A Novel Micropeptide Encoded by Y-Linked LINC00278 Links

Author Manuscript Published OnlineFirst on March 13, 2020; DOI: 10.1158/0008-5472.CAN-19-3440 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 A Novel Micropeptide Encoded by Y-Linked LINC00278 Links

2 Cigarette Smoking and AR Signaling in Male Esophageal Squamous

3 Cell Carcinoma

4 Running title: Role of micropeptide encoded by lncRNA in male ESCC.

6 Siqi Wu 1*, Liyuan Zhang2*, Jieqiong Deng1, Binbin Guo1, Fang Li1, Yirong Wang1, Rui Wu1,

7 Shenghua Zhang1, Jiachun Lu3, Yifeng Zhou1†

8 *Siqi Wu and Liyuan Zhang contributed equally to this work.

10 Author affiliations

11 1Department of Genetics, Medical College of Soochow University, Suzhou 215123, China;

12 2Department of Radiotherapy & Oncology, The Second Affiliated Hospital of Soochow

13 University, San Xiang Road No. 1055, Suzhou 215004, China

14 3The Institute for Chemical Carcinogenesis, The First Affiliated Hospital, The School of Public

15 Health, Guangzhou Medical University, Guangzhou 510182, China

17 Correspondence to: †Dr. Yifeng Zhou, Medical College of Soochow University, Suzhou 215123,

18 China. Tel: 86-512-65884720; Fax: 86-512-65884720; E-mail: [email protected]

20 Competing interests None.

21 Keywords: lncRNAs, micropeptide, m6A, male ESCC, cigarette smoking

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 13, 2020; DOI: 10.1158/0008-5472.CAN-19-3440 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

22 ABSTRACT

23 Long non-coding RNAs (lncRNA) have been shown to play critical roles in many diseases,

24 including esophageal squamous cell carcinoma (ESCC). Recent studies have reported that some

25 lncRNA encode functional micropeptides. However, the association between ESCC and

26 micropeptides encoded by lncRNA remains largely unknown. In this study, we characterized a

27 Y-linked lncRNA, LINC00278, which was downregulated in male ESCC. LINC00278 encoded a

28 Yin Yang 1 (YY1)-binding micropeptide, designated YY1BM. YY1BM was involved in the

29 ESCC progression and inhibited the interaction between YY1 and androgen receptor (AR),

30 which in turn decreased expression of eEF2K through the AR signaling pathway.

31 Downregulation of YY1BM significantly upregulated eEF2K expression and inhibited apoptosis,

32 thus conferring ESCC cells more adaptive to nutrient deprivation. Cigarette smoking decreased

33 m6A modification of LINC00278 and YY1BM translation. In conclusion, these results provide a

34 novel mechanistic link between cigarette smoking and AR signaling in male ESCC progression.

36 SIGNIFICANCE

37 Post-transcriptional modification of a micropeptide-encoding lncRNA is negatively impacted by

38 cigarette smoking, disrupting negative regulation of the AR signaling pathway in male ESCC.

40 INTRODUCTION

41 Esophageal squamous cell carcinoma (ESCC) is two to four times more common in men

42 than in women worldwide(1). Previous studies suggest that several male-specific factors

43 contribute to such gender disparity, including cigarette smoking and sexual hormone. A survey

44 in 2010 indicated that 52.9% of Chinese men while only 2.4% of Chinese women were current

45 smokers(2,3). Expression of androgen receptors has been reported in ESCC as well as

46 association with prognosis(4,5). However, the exact underlying molecular mechanisms in male

47 ESCC progression remain largely unknown.

48 A recent study identified a tumor suppressor gene on Y chromosome for male breast

49 cancer(6), suggesting that genetic material encoded by Y chromosome could be involved in

50 male-dominant tumors. Long non-coding RNAs (lncRNAs) are defined as RNA transcripts

51 longer than 200nt that lack protein-coding potential(7,8). LncRNAs act as master regulators for

52 gene expression, thus play an important role in many biological functions and diseases, including

53 cancer(9). However, no study so far has reported on the involvement of Y-linked lncRNAs in

54 ESCC.

55 Recent computational and genome-wide studies have demonstrated that hundreds of

56 functional micropeptides (less than 100 amino acids) are embedded in lncRNAs. For example,

57 myomixer is an 84-amino acid muscle-specific micropeptide encoded by a lncRNA that controls

58 the critical steps in myofiber formation during muscle development(10); myoregulin is identified

59 as a skeletal muscle-specific lncRNA, which regulates muscle performance by impeding Ca2+

60 uptake into the SR(11). It is still unclear whether micropeptides play a key role in tumor

61 development, although a recent study has identified a micropeptide encoded by HOXB-AS3

62 lncRNA that suppresses colon cancer growth(12).

63 N6-methyladenosine (m6A) is the most abundant post-transcription modification on

64 eukaryotic mRNAs and lncRNAs(13). Recent studies show that m6A modification is dynamic

65 and reversible in cells, whose level is regulated by m6A methyltransferases (also called “writers”:

66 METTL3, METTL14, etc.) and m6A demethylases (also called “erasers”: FTO, ALKBH5, etc.).

67 m6A regulates gene expression through m6A binding proteins (also called “readers”: YTHDF1,

68 YTHDF2, YTHDF3, etc.)(14,15). These m6A-associated proteins play critical roles to regulate

69 the metabolism and functions of m6A-modified mRNAs and lncRNAs(15).

70 In this work, we identified a micropeptide encoded by a Y-linked lncRNA, LINC00278,

71 which is downregulated in male ESCC. The expression of this micropeptide was downregulated

72 by cigarette smoking in ESCC through erasing m6A modification. It specifically bound to Yin

73 Yang 1 (YY1) and blocked the interaction between YY1 and AR, therefore named YY1-

74 blocking micropeptide (YY1BM). YY1BM downregulated eEF2K expression through AR

75 signaling pathway and induced apoptosis in ESCC under nutrient deprivation (ND).

76 Furthermore, YY1BM also acts as a potential anticancer micropeptide for ESCC.

78 MATERIALS AND METHODS

79 Human study subjects

80 A total of 281 pairs of fresh frozen ESCC and adjacent non-cancerous tissue samples were

81 obtained from patients in eastern China who underwent tylectomies at the Affiliate Hospitals of

82 Soochow University (Suzhou cohort). Another 288 pairs of fresh frozen ESCC tissues were

83 collected from patients in southern China at the Cancer Hospitals affiliated with Guangzhou

84 Medical University (Guangzhou cohort). None of the patients received anti-cancer treatment

85 before surgery, including chemotherapy or radiotherapy. The Medical Ethics Committees of

86 Soochow University and Guangzhou Medical College approved this study. The clinical

87 characteristics of patients in this study are listed in Table. S1.

88 Statistical Analysis

89 The data analysis was performed using the SPSS 19.0 software for Windows. The statistical

90 significance between data sets was expressed as P values, and P<0.05 was considered

91 statistically significant. Survival curves were obtained using the Kaplan-Meier method and

92 compared using the log-rank test. Multivariable Cox regression analysis was performed using the

93 R package “survival”. Paired or unpaired Student’s t-test, Pearson correlation coefficients were

94 used for various types of data comparison. Mediation analysis was conducted using the

95 procedure described by Baron and Kenny (16) and a P<0.05 was considered significant.

96 Animals and Cell cultures

97 Male nude mice of 6-8 weeks of age were purchased from the Shanghai Laboratory Animal

98 Center at the Chinese Academy of Sciences (Shanghai, China). All animal studies were

99 conducted with the approval of Soochow University Institutional Animal Care and Use

100 Committee and were performed in accordance with established guidelines

101 All cell lines were purchased from Procell Life Science&Technology Co.,Ltd. These cell

102 lines were all characterized by DNA finger printing analysis and passaged less than 6 months in

103 this study. DMEM, RPMI-1640 and fetal bovine serum (FBS) were purchased from Invitrogen.

104 Eca-109, TE-1, KYSE-30 cells were grown in RPMI-1640 with 10% FBS; Het-1A, 293T cells

105 were grown in DMEM with 10% FBS. All cell lines were grown in penicillin/streptomycin

106 containing medium, at 37°C in a humidified atmosphere with 5% CO2. In addition, cells were

107 treated with 1 nmol/L R1881 (methyltrienolone) to activate AR signaling pathway. To inhibit

108 specific signaling pathways, cells were pretreated with vehicle (DMSO) or 10μM A-484954

109 (EMD Millipore) for 1h at 37°C prior to the experiments.

110 Transplantation of human ESCC tissues

111 Primary viable human ESCC samples were obtained from surgical ESCC specimens (n=50)

112 at the Affiliate Hospitals of Soochow University (Suzhou). During surgery, fresh tumor tissue

113 was collected in transport medium, [RPMI 1640 medium supplemented with

114 penicillin/streptomycin (100U/ml; 100μg/ml), fungizone (1μg/ml) and gentamicin (50μg/ml; all

115 from Life Technologies)] and implanted in mice within 4hr. In parallel, primary tumor tissue

116 fragments were also fresh-frozen and formalin-fixed for further analyses. Before implantation,

117 tumor tissue was rinsed in PBS supplemented with penicillin/streptomycin and fungizone. Each

118 tumor specimen was cut into three small fragments (1.5mm×1.5mm) and grafted subcutaneously

119 into NCG mice. The NCG mice were anesthetized by intraperitoneal of pentobarbitone

120 (10mg/ml) at a dose of 65mg/kg.

121 Microarray data analysis

122 In order to identify male ESCC-associated lncRNAs, differential gene expression analysis

123 was performed on gene expression profiles of 179 pairs of ESCC and matched adjacent normal

124 tissues, and the tissues were separated male and female groups. Differential gene expression

125 analysis was performed by the R package “limma”. The probe which adjusted P-value

126 (adj.P)<0.01 and the absolute value of log2 fold-change (abs.logFC)>1 were defined as

127 differentially expressed probes. The differentially expressed probes were subsequently annotated

128 by mapping onto the genomic coordinates of lncRNAs derived from GENCODE.

129 ChIP-sequencing data analysis

130 ChIP-seq data were obtained from the GEO database. ChIP-seq reads were aligned to the

131 hg19 by Bowtie2 with default parameters; the mapped reads of ChIP-seq were pre-processed by

132 Samtools and then submitted to MACS2 for peaks calling. The peaks were annotated by the R

133 package “ChIPseeker” and visualized by IGV software. Finally, genes that contained peaks at -

134 800bp upstream of transcriptional start sites (TSS) to +200bp downstream of TSS region were

135 defined as genes regulated by corresponding TFs.

136 Overall survival analysis

137 Using the median expression level of LINC00278 among ESCC tissues, we separated ESCC

138 patients into two different groups: patients with high LINC00278 expression (relative expression

139 level>median expression level); and patients with low LINC00278 expression (relative

140 expression level≤median expression level), in both the Suzhou cohort (discovery set, 281

141 patients) and Guangzhou cohort (validation set, 288 patients). Further Kaplan-Meier survival

142 curves and log-rank tests were performed between the high LINC00278 group and the low

143 LINC00278 group.

144 RNA extraction and qRT-PCR

145 Total RNA was isolated from ESCC tissues and corresponding adjacent non-neoplastic

146 tissues using the RNA Isolater Total RNA Extraction Reagent (Vazyme). The purity and

147 concentration of RNA were determined by the ratio of absorbance at 260 nm (A260) and 280 nm

148 (A280) using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). The RNA was

149 considered pure and suitable for downstream experiments when A260/A280 was within the

150 range 1.7-2.0. The RNA integrity was determined by running on a 1.5% denaturing agarose gel.

151 First-strand cDNA was synthesized with the Superscript II-reverse transcriptase kit (Invitrogen,

152 Carlsbad). All qRT-PCR primers are listed in Table. S2.

153 Antibodies and Western blotting

154 ESCC cells were collected and lysed in cell lysis buffer for Western and IP (Beyotime

155 Institute of Biotechnology). Proteins were separated on SDS-polyacrylamide gel and transferred

156 to nitrocellulose membrane. Immunoblotting of the membranes was performed using the

157 following primary antibodies: anti-FLAG (sc-807, Santa Cruz), HA (ab9110, Abcam), YTHDF1

158 (ab99080, Abcam), YTHDF2 (ab170118, Abcam), YTHDF3 (ab103328, Abcam), METTL3

159 (ab195352, Abcam), METTL14 (ab98166, Abcam), WTAP (ab155655, Abcam), ALKBH5

160 (ab69325, Abcam), YY1 (sc-7341, Santa Cruz), CRKL (sc-365092, Santa Cruz), eEF2K (3692,

161 Cell Signaling Technology), APC (sc-393704, Santa Cruz), GOLPH3 (19112-1-AP,

162 Proteintech), KDM4C (ab85454, Abcam), BCAR3 (24032, Cell Signaling Technology), CYCS

163 (sc-13156, Santa Cruz), PON2 (sc-374158), LARP1 (sc-515873, Santa Cruz), PKD2 (sc-28331,

164 Santa Cruz), Cleaved caspase-3 (ab32042, Abcam), eEF2 (2332, Cell Signaling Technology), p-

165 eEF2 (2331, Cell Signaling Technology), and anti-β-actin (A5441, Sigma-Aldrich). Signals were 8

166 revealed after incubation with recommended secondary antibody coupled to peroxidase using

167 enhanced chemiluminescence.

168 DNA Methylation Analysis

169 DNA methylation analysis was performed as previously reported(17). Briefly, we designed

170 primers of the CpG islands in the promoter region of ALKBH5 gene using MethPrimer. After

171 robotically dispensing 22nL of the cleavage reaction onto the silicon matrix preloaded chips

172 (SpectroCHIP; Sequenom), the mass spectra were collected using a MassARRAY Compact

173 MALDI-TOF (Sequenom) and the spectra’s methylation ratios were generated by EpiTYPER

174 software (version 1.0; Sequenom).

175 Anti-YY1BM antibody preparation.

176 Peptide synthesis and anti-YY1BM antibody generation were performed as previously

177 described with some modifications(18). Briefly, a BSA and OVA-coupled peptide

178 CLSGQLQPEGRSALPQPG-NH2 was synthesized, and polyclonal antibody against the

179 YY1BM was obtained from inoculated rabbits. Antibody was purified using affinity

180 chromatography on columns containing the corresponding peptide.

181 Polysome profiling

182 Polysome profiling was performed to measure the translation of YY1BM monitored by

183 qRT-PCR. We performed polysome profiling followed the procedure described before(19). The

184 primers for qRT-PCR were listed in Table. S2.

185 RNA Stability Analysis

186 To determine the stability of LINC00278 and YY1BM transcripts, TE-1 cells were treated

187 with 2μg/ml Actinomycin D (Sigma). The cells were harvested at 0h, 6h, 12h, 18h and 24h post

188 treatment, LINC00278 and YY1BM transcripts were quantified by qRT-PCR.

189 Preparation of cigarette smoke condensate

190 Cigarette smoke condensate (CSC) was prepared as previously reported (20). Briefly,

191 Cigarette smoke was collected from a popular Chinese brand cigarette (12 mg tar per cigarette)

192 by a vacuum machine into a container and frozen with liquid nitrogen. CSC was dissolved in

193 DMSO at a concentration of 235 mg/ml, and aliquots were stored at -80°C until use.

194 Co-immunoprecipitation assay

195 Co-immunoprecipitation assay was performed using Pierce™ Co-Immunoprecipitation Kit

196 (Thermo Scientific) according to the manufacturer’s instructions. The lysates were applied to

197 columns containing 10μg of immobilized antibodies covalently linked to an amine-active resin

198 and incubated overnight at 4°C. Then the co-immunoprecipitate was eluted and analyzed by

199 SDS-PAGE or mass spectrometry along with the controls. Co-immunoprecipitation assays were

200 performed using the following antibodies: anti-FLAG (sc-807, Santa Cruz), anti-HA (ab9110,

201 Abcam), anti-YY1 (sc-7341, Santa Cruz), anti-AR (sc-7305, Santa Cruz).

202 Mass spectrometry analysis

203 The samples were analyzed on Thermo Fisher LTQ Obitrap ETD mass spectrometry.

204 Briefly, the samples were loaded onto an HPLC chromatography system named Thermo Fisher

205 Easy-nLC 1000 equipped with a C18 column (1.8mm, 0.15×1,00mm). Solvent A contained 0.1%

206 formic acid and solvent B contained 100% acetonitrile. The elution gradient was from 4% to

207 18% in solvent A for 182 min, 18% to 90% in solvent B for 13 min at a flow rate of 300nL/min.

208 Mass spectrometry analysis was carried out at the AIMS Scientific Co.,Ltd. (Shanghai, China) in

209 the positive-ion mode with an automated data-dependent MS/MS analysis with full scans (350-

210 1600 m/z) acquired using FTMS at a mass resolution of 30,000 and the ten most intense

211 precursor ions were selected for MS/MS. The MS/MS was acquired using higher-energy

212 collision dissociation at 35% collision energy at a mass resolution of 15,000.

213 Production of YY1BM knockout and FLAG knockin cells

214 The gRNA sequence designed specifically for the ORF of YY1BM start codon inserted to

215 the Cas9/gRNA (puro-GFP) vector (VK001-02, ViewSolid BioTech) was 5′-

216 GACTCCAGGCATGCTATCAGG-3′. The donor oligo was purchased from Cyagen

217 Biosciences Inc. (Suzhou, China). The constructed targeting vector and donor oligo were

218 subsequently transfected into the cells using Lipofectamine 3000 (Thermo Fisher Scientific),

219 after which the cells were cultured under puromycin drug selection (2μg/mL) for 48hr. Viable

220 clones were grown to a larger size and picked up for Western blot analysis or sequencing. The

221 schematic diagram and the sequence of YY1BM-KO cells were present in Fig. S1A. The levels

222 of LINC00278 transcription and YY1BM translation were present in Fig. S1B-S1D.

223 RNA Interference

224 Small interfering RNA (siRNA) targeting the YY1, METTL3, METTL14, WTAP, ALKBH5,

225 FTO and YTHDF1 gene and non-targeting siRNA control (Table. S2) were purchased from 11

226 GenePharma. Transfections with siRNA (75nM) were performed with Lipofectamine 3000 (Life

227 Technologies) and the expression of interfered genes was present in Fig. S1E.

228 Production of LINC00278 overexpression and YY1BM overexpression cells

229 To produce overexpression cells, the full-length human LINC00278 or YY1BM cDNA were

230 synthesized by GeneWiz (Beijing, China) and cloned into the lentiviral expression vector pLVX-

231 IRES-neo (Clontech Laboratories Inc.). To produce lentivirus containing full-length LINC00278

232 or YY1BM targeting sequence, 293T cells were co-transfected with the vector described above

233 and the lentiviral vector packaging system using Lipofectamine 3000. Infectious lentiviruses

234 were collected at 48h and 72h after transfection and filtered through 0.45μm filters. These

235 lentiviruses were respectively designated as LINC00278-overexpression or YY1BM-

236 overexpression. We used an empty plenty-pLVX-IRES-neo vector to generate negative control

237 lentiviruses. Recombinant lentiviruses were concentrated by centrifugation. The virus-containing

238 pellet was dissolved in DMEM, and aliquots were stored at -80℃ until use. Cells were infected

239 with the concentrated virus in the presence of polybrene (Sigma-Aldrich). The supernatant was

240 replaced with complete culture medium after 24h, followed by selection with 800µg/ml G418,

241 and the expression of LINC00278 and YY1BM in infected cells was verified by qRT-PCR.

242 Micropeptide synthesis

243 The micropeptides used in ESCC cell treatment and intratumoral injection were synthesized

244 from ChinaPeptides Co.,Ltd. The micropeptides were purified by high-performance liquid

245 chromatography, and the sequence and structure were confirmed by mass spectrometry. The

246 peptides were ≧95% pure and kept as 100mg/ml stock solution at -20°C. 12

247 Data sharing statement

248 Microarray data are available at the NCBI Gene Expression Omnibus (GEO) repository

249 with accession number GSE53625. The ribosome profiling data are available at the GEO

250 repository with accession number GSE61742. The ChIP-Seq data are available at the GEO

251 repository with accession numbers GSE32465 and GSE62472.

252 RESULTS

253 Identification of ESCC-associated lncRNA LINC00278

254 In order to identify male ESCC-associated lncRNAs, we analyzed differential expressed

255 lncRNAs in a lncRNAs expression profiles of 179 pairs of ESCC and matched adjacent normal

256 tissues which separated into male and female groups(21). In total, 3401 differentially expressed

257 genes were screened from the male group (146 patients) and 3284 differentially expressed genes

258 were screened from the female group (33 patients). In addition, we filtered 254 differentially

259 expressed lncRNAs in the male group and 244 differentially expressed lncRNAs in the female

260 group (Fig. 1A). Among these, we found 51 differentially expressed lncRNAs were present in

261 the male group, but not in the female group, and 3 of them were mapped to Y chromosome (Fig.

262 1B-1D).

263 Next, we measured the expression of these 3 Y-linked lncRNAs by quantitative RT-PCR

264 (qRT-PCR) in 281 pairs of male ESCC tissue samples from an Eastern Chinese population

265 (Suzhou cohort). Only LINC00278 was significantly downregulated in ESCC tissues when

266 compared to adjacent normal tissues (P<0.001) (Fig. 1E; Fig. S2A-S2B). We further validated

267 the downregulation of LINC00278 in ESCC tissues using an independent 288 pairs of male

268 ESCC samples from a Southern Chinese population (Guangzhou cohort) (P<0.001) (Fig. 1E).

269 We also determined whether LINC00278 expression was associated with overall survival

270 (OS) among male ESCC patients. Using the median expression level of LINC00278 among

271 ESCC tissues, we separated ESCC patients into two different groups: patients with high

272 LINC00278 expression (relative expression level>median expression level); and patients with

273 low LINC00278 expression (relative expression level≤median expression level), in both the

274 Suzhou cohort (discovery set, 281 patients) and Guangzhou cohort (validation set, 288 patients).

275 Using the log-rank test and Kaplan-Meier survival curves, we showed that patients with low

276 LINC00278 expression had significantly shorter OS than patients with high LINC00278

277 expression in both the discovery set (median survival time (MST): 29 vs 36 months, log-rank

278 P=0.0004, hazard ratio (HR)=1.848) and the validation set (MST: 27 vs 39 months, log-rank

279 P<0.0001, HR=1.850) (Fig. 1F and 1G). Multivariable Cox regression analysis also indicated

280 that low LINC00278 expression was associated with shorter OS (Fig. 1F and 1G).

281 Since cigarette smoking has been associated with poor OS, we determined whether

282 LINC00278 expression and cigarette smoking acted synergistically in ESCC. We showed that

283 patients with low LINC00278 expression and who were current smokers had worst OS in both

284 the discovery set (MST: 28 vs 40, log-rank P<0.0001, HR=2.818) and the validation set (MST:

285 25 vs 41, log-rank P<0.0001, HR=2.613) (Fig. S2C and S2D). Using Multivariable Cox

286 regression analysis, we also confirmed that smoking was associated with shorter OS in ESCC

287 patients (Fig. 1F-1G; Fig. S2C-S2D).

288 Biological characterization of LINC00278

289 LINC00278 locus is located on the short arm of Y chromosome. It spans from 3,002,887 to

290 3,200,509 and is comprised of four exons. LINC00278 transcript is 537bp long and northern blot

291 analysis has confirmed the expected size of LINC00278 transcript in total RNA from two pairs of

292 male ESCC samples (Fig. S2E). Both nuclear/cytoplasm fractionation experiment and confocal

293 microscopy analysis of fluorescent in situ hybridization (FISH) showed that LINC00278 is a

294 cytoplasmic RNA (Fig. S2F and S2G).

295 LINC00278 encodes a micropeptide

296 Because recent studies suggested that many lncRNAs could encode functional

297 micropeptides (less than 100 amino-acids), we determined whether LINC00278 encoded any

298 micropeptides. We found that LINC00278 could potentially encode four small ORFs (sORFs)

299 (Fig. 2A). We subsequently cloned each sORF with an in-frame FLAG epitope tag at the C

300 terminus and transfected it into male ESCC cell line TE-1. Western blot analysis indicated that

301 LINC00278-sORF1 generated a micropeptide (Fig. 2B). This sORF is located on Y chromosome

302 from 3,003,090 to 3,003,155, inside the first exon of LINC00278, encoding a 21-amino-acid

303 micropeptide (2.12kDa). This is consistent with the presence of a marked ribosome occupancy

304 peak in the first exon of LINC00278 in human lymphoblastoid cells(22), as well as ribosome

305 occupancy data from GWIPS-viz database(23) (Fig. 2C).

306 We next determined whether an in-frame ATG codon of LINC00278-sORF1 could promote

307 the initiation of translation. We fused GFPmut ORF (in which the initiation codon ATGGTG has

308 mutated to ATTGTT) and FLAG-tag to the C terminus of LINC00278-sORF1 to construct

309 expression plasmids and transfected these plasmids into ESCC cells. After 24h, we observed 15

310 substantial expression of LINC00278-sORF1-GFP fusion protein in the transfected cells (Fig.

311 S2H). Meanwhile, LINC00278-sORF1-FLAG was observed in transfected cells using anti-

312 FLAG western blot (Fig. S2I). Our data indicated that LINC00278-sORF1 could produce

313 micropeptide and the initiation codon of LINC00278-sORF1 could be utilized effectively to drive

314 the expression of the fusion protein.

315 LINC00278-sORF1 was endogenously expressed and downregulated in male

316 ESCC

317 To determine LINC00278-sORF1 expression, we generated a rabbit polyclonal antibody

318 (Anti-LINC00278-sORF1). In order to confirm the specificity of Anti-LINC00278-sORF1, we

319 respectively performed western blot to identify LINC00278-sORF1, LINC00278-sORF1-GFP

320 and LINC00278-sORF1-FLAG in ESCC cells (Fig. S2H and S2I). We also performed polysome

321 profiling in the cell lysate of Het-1A (a non-neoplastic squamous esophageal epithelial cell line),

322 TE-1, and KYSE-30. The mRNA-protein particles (mRNPs) were separated into three groups:

323 non-ribosome (mRNPs without any ribosome), 40S-80S (mRNPs associated with ribosome but

324 not being translated) and polysome (mRNPs being actively translated). The presence of

325 LINC00278 was quantitated in polysome fraction via qRT-PCR (Fig. S2J and S2K).

326 To further confirm the existent of endogenous LINC00278-sORF1 micropeptide, we

327 inserted a C-terminal FLAG-tag at the 3’ end of the ORF of LINC00278-sORF1 (FLAG-KI) and

328 detected endogenous LINC00278-sORF1 by western blot in Het-1A cell line, which expresses a

329 higher level of LINC00278 and LINC00278-sORF1 (Fig. 2D; Fig. S2L). In addition, we showed

330 that the LINC00278-sORF1 translation-blocking antisense oligo could block the expression of

331 LINC00278-sORF1 micropeptide (Fig. S2M).

332 Due to the limited number of tumor cells obtained from ESCC tissues, we determined

333 LINC00278-sORF1 translation and expression in male ESCC patient-derived xenograft (PDX)

334 models using polysome profiling and western blot. A total of 50 ESCC PDXs were generated for

335 the experiment. Tumor cells were harvested and lysed from ESCC PDXs. Our data indicated

336 that LINC00278-sORF1 was endogenously expressed and downregulated in male ESCC tissues

337 (Fig. 2E). In addition, the level of LINC00278 in polysome fraction was positively correlated

338 with the transcription level of LINC00278 in the tissues (Fig. 2E).

339 LINC00278-sORF1 knockout promoted ESCC tumor growth

340 We next generated LINC00278-sORF1-knockout cell lines and determined the effect of

341 LINC00278-sORF1-knockout on tumor growth using mouse ESCC xenograft models. We

342 showed that tumor growth from LINC00278-sORF1-knockout ESCC cells was significantly

343 higher than that from wild-type ESCC cells (Fig. 2F). To investigate whether the transcript of

344 LINC00278 is functional in ESCC tumor growth, we knocked down LINC000278 in wild-type

345 and LINC00278-sORF1-knockout ESCC cells. We showed that LINC00278-knockdown

346 promoted tumor growth in wild-type ESCC cells, but not in LINC00278-sORF1-knockout ESCC

347 cells (Fig. 2G and 2H).

348 Further, we showed that reintroducing either full-length-LINC00278 (full-LINC00278-

349 FLAG) or LINC00278-sORF1 (LINC00278-sORF1-FLAG) into the LINC00278-sORF1-

350 knockout could reverse tumor growth (Fig. 2I). In addition, we also showed that reintroducing

351 LINC00278-sORF1 (LINC00278-sORF1-FLAG) into the LINC00278-knockdown cells could

352 reverse tumor growth (Fig. S2N). Interestingly, overexpression of full-length-LINC00278

353 demonstrated stronger suppression of tumor growth than LINC00278-sORF1 in LINC00278-

354 sORF1-knockout ESCC cells (Fig. 2I). Further we detected higher LINC00278-sORF1

355 micropeptide expression by full-LINC00278-FLAG than by LINC00278-sORF1-FLAG (Fig.

356 2J). We also found that the RNA stability of full-length LINC00278 and LINC00278-sORF1

357 were not significantly different (Fig. S2O). Our results suggest that LINC00278-sORF1 had the

358 main effect on ESCC tumor growth while the untranslated region of LINC00278 augmented such

359 effect.

360 m6A modification of LINC00278 promoted LINC00278-sORF1 translation

361 Since m6A modification is the most prevalent post-transcriptional modification of mRNA

362 and lncRNA, and it regulates translation(15), we determined whether m6A modification of

363 LINC00278 regulated LINC00278-sORF1 translation. Using m6A-specific RNA

364 immunoprecipitation (MeRIP), we showed that LINC00278 contained m6A modification in both

365 Het-1A and ESCC cell lines (Fig. 3A).

366 Next, we identified three m6A modification sequence motifs in the untranslated region of

367 LINC00278 using a computation software called SRAMP(24) (Fig. S3A). To determine which

368 m6A sequence motif was modified and facilitating LINC00278-sORF1 translation, we generated

369 full-length LINC00278-FLAG and LINC00278-sORF1-FLAG wild-type construct as well as

370 constructs with each m6A sequence motif mutated (mut1-LINC00278-FLAG, mut2-LINC00278-

371 FLAG and mut3-LINC00278-FLAG), and transfected into Eca-109 and KYSE-150 cells (which

372 are female ESCC cell lines that do not have endogenous LINC00278 transcript and LINC00278-

373 sORF1 micropeptide, Table. S3). The results showed that only mut3-LINC00278-FLAG

374 significantly reduced LINC00278 m6A level compared to full-length LINC00278-FLAG

375 expression, which was approximately equal to LINC00278-sORF1-FLAG (Fig. 3B). This was

376 consistent with the lower LINC00278-sORF1 protein level produced by mut3-LINC00278-FLAG

377 expression (Fig. 3C). Furthermore, we expressed mut3-LINC00278 and LINC00278-sORF1 in

378 LINC00278-sORF1 ESCC cells and implanted them to generate xenograft models. The results

379 showed that mut3-LINC00278 and LINC00278-sORF1 have no significant difference in tumor

380 growth inhibition (Fig. 3D). To prove the GAACU motif mutated in mut3-LINC00278-FLAG

381 was endogenously m6A modified in LINC00278, we used a Morpholino antisense oligo that

382 specifically blocked m6A modification to this motif in Het-1A cell line. We showed that both the

383 levels of LINC00278 m6A modification and LINC00278-sORF1 were decreased (Fig. 3E and

384 3F). Our results indicated that only the GAACU motif was m6A modified in LINC00278.

385 Finally, we conducted a mediation analysis to determine whether m6A modification was the

386 mediator of the LINC00278 transcript level and LINC00278-sORF1 micropeptide level in PDXs.

387 First, we investigated the relationships between LINC00278 expression and LINC00278-sORF1

388 expression using linear regression. Second, we analyzed the relationship between each

389 LINC00278 expression and m6A modification by linear regression. Third, we examined the

390 relationship between m6A modification and LINC00278-sORF1 expression using linear

391 regression. Fourth, we included both the LINC00278 expression and m6A modification in the

392 model examining associations with LINC00278-sORF1 expression to evaluate mediation. As

393 shown in Fig. S3B, the total effect of the LINC00278 transcription (X) on the LINC00278-

394 sORF1 translation (Y) was statistically significant (Y=cX+e1: c=0.675; SE, 0.106; R=0.675;

395 P<0.001); and the m6A modification of LINC00278 (M) had a partial mediation effect on the

396 relationship between LINC00278 transcription and LINC00278-sORF1 translation (M=aX+e2:

397 a=0.796; SE, 0.0875; R=0.796; P<0.001; Y=c’X+bM+e3: c’=0.396, Pc’=0.024; b=0.351,

398 Pb=0.044). 19

399 Identification of regulators of m6A modification in LINC00278

400 We next investigated which proteins were involved in LINC00278 m6A modification. First,

401 we determined which known m6A “reader” proteins were bound to m6A modified LINC00278

402 using RNA pulldown and western blot(25). As shown in Fig. 3G, we showed that only YTHDF1

403 could be pulled down by m6A modified LINC00278. Using electrophoretic mobility shift assay

404 (EMSA) and RIP, we further confirmed that YTHDF1 interacted with m6A modified LINC00278

405 (Fig. 3H and 3I). Finally, we showed that YTHDF1 knockdown significantly downregulated

406 LINC00278-sORF1 translation without changing the LINC00278 m6A modification level (Fig.

407 S3C).

408 Next, we knocked down each known m6A “writer” and “eraser” protein and determined its

409 effect on LINC00278 expression, LINC00278 m6A modification level, and LINC00278-sORF1

410 translation. We showed that METTL3, METTL14, and WTAP knockdown significantly reduced

411 LINC00278 m6A modification level and LINC00278-sORF1 translation, while ALKBH5

412 knockdown significantly increased LINC00278 m6A modification level and LINC00278-sORF1

413 translation. FTO knockdown did not affect the LINC00278 m6A modification level and

414 LINC00278-sORF1 translation (Fig. S3C). None of these proteins affected the expression of

415 LINC00278 (Fig. S3C).

416 Our data suggest that METTL3, METTL14 and WTAP acted as “writers”, ALKBH5 acted

417 as “eraser”, and YTHDF1 acted as “reader” for LINC00278 m6A modification.

418 Cigarette smoking modulates LINC00278-sORF1 translation

419 Because cigarette smoking acted synergistically with low LINC00278 expression to confer

420 worse prognosis in ESCC patients, we investigated whether cigarette smoking affected 20

421 LINC00278-sORF1 translation. When we divided ESCC patients into smoking and non-smoking

422 groups, we did not detect significant difference in LINC00278 expression level between the two

423 groups (Fig. S3D). However, LINC00278 m6A modification and LINC00278-sORF1 translation

424 levels were significantly lower in the smoking group than in the non-smoking group (Fig. 3J and

425 3K).

426 To determine how cigarette smoking affected m6A modification, we exposed Het-1A cells

427 to cigarette smoke condensate (CSC) and measured the expression level of m6A regulators. CSC

428 treatment only increased the level of ALKBH5 protein (Fig. 3L). Meanwhile, CSC treatment

429 decreased the level of LINC00278-sORF1 micropeptide but did not affect LINC00278 expression

430 (Fig. 3L and 3M).

431 To investigate the mechanism of how CSC upregulated the expression of ALKBH5, we

432 analyzed ALKBH5 gene promoter CpG island methylation using the massArray DNA

433 methylation analysis. We found that ALKBH5 CpG island was hypomethylated in CSC treated

434 cells compared to DMSO mock-treated cells (Fig. 3N). Finally, we showed that ALKBH5

435 knockdown completely abolished the effect of CSC treatment on LINC00278 m6A modification

436 and LINC00278-sORF1 translation (Fig. 3O).

437 Taken together, our data suggest that LINC00278 downregulation promoted ESCC

438 progression. LINC00278 encodes a micropeptide, whose expression was modulated by

439 LINC00278 m6A modification. Finally, LINC00278 m6A modification was regulated by cigarette

440 smoking via ALKBH5 hypomethylation

441 LINC00278-sORF1 blocks the interaction between YY1 and AR

442 To determine the function of LINC00278-sORF1 in ESCC progression, we first

443 investigated the LINC00278-sORF1 interacting proteins by co-immunoprecipitation and mass

444 spectrometry analysis in TE-1 and KYSE-30 cells. We sought for the proteins that could be

445 immunoprecipitated by LINC00278-sORF1-FLAG fusion protein but not by IgG in both TE-1

446 and KYSE-30 cells. As shown in Fig. 4A, we identified YY1 as the potential LINC00278-

447 sORF1 binding protein. We validated that YY1 could be immunoprecipitated by LINC00278-

448 sORF1-FLAG fusion protein (Fig. 4B). Furthermore, we co-transfected YY1-HA and

449 LINC00278-sORF1-FLAG into TE-1 cells and performed co-immunoprecipitation using anti-

450 HA. Western blot showed that YY1-HA and LINC00278-sORF1-FLAG proteins were

451 immunoprecipitated (Fig. 4B). Finally, we showed that endogenous LINC00278-sORF1 in TE-1

452 cells could be immunoprecipitated using anti-YY1 antibody (Fig. 4B). We concluded that

453 LINC00278-sORF1 bound to YY1 and we named the micropeptide YY1BM.

454 YY1 is a ubiquitous and multifunction transcriptional factor that plays a regulatory role in

455 tumorigenesis, including ESCC(26,27). YY1 truncation experiment indicated that YY1BM

456 bound to YY1 C-terminal domain (331-414 amino-acid), where has been documented that bound

457 to AR(28) (Fig. 4C). Given that YY1 is a transcriptional coactivator of AR in prostate

458 cancer(28), we tested whether it was also true in ESCC (Fig. 4D). Finally, we showed that the

459 interaction between YY1 and AR was downregulated by LINC00278 overexpression and

460 upregulated by YY1BM knockout in both TE-1 and KYSE-30 cell lines (Fig. 4E). Our data

461 suggested that YY1BM blocking the interaction between YY1 and AR.

462 YY1 promotes AR-regulated eEF2K transcription

463 We first examined whether YY1 and AR occupied the same genomic locations by re-

464 analyzing publically available ChIP-seq data on YY1(29) and AR(30). We found that YY1

465 bound to 2865 gene promoters, while AR bound to 312 gene promoters respectively. Taken

466 together, 33 genes were common in genes regulated by YY1 and AR (Fig. 4F). Finally, we

467 found that 10 out of these 33 genes have been reported repeatedly (>10) to be associated with

468 cancer (Fig. 4G).

469 Next, we determined whether the expression of any of these genes was affected by

470 LINC00278 or YY1BM by western blot analysis. We found that expression of eEF2K was

471 decreased in LINC00278 upregulated cells, but increased in YY1BM knockout cells (Fig. 4H).

472 These expression changes were abolished by YY1 siRNAs treatment (Fig. S4A), indicated that

473 eEF2K expression was regulated by YY1BM via modulated the interaction between YY1 and

474 AR.

475 To identify YY1 and AR binding sites in the eEF2K promoter, we first analyzed the ChIP-

476 seq data and identified overlapping YY1 and AR peaks surrounding the eEF2K TSS (Fig. 4I).

477 Next, we analyzed the binding sites of YY1 and AR in the promoter of eEF2K using PROMO

478 and JASPAR. The results suggested that AR and YY1 potentially co-binding to the -200 to 100

479 region of eEF2K promoter. We subsequently carried out ChIP experiments to fine map YY1 and

480 AR binding sites in the eEF2K promoter and confirmed that YY1 and AR bound to the -200 to

481 100 region of eEF2K promoter (Fig. 4J).

482 Furthermore, we generated an eEF2K promoter luciferase reporter plasmid (pGL3-eEF2K)

483 and an eEF2K mutant promoter luciferase reporter plasmid that deleted the -200 to 100 region

484 (pGL3-eEF2K-mut) to identify eEF2K transcription regulators. The pGL3-eEF2K luciferase 23

485 activity was decreased in cells overexpression of full-length-LINC00278 and increased in cells

486 with YY1BM knockout, whereas pGL3-eEF2K-mut abolished these difference (Fig. 4K).

487 Furthermore, the pGL3-eEF2K luciferase activity was not significantly different when cells were

488 treated with YY1 siRNAs, confirming the involvement of YY1 in AR-regulated eEF2K

489 expression (Fig. S4B).

490 Finally, we determined the levels of testosterone and eEF2K expression in ESCC patients

491 by Electrochemiluminescence immunoassay (ECLI) and qRT-PCR. We found that the

492 testosterone level was positively correlated with eEF2K expression level in males, but not in

493 females (Fig. S4C). To further confirm the involvement of AR signaling pathway, we showed

494 that YY1BM overexpression did not affect the tumor growth in female mice (Fig. S4D),

495 indicating that YY1BM is indeed involved in male ESCC progression via AR signaling pathway.

496 YY1BM decreases survival of ESCC cells under nutrient deprivation through

497 eEF2K signaling pathway

498 Because eEF2K confers cell survival under acute severe nutrient deprivation (ND) by

499 inhibiting eEF2 activity and translation elongation(31), we determined whether YY1BM

500 regulated ESCC cell survival under ND. Compared to wild type cells, YY1BM knockout cells

501 showed increased survival under ND (Fig. S4E). This increased survival was abolished by A-

502 484954 treatment, a known small molecule eEF2K inhibitor that could decrease the phospho-

503 eEF2 level in ESCC cells (Fig. S4E).

504 We also analyzed apoptosis of ESCC cells under ND, using flow cytometry for Annexin V

505 staining (Fig. S4F and S4G) and western blot analysis of caspase-3 cleavage (Fig. S4H and S4I).

506 We showed that YY1BM knockout reduced apoptosis in ESCC cells under ND, which was

507 abolished by A-484954 treatment. Whereas overexpression of full-length LINC00278 induced

508 apoptosis under ND, which was also abolished by A-484954 treatment. Meanwhile, the

509 expression of eEF2K was upregulated by YY1BM knockout and downregulated by LINC00278

510 overexpression under ND, which was also abolished by treatment with A-484954 (Fig. S4H and

511 S4I). Finally, we found that eEF2 phosphorylation was increased in YY1BM knockout cells and

512 decreased in LINC00278 overexpressed cells under ND, which was also abolished by A-484954

513 treatment (Fig. S4H and S4I).

514 Interestingly, we found that A-484954 treatment under ND reduced the speed of YY1BM

515 translation decrease (Fig. S4J), suggesting the presence of a positive-feedback loop between the

516 eEF2K/eEF2 axis and YY1BM.

517 These data suggested that YY1BM inactivated the AR-regulated transcription of eEF2K

518 under ND, thereby enhancing translation elongation and resulting in ESCC cell apoptosis.

519 Low YY1BM expression is associated with reduced apoptosis in ESCC

520 xenografts and tissues

521 We then sought to explore the relationship between YY1BM expression and ESCC

522 apoptosis in xenograft model. YY1BM knockout xenografts showed higher expression of eEF2K

523 and lower expression of cleaved caspase-3 by IHC analysis (Fig. 5A and 5B), consistent with

524 reduced apoptosis by TUNEL staining (Fig. 5C). Moreover, we also found that expression of

525 eEF2K and cleaved caspase-3 were not changed in LINC00278 overexpression xenografts when

526 we implanted female ESCC cells into female mice (Fig. 5D).

527 When we correlated eEF2K and caspase-3 expression (≥30%, strong staining; <30%, weak

528 staining) with YY1BM expression in 50 ESCC tissues, we found that expression of YY1BM was

529 inversely correlated with eEF2K expression, but positively correlated with cleaved caspase-3

530 (Fig. 5E and 5F).

531 YY1BM is a potential anti-cancer micropeptide

532 Since several anticancer peptides have been reported, we investigated whether YY1BM is a

533 novel anticancer micropeptide. We first tested the cytotoxicity of YY1BM in ESCC cells. As

534 shown in Fig. 6A and 6B, we found that YY1BM was cytotoxic to TE-1 and KYSE-30 cells,

535 while scrambled YY1BM (svYY1BM) control micropeptide was not.

536 To probe the anticancer effect of YY1BM in vivo, we injected YY1BM intratumorally into

537 ESCC tumors grafted in nude mice and analyzed the survival time. We found YY1BM injection

538 significantly improved the survival rate of male mice, but not female mice (Fig. 6C and 6D).

539 Furthermore, IHC analysis revealed a higher apoptosis rate and lower eEF2K expression in male

540 mice, but not female mice (Fig. 6Cand 6D), suggesting that YY1BM intratumoral injection

541 downregulated the expression of eEF2K and induced apoptosis, ultimately improved male mice

542 survival.

543

544 DISCUSSION

545 Globally, ESCC is a male dominant malignancy. Both sex hormone and lifestyle factors,

546 such as cigarette smoking, contribute to this gender disparity. In this study, we discovered a 21-

547 amino-acid micropeptide (YY1BM) encoded by Y-linked lncRNA LINC00278. The translation

548 of YY1BM was modulated by cigarette-smoking-mediated LINC00278 m6A modification.

549 YY1BM blocked YY1 binding to AR to activate the expression of eEF2K, which is a key

550 regulator for male ESCC progression.

551 Through mining a large cohort of ESCC lncRNA profiling data, we discovered that

552 LINC00278 might play a critical role in male-specific ESCC progression. LINC00278 is a 537bp

553 transcript, located on Y chromosome and previously annotated as a non-coding RNA. It has been

554 reported that lncRNAs are involved in ESCC progression, such as Linc-POU3F3 promotes

555 methylation of POU3F3 by interacting with EZH2 in ESCC(17). Interestingly, our data indicated

556 that YY1BM, instead of the LINC00278 transcript, plays a major role in ESCC progression. We

557 also found that the m6A modification motif of LINC00278 is close to the stop codon of YY1BM,

558 consistent with its role in the regulation of YY1BM translation(15). m6A is the major reversible

559 post-transcription modification in RNAs(32,33), involved in RNA stability(34) and protein

560 production(15). m6A modification changes have been linked to various disease processes,

561 including tumorigenesis. It has been shown that the physiological functions of m6A modification

562 mainly depend on the “reader” proteins that bind to the m6A modification motif. YTHDF1, a

563 member of YTH family which has been reported to facilitate protein synthesis by interacting

564 with translation machinery(15), is the “reader” for m6A modification of LINC00278. Based on

565 previously reported studies, we speculate that YTHDF1 binds to m6A modified LINC00278 to

566 recruit the translation machinery, therefore promote the translation efficiency of YY1BM. This is

567 consistent with our mediation analysis showing that m6A modified LINC00278 has an

568 incomplete mediating effect on the relation between LINC00278 and YY1BM expression levels

569 in ESCC tissues. Our data showed that CSC treatment leads to ALKBH5 promoter

570 hypomethylation and increased expression of ALKBH5. ALKBH5 is an m6A demethylase that

571 acts as the “eraser” protein of m6A modified LINC00278, which leads to a decreased level of 27

572 m6A modified LINC00278, and in turn reduced YY1BM expression through YTHDF1. In China,

573 current smokers are significantly more prevalent in the male population than in the female

574 population. Cigarette smoking is a key factor in ESCC carcinogenesis. In this study, we found

575 that cigarette smoking contributes to poor prognosis in ESCC in part by regulating LINC00278

576 translation through m6A modification. In summary, we conclude that m6A modification of

577 LINC00278 modulates YY1BM translation and results partially in the sex bias of ESCC.

578 Recently, several lncRNA encoded micropeptides have been identified and reported to play

579 crucial roles in a variety of physiological processes. MLN, a micropeptide encoded by a skeletal

580 muscle-specific lncRNA, has been shown to interact directly with SERCA and impede Ca2+

581 uptake into the sarcoplasmic reticulum (SR), thereby regulating muscle performance(11).

582 Expression of another lncRNA encoded micropeptide Myomixer, together with Myomaker,

583 controls the critical step in myofiber formation during muscle development(10). Furthermore,

584 lncRNA HOXB-AS3 encodes a conserved 53-amino acid micropeptide, which suppresses colon

585 cancer growth by regulating the pyruvate kinase M (PKM) splicing and suppressing glucose

586 metabolism reprogramming(12). In our study, YY1BM is identified as a novel micropeptide

587 encoded by Y-linked LINC00278. It interacts with YY1 and blocks its interaction with AR. YY1

588 is a zinc finger protein belonging to the GLI-Kruppel family that can activate or inactivate gene

589 expression depending on interacting partners, promoter context and chromatin structure(35).

590 YY1 is known to be overexpressed in various cancers, including ESCC(27). Moreover, YY1 acts

591 as a coactivator of several transcription factors that play important roles in carcinogenesis, such

592 as P53, GATA-4 and AR(28,36). Sex hormone, especially androgen, has been documented to be

593 associated with ESCC progression(37). AR promotes ESCC cell invasion and proliferation via

594 matrix metalloproteinase 2(38). AR and IL6 form a reciprocal regulatory circuit to sustain 28

595 STAT3 oncogenic signaling in ESCC(39). High-level AR expression in ESCC predicts poor

596 clinical outcome in tobacco-using ESCC patients(39), suggesting another mechanism of how

597 smoking contributes to poor prognosis in ESCC patients. Our data are consistent with these

598 findings by showing that YY1BM modulates the transcription activity of YY1 and AR, which

599 directly co-regulate the expression of eEF2K.

600 eEF2K is a conserved mediator of the cellular response to ND(31). Activated eEF2K

601 phosphorylates and inactivates eEF2, thereby block the translation elongation of mRNAs(40).

602 eEF2K reduces cancer cell apoptosis and promotes cancer cell survival under ND(31). In the

603 present study, we demonstrated that YY1BM can regulate the eEF2K/eEF2 axis via inhibiting

604 the transcriptional activity of YY1 and AR. Furthermore, inactivation of eEF2 could in turn

605 block the translation of YY1BM, leading to the formation of LINC00278-YY1BM-YY1-AR-

606 eEF2K-eEF2 cycle, which is probably one of the underlying molecular mechanisms for

607 micropeptide encoded by lncRNA to induce tumorigenesis and progression of male ESCC.

608 Since YY1BM has such a critical role in male ESCC, we determined whether YY1BM is a

609 potential anticancer micropeptide. Similar to PNC-27 targeting HDM-2 in the membrane to kill

610 cancer cells(41), we showed that YY1BM is a potent anticancer micropeptide in ESCC.

611 In summary, we found that the Y-linked lncRNA LINC00278 encodes a micropeptide

612 termed YY1BM. YY1BM suppresses the transcription of eEF2K by blocks the interaction

613 between YY1 and AR, thereby promoting the activity of eEF2 and resulting in apoptosis of

614 ESCC. LINC00278 has a classical m6A modification motif close to the stop codon of YY1BM,

615 which interacts with YTHDF1 and facilitates the translation of YY1BM. Cigarette smoking

616 increases ALKBH5 expression and reduces m6A modification of LINC00278, thereby inhibits the

617 translation of YY1BM and induces the ESCC progression. Interestingly, exogenous YY1BM has 29

618 anticancer potential. In conclusion, our study reveals that LINC00278 and its product YY1BM

619 are at the intersection of hormones, lifestyle factors and genetics in male ESCC progression,

620 highlighting the fact that LINC00278 and YY1BM could serve as potential prognostic

621 biomarkers and therapeutic targets for male ESCC.

622

623 ACKNOWLEDGMENTS

624 This work was supported by the National Scientific Foundation of China grants 81772544 and

625 81972649; Science Foundation for Distinguished Young Scholars in Jiangsu (BK20160008); A

626 Project Funded by the Priority Academic Program Development of Jiangsu Higher Education

627 Institutions; National Key R&D Program of China (2016YFC1302100); the Program for

628 Guangdong Introducing Innovative and Entrepreneurial Teams (2017ZT07S096).

629

630

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731 patient prognosis. J Pathol 2017;241:448-62

732 40. Ryazanov AG, Shestakova EA, Natapov PG. Phosphorylation of elongation factor

733 2 by EF-2 kinase affects rate of translation. Nature 1988;334:170-3

734 41. Sarafraz-Yazdi E, Bowne WB, Adler V, Sookraj KA, Wu V, Shteyler V, et al.

735 Anticancer peptide PNC-27 adopts an HDM-2-binding conformation and kills cancer cells

736 by binding to HDM-2 in their membranes. Proc Natl Acad Sci U S A 2010;107:1918-23

737 FIGURE LEGENDS

738 Fig. 1. Y-linked male-specific lncRNA LINC00278 was downregulated in male ESCC

739 samples and associated with the overall survival of male ESCC patients.

740 (A) The Venn diagram depicts the number of lncRNAs that are differentially expressed in male

741 versus female ESCC groups. (B) The distribution of male differentially expressed lncRNAs on

742 each chromosome. (C and D) Y-linked lncRNAs that were differentially expressed in male

743 ESCC samples. (E) The expression of 3 candidate lncRNAs in male ESCC and matched non-

744 tumor esophagus specimens from 281 patients in the Suzhou cohort; Expression of LINC00278

745 was also determined from 288 patients in the Guangzhou cohort. (mean±SD). (F) Left: Kaplan-

746 Meier overall survival curves for male ESCC patients with high or low LINC00278 expression in

747 Suzhou cohort (281, discovery set). Right: Forest plot derived from multivariable Cox regression

748 analyses that adjusted for age and tumor stage. (G) Left: Kaplan-Meier overall survival curves

749 for male ESCC patients with differential LINC00278 expression and smoking history in

750 Guangzhou cohort (288, validation set). Right: Forest plot derived from multivariable Cox

751 regression analyses that adjusted for age and tumor stage. *, p<0.05; **, p<0.01; ***, p<0.001.

752

753 Fig. 2. Micropeptide LINC00278-sORF1 instead of the LINC00278 transcript inhibited the

754 progression of male ESCC.

755 (A) Schematic drawing to show the genomic position of the exons and the predicted sORFs of

756 LINC00278. (B) The sORFs were constructed to pcDNA3.1 vector and transfected to TE-1 cells

757 for 24h. The sORFs-FLAG fusion proteins were determined by western blotting with anti-FLAG

758 antibody. (C) Ribosome occupancy map at the LINC00278 locus. The blue and red tracks

759 indicate reads density that mapped to the region, the green track indicate the predicted sORFs of

760 LINC00278. (D) Upper: diagram of the LINC00278-sORF1 location at the LINC00278 locus and

761 the FLAG tag that inserted to the 3’ end of LINC00278-sORF1. Lower: LINC00278-sORF1-

762 FLAG fusion protein levels were determined by immunofluorescence and western blotting in

763 FLAG-KI Het-1A cells. (E) Upper: correlation analysis of LINC00278 transcription and

764 LINC00278-sORF1 translation. LINC00278 transcription and LINC00278-sORF1 translation

765 levels were determined in fifty male ESCC PDXs using qPCR and polysome profiling-qPCR,

766 respectively. Lower: LINC00278-sORF1 expression in four pairs of male ESCC tissues was 37

767 determined by western blot using anti-LINC00278-sORF1. C, ESCC tissue; N, matched non-

768 tumor esophagus specimen. (F) LINC00278-sORF1 KO in WT ESCC cells increased tumor

769 growth in xenograft mice (mean±SD, n=5). (G) LINC00278 knockdown in WT ESCC cells

770 increased tumor growth in xenograft mice (mean±SD, n=5). (H) LINC00278 knockdown in

771 LINC00278-sORF1 KO ESCC cells cannot increase tumor growth in xenograft mice (mean±SD,

772 n=5). (I) Overexpression of LINC00278-sORF1 (LINC00278-sORF1 OE) or full-length

773 LINC00278 (LINC00278 OE) in LINC00278-sORF1 KO ESCC cells suppressed tumor growth

774 in xenograft mice (mean±SD, n=5). (J) The LINC00278-sORF1-FLAG fusion protein level in

775 xenograft tumors of WT, LINC00278-sORF1 OE and LINC00278 OE ESCC cells was

776 determined by western blot analysis with anti-FLAG antibody. *, p<0.05; **, p<0.01; ***,

777 p<0.001.

778

779 Fig. 3. N6-methyladenosine modification of LINC00278 promoted YY1BM translation

780 which was reduced by cigarette smoking.

781 (A) [m6A]LINC00278 or [m6A]MALAT1 was detected by immunoprecipitation with antibody

782 against m6A followed by RT-qPCR analysis in individual cells (mean±SD, n=3). MALAT1 was

783 used as a positive control. (B) m6A level of LINC00278 in female ESCC cells that transfected

784 with indicated plasmids (mean±SD, n=3). (C) LINC00278-sORF1-FLAG fusion protein levels in

785 female ESCC cells that were transfected with indicated plasmids. (D) Tumor growth in xenograft

786 mice subcutaneously implanted ESCC cells that were transfected with indicated constructs

787 (mean±SD, n=5). (E) m6A level of LINC00278 in Het-1A cells that transfected with Morpholino

788 antisense oligo that specifically blocking the m6A motif of LINC00278. (F) LINC00278-sORF1

789 micropeptide level in Het-1A cells that transfected with Morpholino antisense oligo that 38

790 specifically blocking the m6A motif of LINC00278. (G) The interaction between

791 [m6A]LINC00278 and YTHDF1 was detected by RNA pulldown assays. (H) The interaction

792 between [m6A]LINC00278 and YTHDF1 was detected by EMSA assays. (I) RIP assays

793 indicated that YTHDF1 interacts with LINC00278. PNPLA2 was used as a positive control. (J)

794 Significant [m6A]LINC00278 level difference in male ESCC tissues from smokers (n=34) and

795 nonsmokers (n=16) (mean±SD). (K) Significant LINC00278-sORF1 level difference in male

796 ESCC tissues from smokers (n=34) and nonsmokers (n=16) (mean±SD). (L) METTL3,

797 METTL14, WTAP, ALKBH5, YTHDF1 and LINC00278-sORF1 levels in Het-1A cells were

798 detected by western blotting after treated with cigarette smoke condensate (CSC, 100µg/ml) or

799 DMSO as solvent control. (M) The relative levels of LINC00278, [m6A]LINC00278 and

800 LINC00278-sORF1 in Het-1A cells that treated with CSC (100µg/ml) or DMSO as solvent

801 control for 48h (mean±SD, n=3). (N) Amplicon size and place of CpG sites in the amplicon.

802 Methylation profile of CpG sites for the ALKBH5 gene. The color of the circles is related to the

803 percentage of methylation in each CpG site. Boxes indicate the different methylation patterns

804 between CSC (100 µg/ml, 48h) or DMSO treated Het-1A cells. (O) ALKBH5-knockdown

805 abolished effect of CSC (100 µg/ml, 48h) treatment on levels [m6A]LINC00278 and LINC00278-

806 sORF1 in Het-1A cells (mean±SD, n=3). *, p<0.05; **, p<0.01; ***, p<0.001.

807

808 Fig. 4. YY1BM inhibited the interaction between YY1 and AR, repressed the

809 transcriptional co-activation effect of AR and decreased the expression of eEF2K.

810 (A) LINC00278-sORF1-FLAG plasmid was transfected into TE-1 and KYSE-30 cells, and the

811 LINC00278-sORF1-FLAG complexes were co-immunoprecipitated by anti-FLAG antibody. The

812 Venn diagram of mass spectrometric analysis results for the co-immunoprecipitation 39

813 experiments. (B) Upper: LINC00278-sORF1-FLAG plasmid was transfected into TE-1 and

814 KYSE-30 cells, and the LINC00278-sORF1-FLAG complexes were co-immunoprecipitated by

815 anti-FLAG antibody, and LINC00278-sORF1-FLAG was detected. Middle: LINC00278-sORF1-

816 FLAG and YY1-HA plasmids were transfected into TE-1 cells, and the YY1-HA complexes

817 were co-immunoprecipitated by anti-HA antibody, and LINC00278-sORF1-FLAG was detected.

818 Lower: LINC00278-sORF1 was detected in the complex that was co-immunoprecipitated by

819 anti-YY1 antibody. (C) Co-immunoprecipitation assays revealed the interaction of YY1BM with

820 YY1 via the C-terminal region of YY1. (D) Immunoprecipitation was performed with anti-YY1

821 or anti-AR antibodies. Immunoprecipitated AR or YY1 were then revealed by blotting with anti-

822 AR or anti-YY1 antibodies. (E) Immunoprecipitation was performed with anti-YY1 antibody in

823 LINC00278 overexpressed (OE), YY1BM KO and respective control ESCC cells.

824 Immunoprecipitated AR was then revealed by blotting with anti-AR antibody. (F) The Venn

825 diagram depicts the number of genes regulated by YY1 and AR derived from ChIP-seq data

826 analysis. (G) The histogram indicates the number of searching results of these genes that

827 associated with cancer in PubMed. (H) Western blotting was performed to verify the expression

828 of APC, CRKL, GOLPH3, eEF2K, KDM4C, BCAR3, CYCS, PON2, LARP1 and PKD2 in

829 LINC00278 overexpressed (OE), YY1BM KO and respective control ESCC cells. (I) An

830 overview of ChIP-seq data are illustrated by Integrative Genomics Viewer (IGV) software of the

831 promoter and the first exon of the eEF2K. (J) Chromatin immunoprecipitation (ChIP) assays

832 showed enrichment of YY1 and AR at eEF2K in LINC00278 OE, YY1BM KO and respective

833 control ESCC cells. Co-precipitated DNA was analyzed by qPCR using amplicons C1–C4

834 (mean±SD, n=3). (K) Luciferase reporter assay for eEF2K promoter in LINC00278 OE, YY1BM

835 KO and respective control ESCC cells. The reporter constructs expressing the luciferase gene 40

836 under full-length eEF2K gene promoter or eEF2K promoter deleted -200 to 0 region (mean±SD,

837 n=4). *, p<0.05; **, p<0.01; ***, p<0.001.

838

839 Fig. 5. YY1BM induced apoptosis of ESCC cells.

840 (A) Cleaved caspase-3 and eEF2K immunostaining in xenograft tumors of LINC00278 OE,

841 YY1BM KO and respective control TE-1 cells. (B) Cleaved caspase-3 and eEF2K

842 immunostaining in xenograft tumors of LINC00278 OE, YY1BM KO and respective control

843 KYSE-30 cells. (C) TUNEL staining in xenograft tumors of LINC00278 OE, YY1BM KO and

844 respective control ESCC cells. (D) Cleaved caspase-3 and eEF2K immunostaining in xenograft

845 tumors of female LINC00278 OE and control ESCC cells that implanted into female mice. (E)

846 Left: eEF2K immunostaining in male ESCC samples. Right: the relative level of YY1BM in

847 eEF2K strong and weak samples. (F) Left: cleaved caspase-3 immunostaining in male ESCC

848 samples. Right: the relative level of YY1BM in cleaved caspase-3 strong and weak samples. *,

849 p<0.05; **, p<0.01; ***, p<0.001.

850

851 Fig. 6. YY1BM acts as a potential anti-cancer micropeptide in vivo.

852 (A) Cell proliferation analysis of TE-1 cells treated with different concentrations of YY1BM or

853 svYY1BM under acute ND at different time-points (mean±SD, n=5). (B) Cell proliferation

854 analysis of KYSE-30 cells treated with different concentrations of YY1BM or svYY1BM under

855 acute ND at different time-points (mean±SD, n=5). (C) Upper: survival data for xenograft mice

856 that were subcutaneously implanted with TE-1 or KYSE-30 cells and direct intratumorally

857 injected with 400μg/ml YY1BM or svYY1BM. Lower: cleaved caspase-3 and eEF2K

858 immunostaining in xenograft tumors that were direct intratumorally injected with 400μg/ml 41

859 YY1BM or svYY1BM. (D) Upper: survival data for xenograft female mice that were

860 subcutaneously implanted with female ESCC cells and direct intratumorally injected with

861 400μg/ml YY1BM or svYY1BM. Lower: cleaved caspase-3 and eEF2K immunostaining in

862 xenograft tumors that were direct intratumorally injected with 400μg/ml YY1BM or svYY1BM.

863 *, p<0.05; **, p<0.01; ***, p<0.001.

Chr.Y

LINC00278 sORF

A A METTL3 A METTL14 ALKBH5 eEF2K eEF2K AR YY1 WTAP

m6 LINC00278 sORF A Smoking A AA eEF2

YTHDF1 YY1BM Translation Caspase-3

m 6 sORF A

A AA

Cell death

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer The micropeptide YY1BM functions as a tumor suppressorResearch. in male ESCC cells. Author Manuscript Published OnlineFirst on March 13, 2020; DOI: 10.1158/0008-5472.CAN-19-3440 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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 Published OnlineFirst March 13, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-3440

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2020/03/13/0008-5472.CAN-19-3440.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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