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 regulators 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 2020 American Association for Cancer Research. modification on eukaryotic mRNAs and lncRNAs (13). Recent studies

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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 800 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 defined as genes compared using the log-rank test. Multivariable Cox regression anal- regulated by corresponding transcription factors. ysis was performed using the R package “survival.” Paired or unpaired Student t test and Pearson correlation coefficients were used for Overall survival analysis various types of data comparison. Mediation analysis was conducted Using the median expression level of LINC00278 among ESCC P < using the procedure described by Baron and Kenny (16) and a 0.05 tissues, we separated patients with ESCC into two different groups: fi was considered signi cant. patients with high LINC00278 expression (relative expression level>median expression level); and patients with low LINC00278 Animals and cell cultures expression (relative expression levelmedian expression level), in both – Male nude mice of 6 8 weeks of age were purchased from the the Suzhou cohort (discovery set, 281 patients) and Guangzhou cohort Shanghai Laboratory Animal Center at the Chinese Academy of Sciences (validation set, 288 patients). Furthermore, Kaplan–Meier survival (Shanghai, China). All animal studies were conducted with the approval curves and log-rank tests were performed between the high LINC00278 of Soochow University Institutional Animal Care and Use Committee group and the low LINC00278 group. and were performed in accordance with established guidelines. All cell lines were purchased from Procell Life Science&Technology RNA extraction and qRT-PCR Co., Ltd. These cell lines were all characterized by DNA finger printing Total RNA was isolated from ESCC tissues and corresponding analysis and passaged less than 6 months in this study. DMEM, adjacent nonneoplastic tissues using the RNA Isolater Total RNA RPMI1640, and FBS were purchased from Invitrogen. Eca-109, TE- Extraction Reagent (Vazyme). The purity and concentration of RNA 1, and KYSE-30 cells were grown in RPMI1640 with 10% FBS; Het-1A were determined by the ratio of absorbance at 260 nm (A260) and and 293T cells were grown in DMEM with 10% FBS. All cell lines were 280 nm (A280) using a NanoDrop 2000 spectrophotometer (Thermo grown in penicillin/streptomycin containing medium, at 37Cina Fisher Scientific). The RNA was considered pure and suitable for fi humidi ed atmosphere with 5% CO2. In addition, cells were treated downstream experiments when A260/A280 was within the range

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1.7–2.0. The RNA integrity was determined by running on a 1.5% into a container and frozen with liquid nitrogen. CSC was dissolved in denaturing agarose gel. First-strand cDNA was synthesized with the DMSO at a concentration of 235 mg/mL, and aliquots were stored at Superscript II-Reverse Transcriptase Kit (Invitrogen). All qRT-PCR 80C until use. primers are listed in Supplementary Table S2. Coimmunoprecipitation assay Antibodies and Western blotting Coimmunoprecipitation assay was performed using Pierce Co- ESCC cells were collected and lysed in cell lysis buffer for Western Immunoprecipitation Kit (Thermo Fisher Scientific) according to the blotting and IP (Beyotime Institute of Biotechnology). Proteins were manufacturer's instructions. The lysates were applied to columns separated on SDS-PAGE and transferred to nitrocellulose membrane. containing 10 mg of immobilized antibodies covalently linked to an Immunoblotting of the membranes was performed using the following amine-active resin and incubated overnight at 4C. Then the coim- primary antibodies: anti-FLAG (sc-807, Santa Cruz Biotechnology), munoprecipitate was eluted and analyzed by SDS-PAGE or mass HA (ab9110, Abcam), YTHDF1 (ab99080, Abcam), YTHDF2 spectrometry along with the controls. Coimmunoprecipitation assays (ab170118, Abcam), YTHDF3 (ab103328, Abcam), METTL3 were performed using the following antibodies: anti-FLAG (sc-807, (ab195352, Abcam), METTL14 (ab98166, Abcam), WTAP (ab155655, Santa Cruz Biotechnology), anti-HA (ab9110, Abcam), anti-YY1 Abcam), ALKBH5 (ab69325, Abcam), YY1 (sc-7341, Santa Cruz (sc-7341, Santa Cruz Biotechnology), and anti-AR (sc-7305, Santa Biotechnology), CRKL (sc-365092, Santa Cruz Biotechnology), eEF2K Cruz Biotechnology). (3692, Cell Signaling Technology), APC (sc-393704, Santa Cruz Biotechnology), GOLPH3 (19112-1-AP, Proteintech), KDM4C Mass spectrometry analysis (ab85454, Abcam), BCAR3 (24032, Cell Signaling Technology), CYCS The samples were analyzed on Thermo Fisher LTQ Obitrap ETD (sc-13156, Santa Cruz Biotechnology), PON2 (sc-374158), LARP1 (sc- mass spectrometry. Briefly, the samples were loaded onto an high- 515873, Santa Cruz Biotechnology), PKD2 (sc-28331, Santa Cruz performance liquid chromatography (HPLC) chromatography system Biotechnology), Cleaved caspase-3 (ab32042, Abcam), eEF2 (2332, named Thermo Fisher Easy-nLC 1000 equipped with a C18 column Cell Signaling Technology), p-eEF2 (2331, Cell Signaling Technology), (1.8 mm, 0.15 1,00 mm). Solvent A contained 0.1% formic acid and and anti-b-actin (A5441, Sigma-Aldrich). Signals were revealed after solvent B contained 100% acetonitrile. The elution gradient was from incubation with recommended secondary antibody coupled to per- 4% to 18% in solvent A for 182 minutes and 18% to 90% in solvent B for oxidase using enhanced chemiluminescence. 13 minutes at a flow rate of 300 nL/minute. Mass spectrometry analysis was carried out at the AIMS Scientific Co.,Ltd. (Shanghai, China) in the DNA methylation analysis positive-ion mode with an automated data-dependent MS/MS analysis DNA methylation analysis was performed as reported previous- with full scans (350–1,600 m/z) acquired using Fourier transform mass ly (17). Briefly, we designed primers of the CpG islands in the promoter spectrometer at a mass resolution of 30,000 and the 10 most intense region of ALKBH5 gene using MethPrimer. After robotically dispens- precursor ions were selected for MS/MS. The MS/MS was acquired ing 22 nL of the cleavage reaction onto the silicon matrix preloaded using higher-energy collision dissociation at 35% collision energy at a chips (SpectroCHIP; Sequenom), the mass spectra were collected using mass resolution of 15,000. a MassARRAY Compact MALDI-TOF (Sequenom) and the spectra's methylation ratios were generated by EpiTYPER software (version 1.0; Production of YY1BM knockout and FLAG knockin cells Sequenom). The gRNA sequence designed specifically for the open reading frame (ORF) of YY1BM start codon inserted to the Cas9/gRNA (puro- Anti-YY1BM antibody preparation GFP) vector (VK001-02, ViewSolid BioTech) was 50-GACTCCAGG- Peptide synthesis and anti-YY1BM antibody generation were per- CATGCTATCAGG-30. The donor oligo was purchased from Cyagen formed as described previously with some modifications (18). Briefly, a Biosciences Inc. (Suzhou, China). The constructed targeting vector BSA- and OVA-coupled peptide CLSGQLQPEGRSALPQPG-NH2 and donor oligo were subsequently transfected into the cells using was synthesized, and polyclonal antibody against the YY1BM was Lipofectamine 3000 (Thermo Fisher Scientific), after which, the cells obtained from inoculated rabbits. Antibody was purified using affinity were cultured under puromycin drug selection (2 mg/mL) for 48 hours. chromatography on columns containing the corresponding peptide. Viable clones were grown to a larger size and picked up for Western blot analysis or sequencing. The schematic diagram and the sequence Polysome profiling of YY1BM-KO cells were present in Supplementary Fig. S1A. The Polysome profiling was performed to measure the translation of levels of LINC00278 transcription and YY1BM translation were YY1BM monitored by qRT-PCR. We performed polysome profiling present in Supplementary Fig. S1B–S1D. followed the procedure described before (19). The primers for qRT- PCR were listed in Supplementary Table S2. RNA interference siRNA targeting the YY1, METTL3, METTL14, WTAP, ALKBH5, RNA stability analysis FTO, and YTHDF1 gene and nontargeting siRNA control (Supple- To determine the stability of LINC00278 and YY1BM transcripts, mentary Table S2) were purchased from GenePharma. Transfections TE-1 cells were treated with 2 mg/mL Actinomycin D (Sigma). The cells with siRNA (75 nmol/L) were performed with Lipofectamine 3000 were harvested at 0, 6, 12, 18, and 24 hours posttreatment, LINC00278 (Life Technologies) and the expression of interfered genes was present and YY1BM transcripts were quantified by qRT-PCR. in Supplementary Fig. S1E.

Preparation of cigarette smoke condensate Production of LINC00278 overexpression and YY1BM Cigarette smoke condensate (CSC) was prepared as reported pre- overexpression cells viously (20). Brieﬂy, cigarette smoke was collected from a popular To produce overexpression cells, the full-length human LINC00278 Chinese brand cigarette (12 mg tar/cigarette) by a vacuum machine or YY1BM cDNA were synthesized by GeneWiz and cloned into the

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lentiviral expression vector pLVX-IRES-neo (Clontech Laboratories sion level); and patients with low LINC00278 expression (relative Inc.). To produce lentivirus containing full-length LINC00278 or expression level median expression level), in both the Suzhou YY1BM targeting sequence, 293T cells were cotransfected with the cohort (discovery set, 281 patients) and Guangzhou cohort (vali- vector described above and the lentiviral vector packaging system dation set, 288 patients). Using the log-rank test and Kaplan–Meier using Lipofectamine 3000. Infectious lentiviruses were collected at survival curves, we showed that patients with low LINC00278 48 and 72 hours after transfection and filtered through 0.45-mm filters. expression had significantlyshorterOSthanpatientswithhigh These lentiviruses were, respectively, designated as LINC00278 over- LINC00278 expression in both the discovery set [median survival expression or YY1BM overexpression. We used an empty plenty- time (MST): 29 vs. 36 months, log-rank P ¼ 0.0004, HR ¼ 1.848] pLVX-IRES-neo vector to generate negative control lentiviruses. and the validation set (MST: 27 vs. 39 months, log-rank P < 0.0001, Recombinant lentiviruses were concentrated by centrifugation. The HR ¼ 1.850; Fig. 1F and G). Multivariable Cox regression analysis virus-containing pellet was dissolved in DMEM, and aliquots were also indicated that low LINC00278 expression was associated with stored at 80C until use. Cells were infected with the concentrated shorter OS (Fig. 1F and G). virus in the presence of polybrene (Sigma-Aldrich). The supernatant Because cigarette smoking has been associated with poor OS, we was replaced with complete culture medium after 24 hours, followed determined whether LINC00278 expression and cigarette smoking by selection with 800 mg/mL G418, and the expression of LINC00278 acted synergistically in ESCC. We showed that patients with low and YY1BM in infected cells was verified by qRT-PCR. LINC00278 expression and who were current smokers had worst OS in both the discovery set (MST: 28 vs. 40, log-rank P < 0.0001, HR ¼ Micropeptide synthesis 2.818) and the validation set (MST: 25 vs. 41, log-rank P < 0.0001, The micropeptides used in ESCC cell treatment and intratumoral HR ¼ 2.613; Supplementary Fig. S2C and S2D). Using Multivariable injection were synthesized from ChinaPeptides Co.,Ltd. The micro- Cox regression analysis, we also confirmed that smoking was associ- peptides were purified byHPLC, and the sequence and structure were ated with shorter OS in patients with ESCC (Fig. 1F and G; Supple- confirmed by mass spectrometry. The peptides were ≥95% pure and mentary Fig. S2C and S2D). kept as 100 mg/mL stock solution at 20C. Biological characterization of LINC00278 Data sharing statement LINC00278 locus is located on the short arm of Y chromosome. It Microarray data are available at the NCBI Gene Expression spans from 3,002,887 to 3,200,509 and is comprised of four exons. Omnibus (GEO) repository with accession number GSE53625. The LINC00278 transcript is 537 bp long and Northern blot analysis has ribosome profiling data are available at the GEO repository with confirmed the expected size of LINC00278 transcript in total RNA accession number GSE61742. The ChIP-seq data are available at from two pairs of male ESCC samples (Supplementary Fig. S2E). Both the GEO repository with accession numbers GSE32465 and nuclear/cytoplasm fractionation experiment and confocal microscopy GSE62472. analysis of FISH showed that LINC00278 is a cytoplasmic RNA (Supplementary Fig. S2F and S2G).

Results LINC00278 encodes a micropeptide Identification of ESCC-associated lncRNA LINC00278 Because recent studies suggested that many lncRNAs could To identify male ESCC-associated lncRNAs, we analyzed differen- encode functional micropeptides (<100 amino-acids), we deter- tially expressed lncRNAs in lncRNA expression profiles of 179 pairs mined whether LINC00278 encoded any micropeptides. We found of ESCC and matched adjacent normal tissues, which were separated that LINC00278 could potentially encode four small ORFs into male and female groups (21). In total, 3,401 differentially (sORF; Fig. 2A). We subsequently cloned each sORF with an in- expressed genes were screened from the male group (146 patients) frame FLAG epitope tag at the C terminus and transfected it into and 3,284 differentially expressed genes were screened from the female male ESCC cell line TE-1. Western blot analysis indicated that group (33 patients). In addition, we filtered 254 differentially expressed LINC00278-sORF1 generated a micropeptide (Fig. 2B). This sORF lncRNAs in the male group and 244 differentially expressed is located on Y chromosome from 3,003,090 to 3,003,155, inside the lncRNAs in the female group (Fig. 1A). Among these, we found 51 first exon of LINC00278, encoding a 21-amino-acid micropeptide differentially expressed lncRNAs were present in the male group, but (2.12 kDa). This is consistent with the presence of a marked not in the female group, and 3 of them were mapped to Y chromosome ribosome occupancy peak in the first exon of LINC00278 in human (Fig. 1B–D). lymphoblastoid cells (22), as well as ribosome occupancy data from Next, we measured the expression of these 3 Y-linked lncRNAs by GWIPS-viz database (23; Fig. 2C). qRT-PCR in 281 pairs of male ESCC tissue samples from an Eastern We next determined whether an in-frame ATG codon of Chinese population (Suzhou cohort). Only LINC00278 was signifi- LINC00278-sORF1 could promote the initiation of translation. We cantly downregulated in ESCC tissues when compared with adjacent fused GFPmut ORF (in which the initiation codon ATGGTG has normal tissues (P < 0.001; Fig. 1E; Supplementary Fig. S2A and S2B). mutated to ATTGTT) and FLAG-tag to the C terminus of LINC00278- We further validated the downregulation of LINC00278 in ESCC sORF1 to construct expression plasmids and transfected these plas- tissues using an independent 288 pairs of male ESCC samples from mids into ESCC cells. After 24 hours, we observed substantial expres- a Southern Chinese population (Guangzhou cohort; P < sion of LINC00278-sORF1-GFP fusion protein in the transfected cells 0.001; Fig. 1E). (Supplementary Fig. S2H). Meanwhile, LINC00278-sORF1-FLAG was We also determined whether LINC00278 expression was associated observed in transfected cells using anti-FLAG Western blot analysis with overall survival (OS) among male patients with ESCC. Using the (Supplementary Fig. S2I). Our data indicated that LINC00278-sORF1 median expression level of LINC00278 among ESCC tissues, we could produce micropeptide and the initiation codon of LINC00278- separated patients with ESCC into two different groups: patients with sORF1 could be utilized effectively to drive the expression of the fusion high LINC00278 expression (relative expression level>median expres- protein.

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Figure 1. Y-linked male-speciﬁc lncRNA LINC00278 was downregulated in male ESCC samples and associated with the overall survival of male patients with ESCC. A, The Venn diagram depicts the number of lncRNAs that are differentially expressed in male versus female ESCC groups. B, The distribution of male differentially expressed lncRNAs on each chromosome. C and D, Y-linked lncRNAs that were differentially expressed in male ESCC samples. E, The expression of three candidate lncRNAs in male ESCC and matched nontumor esophagus specimens from 281 patients in the Suzhou cohort. Expression of LINC00278 was also determined from 288 patients in the Guangzhou cohort. Mean SD. F, Left, Kaplan–Meier overall survival curves for male patients with ESCC with high or low LINC00278 expression in Suzhou cohort (281, discovery set). Right, forest plot derived from multivariable Cox regression analyses that adjusted for age and tumor stage. G, Left, Kaplan–Meier overall survival curves for male patients with ESCC with differential LINC00278 expression and smoking history in Guangzhou cohort (288, validation set). Right, forest plot derived from multivariable Cox regression analyses that adjusted for age and tumor stage. , P < 0.05; , P < 0.01; , P < 0.001.

LINC00278-sORF1 was endogenously expressed and ORF of LINC00278-sORF1 (FLAG-KI) and detected endogenous downregulated in male ESCC LINC00278-sORF1 by Western blot analysis in Het-1A cell line, which To determine LINC00278-sORF1 expression, we generated a rabbit expresses a higher level of LINC00278 and LINC00278-sORF1 polyclonal antibody (anti-LINC00278-sORF1). To confirm the spec- (Fig. 2D; Supplementary Fig. S2L). In addition, we showed that the ificity of anti-LINC00278-sORF1, we respectively performed Western LINC00278-sORF1 translation-blocking antisense oligo could block blot analysis to identify LINC00278-sORF1, LINC00278-sORF1-GFP, the expression of LINC00278-sORF1 micropeptide (Supplementary and LINC00278-sORF1-FLAG in ESCC cells (Supplementary Fig. S2H Fig. S2M). and S2I). We also performed polysome profiling in the cell lysate of Because of the limited number of tumor cells obtained from ESCC Het-1A (a nonneoplastic squamous esophageal epithelial cell line), TE- tissues, we determined LINC00278-sORF1 translation and expression 1, and KYSE-30. The mRNA-protein particles (mRNP) were separated in male ESCC patient-derived xenograft (PDX) models using poly- into three groups: nonribosome (mRNPs without any ribosome), 40S– some profiling and Western blot analysis. A total of 50 ESCC PDXs 80S (mRNPs associated with ribosome but not being translated) and were generated for the experiment. Tumor cells were harvested and polysome (mRNPs being actively translated). The presence of lysed from ESCC PDXs. Our data indicated that LINC00278-sORF1 LINC00278 was quantitated in polysome fraction via qRT-PCR (Sup- was endogenously expressed and downregulated in male ESCC tissues plementary Fig. S2J and S2K). (Fig. 2E). In addition, the level of LINC00278 in polysome fraction was To further confirm the existent of endogenous LINC00278-sORF1 positively correlated with the transcription level of LINC00278 in the micropeptide, we inserted a C-terminal FLAG-tag at the 30 end of the tissues (Fig. 2E).

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Figure 2. Micropeptide LINC00278-sORF1 instead of the LINC00278 transcript inhibited the progression of male ESCC. A, Schematic drawing to show the genomic position of the exons and the predicted sORFs of LINC00278. B, The sORFs were constructed to pcDNA3.1 vector and transfected to TE-1 cells for 24 hours. The sORFs-FLAG fusion proteins were determined by Western blotting with anti-FLAG antibody. C, Ribosome occupancy map was at the LINC00278 locus. The blue and red tracks indicate reads density that is mapped to the region, and the green track indicates the predicted sORFs of LINC00278. D, Top, diagram of the LINC00278-sORF1 location at the LINC00278 locus and the FLAG tag that is inserted to the 30 end of LINC00278-sORF1. Bottom, LINC00278-sORF1-FLAG fusion protein levels were determined by immunoﬂuorescence and Western blotting in FLAG-KI Het-1A cells. E, Top, correlation analysis of LINC00278 transcription and LINC00278-sORF1 translation. LINC00278 transcription and LINC00278-sORF1 translation levels were determined in 50 male ESCC PDXs using qPCR and polysome proﬁling-qPCR, respectively. Bottom, LINC00278-sORF1 expression in four pairs of male ESCC tissues was determined by Western blot analysis using anti-LINC00278-sORF1. C, ESCC tissue; N, matched nontumor esophagus specimen. F, LINC00278-sORF1 KO in WT ESCC cells increased tumor growth in xenograft mice (mean SD; n ¼ 5). G, LINC00278 knockdown in WT ESCC cells increased tumor growth in xenograft mice (mean SD, n ¼ 5). H, LINC00278 knockdown in LINC00278-sORF1 KO ESCC cells cannot increase tumor growth in xenograft mice (mean SD; n ¼ 5). I, Overexpression of LINC00278-sORF1 (LINC00278-sORF1 OE) or full-length LINC00278 (LINC00278 OE) in LINC00278-sORF1 KO ESCC cells suppressed tumor growth in xenograft mice (mean SD; n ¼ 5). J, The LINC00278-sORF1-FLAG fusion protein level in xenograft tumors of WT, LINC00278-sORF1 OE, and LINC00278 OE ESCC cells was determined by Western blot analysis with anti-FLAG antibody. , P < 0.05; , P < 0.01; , P < 0.001.

LINC00278-sORF1 knockout promoted ESCC tumor growth down promoted tumor growth in WT ESCC cells, but not in We next generated LINC00278-sORF1-knockout cell lines and LINC00278-sORF1-knockout ESCC cells (Fig. 2G and H). determined the effect of LINC00278-sORF1-knockout on tumor Furthermore, we showed that reintroducing either full-length- growth using mouse ESCC xenograft models. We showed that tumor LINC00278 (full-LINC00278-FLAG) or LINC00278-sORF1 (LINC00278- growth from LINC00278-sORF1-knockout ESCC cells was signiﬁcant- sORF1-FLAG) into the LINC00278-sORF1-knockout could reverse ly higher than that from wild-type (WT) ESCC cells (Fig. 2F). To tumor growth (Fig. 2I). In addition, we also showed that reintroducing investigate whether the transcript of LINC00278 is functional in ESCC LINC00278-sORF1 (LINC00278-sORF1-FLAG) into the LINC00278- tumor growth, we knocked down LINC000278 in WT and LINC00278- knockdown cells could reverse tumor growth (Supplementary sORF1-knockout ESCC cells. We showed that LINC00278-knock- Fig. S2N). Interestingly, overexpression of full-length LINC00278

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demonstrated stronger suppression of tumor growth than partial mediation effect on the relationship between LINC00278 LINC00278- LINC00278 LINC00278- M ¼ a þe a ¼ sORF1 in -sORF1-knockout ESCC cells transcription and sORF1 translation ( X 2: LINC00278- R ¼ P < Y ¼ c0 þb þe c0 ¼ (Fig. 2I). Furthermore, we detected higher sORF1 0.796; SE, 0.0875; 0.796; 0.001; X M 3: 0.396, micropeptide expression by full-length LINC00278-FLAG than by Pc’ ¼ 0.024; b ¼ 0.351, Pb ¼ 0.044). LINC00278-sORF1-FLAG (Fig. 2J). We also found that the RNA stability of full-length LINC00278 and LINC00278-sORF1 were not Identification of regulators of m6A modification in LINC00278 significantly different (Supplementary Fig. S2O). Our results sug- We next investigated which proteins were involved in LINC00278 gest that LINC00278-sORF1 had the main effect on ESCC tumor m6A modification. First, we determined which known m6A “reader” growth while the untranslated region of LINC00278 augmented proteins were bound to m6A modified LINC00278 using RNA pull- such effect. down and Western blot analysis (25). As shown in Fig. 3G, we showed that only YTHDF1 could be pulled down by m6A modified m6A modification of LINC00278 promoted LINC00278-sORF1 LINC00278. Using electrophoretic mobility shift assay (EMSA) and translation RNA immunoprecipitation (RIP), we further confirmed that YTHDF1 Because m6A modification is the most prevalent posttranscriptional interacted with m6A modified LINC00278 (Fig. 3H and I). Finally, we modification of mRNA and lncRNA, and it regulates translation (15), showed that YTHDF1 knockdown significantly downregulated we determined whether m6A modification of LINC00278 regulated LINC00278-sORF1 translation without changing the LINC00278 LINC00278-sORF1 translation. Using m6A-specific RNA immuno- m6A modification level (Supplementary Fig. S3C). precipitation, we showed that LINC00278 contained m6A modification Next, we knocked down each known m6A “writer” and “eraser” in both Het-1A and ESCC cell lines (Fig. 3A). protein and determined its effect on LINC00278 expression, Next, we identified three m6A modification sequence motifs in the LINC00278 m6A modification level, and LINC00278-sORF1 transla- untranslated region of LINC00278 using a computation software called tion. We showed that METTL3, METTL14, and WTAP knockdown SRAMP (24; Supplementary Fig. S3A). To determine which m6A significantly reduced LINC00278 m6A modification level and sequence motif was modified and facilitating LINC00278-sORF1 LINC00278-sORF1 translation, while ALKBH5 knockdown signifi- translation, we generated full-length LINC00278-FLAG and cantly increased LINC00278 m6A modification level and LINC00278- LINC00278-sORF1-FLAG WT construct as well as constructs with sORF1 translation. FTO knockdown did not affect the LINC00278 each m6A sequence motif mutated (mut1-LINC00278-FLAG, mut2- m6A modification level and LINC00278-sORF1 translation (Supple- LINC00278-FLAG, and mut3-LINC00278-FLAG), and transfected mentary Fig. S3C). None of these proteins affected the expression of into Eca-109 and KYSE-150 cells (which are female ESCC cell lines LINC00278 (Supplementary Fig. S3C). that do not have endogenous LINC00278 transcript and LINC00278- Our data suggest that METTL3, METTL14, and WTAP acted as sORF1 micropeptide, Supplementary Table S3). The results showed “writers,” ALKBH5 acted as “eraser,” and YTHDF1 acted as “reader” that only mut3-LINC00278-FLAG significantly reduced LINC00278 for LINC00278 m6A modification. m6A level compared with full-length LINC00278-FLAG expression, which was approximately equal to LINC00278-sORF1-FLAG Cigarette smoking modulates LINC00278-sORF1 translation (Fig. 3B). This was consistent with the lower LINC00278-sORF1 Because cigarette smoking acted synergistically with low protein level produced by mut3-LINC00278-FLAG expression LINC00278 expression to confer worse prognosis in patients with (Fig. 3C). Furthermore, we expressed mut3-LINC00278 and ESCC, we investigated whether cigarette smoking affected LINC00278- LINC00278-sORF1 in LINC00278-sORF1 ESCC cells and implanted sORF1 translation. When we divided patients with ESCC into smoking them to generate xenograft models. The results showed that mut3- and nonsmoking groups, we did not detect significant difference in LINC00278 and LINC00278-sORF1 have no significant difference in LINC00278 expression level between the two groups (Supplementary tumor growth inhibition (Fig. 3D). To prove the GAACU motif Fig. S3D). However, LINC00278 m6A modification and LINC00278- mutated in mut3-LINC00278-FLAG was endogenously m6A modified sORF1 translation levels were significantly lower in the smoking group in LINC00278, we used a Morpholino antisense oligo that specifically than in the nonsmoking group (Fig. 3J and K). blocked m6A modification to this motif in Het-1A cell line. We showed To determine how cigarette smoking affected m6A modification, we that both the levels of LINC00278 m6A modification and LINC00278- exposed Het-1A cells to CSC and measured the expression level of m6A sORF1 were decreased (Fig. 3E and F). Our results indicated that only regulators. CSC treatment only increased the level of ALKBH5 protein the GAACU motif was m6A modified in LINC00278. (Fig. 3L). Meanwhile, CSC treatment decreased the level of Finally, we conducted a mediation analysis to determine whether LINC00278-sORF1 micropeptide but did not affect LINC00278 m6A modification was the mediator of the LINC00278 transcript level expression (Fig. 3L and M). and LINC00278-sORF1 micropeptide level in PDXs. First, we inves- To investigate the mechanism of how CSC upregulated the expres- tigated the relationships between LINC00278 expression and sion of ALKBH5, we analyzed ALKBH5 gene promoter CpG island LINC00278-sORF1 expression using linear regression. Second, we methylation using the massArray DNA methylation analysis. We analyzed the relationship between each LINC00278 expression and found that ALKBH5 CpG island was hypomethylated in CSC- m6A modification by linear regression. Third, we examined the treated cells compared with DMSO mock-treated cells (Fig. 3N). relationship between m6A modification and LINC00278-sORF1 Finally, we showed that ALKBH5 knockdown completely abolished expression using linear regression. Fourth, we included both the the effect of CSC treatment on LINC00278 m6A modification and LINC00278 expression and m6A modification in the model examining LINC00278-sORF1 translation (Fig. 3O). associations with LINC00278-sORF1 expression to evaluate media- Taken together, our data suggest that LINC00278 downregulation tion. As shown in Supplementary Fig. S3B, the total effect of the promoted ESCC progression. LINC00278 encodes a micropeptide, LINC00278 transcription (X) on the LINC00278-sORF1 translation whose expression was modulated by LINC00278 m6A modification. fi ¼ c þe c ¼ R ¼ LINC00278 6 fi (Y) was statistically signi cant (Y X 1: 0.675; SE, 0.106; Finally, m A modi cation was regulated by cigarette 0.675; P < 0.001); and the m6A modification of LINC00278 (M) had a smoking via ALKBH5 hypomethylation.

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Figure 3. m6A modification of LINC00278 promoted YY1BM translation, which was reduced by cigarette smoking. A, [m6A]LINC00278 or [m6A]MALAT1 was detected by immunoprecipitation with antibody against m6A, followed by qRT-PCR analysis in individual cells (mean SD; n ¼ 3). MALAT1 was used as a positive control. B, m6A level of LINC00278 in female ESCC cells that was transfected with indicated plasmids (mean SD; n ¼ 3). C, LINC00278-sORF1-FLAG fusion protein levels in female ESCC cells that were transfected with indicated plasmids. D, Tumor growth in xenograft mice subcutaneously implanted with ESCC cells that were transfected with indicated constructs (mean SD; n ¼ 5). E, m6A level of LINC00278 in Het-1A cells that were transfected with Morpholino antisense oligo, which was specifically blocking the m6A motif of LINC00278. F, LINC00278-sORF1 micropeptide level in Het-1A cells that were transfected with Morpholino antisense oligo, which was specifically blocking the m6A motif of LINC00278. G, The interaction between [m6A]LINC00278 and YTHDF1 was detected by RNA pulldown assays. H, The interaction between [m6A]LINC00278 and YTHDF1 was detected by EMSA assays. I, RIP assays indicated that YTHDF1 interacts with LINC00278. PNPLA2 was used as a positive control. J, Significant [m6A]LINC00278 level difference in male ESCC tissues from smokers (n ¼ 34) and nonsmokers (n ¼ 16; mean SD). K, Significant LINC00278-sORF1 level difference in male ESCC tissues from smokers (n ¼ 34) and nonsmokers (n ¼ 16; mean SD). L, METTL3, METTL14, WTAP, ALKBH5, YTHDF1, and LINC00278-sORF1 levels in Het-1A cells were detected by Western blotting after treatment with CSC (100 mg/mL) or DMSO as solvent control. M, The relative levels of LINC00278,[m6A]LINC00278, and LINC00278-sORF1 in Het-1A cells that were treated with CSC (100 mg/mL) or DMSO as solvent control for 48 hours (mean SD; n ¼ 3). N, Amplicon size and place of CpG sites in the amplicon. Methylation profile of CpG sites for the ALKBH5 gene. The color of the circles is related to the percentage of methylation in each CpG site. Boxes indicate the different methylation patterns between CSC (100 mg/mL; 48 hours) or DMSO-treated Het-1A cells. O, ALKBH5-knockdown abolished effect of CSC (100 mg/mL; 48 hours) treatment on levels [m6A]LINC00278 and LINC00278-sORF1 in Het-1A cells. Mean SD; n ¼ 3. , P < 0.05; , P < 0.01; , P < 0.001.

LINC00278-sORF1 blocks the interaction between YY1 and AR terminal domain (331–414 amino acid), where it has been documented To determine the function of LINC00278-sORF1 in ESCC progres- that bound to AR (28; Fig. 4C). Given that YY1 is a transcriptional sion, we first investigated the LINC00278-sORF1 interacting proteins coactivator of AR in prostate cancer (28), we tested whether it was also by coimmunoprecipitation and mass spectrometry analysis in TE-1 true in ESCC (Fig. 4D). Finally, we showed that the interaction and KYSE-30 cells. We sought for the proteins that could be immu- between YY1 and AR was downregulated by LINC00278 overexpres- noprecipitated by LINC00278-sORF1-FLAG fusion protein but not by sion and upregulated by YY1BM knockout in both TE-1 and KYSE-30 IgG in both TE-1 and KYSE-30 cells. As shown in Fig. 4A, we identified cell lines (Fig. 4E). Our data suggested that YY1BM was blocking the YY1 as the potential LINC00278-sORF1 binding protein. We validated interaction between YY1 and AR. that YY1 could be immunoprecipitated by LINC00278-sORF1-FLAG fusion protein (Fig. 4B). Furthermore, we cotransfected YY1-HA and YY1 promotes AR-regulated eEF2K transcription LINC00278-sORF1-FLAG into TE-1 cells and performed coimmuno- We first examined whether YY1 and AR occupied the same genomic precipitation using anti-HA. Western blot analysis showed that YY1- locations by reanalyzing publically available ChIP-seq data on YY1 (29) HA and LINC00278-sORF1-FLAG proteins were immunoprecipitated and AR (30). We found that YY1 bound to 2,865 gene promoters, while (Fig. 4B). Finally, we showed that endogenous LINC00278-sORF1 in AR bound to 312 gene promoters, respectively. Taken together, TE-1 cells could be immunoprecipitated using anti-YY1 antibody 33 genes were common in genes regulated by YY1 and AR (Fig. 4B). We concluded that LINC00278-sORF1 bound to YY1 and (Fig. 4F). Finally, we found that 10 of these 33 genes have been we named the micropeptide YY1BM. reported repeatedly (>10) to be associated with cancer (Fig. 4G). YY1 is a ubiquitous and multifunction transcriptional factor that Next, we determined whether the expression of any of these genes plays a regulatory role in tumorigenesis, including ESCC (26, 27). YY1 was affected by LINC00278 or YY1BM by Western blot analysis. We truncation experiment indicated that YY1BM bound to YY1 C- found that expression of eEF2K was decreased in LINC00278

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Figure 4. YY1BM inhibited the interaction between YY1 and AR, repressed the transcriptional coactivation effect of AR, and decreased the expression of eEF2K. A, LINC00278- sORF1-FLAG plasmid was transfected into TE-1 and KYSE-30 cells, and the LINC00278-sORF1-FLAG complexes were coimmunoprecipitated by anti-FLAG antibody. The Venn diagram of mass spectrometric analysis shows results for the coimmunoprecipitation experiments. B, Top, LINC00278-sORF1-FLAG plasmid was transfected into TE-1 and KYSE-30 cells, and the LINC00278-sORF1-FLAG complexes were coimmunoprecipitated by anti-FLAG antibody, and LINC00278-sORF1- FLAG was detected. Middle, LINC00278-sORF1-FLAG and YY1-HA plasmids were transfected into TE-1 cells, and the YY1-HA complexes were coimmunoprecipitated by anti-HA antibody, and LINC00278-sORF1-FLAG was detected. Bottom, LINC00278-sORF1 was detected in the complex that was coimmunoprecipitated by anti- YY1 antibody. C, Coimmunoprecipitation assays revealed the interaction of YY1BM with YY1 via the C-terminal region of YY1. D, Immunoprecipitation was performed with anti-YY1 or anti-AR antibodies. Immunoprecipitated AR or YY1 was then revealed by blotting with anti-AR or anti-YY1 antibodies. E, Immunoprecipitation was performed with anti-YY1 antibody in LINC00278 overexpressed (OE), YY1BM KO, and respective control ESCC cells. Immunoprecipitated AR was then revealed by blotting with anti-AR antibody. F, The Venn diagram depicts the number of genes regulated by YY1 and AR derived from ChIP-seq data analysis. G, The histogram indicates the number of search results of these genes that associated with cancer in PubMed. H, Western blotting was performed to verify the expression of APC, CRKL, GOLPH3, eEF2K, KDM4C, BCAR3, CYCS, PON2, LARP1, and PKD2 in LINC00278 overexpressed (OE), YY1BM KO, and respective control ESCC cells. I, An overview of ChIP-seq data is illustrated by Integrative Genomics Viewer software of the promoter and the ﬁrst exon of the eEF2K. J, ChIP assays showed enrichment of YY1 and AR at eEF2K in LINC00278 OE, YY1BM KO, and respective control ESCC cells. Coprecipitated DNA was analyzed by qPCR using amplicons C1– C4 (mean SD; n ¼ 3). K, Luciferase reporter assay for eEF2K promoter in LINC00278 OE, YY1BM KO, and respective control ESCC cells. The reporter constructs expressing the luciferase gene under full-length eEF2K gene promoter or eEF2K promoter deleted 200 to 0 region. Mean SD; n ¼ 4. , P < 0.05; , P < 0.01.

upregulated cells, but increased in YY1BM knockout cells (Fig. 4H). AR peaks surrounding the eEF2K TSS (Fig. 4I). Next, we analyzed the These expression changes were abolished by YY1 siRNAs treatment binding sites of YY1 and AR in the promoter of eEF2K using PROMO (Supplementary Fig. S4A), indicated that eEF2K expression was and JASPAR. The results suggested that AR and YY1 potentially regulated by YY1BM via modulated the interaction between YY1 cobinding to the 200 to 100 region of eEF2K promoter. We subse- and AR. quently carried out ChIP experiments to fine map YY1 and AR binding To identify YY1 and AR binding sites in the eEF2K promoter, we sites in the eEF2K promoter and confirmed that YY1 and AR bound to first analyzed the ChIP-seq data and identified overlapping YY1 and the 200 to 100 region of eEF2K promoter (Fig. 4J).

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Furthermore, we generated an eEF2K promoter luciferase reporter changed in LINC00278 overexpression xenografts when we implanted plasmid (pGL3-eEF2K) and an eEF2K mutant promoter luciferase female ESCC cells into female mice (Fig. 5D). reporter plasmid that deleted the 200 to 100 region (pGL3-eEF2K- When we correlated eEF2K and caspase-3 expression (≥30%, strong mut) to identify eEF2K transcription regulators. The pGL3-eEF2K staining; <30%, weak staining) with YY1BM expression in 50 ESCC luciferase activity was decreased in cells overexpression of full-length tissues, we found that expression of YY1BM was inversely correlated LINC00278 and increased in cells with YY1BM knockout, whereas with eEF2K expression, but positively correlated with cleaved caspase pGL3-eEF2K-mut abolished these difference (Fig. 4K). Furthermore, 3(Fig. 5E and F). the pGL3-eEF2K luciferase activity was not significantly different when cells were treated with YY1 siRNAs, confirming the involvement of YY1BM is a potential anticancer micropeptide YY1 in AR-regulated eEF2K expression (Supplementary Fig. S4B). Because several anticancer peptides have been reported, we inves- Finally, we determined the levels of testosterone and eEF2K expres- tigated whether YY1BM is a novel anticancer micropeptide. We first sion in patients with ESCC by Electrochemiluminescence immuno- tested the cytotoxicity of YY1BM in ESCC cells. As shown in Fig. 6A assay and qRT-PCR. We found that the testosterone level was pos- and B, we found that YY1BM was cytotoxic to TE-1 and KYSE-30 cells, itively correlated with eEF2K expression level in males, but not in while scrambled YY1BM (svYY1BM) control micropeptide was not. females (Supplementary Fig. S4C). To further confirm the involvement To probe the anticancer effect of YY1BM in vivo, we injected of AR signaling pathway, we showed that YY1BM overexpression did YY1BM intratumorally into ESCC tumors grafted in nude mice and not affect the tumor growth in female mice (Supplementary Fig. S4D), analyzed the survival time. We found YY1BM injection significantly indicating that YY1BM is indeed involved in male ESCC progression improved the survival rate of male mice, but not female mice (Fig. 6C via AR signaling pathway. and D). Furthermore, IHC analysis revealed a higher apoptosis rate and lower eEF2K expression in male mice, but not female mice YY1BM decreases survival of ESCC cells under ND through (Fig. 6C and D), suggesting that YY1BM intratumoral injection eEF2K signaling pathway downregulated the expression of eEF2K and induced apoptosis, Because eEF2K confers cell survival under acute severe ND by ultimately improved male mice survival. inhibiting eEF2 activity and translation elongation (31), we determined whether YY1BM regulated ESCC cell survival under ND. Compared with WT cells, YY1BM knockout cells showed increased Discussion survival under ND (Supplementary Fig. S4E). This increased survival Globally, ESCC is a male-dominant malignancy. Both sex hormone was abolished by A-484954 treatment, a known small-molecule eEF2K and lifestyle factors, such as cigarette smoking, contribute to this inhibitor that could decrease the phospho-eEF2 level in ESCC cells gender disparity. In this study, we discovered a 21-amino-acid micro- (Supplementary Fig. S4E). peptide (YY1BM) encoded by Y-linked lncRNA LINC00278. The We also analyzed apoptosis of ESCC cells under ND, using flow translation of YY1BM was modulated by cigarette smoking– cytometry for Annexin V staining (Supplementary Fig. S4F and S4G) mediated LINC00278 m6A modification. YY1BM blocked YY1 bind- and Western blot analysis of caspase-3 cleavage (Supplementary ing to AR to activate the expression of eEF2K, which is a key regulator Fig. S4H and S4I). We showed that YY1BM knockout reduced for male ESCC progression. apoptosis in ESCC cells under ND, which was abolished by A- Through mining a large cohort of ESCC lncRNA profiling data, we 484954 treatment. Whereas overexpression of full-length LINC00278 discovered that LINC00278 might play a critical role in male-specific induced apoptosis under ND, which was also abolished by A-484954 ESCC progression. LINC00278 is a 537 bp transcript, located on Y treatment. Meanwhile, the expression of eEF2K was upregulated by chromosome and previously annotated as a noncoding RNA. It has YY1BM knockout and downregulated by LINC00278 overexpression been reported that lncRNAs are involved in ESCC progression, such as under ND, which was also abolished by treatment with A-484954 Linc-POU3F3 promotes methylation of POU3F3 by interacting with (Supplementary Fig. S4H and S4I). Finally, we found that eEF2 EZH2 in ESCC (17). Interestingly, our data indicated that YY1BM, phosphorylation was increased in YY1BM knockout cells and instead of the LINC00278 transcript, plays a major role in ESCC decreased in LINC00278 overexpressed cells under ND, which was progression. We also found that the m6A modification motif of also abolished by A-484954 treatment (Supplementary Fig. S4H and LINC00278 is close to the stop codon of YY1BM, consistent with its S4I). role in the regulation of YY1BM translation (15). m6A is the major Interestingly, we found that A-484954 treatment under ND reduced reversible posttranscription modification in RNAs (32, 33), involved in the speed of YY1BM translation decrease (Supplementary Fig. S4J), RNA stability (34) and protein production (15). m6A modification suggesting the presence of a positive-feedback loop between the changes have been linked to various disease processes, including eEF2K/eEF2 axis and YY1BM. tumorigenesis. It has been shown that the physiologic functions of These data suggested that YY1BM inactivated the AR-regulated m6A modification mainly depend on the “reader” proteins that bind to transcription of eEF2K under ND, thereby enhancing translation the m6A modification motif. YTHDF1, a member of YTH family that elongation and resulting in ESCC cell apoptosis. has been reported to facilitate protein synthesis by interacting with translation machinery (15), is the “reader” for m6A modification of Low YY1BM expression is associated with reduced apoptosis in LINC00278. Based on previously reported studies, we speculate that ESCC xenografts and tissues YTHDF1 binds to m6A modified LINC00278 to recruit the translation We then sought to explore the relationship between YY1BM machinery, therefore promote the translation efficiency of YY1BM. expression and ESCC apoptosis in xenograft model. YY1BM knockout This is consistent with our mediation analysis showing that m6A xenografts showed higher expression of eEF2K and lower expression of modified LINC00278 has an incomplete mediating effect on the cleaved caspase-3 by IHC analysis (Fig. 5A and B), consistent with relation between LINC00278 and YY1BM expression levels in ESCC reduced apoptosis by TUNEL staining (Fig. 5C). Moreover, we also tissues. Our data showed that CSC treatment leads to ALKBH5 found that expression of eEF2K and cleaved caspase 3 were not promoter hypomethylation and increased expression of ALKBH5.

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Figure 5. YY1BM induced apoptosis of ESCC cells. A, Cleaved caspase-3 and eEF2K immunostaining in xenograft tumors of LINC00278 OE, YY1BM KO, and respective control TE-1 cells. B, Cleaved caspase-3 and eEF2K immunostaining in xenograft tumors of LINC00278 OE, YY1BM KO, and respective control KYSE-30 cells. C, TUNEL staining in xenograft tumors of LINC00278 OE, YY1BM KO, and respective control ESCC cells. D, Cleaved caspase-3 and eEF2K immunostaining in xenograft tumors of female LINC00278 OE and control ESCC cells that were implanted into female mice. E, Left, eEF2K immunostaining in male ESCC samples. Right, the relative level of YY1BM in eEF2K strong and weak samples. F, Left, cleaved caspase-3 immunostaining in male ESCC samples. Right, the relative level of YY1BM in cleaved caspase-3 strong and weak samples. , P < 0.05; , P < 0.001.

ALKBH5 is an m6A demethylase that acts as the “eraser” protein of HOXB-AS3 encodes a conserved 53 amino-acid micropeptide, which m6A modified LINC00278, which leads to a decreased level of m6A suppresses colon cancer growth by regulating the pyruvate kinase M modified LINC00278, and in turn reduced YY1BM expression through (PKM) splicing and suppressing glucose metabolism reprogram- YTHDF1. In China, current smokers are significantly more prevalent ming (12). In our study, YY1BM is identified as a novel micropeptide in the male population than in the female population. Cigarette encoded by Y-linked LINC00278. It interacts with YY1 and blocks its smoking is a key factor in ESCC carcinogenesis. In this study, we interaction with AR. YY1 is a zinc finger protein belonging to the GLI- found that cigarette smoking contributes to poor prognosis in ESCC in Kruppel family that can activate or inactivate gene expression depend- part by regulating LINC00278 translation through m6A modification. ing on interacting partners, promoter context, and chromatin struc- In summary, we conclude that m6A modification of LINC00278 ture (35). YY1 is known to be overexpressed in various cancers, modulates YY1BM translation and results partially in the sex bias of including ESCC (27). Moreover, YY1 acts as a coactivator of several ESCC. transcription factors that play important roles in carcinogenesis, such Recently, several lncRNA-encoded micropeptides have been iden- as P53, GATA-4, and AR (28, 36). Sex hormone, especially androgen, tified and reported to play crucial roles in a variety of physiologic has been documented to be associated with ESCC progression (37). AR processes. MLN, a micropeptide encoded by a skeletal muscle-specific promotes ESCC cell invasion and proliferation via matrix metallo- lncRNA, has been shown to interact directly with SERCA and impede proteinase 2 (38). AR and IL6 form a reciprocal regulatory circuit to þ Ca2 uptake into the SR, thereby regulating muscle performance (11). sustain STAT3 oncogenic signaling in ESCC (39). High-level AR Expression of another lncRNA-encoded micropeptide Myomixer, expression in ESCC predicts poor clinical outcome in tobacco- together with Myomaker, controls the critical step in myofiber for- using patients with ESCC (39), suggesting another mechanism of how mation during muscle development (10). Furthermore, lncRNA smoking contributes to poor prognosis in patients with ESCC. Our

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Figure 6. YY1BM acts as a potential anticancer micropeptide in vivo. A, Cell proliferation analysis of TE-1 cells was treated with different concentrations of YY1BM or svYY1BM under acute ND at different time points (mean SD; n ¼ 5). B, Cell proliferation analysis of KYSE-30 cells that were treated with different concentrations of YY1BM or svYY1BM under acute ND at different time points (mean SD, n ¼ 5). C, Top, survival data for xenograft mice that were subcutaneously implanted with TE-1 or KYSE- 30 cells and directly intratumorally injected with 400 mg/mL YY1BM or svYY1BM. Bottom, cleaved caspase-3 and eEF2K immunostaining in xenograft tumors that were directly intratumorally injected with 400 mg/mL YY1BM or svYY1BM. D, Top, survival data for xenograft female mice that were subcutaneously implanted with female ESCC cells and directly intratumorally injected with 400 mg/mL YY1BM or svYY1BM. Bottom, cleaved caspase-3 and eEF2K immunostaining in xenograft tumors that were directly intratumorally injected with 400 mg/mL YY1BM or svYY1BM. , P < 0.05; , P < 0.01; , P < 0.001.

data are consistent with these findings by showing that YY1BM In summary, we found that the Y-linked lncRNA LINC00278 modulates the transcription activity of YY1 and AR, which directly encodes a micropeptide termed YY1BM. YY1BM suppresses the coregulate the expression of eEF2K. transcription of eEF2K by blocks the interaction between YY1 and eEF2K is a conserved mediator of the cellular response to ND (31). AR, thereby promoting the activity of eEF2 and resulting in apoptosis Activated eEF2K phosphorylates and inactivates eEF2, thereby block of ESCC. LINC00278 has a classical m6A modification motif close to the translation elongation of mRNAs (40). eEF2K reduces cancer cell the stop codon of YY1BM, which interacts with YTHDF1 and facil- apoptosis and promotes cancer cell survival under ND (31). In this itates the translation of YY1BM. Cigarette smoking increases ALKBH5 study, we demonstrated that YY1BM can regulate the eEF2K/eEF2 axis expression and reduces m6A modification of LINC00278, thereby via inhibiting the transcriptional activity of YY1 and AR. Furthermore, inhibits the translation of YY1BM and induces the ESCC progression. inactivation of eEF2 could in turn block the translation of YY1BM, Interestingly, exogenous YY1BM has anticancer potential. In conclu- leading to the formation of LINC00278-YY1BM-YY1-AR-eEF2K- sion, our study reveals that LINC00278 and its product YY1BM are at eEF2 cycle, which is probably one of the underlying molecular the intersection of hormones, lifestyle factors, and genetics in male mechanisms for micropeptide encoded by lncRNA to induce tumor- ESCC progression, highlighting the fact that LINC00278 and YY1BM igenesis and progression of male ESCC. could serve as potential prognostic biomarkers and therapeutic targets Because YY1BM has such a critical role in male ESCC, we deter- for male ESCC. mined whether YY1BM is a potential anticancer micropeptide. Similar to PNC-27 targeting HDM-2 in the membrane to kill cancer cells (41), Disclosure of Potential Conflicts of Interest we showed that YY1BM is a potent anticancer micropeptide in ESCC. No potential conflicts of interest were disclosed.

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Authors’ Contributions Jiangsu (BK20160008); a project funded by the Priority Academic Program Devel- Conception and design: S. Wu, Y. Zhou opment of Jiangsu Higher Education Institutions; National Key R&D Program of Acquisition of data (provided animals, acquired and managed patients, provided China (2016YFC1302100); the Program for Guangdong Introducing Innovative and facilities, etc.): S. Wu, L. Zhang, B. Guo, F. Li, Y. Wang, R. Wu, J. Lu, Y. Zhou Entrepreneurial Teams (2017ZT07S096). Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Wu, L. Zhang, J. Deng, S. Zhang, J. Lu, Y. Zhou The costs of publication of this article were defrayed in part by the Writing, review, and/or revision of the manuscript: S. Wu, L. Zhang, J. Lu, Y. Zhou payment of page charges. This article must therefore be hereby marked Administrative, technical, or material support (i.e., reporting or organizing data, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate constructing databases): S. Wu, J. Deng, Y. Zhou this fact. Study supervision: Y. Zhou

Acknowledgments This work was supported by the National Scientiﬁc Foundation of China grants Received November 2, 2019; revised January 15, 2020; accepted March 10, 2020; 81772544 and 81972649; Science Foundation for Distinguished Young Scholars in published ﬁrst March 13, 2020.

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Role of Micropeptide Encoded by lncRNA in Male ESCC

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AACRJournals.org Cancer Res; 80(13) July 1, 2020 2803

A Novel Micropeptide Encoded by Y-Linked LINC00278 Links Cigarette Smoking and AR Signaling in Male Esophageal Squamous Cell Carcinoma

Siqi Wu, Liyuan Zhang, Jieqiong Deng, et al.

Cancer Res 2020;80:2790-2803. Published OnlineFirst March 13, 2020.

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