, Eterovic AK, Yuan Y, Li J, Nair N, Minelli R, Tsang YH, Cheung LW, Jeong KJ, Roszik J, Ju Z, Woodman SE, Lu Y, Scott KL, Li JB, Mills GB, Liang H. The Genomic Landscape and Clinical Relevance of A-to-I RNA Editing in Human Cancers. Cancer Cell. 2015;28:515–528. - PMC - PubMed

MeSH terms