Gene amplification-associated overexpression of the RNA editing enzyme ADAR1 enhances human lung tumorigenesis

Abstract

The introduction of new therapies against particular genetic mutations in non-small-cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis.

Figure 1

Determination of ADAR1 RNA and protein overexpression in lung cancer cell lines and its association with gene amplification. (a) Assessment of ADAR1 and ADAR2 expression by quantitative reverse-transcription PCR. Determination of ADAR1 protein levels by western blot (AMAb90535; Atlas Antibody AB, Stockholm, Sweden) (b) and immunofluorescence (ab88574; Abcam, Cambridge, UK) (c) confirms overexpression of the ADAR1 protein in NCI-H1395, NCI-H1437 and NCI-H1993 cells. (d) Fluorescence in situ hybridization of the ADAR1 gene. The UCSC genome browser (http://www.genome.ucsc.edu) was used to select the bacterial artificial chromosome (BAC) clone spanning the 1q21.3 region of the ADAR1 gene: RP11-498A2 (1q21.3–1q22). A telomeric BAC clone located in the telomeric 1p36.23 region was used as a control. The BACs were obtained from the BACPAC Resource Center of the Children's Hospital Oakland Research Institute (Oakland, CA, USA). ADAR1 and telomeric probes were labeled with Spectrum Green and Red dUTP (Abbott, Wiesbaden, Germany), respectively, using a CGH Nick Translation Reagent Kit (Abbott Molecular Inc., Des Plaines, IL, USA). The samples were counterstained with 4′,6-diamidino-2-phenylindole in Vectashield antifade solution (Burlingame, CA, USA). Gene amplification was observed in the interphases of NCI-H1395, NCI-H1437 and NCI-H1993 cells. Probes were verified to give a single signal on normal commercial lymphocyte metaphase slides (CGH Reagents; Abbott). (e) Assessment of ADAR1 copy number by quantitative genomic PCR. Amplification frequency of ADAR1 (evaluated with SYBR Green; Bio-Rad, Hercules, CA, USA) was calculated by the standard curve method using the 7900HT SDS program. To define an internal control gene, we chose chromosome 1p36.11 because it is the least aneuploid region among our cell lines (RPL11 gene). Primers are available upon request. DNA from the normal lung was used as the reference standard. Results are reported as n-fold copy number increase relative to the RPL11 gene. Gene amplification was observed in the NCI-H1395, NCI-H1437 and NCI-H1993 cell lines. (f) Multiplex ligation-dependent probe amplification (MLPA) assay. Two probemixes contain one probe for exons 4, 8 and 14 of the ADAR1 gene (in light blue). Twenty-one reference probes are included (in green). MLPA images from one of the two probemixes are shown. Values greater than 1 (equivalent to two copies) were considered to be extra copies. NCI-H1395, NCI-H1437 and NCI-H1993 show ADAR1 gene amplification, while A549, Cal12T and HBEC3KT-p53-K-ras are presented as examples of two ADAR1 copy number cells. (g) Graph depicting the ADAR1 amplified region in 1q21.3, identified with the Illumina Infinium HumanOmni5 microarray that interrogates 4301332 single-nucleotide polymorphisms in the entire genome. NCI-H1395 cell line has the largest amplified region (1502410 bp) that encompasses the ADAR1 gene. NCI-H1437 and NCI-H1993 have an amplified region of 767 275 and 759 573 bp, respectively. For the three cell lines there is a minimal common region of 662 085 bp where ADAR1 is included.

Figure 2

Growth-promoting effects of ADAR1 in lung cancer. (a) Stable downregulation of the ADAR1 gene by short hairpins using two different target sequences for NCI-H1993 (clones sh1_4, sh1_5, sh1_29, sh4_24, sh4_25 and sh4_27) and NCI-H1395 (sh1_24 and sh2_6). ADAR1 shRNA sequences are available upon request. (b) The short hairpin ADAR1-depleted cells were less viable in the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay than in the untransfected or scrambled shRNA-transfected cells. Probabilities are those from permutation test. (c) The colony formation assay showed that NCI-H1993 and NCI-H1395 cells stably transfected with the shRNA against ADAR1 formed significantly fewer colonies than did scrambled shRNA-transfected cells. Probabilities are those from Student's t-test. Results are presented as the mean±s.d., n=8. (d) Effect of ADAR1 shRNA-mediated depletion on the growth of NCI-H1993 xenografts in nude mice. Upper right panel: western-blot showing effective depletion of the ADAR1 protein upon shRNA targeting. Tumor volume was monitored over time and the tumor was excised and weighed at 49 days. There was a significant decrease in tumor weight and volume in the ADAR1 shRNA-stably transfected cells. Probabilities are those from Student's t-test. Results are presented as the mean±s.e.m., n=20. (e) Left: illustrative tumor samples obtained at the end point of the orthotopic growth nude mouse experiment from shRNA scramble and shRNA-ADAR1-depleted NCI-H1993 cells. Right: mean±s.e.m. of tumor weights and volumes of orthotopic tumors for both groups at 60 days. Student's t-test: **P<0.05. (f) Right: direct spleen injection of ADAR1 shRNA-depleted NCI-H1993 cells (n=24 mice) showed fewer liver metastases than with scramble-shRNAcells (n=7 mice). Left: illustrative images of macroscopic and microscopic metastases. (g) Top left: western blot showing ADAR1 protein overexpression upon transfection. Bottom left: effect of ADAR1 on the invasion potential of A549 cells determined by the matrigel invasion assay. Statistical significance was derived from ANOVAs. *P<0.05. Right: ADAR1 transfection in A549 also increased colony formation (top) and migration capacity in the wound-healing assay (below). (h) Right: direct spleen injection of ADAR1-transfected A549 cells (n=10 mice) showed increased liver metastases than with empty-vector transfected cells (n=5 mice). Left: illustrative images of macroscopic and microscopic metastases. (i) Top left: western blot showing ADAR1 protein overexpression upon transfection. Stable transfection of ADAR1 in HBEC3KT-p53-K-ras cells also caused increased colony formation (bottom left) and enhanced migration capability (right). Results are presented as the mean±s.d. from three independent experiments (Student's t-test P<0.01 vs empty vector in all experiments). ***P<0.01.

Figure 3

Effect of ADAR1 on RNA-editing lung cancer cells. (a) Quantification of A-to-I editing at the lysine 242 AAA codon of the NEIL1 transcript determined by cDNA sequencing. Position 1, AIA; Position 2, AII. Left: ADAR1 gene-amplified cells (NCI-H1993 and NCI-H1395) show higher basal levels of NEIL1 editing than ADAR1 non-amplified cells (A549 and HBEC3KT-p53-K-ras). (b) Transfection-mediated overexpression of ADAR1 in A549 and HBEC3KT-p53-K-ras cells increases NEIL1 editing (left), whereas ADAR1-shRNA-mediated depletion in NCI-H1993 and NCI-H1395 cells reduces NEIL1 editing (right). P-values obtained by Fisher's exact test. (c) Depiction of the minigene strategy to study the editing effect of ADAR1 in miR-381. The minigene is cloned in pLVX-shRNA2 vector and inserted into the genome. After its transcription, complementary sequences of the intron fall into a local stem loop that enhance ADAR1 binding and editing on the neighboring pri-miR-381 sequence. The two edited positions are highlighted in blue. (d) ADAR1-shRNA-mediated depletion in NCI-H1993 reduces miR-381 A-to-I editing.

Figure 4

Detection of ADAR1 gene amplification, its associated overexpression and clinical impact in primary tumors from lung cancer patients. (a) Examples of assessment of ADAR1 copy number by MLPA in NSCLC patients with (top) and without (bottom) gene amplification. (b) Association between ADAR1 gene amplification and ADAR1 protein expression measured by immunohistochemistry (Ab126755; Abcam) in 12 studied cases. Fisher's exact test, significant for two-tailed values of P=0.0182. Illustrative immunohistochemistry images for ADAR1 expression in two ADAR1 amplified (1 and 2) and unamplified (3 and 4) cases are shown. x100 magnification. (c) Left: Kaplan–Meier analysis of progression-free survival (PFS) among all clinical stages of NSCLC cases by ADAR1 genomic status. ADAR1 gene amplification is marginally associated with a shorter PFS. Number of events (progression) from 10 to 60 months in both groups. Right: forest plot of Cox multivariate regression, taking clinical features and ADAR1 copy number into account. Parameters with an associated value of P<0.05 were considered to be independent prognostic factors. (d) Left: Kaplan–Meier analysis of PFS in stage I NSCLC cases according to ADAR1 genomic status. ADAR1 gene amplification is significantly associated with a shorter PFS. Number of events (progression) from 10 to 60 months in both groups. Right: forest plot of Cox multivariate regression, taking clinical features of the validation cohort into account. Parameters with an associated value of P<0.05 were considered to be independent prognostic factors. ADAR1 gene amplification was associated with PFS in the NSCLC cohort.