Author Manuscript Published OnlineFirst on August 6, 2021; DOI: 10.1158/0008-5472.CAN-21-0494 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 N6-methyladenosine-mediated upregulation of WTAPP1 promotes WTAP translation and Wnt 2 signaling to facilitate pancreatic cancer progression 3 4 Junge Deng1,#, Jialiang Zhang1,#, Ying Ye1,#, Kaijing Liu1, Lingxing Zeng1, Jingyi Huang1, Ling Pan1, Mei 5 Li2, Ruihong Bai1, Lisha Zhuang1, Xudong Huang1, Guandi Wu1, Lusheng Wei3, Yanfen Zheng1, Jiachun 6 Su1, Shaoping Zhang1, Rufu Chen4, Dongxin Lin1,5,6,*, Jian Zheng1,6,* 7 8 1Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China and 9 Collaborative Innovation Center for Cancer Medicine, Guangzhou, China 10 2Department of Pathology, Sun Yat-sen University Cancer Center, Guangzhou, China 11 3Department of Pancreaticobiliary Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 12 Guangzhou, China 13 4Guangdong Provincial People’s Hospital & Guangdong Academy of Medical Sciences, Guangzhou, 14 China 15 5Department of Etiology and Carcinogenesis, National Cancer Center/National Clinical Research 16 Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 17 Beijing, China 18 6Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing Medical University, 19 Nanjing, China 20 21 # These authors contributed equally to this work. 22 23 *Correspondence should be addressed to Prof. Jian Zheng ([email protected]) or Prof. 24 Dongxin Lin ([email protected]) 25 26 Running title: m6A mediated excessive WTAPP1 RNA promotes PDAC progression 27 28 Key words: Pancreatic cancer; Pseudogene; Wnt pathway. 29 30 Disclosures: The authors have no conflicts to report 31 32 Abbreviations 33 AMT, 4’-aminomethyltrioxalen; ChIRP, chromatin isolation by RNA purification; CIMS, cross-linking 34 induced mutation sites; CITS, crosslinking-induced truncation sites; CNBP, CCHC-type zinc finger 35 nucleic acid binding protein; EMSA, electrophoretic mobility shift assay; FISH, fluorescent in situ 36 hybridization; lncRNA, long non-coding RNA; m6A, N6-methyladenosine; MeRIP, m6A-specific RNA 37 immunoprecipitation; miCLIP, m6A individual-nucleotide resolution cross-linking and 38 immunoprecipitation; PDAC, pancreatic ductal adenocarcinoma; PDX, patient-derived xenograft; RIP, 39 RNA immunoprecipitation; sgRNA, small guide RNA; siRNA, small interfering RNA; WTAP, WT1 40 associated protein. 41 1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 6, 2021; DOI: 10.1158/0008-5472.CAN-21-0494 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 42 Abstract 43 Pseudogenes may play important roles in cancer. Here, we explore the mechanism and function of a 44 pseudogene WTAPP1 in the progress of pancreatic ductal adenocarcinoma (PDAC). WTAPP1 RNA 45 was significantly elevated in PDAC and was associated with poor prognosis in patients. 46 Overexpression of WTAPP1 RNA promoted PDAC proliferation and invasiveness in vitro and in vivo. 47 Mechanistically, N6-methyladenosine (m6A) modification stabilized WTAPP1 RNA via CCHC-type zinc 48 finger nucleic acid binding protein (CNBP), resulting in increased levels of WTAPP1 RNA in PDAC cells. 49 Excessive WTAPP1 RNA bound its protein-coding counterpart WT1 associated protein (WTAP) mRNA 50 and recruited more EIF3 translation initiation complex to promote WTAP translation. Increased 51 WTAP protein enhanced the activation of Wnt signaling and provoked the malignant phenotypes of 52 PDAC. Decreasing WTAPP1 RNA significantly suppressed the in vivo growth and metastasis of PDAC 53 cell lines and patient-derived xenografts. These results indicate that m6A-mediated increases in 54 WTAPP1 expression promotes PDAC progression and thus may serve as a therapeutic target. 55 Statement of Significance 56 This study reveals how aberrant m6A modification of the WTAPP1 pseudogene results in increased 57 translation of its protein-coding counterpart to promote Wnt signaling, which contributes to 58 pancreatic cancer progression. 59 2 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 6, 2021; DOI: 10.1158/0008-5472.CAN-21-0494 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 60 Introduction 61 Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies with only 62 approximately 10% of 5-year survival (1,2). Most PDAC patients eventually die of cancer progression 63 due to lack of effective treatment modalities (2,3), which poses a great medical challenge and 64 requires urgent development of new target therapies. To achieve this, it is important to have better 65 understanding of molecular mechanisms underlying the pathogenesis of PDAC. 66 Recent studies have shown that pseudogene produced RNAs play an important regulatory role 67 in many types of human cancer (4-6). RNAs produced by pseudogene are considered as a subclass 68 of long noncoding RNAs (lncRNAs) that develop from protein-coding genes but harbor multiple 69 mutations which abrogate their translation into functional proteins (7). However, accumulating 70 evidence has demonstrated that RNAs produced by pseudogene may function in regulating their 71 protein-coding counterparts at either transcriptional or post-transcriptional level (5-8). While 72 attempts have been made to explore the mechanism of anomalously expressed pseudogene RNAs 73 in several types of cancer (5,6,8), little has been known about their roles and functions in PDAC. 74 It has been well known that RNAs are substantially modified during their maturation and 75 aberrant modifications might cause their functional alterations and thus implicate in the 76 development of diseases such as cancer (9-13). N6-methyladenosine is the most abundant 77 modification of RNAs that regulates RNAs at different levels such as RNA stability (9,14-16). Recent 78 studies have shown that some long non-coding RNAs are regulated by m6A modifications and are 79 involved in tumor progression (9,17-21). However, there are few reports on the modifications of 80 RNAs produced by pseudogene and their resultant roles in PDAC. 81 In this study, we have demonstrated a m6A modified RNA produced by the pseudogene 82 WTAPP1 as an oncogenic regulator in pancreatic cancer. We have found that CCHC-type Zinc finger 83 nucleic acid binding protein (CNBP) can recognize m6A modified WTAPP1 RNA and increase its 3 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 6, 2021; DOI: 10.1158/0008-5472.CAN-21-0494 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 84 stability. Excessive WTAPP1 RNA enhances WTAP translation through recruiting EIF3B to WTAP RNA. 85 Increased WTAP level may evoke oncogenic Wnt signaling and promote PDAC progression. 86 Furthermore, we have treated mouse models with WTAPP1 inhibitor and the results suggest that 87 WTAPP1 may be a potential therapeutic target for PDAC. 88 89 Materials and Methods 90 Tissue specimens 91 Surgically removed PDAC samples and their corresponding adjacent normal tissues were 92 collected from patients at Sun Yat-sen University Sun Yat-sen Memorial Hospital, Guangzhou, China 93 (N = 158) and Chinese Academy of Medical Sciences Cancer Hospital, Beijing, China (N = 73) 94 between 2010 and 2016 (Supplementary Table S1). PDAC were confirmed histopathologically by 95 three independent pathologists. None of patients included in this study received any preoperative 96 radiotherapy or chemotherapy. The patient survival time was the interval between the date of 97 diagnosis and the date of last contact (death or last follow-up). Death information was obtained 98 from inpatient and outpatient records or follow-up telephone calls. Written informed consent was 99 obtained from each patient, and this study was approved by the Institutional Review Board of the 100 Sun Yat-sen Memorial Hospital and Chinese Academy of Medical Sciences Cancer Hospital. 101 Pseudogene detection and differential expression analysis 102 We performed RNA-sequencing using 65 PDAC tumor and 33 normal tissue samples. Adaptors 103 and low-quality bases of reads were trimmed by Cutadapt (v1.16) (22), and reads shorter than 20 104 nucleotides were discarded. RSEM (23) were used to quantify gene expression. To perform a 105 comprehensive survey of pseudogenes, we obtained the genomic information of human 106 pseudogenes through GTF file from the GENCODE database (version 25) (24) and quantified 107 pseudogene expression as RPKM. The pseudogenes with detectable expression were defined as 4 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 6, 2021; DOI: 10.1158/0008-5472.CAN-21-0494 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 108 those with an RPKM ≥ 0.1 across at least 2 samples. Finally, we identified 1,958 pseudogenes in our 109 PDAC samples. We then used DESeq2 (25) for further differential expression analysis. 110 Cell lines and