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2790.Full.Pdf Published OnlineFirst March 13, 2020; DOI: 10.1158/0008-5472.CAN-19-3440 CANCER RESEARCH | MOLECULAR CELL BIOLOGY A Novel Micropeptide Encoded by Y-Linked LINC00278 Links Cigarette Smoking and AR Signaling in Male Esophageal Squamous Cell Carcinoma Siqi Wu1, Liyuan Zhang2, Jieqiong Deng1, Binbin Guo1, Fang Li1, Yirong Wang1, Rui Wu1, Shenghua Zhang1, Jiachun Lu3, and Yifeng Zhou1 ABSTRACT ◥ Long noncoding RNAs (lncRNA) have been shown to play critical Graphical Abstract: http://cancerres.aacrjournals.org/content/ roles in many diseases, including esophageal squamous cell carcino- canres/80/13/2790/F1.large.jpg. ma (ESCC). Recent studies have reported that some lncRNA encode See related commentary by Banday et al., p. 2718 functional micropeptides. However, the association between ESCC and micropeptides encoded by lncRNA remains largely unknown. In this study, we characterized a Y-linked lncRNA, LINC00278,which was downregulated in male ESCC. LINC00278 encoded a Yin Yang 1 Chr.Y (YY1)-binding micropeptide, designated YY1BM. YY1BM was LINC0027 involved in the ESCC progression and inhibited the interaction 8 sORF A A METTL3 A between YY1 and androgen receptor (AR), which in turn decreased METTL14 eEF2K ALKBH5 eEF2K eEF2K expression of through the AR signaling pathway. Down- AR YY1 regulation of YY1BM significantly upregulated eEF2K expression WTAP m6A Smoking and inhibited apoptosis, thus conferring ESCC cells more adaptive to LINC00278 sORF A A nutrient deprivation. Cigarette smoking decreased m6A modification A eEF2 of LINC00278 and YY1BM translation. In conclusion, these results YTHDF1 provide a novel mechanistic link between cigarette smoking and AR YY1BM Translation Caspase-3 signaling in male ESCC progression. m 6 sORF A A AA Significance: Posttranscriptional modification of a micropeptide- Cell death encoding lncRNA is negatively impacted by cigarette smoking, disrupting negative regulation of the AR signaling pathway in male ESCC. The micropeptide YY1BM functions as a tumor suppressor in male ESCC cells. Introduction receptors has been reported in ESCC as well as association with prognosis (4, 5). However, the exact underlying molecular mechan- Esophageal squamous cell carcinoma (ESCC) is two to four times isms in male ESCC progression remain largely unknown. more common in men than in women worldwide (1). Previous studies A recent study identified a tumor suppressor gene on Y chromosome suggest that several male-specific factors contribute to such gender for male breast cancer (6), suggesting that genetic material encoded by Y disparity, including cigarette smoking and sexual hormone. A survey chromosome could be involved in male-dominant tumors. Long non- in 2010 indicated that 52.9% of Chinese men while only 2.4% of codingRNAs(lncRNA)aredefinedasRNAtranscriptslongerthan200nt Chinese women were current smokers (2, 3). Expression of androgen that lack protein-coding potential (7, 8). LncRNAs act as master regula- tors for gene expression, thus play an important role in many biological functionsanddiseases,includingcancer(9).However,nostudysofarhas 1Department of Genetics, Medical College of Soochow University, Suzhou, China. 2Department of Radiotherapy and Oncology, The Second Affiliated Hospital of reported on the involvement of Y-linked lncRNAs in ESCC. Soochow University, Suzhou, China. 3The Institute for Chemical Carcinogenesis, Recent computational and genome-wide studies have demonstrated The First Affiliated Hospital, The School of Public Health, Guangzhou Medical that hundreds of functional micropeptides (<100 amino acids) are University, Guangzhou, China. embedded in lncRNAs. For example, myomixer is an 84-amino acid fi Note: Supplementary data for this article are available at Cancer Research muscle-speci c micropeptide encoded by a lncRNA that controls the Online (http://cancerres.aacrjournals.org/). critical steps in myofiber formation during muscle development (10); myoregulin is identified as a skeletal muscle-specific lncRNA, which S. Wu and L. Zhang contributed equally to this article. þ regulates muscle performance by impeding Ca2 uptake into the Corresponding Author: Yifeng Zhou, Medical College of Soochow University, No. 199 Ren-ai Rd., Suzhou, Jiangsu 215123, China. Phone: 8651-2658-84720; sarcoplasmic reticulum (SR; ref. 11). It is still unclear whether micro- Fax: 8651-2658-84720; E-mail: [email protected] peptides play a key role in tumor development, although a recent study has identified a micropeptide encoded by HOXB-AS3 lncRNA that Cancer Res 2020;80:2790–803 suppresses colon cancer growth (12). doi: 10.1158/0008-5472.CAN-19-3440 N6-methyladenosine (m6A) is the most abundant posttranscription Ó2020 American Association for Cancer Research. modification on eukaryotic mRNAs and lncRNAs (13). Recent studies AACRJournals.org | 2790 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 13, 2020; DOI: 10.1158/0008-5472.CAN-19-3440 Role of Micropeptide Encoded by lncRNA in Male ESCC show that m6A modification is dynamic and reversible in cells, whose with 1 nmol/L R1881 (methyltrienolone) to activate AR signaling level is regulated by m6A methyltransferases (also called “writers”: pathway. To inhibit specific signaling pathways, cells were pretreated METTL3, METTL14, etc.) and m6A demethylases (also called with vehicle (DMSO) or 10 mmol/L A-484954 (EMD Millipore) for “erasers”: FTO, ALKBH5, etc.). m6A regulates gene expression 1 hour at 37C prior to the experiments. through m6A binding proteins (also called “readers”: YTHDF1, YTHDF2, YTHDF3, etc.; refs. 14, 15). These m6A-associated proteins Transplantation of human ESCC tissues play critical roles to regulate the metabolism and functions of Primary viable human ESCC samples were obtained from surgical m6A-modified mRNAs and lncRNAs (15). ESCC specimens (n ¼ 50) at the Affiliate Hospitals of Soochow In this work, we identified a micropeptide encoded by a Y-linked University (Suzhou, China). During surgery, fresh tumor tissue was lncRNA, LINC00278, which is downregulated in male ESCC. The collected in transport medium, [RPMI1640 medium supplemented expression of this micropeptide was downregulated by cigarette with penicillin/streptomycin (100 U/mL; 100 mg/mL), fungizone smoking in ESCC through erasing m6A modification. It specifically (1 mg/mL), and gentamicin (50 mg/mL; all from Life Technologies)] bound to Yin Yang 1 (YY1) and blocked the interaction between YY1 and implanted in mice within 4 hours. In parallel, primary tumor tissue and androgen receptor (AR), therefore named YY1-blocking micro- fragments were also fresh-frozen and formalin-fixed for further anal- peptide (YY1BM). YY1BM downregulated eEF2K expression through yses. Before implantation, tumor tissue was rinsed in PBS supplemen- AR signaling pathway and induced apoptosis in ESCC under nutrient ted with penicillin/streptomycin and fungizone. Each tumor specimen deprivation (ND). Furthermore, YY1BM also acts as a potential was cut into three small fragments (1.5 mm  1.5 mm) and grafted anticancer micropeptide for ESCC. subcutaneously into NCG mice. The NCG mice were anesthetized by intraperitoneal of pentobarbitone (10 mg/mL) at a dose of 65 mg/kg. Materials and Methods Microarray data analysis Human study subjects To identify male ESCC-associated lncRNAs, differential gene fi A total of 281 pairs of fresh-frozen ESCC and adjacent noncancer- expression analysis was performed on gene expression pro les of ous tissue samples were obtained from patients in Eastern China who 179 pairs of ESCC and matched adjacent normal tissues, and the underwent tylectomies at the Affiliate Hospitals of Soochow University tissues were separated in male and female groups. Differential gene “ ” (Suzhou cohort; Suzhou, China). Another 288 pairs of fresh-frozen expression analysis was performed by the R package limma. The P- P < ESCC tissues were collected from patients in Southern China at the probe that adjusted value ( adj.) 0.01 and the absolute value > fi Cancer Hospitals affiliated with Guangzhou Medical University of log2-fold change (abs.logFC) 1 were de ned as differentially (Guangzhou cohort; Guangzhou, China). None of the patients expressed probes. The differentially expressed probes were subse- received anticancer treatment before surgery, including chemo- quently annotated by mapping onto the genomic coordinates of therapy or radiotherapy. The Medical Ethics Committees of Soochow lncRNAs derived from GENCODE. University (Suzhou, China) and Guangzhou Medical College Chromatin immunoprecipitation–sequencing data analysis (Guangdong, China) approved this study. The clinical characteristics Chromatin immunoprecipitation (ChIP)–sequencing (ChIP-seq) of patients in this study are listed in Supplementary Table S1. data were obtained from the GEO database. ChIP-seq reads were aligned to the hg19 by Bowtie2 with default parameters; the mapped Statistical analysis reads of ChIP-seq were preprocessed by Samtools and then submitted The data analysis was performed using the SPSS 19.0 software for to MACS2 for peaks calling. The peaks were annotated by the R Windows. The statistical significance between datasets was expressed package “ChIPseeker” and visualized by IGV software. Finally, genes as P values, and P < 0.05 was considered statistically significant. that contained peaks at À800 bp upstream of transcriptional start sites Survival curves were obtained using the Kaplan–Meier method and (TSS) to þ200 bp downstream of TSS region were
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