Adenosine-To-Inosine RNA Editing Mediated by Adars in Esophageal Squamous Cell Carcinoma

Published OnlineFirst December 3, 2013; DOI: 10.1158/0008-5472.CAN-13-2545

Cancer Molecular and Cellular Pathobiology Research

Adenosine-to-Inosine RNA Editing Mediated by ADARs in Esophageal Squamous Cell Carcinoma

Yan-Ru Qin1, Jun-Jing Qiao1,6, Tim Hon Man Chan6, Ying-Hui Zhu2, Fang-Fang Li4, Haibo Liu3, Jing Fei6, Yan Li2, Xin-Yuan Guan2,5, and Leilei Chen6

Abstract Esophageal squamous cell carcinoma (ESCC), the major histologic form of esophageal cancer, is a heterogeneous tumor displaying a complex variety of genetic and epigenetic changes. Aberrant RNA editing of adenosine-to-inosine (A-to-I), as it is catalyzed by adenosine deaminases acting on RNA (ADAR), represents a common posttranscriptional modification in certain human diseases. In this study, we investigated the status and role of ADARs and altered A-to-I RNA editing in ESCC tumorigenesis. Among the three ADAR enzymes expressed in human cells, only ADAR1 was overexpressed in primary ESCC tumors. ADAR1 overexpression was due to gene amplification. Patients with ESCC with tumoral overexpression of ADAR1 displayed a poor prognosis. In vitro and in vivo functional assays established that ADAR1 functions as an oncogene during ESCC progression. Differential expression of ADAR1 resulted in altered gene-specific editing activities, as reflected by hyperediting of FLNB and AZIN1 messages in primary ESCC. Notably, the edited form of AZIN1 conferred a gain-of-function phenotype associated with aggressive tumor behavior. Our findings reveal that altered gene-specific A-to-I editing events mediated by ADAR1 drive the development of ESCC, with potential implications in diagnosis, prognosis, and treatment of this disease. Cancer Res; 1–12. 2013 AACR.

Introduction prognosis of patients with ESCC is still poor and the 5-year Esophageal squamous cell carcinoma (ESCC), the leading overall survival rate is ranging from 20% to 30% (2). There- cause of cancer death worldwide, is a heterogeneous tumor fore, it is very important to search for biologic markers, displaying a complex variety of genetic and epigenetic which can diagnose cancer as early as possible, estimate changes. ESCC is characterized by its remarkable geographic reaction to chemotherapy or radiotherapy in those patients distribution and high-risk areas include Northern China, with ESCC, and predict overall survival rate of patients especially in Henan province, Northern Iran, and South undergoing treatment. fi Africa (1). Despite the recent advances in treatment, the In human cancers, aberrant posttranscriptional modi ca- benefit of all kind of treatment is not satisfactory. The tions such as alternative splicing and RNA editing may lead to tumor-specific transcriptome diversity. The best-characterized type of RNA editing found in mammals converts cytosine (C) to uracil (U) and adenosine (A) to inosine (I). In Authors' Affiliations: 1Department of Clinical Oncology, the First Affiliated Hospital, Zhengzhou University, Zhengzhou; 2State Key Laboratory of humans, the most common type of RNA editing is conver- Oncology in Southern China, Sun Yat-Sen University Cancer Centre; 3Key sion of A to I, which is catalyzed by the double-stranded RNA Laboratory for Major Obstetric Disease of Guangdong Province, The Third fi Affiliated Hospital of Guangzhou Medical University, Guangzhou; 4Depart- (dsRNA)-speci c ADAR (adenosine deaminases acting on ment of Clinical Oncology, Nanyang city first people's hospital, Henan; RNA) family of protein (3). As inosine preferentially base 5 Department of Clinical Oncology, The University of Hong Kong, Hong pairs with cytidine, inosines are interpreted as guanosines by Kong, China; and 6Cancer Science Institute of Singapore, National Uni- versity of Singpaore, Singapore the translation and splicing machineries. Thus, the ADARs may recode transcripts, which results in a proteome that is Note: Supplementary data for this article are available at Cancer Research – Online (http://cancerres.aacrjournals.org/). divergent from the genome (4 7) and therefore, modulates the protein sequence and function of gene products. Because Y.-R. Qin and J.-J. Qiao contributed equally to this work. of the diverse impacts of RNA editing on gene expression Corresponding Authors: Yan-Ru Qin, Department of Clinical Oncology, the and function, it is possible that the misregulation of A-to-I First Affiliated Hospital, Zhengzhou University, Zhengzhou, China. Phone: 86-371-66295542; E-mail: [email protected]; Xin-Yuan Guan, State Key RNAeditingmayplayaroleintumorigenesisbyeither Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer inactivating a tumor suppressor or activating genes that Centre, Room 605, 651 East Dongfeng Road, Guangzhou 510050, China. promote tumor progression. Phone: 86-20-87343166; Fax: 86-852-28169126; E-mail: [email protected]; ADAR and Leilei Chen, Cancer Science Institute of Singapore, National University The gene family includes three members, ADAR1 (also of Singapore, 14 Medical Drive, Singapore 117599. Phone: 65-6516-8435; known as ADAR), ADAR2 (ADARB1), and ADAR3 (ADARB2). Fax: 65-6516-1873; E-mail: [email protected] ADAR1 and ADAR2 are expressed in most tissues; ADAR3 is doi: 10.1158/0008-5472.CAN-13-2545 exclusively detected in brain tissue (8). There are two isoforms 2013 American Association for Cancer Research. of ADAR1, a full-length ADAR1 p150 and an N-terminally

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truncated ADAR1 p110 (9). The p150 isoform is produced from Analysis of RNA editing an IFN-inducible promoter and the p110 form is constitutively Direct sequencing was performed on PCR products, and the expressed, which is initiated from a downstream methionine as editing frequency was calculated using software ImageJ the result of the skipping of the exon containing the upstream (http://rsb.info.nih.gov/ij/). The reliability of this method was methionine. Because the p150 isoform of ADAR1 can be further verified by cloning of individual sequences. As induced by IFNs and found in cytoplasm, possibly it exerts described previously (12), PCR products were subcloned into its functions to target viruses that replicate in the cytoplasm. the T-easy vector (Promega), and approximately 50 individual However, the ADAR1 p110 isoform exerts its A-to-I modifica- plasmids were sequenced for each sample. For each sample, tion in the nuclear pre-mRNA. Our recent study has reported three independent real-time PCR (RT-PCR) reactions were that the recoding RNA editing of a gene AZIN1 (antizyme performed. inhibitor 1) is specifically catalyzed by ADAR1, and the hyperediting pattern of AZIN1 gene predisposes to human hepato- FISH cellular carcinoma (10). Until now, the roles of RNA editing The detailed procedure for normal karyotype preparation enzyme ADARs and the edited transcripts of target genes in the was described in "Supplementary Materials and Methods." A development of ESCC have not been studied. Here, we dem- BAC clone at 1q21.3 containing the ADAR1 gene (RP11-61L14) onstrated that the RNA editing enzyme ADAR1, but not ADAR2 was labeled with SpectrumRed (Vysis). The chromosome 1 and ADAR3, was significantly overexpressed in primary ESCC centromere probe was labeled with SpectrumGreen (Vysis). tumor compared with their matched nontumor specimens. FISH reaction was performed according to the method Moreover, the role of ADAR1 and the altered gene-specific described previously (13). editing pattern were further investigated in clinical specimen, cell models, and mice. cDNA synthesis and quantitative real-time PCR As described previously (10), the total RNA was isolated Materials and Methods and the equal amounts of cDNA were synthesized using the Cell lines Advantage RT-for-PCR kit (Clontech) and used for quanti- Six Japanese ESCC cell lines (KYSE140, KYSE410, KYSE180, tative PCR (qPCR) analysis. The sequences of primers are KYSE30, KYSE510, and KYSE520) were obtained from DSMZ, included in "Supplementary Materials and Methods." the German Resource Centre for Biological Material (11). A Chinese ESCC cell line HKESC1 was kindly provided by Cell proliferation assay professor Srivastava (Department of Pathology, The Univer- Cells were seeded in 96-well plate at a density of 0.5 to 1 103 sity of Hong Kong, Hong Kong, China), and two Chinese cells per well. The cell growth rate was measured using Cell ESCC cell lines EC18 and EC109 were kindly provided by Proliferation XTT Kit (Roche Diagnostic) according to the professor Tsao (Department of Anatomy, The University of manufacturer's instruction. Three independent experiments Hong Kong). All nine ESCC cell lines were cultured in RPMI- done in triplicate were performed. 1640 medium (Gibco BRL) supplemented with 10% FBS (Gibco BRL). All cell lines used in this study were regularly Focus formation and colony formation in soft agar authenticated by morphologic observation and tested for Briefly, 1 103 cells were seeded in a 6-well plate. After absence of mycoplasma contamination (MycoAlert, Lonza culture for 12 days, surviving colonies (>50 cells per colony) Rockland). The cells were incubated at 37C in a humidified were counted and stained with crystal violet (Sigma-Aldrich). chamber containing 5% CO2. Triplicate independent experiments were performed and the data were expressed as the mean SD of triplicate wells within Clinical samples the same experiment. For soft agar assay, 2 103 cells in 0.4% Cohort 1. A total of 69 paired primary ESCC tumor tissues low-melting agarose (Lonza Rockland) were placed on the top and their matched nontumorous tissues that were surgically of the bottom layer of 0.6% low-melting agarose in a 6-well removed, snap-frozen in liquid nitrogen (for protein, RNA, and plate. After 2 to 4 weeks, surviving colonies (>80 cells/colony) DNA extraction) were obtained from Linzhou Cancer Hospital were counted. (Henan, China) between 2010 and 2011. Cohort 2. A total of 180 paired primary ESCC tumor Cell migration assay tissues and their matched nontumor tissues that were surgi- The Transwell cell migration assay was performed using cally removed and embedded in a paraffin block [for tissue Bio-coat cell migration chambers (BD Biosciences) con- microarray (TMA) construction] were obtained from Linzhou taining polyethylene terephthalate membranes (PET) of Cancer Hospital along with clinicopathological summaries 8 mm pore size according to the manufacturer's instruc- between 2001 and 2005. tions. Briefly, 1 to 2 105 cells in FBS-free RPMI were None of these patients received preoperative chemotherapy added. RPMI supplemented with 10% FBS was added to the and radiotherapy. All clinical samples used in this study were bottom chamber as a chemoattractant. After 24 to 36 approved by the committees for ethical review of research hours, the number of cells that had migrated through involving human subjects at Zhengzhou University (Zhengz- the filter pores was stained with crystal violet (Sigma- hou, China), The University of Hong Kong, and National Aldrich), counted, and imaged using SPOT imaging soft- University of Singapore (Singapore). ware (Nikon).

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Matrigel invasion assay ing in <25% of tumor cells), moderate expression (2; moderate We performed invasion assays using 24-well BioCoat Matri- staining in > ¼ 25% to <75%, or strong staining in <25% of gel Invasion Chambers (BD Biosciences) according to the tumor cells), and strong expression (3; moderate staining in > manufacturer's instructions. Briefly, 1 to 2 105 cells in ¼ 75%, or strong staining in > ¼ 25% of tumor cells). FBS-free RPMI were added to the top chamber, and 10% FBS in RPMI was added to the bottom chamber as a chemoat- Statistical analysis tractant. After 36 to 48 hours of incubation, cells that invaded Unless otherwise indicated, the data are presented as the the Matrigel were fixed and stained with crystal violet (Sigma- mean SD of three independent experiments. The SPSS Aldrich). The number of cells was counted and imaged using statistical package for Windows (version 16; SPSS) was used SPOT imaging software (Nikon). to perform the data analyses. The ADAR1 or ADAR2 expression levels in any two groups of clinical samples (e.g., ESCC tumors In vivo tumorigenicity assay and matched nontumor tissues) were compared using a Wil- We subcutaneously injected approximately 2 106 of coxon signed rank test. Kaplan–Meier plots and log-rank tests EC109-CTL or EC109-AR1 cells into the left or right flank of were used for overall survival analysis. For the TMA analysis, 4- to 5-week-old male severe combined immunodeficient which was based on the scores of IHC staining, ADAR1 (SCID) mice (n ¼ 5), respectively. To compare the tumorigenic expression levels in the primary ESCC tissues and their abilities of the wild-type or edited AZIN1 gene, we subcutane- matched nontumor tissues were compared using the Wilcoxon ously injected approximately 2 106 of EC109-LacZ, EC109- signed rank test. A paired Student t test was used to compare wt/AZI, or EC109-edt/AZI cells into the right flank of SCID the editing levels as well as the total mRNA levels of FLNB or mice (n ¼ 6). We monitored tumor formation in the SCID mice AZIN1 in ESCC and their matched nontumor specimens of over a 4-week period and calculated the tumor volume weekly patients. Spearman correlation coefficients were used to eval- by the formula V (volume) ¼ 0.5 L (length) W (width) W. uate the positive correlation between the expression of ADAR1 All animal experiments were approved by and performed in and the editing level of AZIN1 or FLNB in clinical samples. An accordance with the Institutional Animal Care and Use Com- unpaired two-tailed Student t test was used to compare the mittees of National University of Singapore. number of colonies, the number of migrative and invasive cells, and tumor volume between any two preselected groups. Antibodies and Western blot analysis P < 0.05 was considered to be statistically significant. Mouse anti-ADAR1, anti-AZIN1, and anti-b-actin antibodies were purchased from Abcam. The mouse anti-ADAR2 and anti- Results GAPDH antibodies were purchased from Sigma-Aldrich and RNA editing enzyme ADAR1 is significantly upregulated Santa Cruz Biotechnology. Protein lysates were quantified and in primary ESCCs and its clinical implications resolved on a SDS-PAGE gel, transferred onto a polyvinylidene A-to-I RNA editing is a posttranscriptional modification in difluoride membrane (Millipore), and immunoblotted with a stem-loop structures within precursor mRNA, which is cata- primary antibody, followed by incubation with a secondary lyzed by dsRNA-specific ADAR enzymes (14). To investigate the antibody. The blots were visualized by enhanced chemilumi- expression profiles of the three RNA editing enzymes in ESCC nescence (GE Healthcare). specimens, we examined the expression levels of ADAR1, ADAR2, and ADAR3 in 69 pairs of primary ESCC tumor and Immunohistochemical staining their corresponding nontumor tissues that were obtained from The TMA blocks were sectioned (5 mm thick) for immuno- Linzhou Cancer Hospital (cohort 1). As detected by the qRT- histochemical (IHC) staining. Briefly, sections were deparaffi- PCR, only ADAR1 was significantly upregulated in ESCC sam- nized and rehydrated. The endogenous peroxidase activity was ples (P ¼ 0.0045) and approximately 48 of 69 (69.57%) ESCC blocked with 3% hydrogen peroxide for 10 minutes. For antigen specimens demonstrated the higher expression of ADAR1 than retrieval, the slides were immersed in 10 mmol/L citrate buffer their matched nontumor specimens (Fig. 1A, left). There was (pH 6.0) and boiled for 15 minutes in a microwave oven. no significant difference in ADAR2 expression between pri- Nonspecific binding was blocked with 5% normal goat serum mary ESCCs and their matched nontumor specimens (P ¼ for 10 minutes. The slides were incubated in a 1:100 dilution of 0.1465; Fig. 1A, right). In addition, ADAR3 was undetectable in anti-ADAR1 (Abcam) at 4C overnight in a humidified cham- all of the samples (data not shown). To confirm our findings, ber. The slides were then sequentially incubated with biotiny- Western blot analysis of ADAR1 and ADAR2 expression was lated goat anti-mouse immunoglobulin G (IgG; 1:100 dilution, performed in the paired ESCC and nontumor specimens of 35 Santa Cruz Biotechnology) for 30 minutes at room tempera- randomly selected ESCC cases from the cohort 1. Consistently, ture and streptavidin-peroxidase conjugate for 30 minutes at the upregulation of ADAR1 protein (particularly the p110 room temperature. Isotope-matched human IgG was used in isoform) in tumors was observed in 19 of 35 (54.29%) ESCCs, each case as a negative control. Finally, the 3,30-diaminoben- and there was no obvious difference in ADAR2 protein expres- zidine (DAB) Substrate Kit (Dako Ltd.) was used for color sion between ESCC and their matched nontumor specimens development followed by Mayer's hematoxylin counterstain- (Fig. 1B). ing. On the basis of staining intensities, the ADAR1 immuno- To investigate the clinical implication of ADAR1 during the reactivity was scored as negative (0; total absence of staining), development of ESCC, we constructed a panel of TMAs con- weak expression (1; faint staining in <50%, or moderate stain- sisting of 180 matched pairs of primary ESCC and nontumor

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Figure 1. ADAR1 is significantly overexpressed in primary ESCC samples and its clinical implication. A, box plots represent the relative ADAR1 (left) and ADAR2 (right) expression levels in 69 matched pairs of ESCC and nontumor specimens in cohort 1. The data are presented as box plots with the median (horizontal line), 25% to 75% (box), and 5% to 95% (error bar) percentiles for each group. B, Western blot analyses of ADAR1 and ADAR2 expression levels in six paired ESCC and nontumor specimens in cohort 1. GAPDH was used as a loading control. C, example of the ADAR1 expression level detected in a matched pair of primary ESCC and nontumor tissue in cohort 2. The boxed regions are magnified and displayed in the bottom panels. Scale bar: 200 mm. D, Kaplan–Meier plots for the overall survival rate of patients with (n ¼ 90, red line) or without (n ¼ 46, blue line) the tumoral overexpression of ADAR1. E, FISH analysis of the ADAR1 gene (red signal) and the control chromosome 1 centromere probe (green signal) specifically hybridized to the chromosome 1 (indicated by white arrow) of normal human karyotype (left). A representative example of ADAR1 gene amplification (red signals) in a matched pair of primary ESCC and nontumor tissue (middle and right panels). Scale bar, 500 mm.

specimens that were collected from Linzhou Cancer Hospital informative samples included lost samples, inappropriately (cohort 2). Informative results of IHC staining were observed in stained samples, and samples with too few cells; such were not 136 matched pairs of ESCC and nontumor specimens. Non- used as valid data. By performing IHC staining, we observed the

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differential nuclear expression of ADAR1 between the primary using a lentiviral system (Fig. 2A and Supplementary Fig. S1). ESCC tumor and their matched nontumor tissues (Fig. 1C). A As detected by in vitro functional assays, cells transduced with detailed analysis of the IHC data revealed that the ADAR1 was the ADAR1 lentivirus (180-AR1 and EC109-AR1) had the accel- overexpressed in 66.18% (90/136) of the informative ESCC erated growth rates and higher frequencies of focus formation tumor tissues (P < 0.001, Wilcoxon signed rank test; Fig. 1C and colony formation in soft agar than cells transduced with and Supplementary Table S1). On the basis of the TMA analysis the control LacZ lentivirus (180-CTL and EC109-CTL; Fig. 2B– of ADAR1, the patients with tumoral overexpression of ADAR1 D). Moreover, 180-AR1 and EC109-AR1 cells demonstrated the demonstrated the shorter overall survival time than the increased migrative and invasive capabilities compared with patients without overexpression (log rank: 5.738, P ¼ 0.017; Fig. 180-CTL and EC109-CTL cells, respectively (Fig. 2E). Xenograft 1D). In the univariate Cox analyses, the statistically significant studies in mice demonstrated that the growth rate of tumors predictors for a patient's overall survival were cell differenti- derived from EC109-AR1 cells was markedly higher than those ation (P ¼ 0.036), tumor invasion (P ¼ 0.003), clinical stage (P ¼ derived from EC109-CTL cells (Fig. 2F). 0.001), and the tumoral overexpression of ADAR1 (P ¼ To further con firm our findings, RNA interference (RNAi) 0.020; Table 1). In the multivariate Cox analyses, cell differen- was used to silence the expression of ADAR1 in an ESCC cell tiation (P ¼ 0.029), clinical stage (P ¼ 0.004), and the tumoral line KYSE510 with the highest endogenous ADAR1 expression overexpression of ADAR1 (P ¼ 0.005) were shown to be the (Supplementary Fig. S1). Western blot analysis of ADAR1 independent prognostic factors for the overall survival (Table demonstrated that the expression of ADAR1 could be effec- 1). Altogether, we conclude that the tumoral overexpression of tively silenced by the introduction of two specific short hairpin ADAR1 predicts a poor prognosis. RNAs (shRNA) against ADAR1 gene (Fig. 3A). Compared with As described previously (15), chromosome 1q is one of the cells transfected with the control scramble shRNA (510-CTL), frequent regions with chromosomal abnormalities during the cells transfected with two shRNAs targeting ADAR1 (510- development of ESCC. To investigate whether the tumoral shAR1 #5 and 510-shAR1 #7) were found to be less tumorigenic, overexpression of ADAR1 is due to the genomic amplification as manifested by the decreased cell growth rates, the decreased of the ADAR1 gene, FISH was used to detect DNA copy number frequencies of focus formation, and the reduced migrative and of ADAR1 in the same TMA as described above. Using a invasive capabilities (Fig. 3B–D). All these data suggest that bacterial artificial chromosome (BAC) probe containing ADAR1 functions as an oncogene during ESCC progression. ADAR1 gene, the gain of ADAR1 copy number was detected in 39 out of 55 (70.9%) ESCC cases (Fig. 1E), and there was a Tumoral overexpression of ADAR1 contributes to the significantly positive correlation between the overexpression hyperediting patterns of editing targets of ADAR1 and the genomic amplification of ADAR1 gene in As reported previously, ADAR1 protein (the p110 isoform) ESCC specimens (P < 0.001, Spearman r ¼ 0.582; Supplemen- has a number of well-known target transcripts, such as sero- tary Table S2). tonin receptor subunit 2C (5-HT2CR), filamin B (FLNB), antizyme inhibitor 1 (AZIN1), and bladder cancer-associated pro- ADAR1 functions as an oncogene during ESCC tein (BLCAP; refs. 10, 12, 16–18). progression In this study, we examined the editing levels of two repre- As upstream manipulators of the A-to-I RNA editing of the sentative targets, AZIN1 and FLNB, in primary ESCC tumor and nuclear pre-mRNA, we are particularly interested in the func- their matched nontumor specimens of 69 patients with ESCC tional role of ADAR1 (the p110 isoform) during ESCC progres- (cohort 1) as well as nine ESCC cell lines. Among nine ESCC cell sion. For this purpose, we introduced ADAR1 (p110) construct lines, there was a positive correlation between the relative into two ESCC cell lines (KYSE180 and EC109) expressing the quantification value of ADAR1 and the editing frequency of relative low endogenous ADAR1 among nine ESCC cell lines AZIN1 or FLNB (AZIN1: Spearman r ¼ 0.9139, P ¼ 0.0013;

Table 1. Cox proportional hazard regression analyses for overall survival

Univariate analysis Multivariate analysis

Clinical features HR (95% CI) P HR (95% CI) P Age 1.160 (0.716–1.878) 0.547 —— Gender 1.364 (0.857–2.170) 0.190 —— LN metastasis 1.141 (0.601–2.164) 0.688 —— Differentiation 0.759 (0.587–0.982) 0.036a 0.749 (0.577–0.971) 0.029a Clinical stage 10.227 (2.506–41.733) 0.001a 8.534 (1.949–37.358) 0.004a Tumor invasion 2.995 (1.435–6.248) 0.003a 1.599 (0.731–3.501) 0.240 ADAR1 overexpression overregulation 0.577 (0.364–0.916) 0.020a 0.513 (0.321–0.822) 0.005a

Abbreviation: CI, conﬁdence interval. aStatistical signiﬁcance (P < 0.05) is shown in bold.

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Figure 2. ADAR1 functions as an oncogene during ESCC progression. A, Western blot analyses showing expression of ADAR1 and ADAR2 proteins in the indicated cell lines. b-actin was the loading control. B, cell growth rates of the indicated cell lines were compared by XTT assays. The results are expressed as the mean SD of triplicate wells within the same experiment (, P < 0.05; , P < 0.01; , P < 0.001, unpaired, two-tailed Student t test). C, quantiﬁcation of foci formation induced by the indicated stable cell lines. Triplicate independent experiments were performed and the data were expressed as the mean SD of triplicate wells within the same experiment (, P < 0.05; , P < 0.001, unpaired, two-tailed Student t test). Scale bar, 0.5 cm. D, quantiﬁcation of colonies (formed in soft agar) that were induced by the indicated cell lines. (Continued on the following page.)

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Figure 3. Silencing ADAR1 by RNAi inhibits its tumorigenicity. A, Western blot analyses showing expression of ADAR1 and ADAR2 proteins in the indicated cell lines. b-actin was the loading control. B, cell growth rates of the indicated cell lines were compared by XTT assays. The results are expressed as the mean SD of triplicate wells within the same experiment (, P < 0.05; , P < 0.01; , P < 0.001, unpaired, two-tailed Student t test). C, quantiﬁcation of foci formation induced by the indicated stable cell lines. Triplicate independent experiments were performed and the data were expressed as described above (, P < 0.01; , P < 0.001, unpaired, two-tailed Student t test). Scale bar, 0.5 cm. D, quantiﬁcation of cells from the indicated cells that migrated through the PET-membrane or invaded through the Matrigel-coated membrane. (, P < 0.001, unpaired, two-tailed Student t test). Scale bar, 100 mm.

FLNB: spearman r ¼ 0.75, P ¼ 0.025; Fig. 4A and Supplementary affected by the low endogenous transcript levels. Furthermore, Fig. S2). In clinical samples, the editing levels of AZIN1 and we defined the fold-change of ADAR1 in ESCC cases with the FLNB in ESCC tumors were significantly higher than those in ratio of the relative quantification values of tumors to that of nontumor specimens (AZIN1: P < 0.0001; FLNB: P < 0.0001, the corresponding nontumor specimens. In addition, the fold- paired Student t test; Fig. 4B), suggesting that the hyperediting change of AZIN1 or FLNB editing level in ESCC cases was patterns of AZIN1 and FLNB might play a role in ESCC characterized by the ratio of the editing frequency of tumors to progression. We also determined the total mRNA levels of that of their matched nontumor specimens. As expected, there AZIN1 and FLNB in the same clinical cases as described above. was a positive correlation between the editing level of AZIN1 or The mRNA levels of AZIN1 and FLNB were significantly FLNB and the expression level of ADAR1 in ESCC cases (AZIN1: increased in ESCC tumors than their matched nontumor Spearman r ¼ 0.3689, P ¼ 0.0018; FLNB: Spearman r ¼ 0.4186, P specimens (AZIN1: P ¼ 0.043; FLNB: P ¼ 0.023, paired Student ¼ 0.0003; Fig. 4C). To provide direct evidence that ADAR1 t test; Supplementary Fig. S3), indicating that the hyperediting regulates AZIN1 and FLNB editing, the introduction of the phenotypes of AZIN1 and FLNB in tumors would not be ADAR1 p110 expression constructs into EC109 and KYSE180

(Continued.) Triplicate independent experiments were performed and the data were expressed as the mean SD of triplicate wells within the same experiment (, P < 0.01, unpaired, two-tailed Student t test). Scale bar, 200 mm. E, quantiﬁcation of cells from the indicated cells that migrated through the PET membrane or invaded through the Matrigel-coated membrane. (, P < 0.05; , P < 0.001, unpaired, two-tailed Student t test). Scale bar, 200 mm. F, representative images of the tumors derived from the indicated cell lines at 4 weeks post injection (left). Right, growth curves of tumors over a period of 4 weeks. Data are presented as the mean SD (n ¼ 5, , P < 0.05, unpaired, two-tailed Student t test). Scale bar, 1 cm.

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Figure 4. The hyperediting patterns of AZIN1 and FLNB transcripts induced by the upregulation of ADAR1 in ESCC tumors. A, line-bar chart showing the editing levels of AZIN1 and FLNB as well as the expression level of ADAR1 in nine ESCC cell lines. B, the AZIN1 (left) and FLNB (right) editing levels in 69 paired ESCC and matched nontumor specimens in cohort 1 (paired Student t test). C, correlation between the expression level of ADAR1 and the editing level of AZIN1 (left) or FLNB (right) in 69 paired ESCC and matched nontumor specimens in cohort 1. D and E, sequence chromatograms of the AZIN1 and FLNB transcripts in EC109 (D) or KYSE180 cells (E) that transiently transduced with an ADAR1 p110 lentivirus (þADAR1 p110) or LacZ control lentivirus (þcontrol). The percentages of edited AZIN1 and FLNB transcripts were detected as described in the Materials and Methods section. An arrow indicates the editing position. F, sequence chromatograms of the AZIN1 and FLNB transcripts in KYSE510 cells (E) that transiently transfected with two shRNAs against ADAR1 gene (shAR1 #5 and shAR1 #7) or the control shRNA (control). An arrow indicates the editing position.

cell lines led to the increased editing frequencies of AZIN1 and substitution from serine (Ser) to glycine (Gly) at residue FLNB transcripts (Fig. 4D and E). Consistently, silencing ADAR1 367 (10). In this study, the functional difference between the by two shRNAs targeting ADAR1 gene (shAR1 #5 and #7) in wild-typeandtheeditedAZIN1 protein was further studied. KYSE510 cells resulted in the reduced editing levels of AZIN1 For this purpose, we introduced V5-tagged wild-type AZIN1 and FLNB (Fig. 4F). Together, these results suggest that the (wt/AZI) or edited AZIN1 (edt/AZI) expression constructs upregulation of ADAR1 in ESCC tumors contributes to the into two ESCC cell lines (KYSE180 and EC109; Supplemen- gene-speciﬁc hyperediting pattern. tary Fig. S4A). Cells transduced with the edt/AZI lentivirus (180-edt/AZI and EC109-edt/AZI) with 100% of their AZIN1 The functional alteration of AZIN1 transcript as a result transcripts edited (Fig. 5A) had accelerated growth rates of the A-to-I RNA editing during ESCC progression and higher frequencies of focus and colony formation in As a speciﬁc RNA editing target of ADAR1 protein, AZIN1 soft agar than cells transduced with the wt/AZI (180-wt/AZI gene encodes a protein that undergoes an amino acid and EC109-wt/AZI) or control LacZ lentivirus (180-LacZ

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Figure 5. The edited AZIN1 confers more aggressive tumorigenic phenotypes during ESCC progression. A, sequence chromatograms of the AZIN1 transcript in the indicated cell lines. An arrow indicates the editing position. B, cell growth rates of the indicated cell lines were compared by XTT assays. The results are expressed as described above (, P < 0.05; , P < 0.001). C, quantification of foci formation induced by the indicated stable cell lines. Triplicate independent experiments were performed and the data were expressed as described above (, P < 0.01; , P < 0.001). Scale bar, 0.5 cm. D, quantification of colonies (formed in soft agar) that were induced by the indicated cell lines. Triplicate independent experiments were performed and the data were expressed as described above (, P < 0.05; , P < 0.01). Scale bar, 200 mm. E, quantification of cells from the indicated cells that migrated through the PET-membrane or invaded through the Matrigel-coated membrane. (, P < 0.05; , P < 0.01; , P < 0.001, unpaired, two-tailed Student t test). Scale bar, 200 mm. F, growth curves of tumors derived from the indicated cell lines over a period of 4 weeks. Data are presented as the mean SD (n ¼ 6, , P < 0.05; , P < 0.001; unpaired, two-tailed Student t test).

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and EC109-LacZ; Fig. 5B–D). Moreover, 180-edt/AZI and leading to deamination of select adenosine residues, or it can EC109-edt/AZI cells had the increased migrative and inva- be almost random and lead to nonselective conversion of many sive capabilities (Fig. 5E and Supplementary Fig. S4B). adenosines. For long dsRNA (<100 bp) within 30 untranslational Xenograft studies in mice demonstrated that tumors derived region (30-UTR), many adenosine residues are edited promis- from EC109-edt/AZI cells grew significantly faster than cuously, leading to approximately 50% of adenosines being those derived from EC109-wt/AZI and EC109-LacZ cells converted to inosines. However, in terms of A-to-I editing (Fig. 5F). within protein-coding sequences, it is highly selective, and an All these data indicate that the edited AZIN1 confers "gain- imperfect fold-back dsRNA structure is formed between the of-function" phenotypes and promotes the tumorigenicity exon sequence surrounding the editing site(s) and a down- during ESCC progression, suggesting that AZIN1 is likely to stream intronic complementary sequence termed editing-site be one of the downstream editing targets that are responsible complementary sequence (8). Until now, only a few recoding for the oncogenic function of ADAR1. RNA editing events (e.g., Q/R site editing in the glutamate receptor) have been verified. The editing events within 30UTRs Discussion can affect transcript stability via affecting microRNA targeting RNA editing is broadly defined as the posttranscriptional or the nuclear retention of transcripts (26–29). However, the process that changes the sequence of primary RNA transcripts. editing targets within coding region will cause amino acid It is becoming apparent that A-to-I editing is not a rare change and affect protein function rather than protein level, phenotype, but instead it is the most common type of RNA suggesting that the recoding editing events are of high poten- editing in mammalian transcriptome. ADARs, the enzymes tials to play a role in tumorigenesis by either inactivating a responsible for the conversion of A-to-I in dsRNA, were first tumor suppressor or activating genes that promote tumor noticed as cellular RNA unwinding activity (19, 20), as they lead progression. As reported previously, two recoding editing to destabilization of RNA duplexes by introducing I.U mis- events at codon 2269 (Met ! Val) of the FLNB gene and codon match. ADARs exhibit strict tissue-specific and environment- 367 (Ser ! Gly) of the AZIN1 gene are catalyzed by ADAR1 dependent expression patterns (21, 22). There are three ortho- protein (10, 12). In this study, we proved that ADAR1 was logs: ADAR1 and ADAR2 occur in most animals, whereas responsible for the editing of FLNB and AZIN1 in ESCC cells and ADAR3 is vertebrate and brain specific. The ADAR1 p150 clinical samples, suggesting that as a result of the differential isoform is presumably responsible for the A-to-I editing of expression of ADAR1 in ESCC tumors, the editing alterations in viral RNAs produced by viruses (14, 23), but not of the nuclear the specific genes may be responsible for the ADAR1-induced pre-mRNAs (24). It has been reported all the three editing malignant phenotypes during ESCC progression. Therefore, we enzymes ADAR1, ADAR2, and ADAR3 were downregualted in selected one of the recoding editing targets, AZIN1, for further brain tumors. Overepxression of ADAR1 and ADAR2 in the U87 study. Intriguingly, we found the editing frequency of AZIN1 glioblastoma multiforme cell line resulted in a decreased was significantly increased in primary ESCC tumors com- proliferation rate, suggesting that the reduced A-to-I editing pared with their matched nontumor specimen, suggesting in brain tumors is involved in the pathogenesis of cancer (18). there is a hyperediting phenotype of the AZIN1 editing event On the contrary, ADAR1 or/and ADAR2 were also found to be during ESCC progression. AZIN1, termed antizyme inhibitor upregulated in tumor tissues, such as prostate cancer and 1, blocks the effects of antizyme on omithine decarboxylase breast cancer (25). In this study, we investigated the expression (ODC). This protein has substantial similarity to ODC itself profiles of all three RNA editing enzymes in ESCC clinical but has no ODC activity (30). It binds to antizyme more samples. ADAR1 and ADAR2 were abundantly expressed in tightly than ODC and thus releases ODC from the antizyme– ESCC samples, but ADAR3 was undetectable in all samples. ODC complex (30, 31). Although the physiologic importance Because of the genomic amplification of the ADAR1 gene, of AZIN1 is not yet fully understood, a strong case can be which is mapped to chromosome 1q21, ADAR1 was the only made for it to be included as a component of the polyamine RNA editing enzyme found to be significantly upregulated in pathway. Downregulation of antizyme inhibitor in lung primary ESCC tumors compared with their matched nontu- cancer cells reduced ODC levels and led to growth inhibition mor specimens in two individual cohorts (cohort 1 and 2). (32). Also, it has been reported that overexpression of AZIN1 Clinically, the tumoral overexpression of ADAR1 was correlat- in NIH3T3 fibroblasts provides growth advantage through ed with the shorter overall survival time of patients with ESCC, neutralization of antizyme functions (33). In the present which could be used independently for the prediction of a poor study, we demonstrated that although the wild-type AZIN1 prognosis. Consistently, our functional assays have indicated could promote the tumorigenicity, the edited AZIN1 con- that ADAR1 could induce tumorigenic phenotypes in cell ferred "gain-of-function" phenotypes that were manifested models and animals. by more aggressive behaviors during ESCC progression, ADARs can change the structure of RNA by changing an whichhasalsobeenfoundinhuman hepatocellular carci- Adenosine Uracil (AU) base pair to an Inosine Uracil (IU) noma as reported recently (10). All these findings suggest mismatch. Conceivably, ADARs could affect any biologic pro- that the ADAR1-induced editing alteration may play a piv- cess that is associated with the sequence- or structure-specific otal role in human solid tumors and the precise regulation of interaction of RNA (14). So far, ADARs have been shown to alter the expression level of ADARs is essential for accurate the protein coding, create or delete splice sites, and modulate editing and the altered expression of ADARs could be at transcript stability. The A-to-I editing can be very specific, the origin of cell transformation.

OF10 Cancer Res; 2013 Cancer Research

A-to-I Editing Mediated by ADARs in ESCC

The characterization of editing events is a necessary step Disclosure of Potential Conflicts of Interest toward fully understanding the function and regulation of No potential conflicts of interest were disclosed. transcriptome, and investigating the connection between RNA editing and cancer progression is only the initial step in this Authors' Contributions Conception and design: Y.-R. Qin, J.-J. Qiao, T.H.M. Chan, X.-Y. Guan, L. Chen research. The existence of RNA editing, as opposed to hard- Development of methodology: L. Chen wired genomic mutation, indicates that it can be spatiotem- Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y.-R. Qin, J.-J. Qiao, T.H.M. Chan, Y.-H. Zhu, F.-F. Li, H. porally controlled. Indeed, there is a great excitement that we Liu, Y. Li, L. Chen could develop potential therapeutic approaches for targeting Analysis and interpretation of data (e.g., statistical analysis, biostatistics, this epigenetic process. Here, we propose two feasible computational analysis): Y.-R. Qin, J.-J. Qiao, T.H.M. Chan, L. Chen Writing, review, and/or revision of the manuscript: Y.-R. Qin, J.-J. Qiao, T.H. approaches: (i) modulating the expression of RNA editing M. Chan, L. Chen enzyme ADARs by overexpressing or silencing ADARs and (ii) Administrative, technical, or material support (i.e., reporting or orga- fi nizing data, constructing databases): Y.-R. Qin, J.-J. Qiao, J. Fei, L. Chen reinstating a speci c hyperedited or hype-edited transcript Study supervision: Y.-R. Qin, J.-J. Qiao, X.-Y. Guan, L. Chen (34). In this study, we reported that one of the RNA editing enzyme ADAR1 was overexpressed in ESCC tumors, which can Grant Support be used as an independent factor for prognosis prediction. As a This work was supported by the Singapore Translational Research (STaR) result of the tumoral overexpression of ADAR1, the edited Award (NMRC/STaR/0001/2008), the "Hundred Talents Program" at Sun Yat-Sen AZIN1 University (Guangzhou, China; 85000-3171311), the National Natural Science transcript is more abundant in tumors, leading to "gain- Foundation of China (81150010 and 81272227), and the National Key Sci-Tech of-function" phenotypes during ESCC progression. Therefore, Special Project of Infectious Diseases (2012ZX10002-013). The costs of publication of this article were defrayed in part by the payment of we speculate that monitoring the expression level of ADAR1 or page charges. This article must therefore be hereby marked advertisement in the editing activities of key editing targets represent a useful accordance with 18 U.S.C. Section 1734 solely to indicate this fact. biomarker for the detection of disorders in ESCC and our study may also provide the a key biologic process for rationale drug Received September 4, 2013; revised October 18, 2013; accepted November 6, development. 2013; published OnlineFirst December 3, 2013.

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OF12 Cancer Res; 2013 Cancer Research

Adenosine-to-Inosine RNA Editing Mediated by ADARs in Esophageal Squamous Cell Carcinoma

Yan-Ru Qin, Jun-Jing Qiao, Tim Hon Man Chan, et al.

Cancer Res Published OnlineFirst December 3, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-13-2545

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