Over-expression of ANP32E is associated with poor prognosis of pancreatic cancer and promotes cell proliferation and migration through regulating β-catenin

Over-expression of ANP32E is associated with poor prognosis of pancreatic cancer and promotes cell proliferation and migration through regulating β-catenin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Over-expression of ANP32E is associated with poor prognosis of pancreatic cancer and promotes cell proliferation and migration through regulating β-catenin Jianwei Zhang, Zhongmin Lan, Guotong Qiu, Hu Ren, Yajie Zhao, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-27702/v3 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Nov, 2020 Read the published version in BMC Cancer → Version 3 posted 4 You are reading this latest preprint version Show more versions Abstract Background: Pancreatic cancer is a malignant tumor with high mortality. Acidic nuclear phosphoprotein 32 family member E (ANP32E), a specific H2A.Z chaperone, has been shown to contribute to breast cancer development. However, the significance of ANP32E in pancreatic cancer is poorly understood. This study aimed to investigate the role of ANP32E in pancreatic cancer. Methods: The expression of ANP32E in 179 pancreatic cancer tissues and 171 normal tissues, and the correlation between ANP32E expression and patients’ survival were analyzed from the TCGA database. ANP32E was over-expressed and silenced using lentivirus. siRNA was used to knock down β-catenin. CCK8, colony formation, cell cycle and transwell experiments were performed to determine cell proliferation and migration. qRT-PCR and Western blot were conducted to detect mRNA and protein expression. Results: ANP32E was up-regulated in pancreatic cancer tissues and cells. Up-regulation of ANP32E predicted poor prognosis in pancreatic cancer patients. Lentivirus-mediated knockdown of ANP32E suppressed the proliferation, colony growth and migration of PANC1 and MIA cells. By contrast, ANP32E over-expression promoted the proliferation and migration of both cells. In addition, ANP32E accelerated the cell cycle progression in PANC1 and MIA cells. Molecular experiments showed that ANP32E activated β-catenin/cyclin D1 signaling. Silencing of β-catenin reduced cell proliferation and migration in ANP32E over-expressed cells. Conclusion: Our results propose that ANP32E functions as an oncogene in pancreatic cancer via activating β-catenin. Cancer Biology Oncology pancreatic cancer ANP32E proliferation migration β-catenin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Pancreatic ductal adenocarcinoma (PDAC) is the commonest malignancy of the pancreas, with approximately 367,000 newly diagnosed cases and 359,000 PDAC-related deaths worldwide in 2015 [1]. The major risk factors for pancreatic cancer include tobacco smoking, alcohol use, type II diabetes, chronic pancreatitis [2].Various genetic studies and fundamental experiments have shown that genetic variants of KRAS, CDKN2A, TP53 and SMAD4 contribute to the disease progression of pancreatic cancer. However, based on current information from the pharmacy companies or clinical studies, none of these genes are easily targeted. Thus, identifying new driver genes may help develop effective drugs against this deadly disease. H2A.Z is a pivotal histone variant involved in regulating transcriptional activation or inhibition. It is essential for the survival and development in Drosophila and mouse [3, 4]. As a specific H2A.Z histone chaperon, acidic nuclear phosphoprotein 32 family member E (ANP32E) removes the H2A.Z from transcriptional region of the target genes to regulate their expression [5, 6]. ANP32E plays an important role in cerebellar development and synaptogenesis [7, 8]. Dys-regulation of ANP32E contributes to the migration and invasion of breast cancer cells [9]. Overexpression of ANP32E promotes the triple-negative breast carcinogenesis through up-regulating E2F1 [10]. However, the involvement of ANP32E in pancreatic cancer is unclear. Here in this study, we explored the expression and function of ANP32E in pancreatic cancer. ANP32E was up-regulated in pancreatic cancer tissues. Up-regulation of ANP32E promoted the proliferation, colony growth and migration of pancreatic cancer cells. The apoptosis was suppressed by ANP32E. Mechanistically, ANP32E induced the expression of β-catenin and its down-stream target cyclin D1. Overexpression of β-catenin rescued the viability inhibited by ANP32E knockdown. Our study highlights the oncogenic role ANP32E in pancreatic cancer. Methods TCGA analysis of ANP32E expression in pancreatic patients The transcript level of ANP32E in pancreatic cancer tissues and normal tissues and the survival information of the patients were downloaded from The Cancer Genome Atlas ( http://cancergenome.nih.gov ) database. A total of 179 tumor tissues and 175 normal tissues were included to analyze the expression of ANP32E. A total of 89 patients with ANP32E high expression and 89 patients with low expression was included to determine the correlation between ANP32E expression and patients’ survival. Cell culture Human normal ductal epithelial cells of the pancreas HPDE and pancreatic cancer cells AsPC1, PANC1 and MIA were obtained from ATCC. The cells were cultured in Dulbecco modified Eagle’s medium (DMEM, Hyclone), which contained 10% fetal bovine serum (FBS, Gibco) and 1% penicillin and streptomycin (Corning). The cell culture was maintained in a 37 o C incubator with 5% CO 2 . ANP32E knockdown Lentivirus vector system pGCSIL-GFP (with a GFP marker), Helper2.0 (VSVG element) and pHelper1.0 (gag/pol element) was used to knock down ANP32E in PANC1 and MIA cells. Targeting sequence of the Ctrl, ANP32E#1 and ANP32E#2 was 5’-TTCTCCGAACGTGTCACGT-3’, 5’-GTCCACCGGAAGGATATGA-3’ and 5’-GCCTCTCATACTTAATGAA-3’. The lentivirus was packaged by co-transfecting the pGCSIL-GFP, Helper2.0 and pHelper1.0 to 293FT cells. The knockdown efficiency was detected by qRT-PCR and Western blot. ANP32E overexpression Lentivirus vector system pCDH, PSPAX2 and PDM2G was used to over-express ANP32E in PANC1 and MIA cells. Coding sequence of ANP32E was cloned into the pCDH vector, which contained a GFP marker. Lentivirus was packaged by co-transfecting the pCDH, PSPAX2 and PDM2G to 293FT cells. Overexpression abundance was detected by qRT-PCR and Western blot. β-catenin interference The siRNAs against CTNNB or negative control were synthesized from GenePharma company. The transfection of the siRNAs were conducted using RNAimax (Invitrogen), following the following to the manufacturer’s instructions. The targeted sequence of siRNA was as followed: siCTNNB, 5’-CCCACTAATGTCCAGCGTT-3’; and siCtrl, 5’-TTCTCCGAACGTGTCACGT-3’. RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) PANC1 and MIA cells were lysed using Trizol regent. Total RNA was extracted from the cells and subjected to reverse transcription reaction with M-MLV reverse transcriptase (Promega). Quantification of the cDNA was performed with SYBR master mixture on the Biorad machine. The qPCR primer sequence was as followed: ANP32E forward, 5’-TGCCTGTGTGTCAATGGGG-3’, and reverse, 5’-GCAGAGCTTCTACTGTACTGAGA-3’; and GAPDH forward, 5’-TGACTTCAACAGCGACACCCA-3’, and reverse, 5’-CACCCTGTTGCTGTAGCCAAA-3. The expression ANP32E was normalized to GAPDH. Western blot Total protein was extracted from PANC1 and MIA cells with RIPA lysis buffer (Beyotime). BCA experiment (Beyotime) was conducted to measure the protein concentration. Equal amount of the protein was separated on 10-12% SDS-PAGE gels. Subsequently, the proteins were transferred onto PVDF membranes. 5% skim milk was used to block the membranes for 1 hour at room temperature. The membranes were incubated with indicated primary antibodies at 4℃ overnight. Antibody against ANP32E was from Abcam. β-actin primary antibody and the secondary antibodies were obtained from SantaCruz. CCK8 assay Cell proliferation was determined by CCK8 assay. Briefly, ANP32E silenced or over-expressed and its control cells were seeded into 96-well plates at the density of 2000 cells per well. The cells were cultured for 4 days. 1, 2, 3 and 4 days later, 10% the CCK8 regent (YEASEN) was added into each well and the plates were maintained at 37 o C. 3 hours later, the OD value at 450 nm was measured on the micro-plate machine. The cell viability was normalized to the OD value of the first day. Colony formation assay A total of 500 shCtrl, shANP32E#1 and shANP32E#2, OE-Ctrl and OE-ANP32E PANC1 and MIA cells were seeded into the 6-well plates. 10 days later, colonies were formed and the cell culture was removed. After washed by PBS for three times, the colonies were fixed by methanol and stained by crystal violet. Subsequently, the plates were washed by clean water and dried at room temperature. Then the colonies were photographed by the camera. Cell cycle analysis PI staining was used to analyze the cell cycle in PANC1 and MIA cells. Indicated cells were seeded in triplicate in 6-well plates. 2 days later, the cells were re-suspended in FBS free culture medium and fixed by 70% ethanol. After stained by PI regent, flow cytometer was used to analyze the cell cycle distribution in PANC1 and MIA cells. Transwell assay A total of 3×10 4 PANC1 or MIA cells in 200ul FBS free culture medium were seeded onto the upper surface of 8.0-μm filter migration chambers. The lower compartment contained 500ul DMEM medium with 10% FBS. 24 hours later, the cells attached on the upper surface were removed using cotton tips. The cells on the lower surface were fixed by methanol and stained by crystal violet. The images were photographed under a microscope. Statistical analysis Graphpad prism software was used to analyze the data as shown in the Figure. Students’t tests were used to determine the difference between two groups. One-way ANOVA was used to determine the difference when more than two groups. Statistical difference was considered significant when p<0.05. Results ANP32E is over-expressed in pancreatic cancers and predicts the survival of patients To study the clinical relevance of ANP32E in pancreatic cancer, we downloaded the transcript abundance of ANP32E and the patients’ survival information from TCGA database. We found that ANP32E was over-expressed in pancreatic cancer tissues comparing to normal pancreatic tissues (Fig. 1A). The pancreatic cancer patients were divided into two groups: ANP32E low expression and high expression group. Survival curve showed that ANP32E high expression patients exhibited poorer overall and disease free survival than those with ANP32E low expression (Fig. 1B and 1C). We also checked the abundance of ANP32E in human normal ductal epithelial cells of the pancreas HPDE and pancreatic cancer cells AsPC1, PANC1 and MIA. qRT-PCR and Western blot results showed HPDE had lowest ANP32E expression comparing with AsPC1, PANC1 and MIA (Fig. 1D). These results suggest that ANP32E is correlated with the disease progression of pancreatic cancer. ANP32E promotes the proliferation and growth of pancreatic cancer cells To determine whether ANP32E functions as an oncogene in pancreatic cancer, we used lentivirus to knock down ANP32E in pancreatic cancer cells PANC1 and MIA. qRT-PCR and Western blot results showed that ANP32E was efficiently knocked down in PANC1 and MIA cells (Fig. 2A and 2B). We also used lentivirus vector system to over-express ANP32E. Comparing with Ctrl cells, ANP32E over-expressed cells had significantly higher mRAN and protein abundance of ANP32E (Fig. 2C and 3D). These results suggest that ANP32E is efficiently silenced and over-expressed in PANC1 and MIA cells. To examine the effect of ANP32E on cell proliferation, CCK8 assays were performed in ANP32E knockdown and over-expressed cells. We found that down-regulation of ANP32E suppressed the proliferation of PANC1 and MIA cells (Fig. 3A). By contrast, up-regulation of ANP32E enhanced the cell viability of both cells (Fig. 3B). Consistently, ANP32E silencing and overexpression reduced and enhanced the colony formation capacity in PANC1 and MIA cells, respectively (Fig. 3C-3F). Collectively, ANP32E functions as an oncogene in pancreatic cancer. ANP32E promotes the migration of pancreatic cancer cells Next, we assessed the role of ANP32E on pancreatic cancer cell migration. Transwell assay was conducted to evaluate the migration capacity in ANP32E knockdown and over-expressed PANC1 and MIA cells. The results showed that ANP32E overexpression promoted the migration in PANC1 and MIA cells (Fig. 4A and 4B). Moreover, comparing to shCtrl PANC1 and MIA cells, shANP32E#1 and shANP32E#2 cells exhibited reduced migration capacity (Fig. 4C).Collectively, ANP32E enhanced the migration of pancreatic cancer cells. ANP32E promotes the cell cycle progression in pancreatic cancer cells Accelerated cell cycle process is a hallmark of cancer cell. We examined the effect of ANP32E on cell cycle distribution. PANC1 and MIA cells infected with ANP32E overexpression lentivirus were stained with PI and subjected to flow cytometer analysis of cell cycle. Ectopic expression of ANP32E led to reduced G0 phase and increased S and G2/M phase (Fig. 5A and 5B). These results indicate that ANP32E promotes cell cycle progression. ANP32E up-regulates β-catenin to promote the proliferation and migration of pancreatic cancer cells We attempted to explore the down-stream targets of ANP32E using Western blot assays. We found the expression of β-catenin and cyclin D1 was increased in ANP32E over-expressed MIA cells (Fig. 6A). Oppositely, ANP32E down-regulation inhibited the expression of β-catenin and cyclin D1 (Fig. 6A). Consistent results were observed in ANP32E over-expressed and silenced PANC1 cells (Fig. 6B). To evaluate whether β-catenin contributes to the oncogenic role of ANP32E in pancreatic cancer, we knocked down CTNNB, which encodes β-catenin, in ANP32E over-expressed PANC1 and MIA cells. Western blot showed that β-catenin was efficiently silenced in PANC1 cells (Fig. 6C). Down-regulation of β-catenin significantly repressed the proliferation and migration of PANC1 and MIA cells (Fig. 6D and 6E). These results suggest that ANP32E promotes pancreatic cancer through potentiating β-catenin signaling. Discussion Pancreatic cancer is a lethal malignancy. Once diagnosed, the disease progresses fast and the 5-year survival rate is very low. To identify novel oncogenes or tumor suppressors may help develop effective drugs to cure this malignancy. In this study, we found that ANP32E was an oncogene in pancreatic cancer. High expression of ANP32E was found in PAAD tissues and predicted poor survival of PAAD patients. In vitro , ANP32E promoted the proliferation, colony formation and migration of pancreatic cancer cells. ANP32E also regulated cell cycle and apoptosis in these cells. β-catenin/cyclin D1 signaling was positively regulated by ANP32E. Inhibition of β-catenin reduced the proliferation of pancreatic cancer cells. These findings indicate that ANP32E promotes pancreatic cancer through regulating β-catenin/cyclin D1 signaling . Acidic nuclear phosphoprotein 32 family member E (ANP32E) is a specific H2A.Z histone chaperon. It regulates the expression of target genes by promoting the removal of H2A.Z from transcriptional region of the genes [5, 6]. Anp32e also plays a pivotal role in nucleosome reorganization and DNA repair by removing H2A.Z from DNA double-strand breaks [11]. Several studies have investigated the role of ANP32E in cancer development. For example, Li et.al showed that miR-141 repressed the proliferation and migration of breast cancer cells and the expression of ANP32E. Knockdown of ANP32E reduced cell viability [9]. Thus, ANP32E contributes to the tumor suppressive role of miR-141 in breast cancer. In addition, ANP32E promotes the development of triple-negative breast cancer via transcriptionally activating E2F1 [10]. These results indicate that ANP32E maybe an oncogene in cancers. In this study, we observed that ANP32E was up-regulated in PAAD tissues. ANP32E expression was also inversely correlated with the prognosis of PAAD patients. In vitro , ANP32E overexpression promoted the proliferation, colony formation and migration of PANC1 and MIA cells. Opposite results were found in ANP32E silenced cells. Furthermore, ANP32E suppressed apoptosis and promoted cell cycle progression in PANC1 and MIA cells. Our results demonstrate that ANP32E functions as an oncogene in pancreatic cancer. Due to alterations of any component of Wnt/β-catenin signaling pathway, it is frequently activated in various cancers, including colorectal cancer, hepatocellular carcinoma and pancreatic cancer [12-14]. In brief, Wnt ligand binds to Frizzled receptor family, leading to inactivation of adenomatous polyposis coli (APC) or axin, which in turn promotes the cytoplasmic accumulation and nuclear localization of β-catenin. β-catenin can also be regulated by other factors to promote cancer development. For instance, TYRO3 promotes the prolifieration and metastasis of gastric cancer cells through activation of Wnt/β-catenin signaling pathway [15]. LncCCAT1 enhances the functions of breast cancer stem cell via activating WNT/β-catenin signaling [16]. β-catenin is a transcriptional factor that interacts with TCF/LEF factors to activate the expression of target genes. The well-known downstream targets for β-catenin are c-myc and cyclin D1 [17, 18]. However, the correlation between ANP32E and β-catenin is unclear in pancreatic cancer. Here, our molecular experiments showed that ANP32E up-regulated β-catenin and its downstream target cyclin D1. We also checked other oncogenes, such as PKM2 and HK2, or tumor suppressors, including P53 and PTEN. No difference was observed after knocking down ANP32E. We also analyzed the role of β-catenin in ANP32E over-expressed cells. The results showed that knockdown of β-catenin reduced the viability of pancreatic cancer cells with highly expressed ANP32E. These results reveal that ANP32E promotes pancreatic cancer through activating β-catenin. Conclusions We demonstrate that ANP32E is an oncogene in pancreatic cancer. ANP32E promotes the proliferation and migration through up-regulation and activation of β-catenin/cyclin D1 signaling. Up-regulation of ANP32E in PAAD tissues and the inverse relationship between ANP32E expression and patients’ survival indicate that ANP32E maybe a diagnosed biomarker for PAAD. Our findings suggest that ANP32E is a promising therapeutic target for PAAD. Abbreviations ANP32E, Acidic nuclear phosphoprotein 32 family member E; PDAC, pancreatic ductal adenocarcinoma; TCGA, The Cancer Genome Atlas; DMEM, Dulbecco modified Eagle’s medium; FBS, fetal bovine serum; qRT-PCR, quantitative real-time polymerase chain reaction; APC, adenomatous polyposis coli. Declarations Ethics approval and consent to participate Ethics approval is not required for the use of the cells in our study. Consent for publication No consent is required in this study. Availability of data and material All data generated or analyzed during this study are included in this published article. Competing interests These authors declare no conflicts of interest. Funding The research was financially supported by National High Technology Research and Development Program of China (2012AA022701) and CAMS Initiation Fund for Mediacal Sciences (2016-I2M-1-001). The funders mentioned were used for purchasing the experiments materials but not for study design and data analysis. Authors' contributions JWZ, JH and CFW designed the study. JWZ, ZML, GTQ and HR performed the experiments. JWZ, YJZ, ZTG, ZZL, LF and CFW analyzed the data. JWZ, JH and CFW wrote the manuscript draft and revised the draft. All authors have read and approved the manuscript Acknowledgements Not Applicable. References RL S, KD M, A J: Cancer statistics, 2015 . CA Cancer J Clin 2015, 65 (1):5-29. J K, M K, M A, C LV, CD J, AV B, RE N, M T, DA T, RH H et al : Pancreatic cancer . Nature reviews Disease primers 2016, 2 :16022. MJ C, JR W, F G, R S, DJ T: Regions of variant histone His2AvD required for Drosophila development . Nature 1999, 399 (6737):694-697. Faast R, Thonglairoam V, Schulz TC, Beall J, Wells JR, Taylor H, Matthaei K, Rathjen PD, Tremethick DJ, Lyons I: Histone variant H2A.Z is required for early mammalian development . Curr Biol 2001, 11 (15):1183-1187. Farris SD, Rubio ED, Moon JJ, Gombert WM, Nelson BH, Krumm A: Transcription-induced chromatin remodeling at the c-myc gene involves the local exchange of histone H2A.Z . J Biol Chem 2005, 280 (26):25298-25303. Mao Z, Pan L, Wang W, Sun J, Shan S, Dong Q, Liang X, Dai L, Ding X, Chen S et al : Anp32e, a higher eukaryotic histone chaperone directs preferential recognition for H2A.Z . Cell Res 2014, 24 (4):389-399. Jiang M, Ma Y, Ni X, Cao G, Ji C, Cheng H, Tang R, Xie Y, Mao Y: Molecular cloning and characterization of a novel human gene (ANP32E alias LANPL) from human fetal brain . Cytogenet Genome Res 2002, 97 (1-2):68-71. Radrizzani M, Vila-Ortiz G, Cafferata EG, Di Tella MC, Gonzalez-Guerrico A, Perandones C, Pivetta OH, Carminatti H, Idoyaga Vargas VP, Santa-Coloma TA: Differential expression of CPD1 during postnatal development in the mouse cerebellum . Brain Res 2001, 907 (1-2):162-174. Li P, Xu T, Zhou X, Liao L, Pang G, Luo W, Han L, Zhang J, Luo X, Xie X et al : Downregulation of miRNA-141 in breast cancer cells is associated with cell migration and invasion: involvement of ANP32E targeting . Cancer Med 2017, 6 (3):662-672. Xiong Z, Ye L, Zhenyu H, Li F, Xiong Y, Lin C, Wu X, Deng G, Shi W, Song L et al : ANP32E induces tumorigenesis of triple-negative breast cancer cells by upregulating E2F1 . Mol Oncol 2018, 12 (6):896-912. Gursoy-Yuzugullu O, Ayrapetov MK, Price BD: Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair . Proc Natl Acad Sci U S A 2015, 112 (24):7507-7512. Fodde R, Smits R, Clevers H: APC, Signal transduction and genetic instability in colorectal cancer . Nature Reviews Cancer 2001, 1 (1):55-67. Klaus A, Birchmeier W: Wnt signalling and its impact on development and cancer . Nature Reviews Cancer 2008, 8 (5):387-398. Morris JP, Wang SC, Hebrok M: KRAS, Hedgehog, Wnt and the twisted developmental biology of pancreatic ductal adenocarcinoma . Nature Reviews Cancer 2010, 10 (10):683-695. D C, Q L, G C, W Z: TYRO3 facilitates cell growth and metastasis via activation of the Wnt/β-catenin signaling pathway in human gastric cancer cells . Aging 2020, 12 . T T, C G, T X, R Z, K Z, Y P, L J: LncCCAT1 Promotes Breast Cancer Stem Cell Function through Activating WNT/β-catenin Signaling . Theranostics 2019, 9 (24):7384-7402. N Z, X C: A positive feedback loop involving the LINC00346/β-catenin/MYC axis promotes hepatocellular carcinoma development . Cell Oncol (Dordr) 2020, 43 (1):137-153. S G, A G, S X, Z Y, S N, Y Z, D P: PSAT1 is regulated by ATF4 and enhances cell proliferation via the GSK3β/β-catenin/cyclin D1 signaling pathway in ER-negative breast cancer . Journal of experimental & clinical cancer research : CR 2017, 36 (1):179. Supplementary Files SupplementaryfileWesternblot.pdf SupplementaryFigure.docx Cite Share Download PDF Status: Published Journal Publication published 04 Nov, 2020 Read the published version in BMC Cancer → Version 3 posted Editorial decision: Accept 21 Oct, 2020 Editor assigned by journal 19 Oct, 2020 Submission checks completed at journal 18 Oct, 2020 Editor invited by journal 18 Oct, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-27702","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":3716145,"identity":"a66466fc-5dfe-4095-bb66-de0cdc930878","order_by":0,"name":"Jianwei Zhang","email":"","orcid":"","institution":"National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jianwei","middleName":"","lastName":"Zhang","suffix":""},{"id":3716146,"identity":"3e12af09-18c2-4e42-a858-ebb4ada3065b","order_by":1,"name":"Zhongmin Lan","email":"","orcid":"","institution":"National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhongmin","middleName":"","lastName":"Lan","suffix":""},{"id":3716147,"identity":"90e73bd9-8d12-4d3a-8add-a09165b46d0c","order_by":2,"name":"Guotong Qiu","email":"","orcid":"","institution":"National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guotong","middleName":"","lastName":"Qiu","suffix":""},{"id":3716148,"identity":"3baaf0c1-08d5-4a02-b486-e4f25695ad18","order_by":3,"name":"Hu Ren","email":"","orcid":"","institution":"National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hu","middleName":"","lastName":"Ren","suffix":""},{"id":3716149,"identity":"d4f2ec17-1a9f-443a-943d-b44f32214661","order_by":4,"name":"Yajie Zhao","email":"","orcid":"","institution":"National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yajie","middleName":"","lastName":"Zhao","suffix":""},{"id":3716150,"identity":"0291516b-6a90-408b-91a7-37bc5eaacad6","order_by":5,"name":"Zongting Gu","email":"","orcid":"","institution":"National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zongting","middleName":"","lastName":"Gu","suffix":""},{"id":3716151,"identity":"df3832dd-b567-42db-bb95-1a618b03376d","order_by":6,"name":"Zongze Li","email":"","orcid":"","institution":"National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zongze","middleName":"","lastName":"Li","suffix":""},{"id":3716152,"identity":"d6452af5-c3ee-4daf-b1d1-88f95590526a","order_by":7,"name":"Lin Feng","email":"","orcid":"","institution":"National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Feng","suffix":""},{"id":3716153,"identity":"aa7da1fa-cbb2-46ea-80ac-e67a7545b202","order_by":8,"name":"Jin He","email":"","orcid":"","institution":"Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"He","suffix":""},{"id":3716154,"identity":"4b772c80-91ce-45f8-ac3d-a3505487f2dd","order_by":9,"name":"Chengfeng Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIiWNgGAWjYLACxgYgwXwASFTYyLGxtx8gUgtbApA4k2bMx3MmgQQtjC2HE+dJOBjgVW1w/OzhFz932OTJuzEfe/i1gTm9TYIhgeFHxTbcWs7kpVn2nkkrNjzGlm4su4Mtt0268QBjz5nbOLWYHcgxM2ZsO5y4cX6PmbTkGZ7cNpkDCcyMbXi0nH8D0vI/cWMb/zdpyTaJdDaJBAP8Wm7kGD9mbDuQOJ+Nh03yY5tBAkEt9jfemDH2tiUnbmBjM5NmOJNg2AYM5IP4/CLZn2P84WebXeL8NuZnkj8q/svLt7cffPCjArcWIGCTAJEGB4AJgAcqdACfeiBg/gAi5RuAMfmDgNJRMApGwSgYmQAAfO9dilNHqpcAAAAASUVORK5CYII=","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College Hospital of Skin Diseases and Institute of Dermatology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Chengfeng","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2020-05-08 08:10:45","currentVersionCode":3,"declarations":"","doi":"10.21203/rs.3.rs-27702/v3","doiUrl":"https://doi.org/10.21203/rs.3.rs-27702/v3","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12885-020-07556-z","type":"published","date":"2020-11-04T15:01:33+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":3206386,"identity":"7de275d9-a09c-4d60-b422-5dd338045d71","added_by":"auto","created_at":"2020-10-26 20:21:00","extension":"tif","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":484704,"visible":true,"origin":"","legend":"Clinical relevance of ANP32E in pancreatic cancer patients.\n(A) The transcript abundance of ANP32E in PAAD (n=179, red box) and normal tissues (n=175, gray box) was analyzed from TCGA database. p\u003c0.001. (B) PAAD patients were divided into ANP32E high expression (n=89, green line) and low expression (n=89, yellow line) group. Overall survival for ANP32E was analyzed. p=0.049. (C) PAAD patients were divided into ANP32E high expression (n=45, red line) and low expression (n=45, blue line) group. Disease free survival for ANP32E was analyzed. p=0.045. (D) qRT-PCR and Western blot analysis of ANP32E in human normal ductal epithelial cells of the pancreas HPDE and pancreatic cancer cells AsPC1, PANC1 and MIA. *p\u003c0.05. **p\u003c0.01. Full-length gels are presented in Supplementary Figure 2.\n","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-27702/v3/af3ec990685c7b999615fffc.tif"},{"id":3206389,"identity":"43a1ca23-aac3-4209-bed4-a73b7831f1b2","added_by":"auto","created_at":"2020-10-26 20:21:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":283594,"visible":true,"origin":"","legend":"ANP32E is efficiently knocked down and over-expressed in MIA and PANC1 cells.\n(A and B) qRT-PCR (A) and Western blot (B) analysis of ANP32E in shCtrl, shANP32E#1 and shANP32E#2 MIA and PANC1 cells. **p\u003c0.01. (C and D) qRT-PCR (C) and Western blot (D) results of ANP32E in Ctrl and ANP32E over-expressed MIA and PANC1 cells. **p\u003c0.01.","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-27702/v3/3a278b970c3ef93df151dcad.png"},{"id":3206390,"identity":"b1741918-5afb-432c-ae25-a11353650079","added_by":"auto","created_at":"2020-10-26 20:21:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":829096,"visible":true,"origin":"","legend":"ANP32E promotes the migration of pancreatic cancer cells.\n(A) Transwell assay was performed to analyze the migration in Ctrl and ANP32E over-expressed MIA cells. Left, cell images. Right, quantification result. **p\u003c0.01. (B) Transwell assay was performed to analyze the migration in Ctrl and ANP32E over-expressed PANC1 cells. Left, cell images. Right, quantification result. **p\u003c0.01. (C) Transwell assay was performed to analyze the migration in shCtrl, shANP32E#1 and shANP32E#2 MIA and PANC1 cells. Left, cell images. Right, quantification result. **p\u003c0.01. Results are mean ± SEM (n=3). Scale bar, 200 um.\n","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-27702/v3/ef0d4fb6aaedebc49de72bd2.jpg"},{"id":3206391,"identity":"b990f68d-ba8f-47fd-9ff0-56ef6c32e731","added_by":"auto","created_at":"2020-10-26 20:21:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3198265,"visible":true,"origin":"","legend":"ANP32E promotes migration of the pancreatic cancer cells.\n(A) Transwell assay was performed to analyze the migration in Ctrl and ANP32E over-expressed MIA cells. Left, cell images. Right, quantification result. **p\u003c0.01. (B) Transwell assay was performed to analyze the migration in Ctrl and ANP32E over-expressed PANC1 cells. Left, cell images. Right, quantification result. **p\u003c0.01. (C) Transwell assay was performed to analyze the migration in shCtrl, shANP32E#1 and shANP32E#2 MIA and PANC1 cells. Left, cell images. Right, quantification result. **p\u003c0.01.","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-27702/v3/f01c728cec06385bf836fc7f.png"},{"id":3206392,"identity":"44a9f7e5-f78a-4b19-b67c-8f7dcdbfd534","added_by":"auto","created_at":"2020-10-26 20:21:02","extension":"tif","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1333841,"visible":true,"origin":"","legend":"ANP32E activates β-catenin to promote the proliferation and migration of pancreatic cancer cells.\n(A and B) Western blot results of β-catenin and cyclin D1 in Ctrl and ANP32E over-expressed, shCtrl and shANP32E MIA (A) and PANC1 (B) cells. (C) Western blot results of β-catenin in siCtrl and siCTNNB PANC1 cells with highly expressed ANP32E. (D) siCtrl and siCTNNB PANC1 cells were subjected to CCK8 analysis of cell proliferation. *p\u003c0.05. **p\u003c0.01. (E) siCtrl and siCTNNB PANC1 cells were subjected to Transwell analysis of migration. **p\u003c0.01. Results are mean ± SEM (n=3). Full-length gels are presented in Supplementary Figure 2. Scale bar, 200 um.\n","description":"","filename":"Figure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-27702/v3/4d0c4fc8f5e96e7302ff5a1b.tif"},{"id":13606198,"identity":"ce0a59eb-3e7a-4ea2-b348-d8c36c35b253","added_by":"auto","created_at":"2021-09-17 06:06:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2883434,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-27702/v3/c4a9844e-0ee3-4c9a-a0be-a6f740beb712.pdf"},{"id":3206387,"identity":"a0f29a2c-240c-4a4a-b529-77ae3c958b37","added_by":"auto","created_at":"2020-10-26 20:21:01","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":73778,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfileWesternblot.pdf","url":"https://assets-eu.researchsquare.com/files/rs-27702/v3/eb7f06149fb9d4d213d1aae4.pdf"},{"id":3206388,"identity":"b5231295-3f57-4127-94b7-730754c8612d","added_by":"auto","created_at":"2020-10-26 20:21:01","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":482752,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-27702/v3/180b6cc062aa675ac9e3a349.docx"}],"financialInterests":"","formattedTitle":"Over-expression of ANP32E is associated with poor prognosis of pancreatic cancer and promotes cell proliferation and migration through regulating β-catenin","fulltext":[{"header":"Background","content":"\u003cp\u003ePancreatic ductal adenocarcinoma (PDAC) is the commonest malignancy of the pancreas, with approximately 367,000 newly diagnosed cases and 359,000 PDAC-related deaths worldwide in 2015 [1]. The major risk factors for pancreatic cancer include tobacco smoking, alcohol use, type II diabetes, chronic pancreatitis [2].Various genetic studies and fundamental experiments have shown that genetic variants of KRAS, CDKN2A, TP53 and SMAD4 contribute to the disease progression of pancreatic cancer. However, based on current information from the pharmacy companies or clinical studies, none of these genes are easily targeted. Thus, identifying new driver genes may help develop effective drugs against this deadly disease.\u003c/p\u003e\n\u003cp\u003eH2A.Z is a pivotal histone variant involved in regulating transcriptional activation or inhibition. It is essential for the survival and development in Drosophila and mouse [3, 4]. As a specific H2A.Z histone chaperon, acidic nuclear phosphoprotein 32 family member E (ANP32E) removes the H2A.Z from transcriptional region of the target genes to regulate their expression [5, 6]. ANP32E plays an important role in cerebellar development and synaptogenesis [7, 8]. Dys-regulation of ANP32E contributes to the migration and invasion of breast cancer cells [9]. Overexpression of ANP32E promotes the triple-negative breast carcinogenesis through up-regulating E2F1 [10]. However, the involvement of ANP32E in pancreatic cancer is unclear.\u003c/p\u003e\n\u003cp\u003eHere in this study, we explored the expression and function of ANP32E in pancreatic cancer. ANP32E was up-regulated in pancreatic cancer tissues. Up-regulation of ANP32E promoted the proliferation, colony growth and migration of pancreatic cancer cells. The apoptosis was suppressed by ANP32E. Mechanistically, ANP32E induced the expression of \u0026beta;-catenin and its down-stream target cyclin D1. Overexpression of \u0026beta;-catenin rescued the viability inhibited by ANP32E knockdown. Our study highlights the oncogenic role ANP32E in pancreatic cancer.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eTCGA analysis of ANP32E expression in pancreatic patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transcript level of ANP32E in pancreatic cancer tissues and normal tissues and the survival information of the patients were downloaded from The Cancer Genome Atlas (\u003ca href=\"http://cancergenome.nih.gov\"\u003ehttp://cancergenome.nih.gov\u003c/a\u003e) database. A total of 179 tumor tissues and 175 normal tissues were included to analyze the expression of ANP32E. A total of 89 patients with ANP32E high expression and 89 patients with low expression was included to determine the correlation between ANP32E expression and patients\u0026rsquo; survival.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman normal ductal epithelial cells of the pancreas HPDE and pancreatic cancer cells AsPC1, PANC1 and MIA were obtained from ATCC. The cells were cultured in Dulbecco modified Eagle\u0026rsquo;s medium (DMEM, Hyclone), which contained 10% fetal bovine serum (FBS, Gibco) and 1% penicillin and streptomycin (Corning). The cell culture was maintained in a 37\u003csup\u003eo\u003c/sup\u003eC incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eANP32E knockdown\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLentivirus vector system pGCSIL-GFP (with a GFP marker), Helper2.0 (VSVG element) and pHelper1.0 (gag/pol element) was used to knock down ANP32E in PANC1 and MIA cells. Targeting sequence of the Ctrl, ANP32E#1 and ANP32E#2 was 5\u0026rsquo;-TTCTCCGAACGTGTCACGT-3\u0026rsquo;, 5\u0026rsquo;-GTCCACCGGAAGGATATGA-3\u0026rsquo; and 5\u0026rsquo;-GCCTCTCATACTTAATGAA-3\u0026rsquo;. The lentivirus was packaged by co-transfecting the pGCSIL-GFP, Helper2.0 and pHelper1.0 to 293FT cells. The knockdown efficiency was detected by qRT-PCR and Western blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eANP32E overexpression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLentivirus vector system pCDH, PSPAX2 and PDM2G was used to over-express ANP32E in PANC1 and MIA cells. Coding sequence of ANP32E was cloned into the pCDH vector, which contained a GFP marker. Lentivirus was packaged by co-transfecting the pCDH, PSPAX2 and PDM2G to 293FT cells. Overexpression abundance was detected by qRT-PCR and Western blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026beta;-catenin interference\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe siRNAs against CTNNB or negative control were synthesized from GenePharma company. The transfection of the siRNAs were conducted using RNAimax (Invitrogen), following the following to the manufacturer\u0026rsquo;s instructions. The targeted sequence of siRNA was as followed: siCTNNB, 5\u0026rsquo;-CCCACTAATGTCCAGCGTT-3\u0026rsquo;; and siCtrl, 5\u0026rsquo;-TTCTCCGAACGTGTCACGT-3\u0026rsquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePANC1 and MIA cells were lysed using Trizol regent. Total RNA was extracted from the cells and subjected to reverse transcription reaction with M-MLV reverse transcriptase (Promega). Quantification of the cDNA was performed with SYBR master mixture on the Biorad machine. The qPCR primer sequence was as followed: ANP32E forward, 5\u0026rsquo;-TGCCTGTGTGTCAATGGGG-3\u0026rsquo;, and reverse, 5\u0026rsquo;-GCAGAGCTTCTACTGTACTGAGA-3\u0026rsquo;; and GAPDH forward, 5\u0026rsquo;-TGACTTCAACAGCGACACCCA-3\u0026rsquo;, and reverse, 5\u0026rsquo;-CACCCTGTTGCTGTAGCCAAA-3. The expression ANP32E was normalized to GAPDH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal protein was extracted from PANC1 and MIA cells with RIPA lysis buffer (Beyotime). BCA experiment (Beyotime) was conducted to measure the protein concentration. Equal amount of the protein was separated on 10-12% SDS-PAGE gels. Subsequently, the proteins were transferred onto PVDF membranes. 5% skim milk was used to block the membranes for 1 hour at room temperature. The membranes were incubated with indicated primary antibodies at 4℃ overnight. Antibody against ANP32E was from Abcam. \u0026beta;-actin primary antibody and the secondary antibodies were obtained from SantaCruz.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCK8 assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell proliferation was determined by CCK8 assay. Briefly, ANP32E silenced or over-expressed and its control cells were seeded into 96-well plates at the density of 2000 cells per well. The cells were cultured for 4 days. 1, 2, 3 and 4 days later, 10% the CCK8 regent (YEASEN) was added into each well and the plates were maintained at 37\u003csup\u003eo\u003c/sup\u003eC. 3 hours later, the OD value at 450 nm was measured on the micro-plate machine. The cell viability was normalized to the OD value of the first day.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 500 shCtrl, shANP32E#1 and shANP32E#2, OE-Ctrl and OE-ANP32E PANC1 and MIA cells were seeded into the 6-well plates. 10 days later, colonies were formed and the cell culture was removed. After washed by PBS for three times, the colonies were fixed by methanol and stained by crystal violet. Subsequently, the plates were washed by clean water and dried at room temperature. Then the colonies were photographed by the camera.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePI staining was used to analyze the cell cycle in PANC1 and MIA cells. Indicated cells were seeded in triplicate in 6-well plates. 2 days later, the cells were re-suspended in FBS free culture medium and fixed by 70% ethanol. After stained by PI regent, flow cytometer was used to analyze the cell cycle distribution in PANC1 and MIA cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranswell assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e PANC1 or MIA cells in 200ul FBS free culture medium were seeded onto the upper surface of 8.0-\u0026mu;m filter migration chambers. The lower compartment contained 500ul DMEM medium with 10% FBS. 24 hours later, the cells attached on the upper surface were removed using cotton tips. The cells on the lower surface were fixed by methanol and stained by crystal violet. The images were photographed under a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraphpad prism software was used to analyze the data as shown in the Figure. Students\u0026rsquo;t tests were used to determine the difference between two groups. One-way ANOVA was used to determine the difference when more than two groups. Statistical difference was considered significant when p\u0026lt;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eANP32E is over-expressed in pancreatic cancers and predicts the survival of patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the clinical relevance of ANP32E in pancreatic cancer, we downloaded the transcript abundance of ANP32E and the patients\u0026rsquo; survival information from TCGA database. We found that ANP32E was over-expressed in pancreatic cancer tissues comparing to normal pancreatic tissues (Fig. 1A). The pancreatic cancer patients were divided into two groups: ANP32E low expression and high expression group. Survival curve showed that ANP32E high expression patients exhibited poorer overall and disease free survival than those with ANP32E low expression (Fig. 1B and 1C). We also checked the abundance of ANP32E in human normal ductal epithelial cells of the pancreas HPDE and pancreatic cancer cells AsPC1, PANC1 and MIA. qRT-PCR and Western blot results showed HPDE had lowest ANP32E expression comparing with AsPC1, PANC1 and MIA (Fig. 1D). These results suggest that ANP32E is correlated with the disease progression of pancreatic cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eANP32E promotes the proliferation and growth of pancreatic cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether ANP32E functions as an oncogene in pancreatic cancer, we used lentivirus to knock down ANP32E in pancreatic cancer cells PANC1 and MIA. qRT-PCR and Western blot results showed that ANP32E was efficiently knocked down in PANC1 and MIA cells (Fig. 2A and 2B). We also used lentivirus vector system to over-express ANP32E. Comparing with Ctrl cells, ANP32E over-expressed cells had significantly higher mRAN and protein abundance of ANP32E (Fig. 2C and 3D). These results suggest that ANP32E is efficiently silenced and over-expressed in PANC1 and MIA cells.\u003c/p\u003e\n\u003cp\u003eTo examine the effect of ANP32E on cell proliferation, CCK8 assays were performed in ANP32E knockdown and over-expressed cells. We found that down-regulation of ANP32E suppressed the proliferation of PANC1 and MIA cells (Fig. 3A). By contrast, up-regulation of ANP32E enhanced the cell viability of both cells (Fig. 3B). Consistently, ANP32E silencing and overexpression reduced and enhanced the colony formation capacity in PANC1 and MIA cells, respectively (Fig. 3C-3F). Collectively, ANP32E functions as an oncogene in pancreatic cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eANP32E promotes the migration of pancreatic cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we assessed the role of ANP32E on pancreatic cancer cell migration. Transwell assay was conducted to evaluate the migration capacity in ANP32E knockdown and over-expressed PANC1 and MIA cells. The results showed that ANP32E overexpression promoted the migration in PANC1 and MIA cells (Fig. 4A and 4B). Moreover, comparing to shCtrl PANC1 and MIA cells, shANP32E#1 and shANP32E#2 cells exhibited reduced migration capacity (Fig. 4C).Collectively, ANP32E enhanced the migration of pancreatic cancer cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eANP32E promotes the cell cycle progression in pancreatic cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccelerated cell cycle process is a hallmark of cancer cell. We examined the effect of ANP32E on cell cycle distribution. PANC1 and MIA cells infected with ANP32E overexpression lentivirus were stained with PI and subjected to flow cytometer analysis of cell cycle. Ectopic expression of ANP32E led to reduced G0 phase and increased S and G2/M phase (Fig. 5A and 5B). These results indicate that ANP32E promotes cell cycle progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eANP32E up-regulates \u0026beta;-catenin to promote the proliferation and migration of pancreatic cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe attempted to explore the down-stream targets of ANP32E using Western blot assays. We found the expression of \u0026beta;-catenin and cyclin D1 was increased in ANP32E over-expressed MIA cells (Fig. 6A). Oppositely, ANP32E down-regulation inhibited the expression of \u0026beta;-catenin and cyclin D1 (Fig. 6A). Consistent results were observed in ANP32E over-expressed and silenced PANC1 cells (Fig. 6B). To evaluate whether \u0026beta;-catenin contributes to the oncogenic role of ANP32E in pancreatic cancer, we knocked down CTNNB, which encodes \u0026beta;-catenin, in ANP32E over-expressed PANC1 and MIA cells. Western blot showed that \u0026beta;-catenin was efficiently silenced in PANC1 cells (Fig. 6C). Down-regulation of \u0026beta;-catenin significantly repressed the proliferation and migration of PANC1 and MIA cells (Fig. 6D and 6E). These results suggest that ANP32E promotes pancreatic cancer through potentiating \u0026beta;-catenin signaling.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePancreatic cancer is a lethal malignancy. Once diagnosed, the disease progresses fast and the 5-year survival rate is very low. To identify novel oncogenes or tumor suppressors may help develop effective drugs to cure this malignancy. In this study, we found that ANP32E was an oncogene in pancreatic cancer. High expression of ANP32E was found in PAAD tissues and predicted poor survival of PAAD patients. \u003cem\u003eIn vitro\u003c/em\u003e, ANP32E promoted the proliferation, colony formation and migration of pancreatic cancer cells. ANP32E also regulated cell cycle and apoptosis in these cells. \u0026beta;-catenin/cyclin D1 signaling was positively regulated by ANP32E. Inhibition of \u0026beta;-catenin reduced the proliferation of pancreatic cancer cells. These findings indicate that ANP32E promotes pancreatic cancer through regulating \u0026beta;-catenin/cyclin D1 signaling\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAcidic nuclear phosphoprotein 32 family member E (ANP32E) is a specific H2A.Z histone chaperon. It regulates the expression of target genes by promoting the removal of H2A.Z from transcriptional region of the genes [5, 6]. Anp32e also plays a pivotal role in nucleosome reorganization and DNA repair by removing H2A.Z from DNA double-strand breaks [11]. Several studies have investigated the role of ANP32E in cancer development. For example, Li et.al showed that miR-141 repressed the proliferation and migration of breast cancer cells and the expression of ANP32E. Knockdown of ANP32E reduced cell viability [9]. Thus, ANP32E contributes to the tumor suppressive role of miR-141 in breast cancer. In addition, ANP32E promotes the development of triple-negative breast cancer via transcriptionally activating E2F1 [10]. These results indicate that ANP32E maybe an oncogene in cancers. In this study, we observed that ANP32E was up-regulated in PAAD tissues. ANP32E expression was also inversely correlated with the prognosis of PAAD patients. \u003cem\u003eIn vitro\u003c/em\u003e, ANP32E overexpression promoted the proliferation, colony formation and migration of PANC1 and MIA cells. Opposite results were found in ANP32E silenced cells. Furthermore, ANP32E suppressed apoptosis and promoted cell cycle progression in PANC1 and MIA cells. Our results demonstrate that ANP32E functions as an oncogene in pancreatic cancer.\u003c/p\u003e\n\u003cp\u003eDue to alterations of any component of Wnt/\u0026beta;-catenin signaling pathway, it is frequently activated in various cancers, including colorectal cancer, hepatocellular carcinoma and pancreatic cancer [12-14]. In brief, Wnt ligand binds to Frizzled receptor family, leading to inactivation of adenomatous polyposis coli (APC) or axin, which in turn promotes the cytoplasmic accumulation and nuclear localization of \u0026beta;-catenin. \u0026beta;-catenin can also be regulated by other factors to promote cancer development. For instance, TYRO3 promotes the prolifieration and metastasis of gastric cancer cells through activation of Wnt/\u0026beta;-catenin signaling pathway [15]. LncCCAT1 enhances the functions of breast cancer stem cell via activating WNT/\u0026beta;-catenin signaling [16]. \u0026beta;-catenin is a transcriptional factor that interacts with TCF/LEF factors to activate the expression of target genes. The well-known downstream targets for \u0026beta;-catenin are c-myc and cyclin D1 [17, 18]. However, the correlation between ANP32E and \u0026beta;-catenin is unclear in pancreatic cancer. Here, our molecular experiments showed that ANP32E up-regulated \u0026beta;-catenin and its downstream target cyclin D1. We also checked other oncogenes, such as PKM2 and HK2, or tumor suppressors, including P53 and PTEN. No difference was observed after knocking down ANP32E. We also analyzed the role of \u0026beta;-catenin in ANP32E over-expressed cells. The results showed that knockdown of \u0026beta;-catenin reduced the viability of pancreatic cancer cells with highly expressed ANP32E. These results reveal that ANP32E promotes pancreatic cancer through activating \u0026beta;-catenin.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe demonstrate that ANP32E is an oncogene in pancreatic cancer. ANP32E promotes the proliferation and migration through up-regulation and activation of \u0026beta;-catenin/cyclin D1 signaling. Up-regulation of ANP32E in PAAD tissues and the inverse relationship between ANP32E expression and patients\u0026rsquo; survival indicate that ANP32E maybe a diagnosed biomarker for PAAD. Our findings suggest that ANP32E is a promising therapeutic target for PAAD.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANP32E, Acidic nuclear phosphoprotein 32 family member E; PDAC, pancreatic ductal adenocarcinoma; TCGA, The Cancer Genome Atlas; DMEM, Dulbecco modified Eagle\u0026rsquo;s medium; FBS, fetal bovine serum; qRT-PCR, quantitative real-time polymerase chain reaction; APC, adenomatous polyposis coli.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval is not required for the use of the cells in our study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo consent is required in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was financially supported by National High Technology Research and Development Program of China (2012AA022701) and CAMS Initiation Fund for Mediacal Sciences (2016-I2M-1-001). The funders mentioned were used for purchasing the experiments materials but not for study design and data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJWZ, JH and CFW designed the study. JWZ, ZML, GTQ and HR performed the experiments. JWZ, YJZ, ZTG, ZZL, LF and CFW analyzed the data. JWZ, JH and CFW wrote the manuscript draft and revised the draft. All authors have read and approved the manuscript\u003cstrong\u003e\u003cbr /\u003e Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRL S, KD M, A J: \u003cstrong\u003eCancer statistics, 2015\u003c/strong\u003e. \u003cem\u003eCA Cancer J Clin \u003c/em\u003e2015, \u003cstrong\u003e65\u003c/strong\u003e(1):5-29.\u003c/li\u003e\n\u003cli\u003eJ K, M K, M A, C LV, CD J, AV B, RE N, M T, DA T, RH H\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003ePancreatic cancer\u003c/strong\u003e. \u003cem\u003eNature reviews Disease primers \u003c/em\u003e2016, \u003cstrong\u003e2\u003c/strong\u003e:16022.\u003c/li\u003e\n\u003cli\u003eMJ C, JR W, F G, R S, DJ T: \u003cstrong\u003eRegions of variant histone His2AvD required for Drosophila development\u003c/strong\u003e. \u003cem\u003eNature \u003c/em\u003e1999, \u003cstrong\u003e399\u003c/strong\u003e(6737):694-697.\u003c/li\u003e\n\u003cli\u003eFaast R, Thonglairoam V, Schulz TC, Beall J, Wells JR, Taylor H, Matthaei K, Rathjen PD, Tremethick DJ, Lyons I: \u003cstrong\u003eHistone variant H2A.Z is required for early mammalian development\u003c/strong\u003e. \u003cem\u003eCurr Biol \u003c/em\u003e2001, \u003cstrong\u003e11\u003c/strong\u003e(15):1183-1187.\u003c/li\u003e\n\u003cli\u003eFarris SD, Rubio ED, Moon JJ, Gombert WM, Nelson BH, Krumm A: \u003cstrong\u003eTranscription-induced chromatin remodeling at the c-myc gene involves the local exchange of histone H2A.Z\u003c/strong\u003e. \u003cem\u003eJ Biol Chem \u003c/em\u003e2005, \u003cstrong\u003e280\u003c/strong\u003e(26):25298-25303.\u003c/li\u003e\n\u003cli\u003eMao Z, Pan L, Wang W, Sun J, Shan S, Dong Q, Liang X, Dai L, Ding X, Chen S\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eAnp32e, a higher eukaryotic histone chaperone directs preferential recognition for H2A.Z\u003c/strong\u003e. \u003cem\u003eCell Res \u003c/em\u003e2014, \u003cstrong\u003e24\u003c/strong\u003e(4):389-399.\u003c/li\u003e\n\u003cli\u003eJiang M, Ma Y, Ni X, Cao G, Ji C, Cheng H, Tang R, Xie Y, Mao Y: \u003cstrong\u003eMolecular cloning and characterization of a novel human gene (ANP32E alias LANPL) from human fetal brain\u003c/strong\u003e. \u003cem\u003eCytogenet Genome Res \u003c/em\u003e2002, \u003cstrong\u003e97\u003c/strong\u003e(1-2):68-71.\u003c/li\u003e\n\u003cli\u003eRadrizzani M, Vila-Ortiz G, Cafferata EG, Di Tella MC, Gonzalez-Guerrico A, Perandones C, Pivetta OH, Carminatti H, Idoyaga Vargas VP, Santa-Coloma TA: \u003cstrong\u003eDifferential expression of CPD1 during postnatal development in the mouse cerebellum\u003c/strong\u003e. \u003cem\u003eBrain Res \u003c/em\u003e2001, \u003cstrong\u003e907\u003c/strong\u003e(1-2):162-174.\u003c/li\u003e\n\u003cli\u003eLi P, Xu T, Zhou X, Liao L, Pang G, Luo W, Han L, Zhang J, Luo X, Xie X\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eDownregulation of miRNA-141 in breast cancer cells is associated with cell migration and invasion: involvement of ANP32E targeting\u003c/strong\u003e. \u003cem\u003eCancer Med \u003c/em\u003e2017, \u003cstrong\u003e6\u003c/strong\u003e(3):662-672.\u003c/li\u003e\n\u003cli\u003eXiong Z, Ye L, Zhenyu H, Li F, Xiong Y, Lin C, Wu X, Deng G, Shi W, Song L\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eANP32E induces tumorigenesis of triple-negative breast cancer cells by upregulating E2F1\u003c/strong\u003e. \u003cem\u003eMol Oncol \u003c/em\u003e2018, \u003cstrong\u003e12\u003c/strong\u003e(6):896-912.\u003c/li\u003e\n\u003cli\u003eGursoy-Yuzugullu O, Ayrapetov MK, Price BD: \u003cstrong\u003eHistone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair\u003c/strong\u003e. \u003cem\u003eProc Natl Acad Sci U S A \u003c/em\u003e2015, \u003cstrong\u003e112\u003c/strong\u003e(24):7507-7512.\u003c/li\u003e\n\u003cli\u003eFodde R, Smits R, Clevers H: \u003cstrong\u003eAPC, Signal transduction and genetic instability in colorectal cancer\u003c/strong\u003e. \u003cem\u003eNature Reviews Cancer \u003c/em\u003e2001, \u003cstrong\u003e1\u003c/strong\u003e(1):55-67.\u003c/li\u003e\n\u003cli\u003eKlaus A, Birchmeier W: \u003cstrong\u003eWnt signalling and its impact on development and cancer\u003c/strong\u003e. \u003cem\u003eNature Reviews Cancer \u003c/em\u003e2008, \u003cstrong\u003e8\u003c/strong\u003e(5):387-398.\u003c/li\u003e\n\u003cli\u003eMorris JP, Wang SC, Hebrok M: \u003cstrong\u003eKRAS, Hedgehog, Wnt and the twisted developmental biology of pancreatic ductal adenocarcinoma\u003c/strong\u003e. \u003cem\u003eNature Reviews Cancer \u003c/em\u003e2010, \u003cstrong\u003e10\u003c/strong\u003e(10):683-695.\u003c/li\u003e\n\u003cli\u003eD C, Q L, G C, W Z: \u003cstrong\u003eTYRO3 facilitates cell growth and metastasis via activation of the Wnt/\u0026beta;-catenin signaling pathway in human gastric cancer cells\u003c/strong\u003e. \u003cem\u003eAging \u003c/em\u003e2020, \u003cstrong\u003e12\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eT T, C G, T X, R Z, K Z, Y P, L J: \u003cstrong\u003eLncCCAT1 Promotes Breast Cancer Stem Cell Function through Activating WNT/\u0026beta;-catenin Signaling\u003c/strong\u003e. \u003cem\u003eTheranostics \u003c/em\u003e2019, \u003cstrong\u003e9\u003c/strong\u003e(24):7384-7402.\u003c/li\u003e\n\u003cli\u003eN Z, X C: \u003cstrong\u003eA positive feedback loop involving the LINC00346/\u0026beta;-catenin/MYC axis promotes hepatocellular carcinoma development\u003c/strong\u003e. \u003cem\u003eCell Oncol (Dordr) \u003c/em\u003e2020, \u003cstrong\u003e43\u003c/strong\u003e(1):137-153.\u003c/li\u003e\n\u003cli\u003eS G, A G, S X, Z Y, S N, Y Z, D P: \u003cstrong\u003ePSAT1 is regulated by\u0026nbsp;ATF4 and enhances cell proliferation via the GSK3\u0026beta;/\u0026beta;-catenin/cyclin D1 signaling pathway in ER-negative breast cancer\u003c/strong\u003e. \u003cem\u003eJournal of experimental \u0026amp; clinical cancer research : CR \u003c/em\u003e2017, \u003cstrong\u003e36\u003c/strong\u003e(1):179.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"pancreatic cancer, ANP32E, proliferation, migration, β-catenin","lastPublishedDoi":"10.21203/rs.3.rs-27702/v3","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-27702/v3","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Background: Pancreatic cancer is a malignant tumor with high mortality. Acidic nuclear phosphoprotein 32 family member E (ANP32E), a specific H2A.Z chaperone, has been shown to contribute to breast cancer development. However, the significance of ANP32E in pancreatic cancer is poorly understood. This study aimed to investigate the role of ANP32E in pancreatic cancer.\nMethods: The expression of ANP32E in 179 pancreatic cancer tissues and 171 normal tissues, and the correlation between ANP32E expression and patients’ survival were analyzed from the TCGA database. ANP32E was over-expressed and silenced using lentivirus. siRNA was used to knock down β-catenin. CCK8, colony formation, cell cycle and transwell experiments were performed to determine cell proliferation and migration. qRT-PCR and Western blot were conducted to detect mRNA and protein expression.\nResults: ANP32E was up-regulated in pancreatic cancer tissues and cells. Up-regulation of ANP32E predicted poor prognosis in pancreatic cancer patients. Lentivirus-mediated knockdown of ANP32E suppressed the proliferation, colony growth and migration of PANC1 and MIA cells. By contrast, ANP32E over-expression promoted the proliferation and migration of both cells. In addition, ANP32E accelerated the cell cycle progression in PANC1 and MIA cells. Molecular experiments showed that ANP32E activated β-catenin/cyclin D1 signaling. Silencing of β-catenin reduced cell proliferation and migration in ANP32E over-expressed cells.\nConclusion: Our results propose that ANP32E functions as an oncogene in pancreatic cancer via activating β-catenin.","manuscriptTitle":"Over-expression of ANP32E is associated with poor prognosis of pancreatic cancer and promotes cell proliferation and migration through regulating β-catenin","msid":"","msnumber":"","nonDraftVersions":[{"code":3,"date":"2020-10-26 20:20:59","doi":"10.21203/rs.3.rs-27702/v3","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2020-10-21T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-10-19T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-10-18T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-10-18T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":2,"date":"2020-08-27 18:03:13","doi":"10.21203/rs.3.rs-27702/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2020-09-22T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-09-14T12:00:00+00:00","index":1,"fulltext":"Recommendation: Accept without revision\nForm responses:\n---\n\nComments to Author:\n---\nThe authors have satisfactorily responded to all my questions and made the necessary changes to the manuscript. The revised version of the manuscript appears to be good.* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **Yes**\n* Declaration of competing interests: **I declare that I have no competing interests.**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **Yes**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **Yes**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"reviewerAgreed","content":"","date":"2020-09-08T12:00:00+00:00","index":2,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-09-08T12:00:00+00:00","index":2,"fulltext":"Recommendation: Accept without revision\nForm responses:\n---\n\nComments to Author:\n---\nI believe after all corrections were performed, the article is suitable for publication.* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **Yes**\n* Declaration of competing interests: **I declare that I have no competing interests'**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **Yes**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **Yes**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"reviewerAgreed","content":"","date":"2020-09-04T12:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-09-03T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-08-23T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-08-22T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-08-22T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2020-06-29 13:01:05","doi":"10.21203/rs.3.rs-27702/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2020-08-07T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-08-06T12:00:00+00:00","index":3,"fulltext":"Recommendation: Accept after minor essential revisions\nForm responses:\n---\n\nComments to Author:\n---\nComments to the authors:\n\nIn the present manuscript, the authors found that ANP32E is overexpressed in pancreatic adenocarcinoma (PDAC) in comparison to normal tissue and its levels correlates with poor disease-free and overall survival. At cellular level, the authors demonstrated that ANP32E positively regulates proliferation, clonogenicity and migration probably through beta-catenin, suggesting that ANP32E acts an oncogene in this tumor. The manuscript presents novel data; however, some major points need to be addressed as mentioned bellow.\n\nMajor comments:\n- Regarding methodology, siRNA, primers and antibody concentrations should be mentioned in the methods section. It would be also desirable to mention the amount of protein loaded onto SDS-PAGE. Regarding the RT-qPCR assays, did the authors test primer efficiency? Moreover, it is highly desirable to use more than one endogenous gene for gene expression analysis in tumor cells. In addition, the authors should provide more information about the cell lines. Are they derived exactly from pancreatic adenocarcinoma tumors?\n\n-In Fig 1, the authors should indicate clearly in the histograms which one refers to normal samples and which one refers to tumor samples. B shows the overall survival (instead of disease-free survival which is shown in C). The authors should describe which color refers to high levels and low levels of ANP32E. In C, what do the other dotted lines represent?\n\n- Regarding Fig 2, it could be presented as a supplementary figure since it shows that both techniques worked well and does not present new data.\n\n- In Fig 3A and B, y axis means cell proliferation or viability? Cell viability and proliferation are two different parameters and the authors used these terms interchangeably. The authors should clarify this point. The increase in cell viability along time is linear with proliferation ratio? In C, it is not possible to visualize the colonies.\n\n- Regarding the results presented in Fig5, which software was used for cell cycle analysis? Were cell doublets gated out? The authors stated that \"Ectopic expression of ANP32E led to reduced G0 phase and increased S and G2/M 191 phase (Fig. 5A and 5B). These results indicate that ANP32E promotes cell cycle progression\". However, it could be also a result from cell cycle delay or arrest at S or G2/M phases and the suggestion that ANP32E induces proliferation is evidenced by the proliferative assay instead. Did the authors analyze the cell cycle distribution in silenced cells? It would be interesting to verify whether there is any difference regarding cell cycle phases distribution caused by reduced levels of ANP32E.\n\n- In Fig 6, why did the authors decide to analyze beta-catenin and cyclin D1 in particular? Did the authors check beta-catenin subcellular localization under ASNP32E overexpression and silencing conditions? In fact, it would reinforce the suggestion that beta-catenin mediates ASPN32E oncogenic action in pancreatic tumor cells and should be considered by the authors. Moreover, are beta-catenin levels also increased in PDAC? It is not clear whether beta-catenin silencing was performed in ASNP32-overexpressing cells as mentioned in the text. In Fig6 legend, the authors mention \"ANC1 cells with highly expressed ANP32E\". What does it mean exactly?\n\n- In the discussion, the authors mentioned that ANP32 regulated apoptosis in pancreatic tumor cells. Did the authors evaluate apoptosis? I did not find any results showing this.\n\n- The authors concluded that \"ANP32E maybe a diagnosed biomarker for PAAD\". If ANP32E is also expressed by normal cells/tissue, how it can be a good diagnostic biomarker?\n\n- In general, legends could be more informative, including the duration of the experiment and sample size. How many times each experiment was repeated?\n\nMinor comments:\nThere are few typo errors.\n* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **Yes**\n* Declaration of competing interests: **I declare that I have no competing interests.**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **Yes**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **No**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"editorInvitedReview","content":"","date":"2020-08-05T12:00:00+00:00","index":2,"fulltext":"Recommendation: Accept after minor essential revisions\nForm responses:\n---\n\nComments to Author:\n---\nThe present study aimed to investigate the clinical relevance and function of ANP32E in pancreatic cancer. I believe the authors managed to show relevant data to confirm the objectives and explain the results. Some points should be addressd:\n\n\n- The abstract should be revised and improved. I believe it is a bit limited.\n- Please check the figures again and legends in order to match the journal guidelines.\n- The letters that describe the images are variable between figures, please correct.\n- Check the text for grammar and typo errors* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **Yes**\n* Declaration of competing interests: **'I declare that I have no competing interests'**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **Yes**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **Yes**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"editorInvitedReview","content":"","date":"2020-07-28T12:00:00+00:00","index":1,"fulltext":"Recommendation: Accept without revision\nForm responses:\n---\n\nComments to Author:\n---\n\nThis manuscript is focused on investigating the role of ANP32E in pancreatic cancer. Results are indicating that ANP32E overexpressed in pancreatic cancer tissues with poor survival. Knockdown of ANP32E decreased proliferation, colony growth, and migration pancreatic cancer cells, and the overexpression of ANP32E increased these functions. They identified that ANP32E induce the β-catenin/cyclin D1 signaling in pancreatic cancer cells. The manuscript is well-written, and it concludes that ANP32E plays an oncogenic role in pancreatic cancer via the β-catenin mechanism. The revised manuscript is acceptable for publication.\n* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **Yes**\n* Declaration of competing interests: **No competing intrest**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **Yes**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **Yes**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"reviewerAgreed","content":"","date":"2020-07-16T12:00:00+00:00","index":3,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-07-14T12:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-07-07T12:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2020-07-02T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-07-02T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-06-26T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-06-26T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"100b1f8a-31f3-469e-bdc8-94af6c0c9b97","owner":[],"postedDate":"October 26th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":378265,"name":"Cancer Biology"},{"id":378266,"name":"Oncology"}],"tags":[],"updatedAt":"2020-11-08T15:02:52+00:00","versionOfRecord":{"articleIdentity":"rs-27702","link":"https://doi.org/10.1186/s12885-020-07556-z","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2020-11-04 15:01:33","publishedOnDateReadable":"November 4th, 2020"},"versionCreatedAt":"2020-10-26 20:20:59","video":"","vorDoi":"10.1186/s12885-020-07556-z","vorDoiUrl":"https://doi.org/10.1186/s12885-020-07556-z","workflowStages":[]},"version":"v3","identity":"rs-27702","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-27702","identity":"rs-27702","version":["v3"]},"buildId":"i_I_jhV-QviBjPLXJga9V","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}