EBF1-Mediated Upregulation of Ribosome Assembly Factor PNO1 Contributes to Cancer Progression by Negatively Regulating the P53 S

Published OnlineFirst March 12, 2019; DOI: 10.1158/0008-5472.CAN-18-3238

Cancer Molecular Cell Biology Research

EBF1-Mediated Upregulation of Ribosome Assembly Factor PNO1 Contributes to Cancer Progression by Negatively Regulating the p53 Signaling Pathway Aling Shen1,2, Youqin Chen1,2,3, Liya Liu1,2,3, Yue Huang1,2, Hongwei Chen1,2,FeiQi1,2, Jiumao Lin1,2, Zhiqing Shen1,2, Xiangyan Wu1,2, Meizhu Wu1,2, Qiongyu Li1,2, Liman Qiu1,2, Na Yu1,2, Thomas J. Sferra3, and Jun Peng1,2

Abstract

The RNA-binding protein PNO1 is critical for ribosome stress and inhibiting MDM2-mediated ubiquitination and biogenesis, but its potential role in cancer remains unknown. p53 degradation. Overexpressing EBF1 suppressed PNO1 pro- In this study, online data mining, cDNA, and tissue micro- moter activity and decreased PNO1 mRNA and protein, inhi- arrays indicated that PNO1 expression was higher in colorectal biting cell proliferation and inducing cell apoptosis through cancer tissue than in noncancerous tissue, and its overexpres- the p53/p21 pathway. In colorectal cancer tissues, the expression was associated with worse patient survival. Gain-of- sion of EBF1 correlated inversely with PNO1. Data mining of function and loss-of-function studies demonstrated that online breast and lung cancer databases showed increased PNO1 knockdown suppressed growth of colorectal cancer PNO1 expression and association with poor patient survival; cells in vitro and in vivo, while PNO1 overexpression promoted PNO1 knockdown reduced cell viability of cultured breast and colorectal cancer cell proliferation in vitro. In colorectal cancer lung cancer cells. Taken together, these findings indicate that cells expressing wild-type p53, PNO1 knockdown enhanced PNO1 is overexpressed in colorectal cancer and correlates with expression of p53 and its downstream gene p21, and reduced poor patient survival, and that PNO1 exerts oncogenic effects, cell viability; these effects were prevented by p53 knockout and at least, in part, by altering ribosome biogenesis. attenuated by the p53 inhibitor PFT-a. Moreover, PNO1 knockdown in HCT116 cells decreased levels of 18S rRNA, Significance: This study identifies the ribosome assembly of 40S and 60S ribosomal subunits, and of the 80S ribosome. factor PNO1 as a potential oncogene involved in tumor It also reduced global protein synthesis, increasing nuclear growth and progression of colorectal cancer.

Introduction diagnostic and prognostic biomarkers. In this study, cDNA microarray analysis of paired cancerous and noncancerous tissues from Colorectal cancer is the third most common cancer and fourth patients with colorectal cancer was followed by high-content leading cause of cancer-related death worldwide, with 1.2 million screening using lentivirus-delivered short hairpin (sh)RNA inter- new cases and over 600,000 deaths each year (1, 2). Despite recent ference in colorectal cancer cells. This led to the ﬁnding that progress in treatment, outcomes in colorectal cancer remain poor. ribosome assembly factor PNO1 was overexpressed in cancer Therefore, a better understanding of the molecular mechanisms of tissues, while PNO1 knockdown inhibited the growth of colo- colorectal cancer is urgently required, as is the discovery of new rectal cancer cells. The ribosome is a supramolecular ribonucleoprotein complex responsible for translating mRNA into proteins. Ribosome bio- 1Academy of Integrative Medicine, Fujian University of Traditional Chinese genesis is a complicated, well-orchestrated process that involves 2 Medicine, Fuzhou, Fujian, China. Fujian Key Laboratory of Integrative Medicine the transcription and processing of ribosomal RNAs, the produc- in Geriatrics, Fujian University of Traditional Chinese Medicine, Fujian, China. tion of ribosomal proteins, as well as the assembly and nuclear 3Department of Pediatrics, Rainbow Babies and Children's Hospital, Case Western Reserve University School of Medicine, Cleveland, Ohio. export of ribosome subunits. In eukaryotes, ribosome biogenesis is facilitated by the coordinated function of over 200 assembly Note: Supplementary data for this article are available at Cancer Research factors including helicases, ATPases, GTPases, and kinases, which Online (http://cancerres.aacrjournals.org/). join and are released from preribosomal particles at different A. Shen and Y. Chen contributed equally to this article. times during ribosome maturation. Because ribosome biogenesis Corresponding Author: Jun Peng, Academy of Integrative Medicine, Fujian determines the capacity of a cell to synthesize proteins and hence University of Traditional Chinese Medicine, 1 Huatuo Road, Minhou Shangjie, plays a crucial role in cell growth and proliferation, dysregulation Fuzhou, Fujian 350122, China. Phone: 8659-1228-61303; Fax: 8659-1228-61157; of this vital process is associated with many diseases including E-mail: [email protected] cancer (3–5). doi: 10.1158/0008-5472.CAN-18-3238 Cancer cells are characterized by an uncontrolled increase in 2019 American Association for Cancer Research. cell proliferation (6), which requires extensive protein synthesis

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and thus increased ribosome biogenesis (7). In several types examined. Potential roles of PNO1 in breast and lung cancers of human malignancies, overexpression of specific ribosomal were also examined. proteins and ribosome assembly factors results in increased ribosome biogenesis and is associated with poor prognosis Patients and specimens (8–16). If upregulated ribosome biogenesis plays an important Fifty pairs of cancerous and matched noncancerous tissues (at role in malignant transformation by promoting cancer cell pro- least 5-cm away) were obtained from patients with colorectal liferation, then defects in ribosome assembly can cause cell-cycle cancer who underwent surgical resection between 2013 and 2014 arrest, inhibit cell proliferation, and induce cell apoptosis, ulti- at Fujian Provincial Hospital and the First People's Hospital mately suppressing tumor growth (17–21). Affiliated to Fujian University of Traditional Chinese Medicine In this way, a promising anticancer strategy may be to suppress (Fujian, China). Clinicopathologic characteristics of patients are ribosome biogenesis by targeting ribosome assembly factors. The summarized in Supplementary Table S1. The specimens were RNA-binding protein "partner of NOB1" (PNO1), also known as used with written informed consent from the patients and approv- Dim2 or Rrp2, is a ribosome neogenesis factor highly conserved al by the local Ethics Committees. No patients received radio- or from yeast to mammals. The human PNO1 gene is located on chemotherapy prior to surgery. chromosome 2q14 and includes seven exons (22). The full-length Samples were processed using routine methods for IHC, or they cDNA sequence of PNO1 (1637 bp) contains a 759-bp open were snap-frozen and stored in liquid nitrogen for other use. reading frame encoding a protein of 252 or 248 residues, which Samples stained with hematoxylin and eosin were examined by contains a conserved C-terminal K homolog (KH) domain experienced pathologists using the pathologic tumor–node– responsible for RNA binding (23, 24). Previous studies in yeast metastasis (p-TNM) classification of the International Union have revealed that PNO1 participates in ribosome biogenesis. Against Cancer. PNO1 is one of six assembly factors required for cytoplasmic maturation of the 20S pre-rRNA to 18S rRNA; it binds to NOB1 Microarray analysis and increases NOB1 affinity for RNA, stimulating NOB1 to cleave Microarray assays were performed to identify DEGs between at the 30 end of pre-18S rRNA. Loss of PNO1 results in a decrease of colorectal cancer tissues (n ¼ 14) and matched adjacent normal 18S rRNA and defective assembly of pre-40S ribosomal subu- tissues (n ¼ 14), as well as between HCT116 cells transduced with nits (25–27). lentivirus encoding anti-PNO1 shRNA (n ¼ 3) or control shRNA In contrast to extensive studies of the yeast homolog of PNO1 (n ¼ 3). These assays were carried out by Shanghai GeneChem or called Yor145, little is known about the functions of PNO1 in CapitalBio. Briefly, total RNA was extracted with TRIzol reagent mammalian cells. In particular, a role in cancer progression has (Thermo Fisher Scientific). Complementary DNA was synthe- never been reported. Therefore in this study, we applied a com- sized, labeled, and hybridized to the human GeneChip Primeview bination of high-throughput "omics" technologies, online data array (Affymetrix). Scanning was conducted using a GeneChip mining, biochemistry, and molecular biology to evaluate PNO1 Scanner 3000 and analyzed using GeneChip GCOS 1.4 software expression in human cancers, its association with cancer progno- (Affymetrix). Genes were classified as DEGs if their expression sis, as well as its potential oncogenic activity and the underlying differed at least 2-fold between the two conditions, and if the mechanisms. difference was associated with P < 0.05. DEGs were identified using volcano plots and hierarchical clustering plots. Kyoto Ency- clopedia of Genes and Genomes (KEGG) pathway enrichment analysis was used to identify pathways represented among the Materials and Methods DEGs. Summary of the experimental design A cDNA microarray was used to identify differentially Cell culture expressed genes (DEG) in paired cancerous and noncancerous The following cell lines were obtained from the Cell Bank of the tissues from patients with colorectal cancer. Cell-based high- Chinese Academy of Sciences (Shanghai, China): human colo- content screening and shRNA-mediated knockdown led to rectal cancer lines RKO, HCT-8, HT-29, HCT116, and Caco2; lung identification of PNO1. Prognostic value of PNO1 was ana- carcinoma cell line A549; breast cancer cell line MCF-7; and lyzed in both clinical colorectal cancer samples and online embryonic kidney HEK293T cells. Wild-type HCT116 cells þ þ databases. Effects of PNO1 on tumor growth in vitro and (HCT116/p53 / ) and HCT116 cells lacking the p53 gene in vivo, as well as on cell proliferation and cell apoptosis, were (HCT116/p53 / ) were a gift from Dr. Yao Lin (Fujian Normal assessed in colorectal cancer cells after PNO1 knockdown University, Fujian, China), which were originally obtained from and/or overexpression. A cDNA microarray was used after Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD). þ þ PNO1 knockdown to identify downstream regulatory mechan- RKO, HCT116, HCT-8, HCT116/p53 / , and HCT116/p53 / isms. Bioinformatics analysis of DEGs identified the p53/p21 cells were maintained in RPMI1640 (Thermo Fisher Scientific); pathway as one of the most enriched signaling pathways. HT-29 cells, in M50A medium (KeyGEN); Caco2, MCF-7, and Effects of p53/p21 signaling on oncogenic activities of PNO1 A549 cells in DMEM (Thermo Fisher Scientific); and HEK293T were investigated in HCT116 cells that were exposed to a p53 cells in MEM (Thermo Fisher Scientific). All media contained inhibitor or in which the p53 gene was deleted. The putative 10% FBS (Thermo Fisher Scientific), 100 U/mL penicillin, and mechanism by which PNO1 knockdown activated p53 signal- 100 mg/mL streptomycin (Hyclone). Cells were cultured at 37C ing was evaluated in colorectal cancer cells by examining in a humidified atmosphere of 5% CO2. For long-term use of ribosome biogenesis and protein synthesis, nuclear morpho- HCT116 and RKO cells, cells were verified using short tandem logy, and levels of p53 in nucleus, as well as ubiquitination repeat genotyping and examined for Mycoplasma contamination and degradation of p53. Regulation of PNO1 by EBF1 was using RT-PCR analysis.

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Lentiviral transduction IHC staining Three shRNAs for each gene were cloned into the GV115 Tissue sections were incubated with an anti-PNO1 antibody lentiviral vector (Shanghai GeneChem), which encoded (1:800; catalog no. LS-C179090, LSBio). Background staining was enhanced GFP (EGFP) under control of the CMV promoter. assessed by omitting the primary antibody. The intensity and The double-stranded shRNAs targeting each gene are summarized percentage of positive cells from five fields in each sample were in Supplementary Table S2. Lentivirus encoding shRNA against determined independently by two experienced pathologists a target gene or a nonsilencing control shRNA was added to blinded to the clinical and pathologic data. Staining intensity cultured cells at a multiplicity of infection (MOI) of 10, as was assessed using a 4-point scale (0, undetectable; 1, weak; 2, recommended by the manufacturer. To generate a cell line stably moderate; 3, strong). Percentage of positively stained cells overexpressing PNO1, cultures of HT-29 and HCT-8 cells were was expressed as one of four categories: 1, 0%–25% cells stained; transduced for 72 hours with a lentiviral vector (MOI: 10) encod- 2, 26%–50% stained; 3, 51%–75% stained; 4, 76%–100% ing full-length human PNO1 (coding region of 756 bp; Shanghai stained (28). PNO1 expression was calculated by multiplying the GeneChem). Transductants were selected for 2 weeks using puro- intensity and percentage scores together. mycin (Thermo Fisher Scientific) at 1.5 mg/mL for HCT-8 cells or 1 mg/mL for HT-29 cells. In experiments to examine effects of EBF1 Tissue microarray and survival analysis on expression of PNO1 and its downstream targets, and on cell Tissue microarray (TMA) slides (Shanghai Outdo Biotech) proliferation and apoptosis, RKO cells were transduced for contained 90 pairs of tissue samples and were hybridized with 72 hours with a lentiviral vector (MOI: 10) encoding full-length primary antibody against PNO1 (1:500) using standard techni- human EBF1 (coding region of 1776 bp; Shanghai GeneChem). ques (29). The 90 pairs of specimens were obtained from Taizhou Transductants were selected for 2 weeks using 1 mg/mL of Hospital of Zhejiang Province (Zhejiang, China) from July 2007 puromycin. to October 2008; all subjects had reliable information on survival, and there was no data censoring prior to 7 years of follow-up. High-content screening for cell growth Images were captured using a Nano Zoomer 2.0 HT slide Growth of cultured cells was assessed using multiparametric scanner (Hamamatsu Photonics) and processed using Nano high-content screening. At 72 hours after shRNA transduction, Zoomer Digital Pathology View 1.6 software. IHC score was cells were seeded into 96-well plates at a density of 2,000 cells/ determined independently by two experienced pathologists well in 100 mL of medium. Cell growth was monitored every day blinded to the clinical and pathologic data. PNO1 expression for 5 days using the Cellomics ArrayScanV(TI) high-content image was scored as described in the "IHC staining" section. PNO1 analysis platform and analyzed using HCS Studio Cell Analysis expression was considered high for scores of 4–12 and low for Software (Thermo Fisher Scientific). scores of 0–3. The relationship between PNO1 expression (low or high) and patient overall survival was analyzed using Kaplan– Quantitative real-time PCR and tissue cDNA array analysis Meier analysis and assessed for significance using the log-rank test. Total RNA was extracted from the cell line samples or tissue samples using RNAiso Plus reagent (Takara). Reverse transcrip- Cell transfection tion was performed using the PrimeScript RT reagent kit (Takara). Three nonoverlapping anti-PNO1 siRNA oligonucleotides (si- The resulting cDNA, or a commercial tissue cDNA array (Shanghai PNO1, 25-mer Stealth RNAi duplexes) and control siRNAs (si- Outdo Biotech), was used to examine levels of mRNAs encoding Ctrl) were designed using the BLOCK-iT RNAi design program PNO1, CDKN1A/p21, THBS1, MAPK1, MCM2, CDC42, EBF1, or (Thermo Fisher Scientific; Supplementary Table S2). Cells were GAPDH using an ABI 7500 Fast Real-Time PCR System (Applied transfected with siRNAs or si-Ctrl at a concentration of 10 nmol/L Biosystems) and the SYBR Premix Ex Tag (Takara). The conditions using Lipofectamine RNAiMax (Thermo Fisher Scientific). for real-time PCR were as follows: predenaturation (95C for 10 minutes), denaturation (95C for 15 seconds), annealing and Cell proliferation assay extension (60C for 60 seconds) for a total 40 cycles. GAPDH was Cell proliferation was determined using CFDA or CCK-8 assays. used as an internal control. Primer sequences are shown in In the CFDA assay, cells were transfected with anti-PNO1 or DDC Supplementary Table S3. mRNA levels are presented as: 2 t control siRNA for 24–72 hours, washed with PBS, and incubated (with Ct being the cycle threshold), where DCt ¼ [Ct (target gene) in fresh culture medium until the indicated time points, when 200 Ct (GAPDH)]. mL CFDA (25 mmol/L; Thermo Fisher Scientific) was added to each well. Plates were incubated for an additional 2 hours at 37Cin Western blot analysis the dark. Fluorescence intensity was measured at 480 nm using a Proteins were extracted using RIPA lysis buffer (Thermo Fisher microplate reader (Tecan). In the CCK-8 assay, cells were trans- Scientific), separated by SDS-PAGE, transferred onto a nitrocel- fected with siRNA or transduced with lentivirus and cultured for lulose membrane, and then blocked with a blocking buffer the indicated duration, when 10 mL CCK-8 (Cell Counting Kit-8, (Thermo Fisher Scientific) prior to overnight incubation at 4C Dojindo) was added to each well. Plates were incubated for with a primary antibody (Supplementary Table S4). Membranes an additional 2 hours at 37C and absorbance was measured at were washed extensively and incubated with a horseradish per- 450 nm. oxidase–conjugated goat anti-rabbit or anti-mouse secondary antibody. The blots were visualized using a chemiluminescence Colony-forming assay method (Thermo Fisher Scientific), and band intensities were For cell survival analysis, transduced cells were seeded into quantified relative to intensity of b-actin using ImageJ software. 12-well plates (500 cells/well) and incubated in humidified air Levels of target protein were expressed relative to levels in control containing 5% CO2 at 37 C for 10–14 days to allow colony cells, defined as 1.00. formation. Medium was replaced every 2–3 days. Cells were

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washed with PBS, fixed with 4% paraformaldehyde, and stained under the control of a p53-driven promoter. Quantitation of with crystal violet. After staining, photographs were taken and luciferase activity allowed assessment of promoter activity. Briefly, numbers of colonies counted. Data were normalized to results for cells were transduced for 48 hours, washed twice with PBS, and control cells. harvested with 1 Passive Lysis Buffer from the Dual-Luciferase For cell colony formation in agarose, transduced cells were Reporter Assay System (Promega). Cells were lysed and centri- suspended in medium containing 0.35% low-melting-point aga- fuged, and the supernatant was collected. Aliquots of supernatant rose (Thermo Fisher Scientific), and 500 cells were seeded in plates (40 mL) were added to 96-well plates, followed by 20 mL luciferase containing medium with 0.6% solidified agarose. After incuba- assay reagent (Promega) at room temperature. Luciferase activity tion for 2–3 weeks, photographs were taken and numbers of was measured immediately using a luminometer (Orion II Micro- colonies counted. Three independent experiments were per- plate Luminometer, Berthold Detection Systems). Data were formed, and data were normalized to results for control cells. normalized to the results obtained for the internal control Renilla luciferase. Cell-cycle analysis To examine whether the 10 predicted transcription factors Cells were fixed with 70% ethanol at 4C overnight, washed indeed modulated PNO1 transcription, plasmids were con- extensively, and incubated with FxCycle PI/RNase Staining Solu- structed encoding the full-length predicted factors in the GV141 tion (Thermo Fisher Scientific) for 30 minutes. DNA content was vector (Shanghai GeneChem), which contains a multiple cloning analyzed by FACS (FACSCalibur, Becton Dickinson). The pro- site followed by a 3FLAG tag downstream of the CMV promoter, portion of DNA in different phases was analyzed using ModfitLT as well as the neomycin gene downstream of the SV40 promoter. A version 3.0 (Verity Software House). separate plasmid was constructed from vector GV238 (Shanghai GeneChem) encoding luciferase reporter downstream of a 2.0-kb Apoptosis analysis fragment of the PNO1 promoter. Plasmid integrity was confirmed Cells were washed with ice-cold PBS followed by binding by DNA sequencing. Briefly, HEK293T or RKO cells were cotrans- buffer, and then stained for 15 minutes with Annexin V-APC fected with each of 10 different overexpression plasmids or (KeyGEN). The percentage of apoptosis was analyzed by FACS EBF1 overexpression plasmid (500 ng), Renilla luciferase reporter (FACSCalibur, Becton Dickinson). (20 ng), and luciferase reporter (500 ng) using Lipofectamine 3000 Transfection reagent (Thermo Fisher Scientific). Promoter Caspase activity assay activities were measured after 48-hour transfection. The activity of caspase-3 and -9 was determined by commercial colorimetric assays (KeyGEN). Cells were lysed with lysis buffer Northern blot analysis of 18S rRNA for 30 minutes on ice, and centrifuged at 14,000 rpm for 10 Extracted total RNA (10 mg per sample) was fractionated on a minutes. Samples containing 100 mg total protein were incubated 1.2% agarose–formaldehyde gel and transferred to Hybond NÞ at 37C in the dark for 2 hours with 50 mL caspase-3 substrate membranes (Amersham). BIO-labeled probe (summarized in Asp-Glue-Val-Asp (DEAD)-pNA, or with 50 mL caspase-9 sub- Supplementary Table S3) was purchased from Thermo Fisher strate Leu-Glu-His-Asp (LEHD)-pNA. Absorbance was measured Scientific. The membrane was hybridized overnight in hybridiza- at 405 nm. tion buffer (Thermo Fisher Scientific), washed twice (5 minutes each) under low-stringency conditions (2 SSC, 0.1% SDS), In vivo experiments blocked with blocking buffer at 50C for 30 minutes, then Animal care and experiments were performed in strict accor- incubated at 50C for 20 minutes with stabilized streptavidin– dance with the "Guide for the Care and Use of Laboratory horseradish peroxidase conjugate (1:300). Membranes were Animals" and the "Principles for the Utilization and Care of exposed to a phosphor storage screen and visualized using the Vertebrate Animals," and approved by the Committee of Fujian Chemi Doc XRSþ System (Bio-Rad). University of Traditional Chinese Medicine (Fujian, China). Male BALB/c nude mice (6–8 weeks of age; 20–22 g) were obtained Ribosome profile analysis from Shanghai SLAC Laboratory Animal Co. and maintained in a Transfected HCT116 cells were treated with 100 mg/mL cyclo- specific pathogen-free facility. HCT116 or RKO cells (1 106)in heximide for 15 minutes, washed with ice-cold PBS containing 100 mL RPMI1640 medium containing 50% Matrigel were 100 mg/mL cycloheximide, and then resuspended in hypotonic injected subcutaneously into the flank of nude mice (n ¼ 8). buffer [10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2,10 Starting on day 7 after the first injection, tumor growth was mmol/L KCl, 0.5 mmol/L dithiothreitol (DTT), 100 mg/mL cyclo- monitored once every other day for 19 days. Tumor volume heximide, 40 U/mL RNase inhibitor, 1 protease inhibitor (mm3) was calculated as (1/2) (length width2), where length cocktail]. Cells were centrifuged at 14,000 rpm for 15 minutes and width refer to the longest longitudinal and transverse dia- at 4C. Total protein (1 mg) was loaded onto a linear sucrose meters, respectively. gradient (5%–50%) and centrifuged for 3 hours at 38,000 rpm at At the end of the experiment, mice were anesthetized with 4C. Fractions (100 mL) were collected and precipitated with isoflurane. Tumor images were captured using an IVIS Spectrum trichloroacetic acid and analyzed by Western blotting. whole live-animal imaging system (PerkinElmer). Mice were then sacrificed for tissue collection. Protein synthesis assay Global protein synthesis was assayed using a commercial kit Promoter activity assay (Cayman Chemical). Transfected HCT116 cells were incubated HCT116 cells were transduced for 4–6 hours with lentivirus for 30 minutes with o-propargyl-puromycin in complete medium encoding anti-PNO1 or control shRNAs. Then cells were trans- in 96-well plates (100 mL/well). Cells were fixed with cell-based fected for 4–6 hours with a plasmid expressing luciferase reporter assay fixative (100 mL/well), stained with 5 FAM-azide (Cayman

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PNO1 Knockdown Inhibits Colorectal Cancer by Activating p53

Chemical), and photographed under a fluorescence microscope Data availability (Accu-Scope). Images were collected using MetaMorph image The authors declare that all data supporting the findings of this acquisition software (Molecular Devices). Fluorescence intensity study are available within the article, in the Supplementary in single cells was quantitated using Image Pro Plus software Information Files, and from the authors upon request. (Media Cybernetics). Results Immunofluorescence staining Cells were fixed with 10% formalin for 15 minutes, permea- Identification of PNO1 as a potential target in colorectal cancer bilized with 0.25% Triton X-100 for 5 minutes, blocked with Microarray experiments comparing 14 pairs of colorectal cancer 10% goat serum and 5% BSA in PBS for 1 hour at room primary lesions and noncancerous surrounding tissue revealed temperature, then incubated overnight at 4C with primary 1,868 DEGs, of which, 778 in primary lesions were upregulated antibody diluted in PBS (anti-p53, 1:200; anti-nucleolin, and 1,090 were downregulated (Fig. 1A and B; GEO Submission: 1:200; Supplementary Table S4). Next, cells were further incu- GSE113513). To identify novel oncogenes, we focused on 16 bated with Alexa Fluor 647–conjugated anti-rabbit secondary upregulated genes that have not been extensively investigated for antibody (1:1,000) in 10% goat serum and 5% BSA in PBS for potential association with cancer, including PNO1, EFNA3, CKS2, 1 hour at room temperature in the dark. Finally, cells were CDCA5, NUF2, and DACH1 (Fig. 1C; Supplementary Table S5). stained with DAPI. Images were acquired on a confocal micro- High-content screening in which lentivirus was used to deliver scope (Ultraview Vox, PerkinElmer). shRNAs into RKO cells in culture showed that PNO1, CDCA5, NUF2, and DACH1 strongly inhibited cancer cell growth (Fig. 1D–F). Considering the critical role of ribosome assembly In vivo ubiquitination assay in oncogenesis (8–16), we focused on PNO1 in subsequent After transduction with sh-PNO1 lentivirus or sh-Ctrl lentivirus studies. for 72 hours, cells were reseeded in 100-mm dishes, and then transfected with 2 mg of Ub-HA overexpression plasmid. After transfection for 72 hours, cells were treated with 10 mmol/L PNO1 is highly expressed in colorectal cancer and is associated proteasome inhibitor MG132 (MCE) for 6 hours and then lysed with poor prognosis using Western and IP lysis buffer (Beyotime Biotechnology) Quantitative PCR of an independent sample of 50 colorectal containing a proteinase inhibitor cocktail and phenylmethylsul- cancer cases previously collected from our laboratory showed that fonylfluoride. A total of 1 mg of soluble proteins was incubated levels of PNO1 mRNA were elevated in colorectal cancer (Fig. 2A), fi overnight at 4C with 5 mg of anti-p53 (MBL) and Protein A&G and these results were con rmed by Western blot analysis (Fig. 2B; n ¼ n ¼ sepharose beads (Santa Cruz Biotechnology). Beads were washed 12) and IHC (Fig. 2C; 10). Quantitative PCR analysis of a three times with Western and IP lysis buffer and the proteins were cDNA array based on a commercially available set of 80 colorectal separated by 15% SDS-PAGE and analyzed by Western blotting cancer primary lesions and 15 noncancerous surrounding tissues using anti-HA antibody. (Shanghai Outdo Biotech) showed increased PNO1 expression in colorectal cancer (Fig. 2D), but no significant relationship was observed between PNO1 mRNA and patient survival. Analysis of Bioinformatics analysis levels of PNO1 protein in an IHC-based tissue microarray based Levels of PNO1 mRNA were analyzed in various types of tumor on 90 colorectal cancer samples (Shanghai Outdo Biotech) tissues from multiple cohorts in the Oncomine database (www. showed increased PNO1 expression in colorectal cancer primary oncomine.org). Correlation between PNO1 mRNA level and lesions (P < 0.05 vs. noncancerous surrounding tissues; Fig. 2E), as survival in patients with breast or lung cancer was analyzed using well as an association between higher PNO1 expression and a Kaplan–Meier method (http://kmplot.com/analysis/). The poorer overall survival (P < 0.05; Fig. 2F and G). threshold of significance was set at P < 0.05. We used the web-based prediction program TFBIND (http:// PNO1 promotes colorectal cancer cell proliferation tfbind.hgc.jp) to search for potential transcription factors with In cultured RKO and HCT116 cells, which constitutively binding sites in the PNO1 promoter. Expression of PNO1 and express relatively high levels of endogenous PNO1 (Supplemen- EBF1 in colorectal cancer samples was analyzed using two tary Fig. S1A and S1B), PNO1 knockdown based on lentivirus- datasets (GEO ID: GSE3629 and GSE23878) with the aid of delivered shRNA or on transfected siRNA (Fig. 3A and B; Sup- the R2 web application (http://r2.amc.nl). Correlation between plementary Fig. S1C and S1D) decreased cell viability (Fig. 3C; levels of PNO1 and EBF1 expression was analyzed using the Supplementary Fig. S1E) and colony formation (Fig. 3D; Sup- Pearson test. plementary Fig. S1F). It also increased the percentage of cells in G0–G1 phase, with a concomitant decrease in the percentage of Statistical analysis cells in S phase (Fig. 3E). PNO1 knockdown increased the per- Statistical analysis was performed using SPSS 20.0 (IBM). Data centage of cells undergoing apoptosis (Fig. 3F), and it increased are presented as mean SD. Differences between two groups were activity of caspases-3 and -9 (Fig. 3G and H). assessed for significance using the independent Student t test, and In contrast, ectopic PNO1 expression in HT-29 and HCT-8 cells differences among three or more groups were assessed using one- (Fig. 3I and J), which constitutively express low levels of endog- way ANOVA. Kaplan–Meier survival differences were assessed enous PNO1, increased cell viability and colony formation using the log-rank test. Correlation between PNO1 and EBF1 (Fig. 3K and L). Xenografts of wild-type HCT116 or RKO cells expression was analyzed using Pearson rank correlation. P < 0.05 grew significantly faster in nude mice than xenografts in which was considered significant. PNO1 was knocked down prior to inoculation (Fig. 4A and B).

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A Color key D RKO Cells

Day 1 Day 2 Day 3 Day 4 Day 5 sh-Ctrl sh-PNO1 sh-CDCA5 sh-DACH1 A3791 A2801 A2799 A2973 A3831 A3797 A2781 A3821 A3817 A3829 A3975 A3837 A2977 A2793 A3790 A2974 A3836 A3816 A3820 A3792 A2798 A2800 A3830 A2976 A2972 A3796 A3828 A2780

TN Volcano plot B sh-NUF2 15

E 10 sh-Ctrl ( P ) 6 10 − log 5 sh-CDCA5 0 sh-DACH1 4 −6 −4 −2 0 2 4 6

Log2 (fold change)

Color key sh-PNO1 C 2 sh-NUF2

1 2 3 4 5 F Days after seeding EROIL EFNA3 8 ARID3A sh-Ctrl DSCC1 7 sh-PNO1 GINS4 sh-CDCA5 GINS2 6 CDCA5 sh-DACH1 CKS2 5 sh-NUF2 KIF14 NUF2 4 TEAD4 3 BYSL GCSH 2 PNO1 Cell number (fold of change) PHLDA1 1 DACH1 0 1 2 3 4 5 A2801 A2799 A3791 A3831 A2793 A2973 A3797 A2977 A3821 A3829 A3837 A3817 A3975 A2781 A2974 A2976 A3796 A3820 A2972 A3836 A3790 A2798 A3828 A3816 A2972 A2780 A3830 A2800 T N Days after seeding

Figure 1. Gene expression profile analysis and high-content screening suggest an oncogenic role for PNO1. cDNA microarray analysis was performed to identify DEGs between 14 pairs of colorectal cancer tissues (T) and adjacent normal tissues (N). High-content screening and lentivirus-delivered, shRNA-based interference were used to assess the effects of candidate genes on colorectal cancer cell growth. A and B, Hierarchical clustering plots (A) and volcano plots (B) were used to compare gene expression profiles (fold change >2or2.0, P < 0.05). C, Heatmap of 16 selected DEGs. RKO cells were transduced with lentivirus encoding shRNAs specific against these 16 DEGs, and cell growth was measured using multiparametric high-content screening. D, Effects of PNO1, CDCA5, NUF2, and DACH1 on growth of RKO cells. Representative images of RKO cell growth are shown. E and F, Heatmap (E) and growth curves (F) showing the growth of RKO cells. Data were normalized to cell number on day 1 and are represented as fold change. , P < 0.05 versus control shRNA.

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A B N1 T1 N2 T2 N3 T3 N4 T4 8 PNO1 35 kDa 4 b-Actin 42 kDa 6 3 N5 T5 N6 T6 N7 T7 N8 T8

4 PNO1 35 kDa 2 b-Actin 42 kDa 2 N9 T9 N10 T10 N11 T11 N12 T12 1 PNO1 35 kDa

Relative mRNA expression Relative mRNA 0 0 NTb-Actin 42 kDa Relative protein expression NT C D 10 14

8 13 6 6 5 4 PNO1 4 3 IHC Score 2 2 1

0 expression Relative mRNA 0 N T NT NT

100 E 10 F 90 80 8 70 60 6 50

PNO1 40 4 30

IHC Score Low expression 2 20 10 High expression

0 Survival probability (%) 0 N T NT 0 24 48 72 96 Months after surgery G Low expression High expression 40× 200× 40× 200× PNO1

Figure 2. PNO1 expression is upregulated in colorectal cancer tissues and associated with poor prognosis of colorectal cancer patients. A, PNO1 mRNA levels in tissues from 50 patients with colorectal cancer were analyzed using quantitative PCR. GAPDH was used as an internal control. B, PNO1 protein expression in 12 pairs of colorectal cancer tissues and adjacent normal tissues was determined by Western blotting. PNO1 bands were quantitated using ImageJ software, then normalized to b-actin. C, PNO1 protein levels in 10 pairs of colorectal cancer tissues and adjacent normal tissues were determined by IHC. Representative images were taken at a magnification of 200. D, PNO1 mRNA levels in colorectal cancer samples were determined using a quantitative PCR-based cDNA array (MecDNA-HColA095Su01). GAPDH was used as an internal control. E, PNO1 protein levels in 90 pairs of colorectal cancer tissues (T) and adjacent normal tissues (N) were determined using an IHC-based tissue microarray. Representative images were taken at a magnification of 40 or 200. , P < 0.05, tumor versus normal tissue. F, Correlation between protein expression of PNO1 and survival of patients with colorectal cancer was analyzed in Kaplan–Meier plots generated on the basis of results from the IHC-based tissue microarray containing 90 samples (, P ¼ 0.0182). G, Representative images of colorectal cancer tissues showing high or low PNO1 expression were taken at a magnification of 40 or 200.

Oncogenic activities of PNO1 depend on p53 signaling increased luciferase activity in the p53-driven luciferase reporter Microarray analysis of HCT116 cells in which PNO1 was assay (Fig. 5F). Western blot analysis confirmed increased expres- knocked down revealed 253 DEGs (Fig. 5A; GEO submission: sion of both p53 and p21 at the protein level (Fig. 5G). The GSE113514). Many of these DEGs, including CDKN1A (also inhibitory effects of PNO1 knockdown were blocked when p53 known as p21), THBS1, MAPK1, MCM2, and CDC42 (Fig. 5B, was knocked out (Fig. 5H and I), and the inhibitory effects were P < 0.05), have been reported to play essential roles in cell attenuated when cells were treated with the p53 inhibitor PFT-a proliferation and apoptosis (30–34). KEGG pathway enrichment (Fig. 5J). analysis of these DEGs indicated that p53 pathways were among the 10 most enriched signaling pathways (Fig. 5C); one of the PNO1 knockdown activates p53 through the ribosomal stress genes most strongly upregulated in response to PNO1 knock- pathway by inhibiting MDM2-mediated ubiquitination and down was the downstream effector p21 (Fig. 5B). degradation of p53 Microarray analysis (Fig. 5D) and quantitative PCR (Fig. 5E) Consistently with studies in yeast (26, 27), we confirmed that showed a slight increase in p53 mRNA. PNO1 knockdown PNO1 knockdown in HCT116 cells significantly decreased levels

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A C 1.2 HCT116 1.2 RKO 12 HCT116 7 RKO sh-Ctrl sh-Ctrl sh-PNO1 sh-PNO1 1.0 1.0 10 6 5 0.8 0.8 8 4 0.6 0.6 6 3 0.4 0.4 4 2

0.2 0.2 2 1

0.0 0.0 0 0

12345 Cell viability (fold change)

Relative mRNA expression Relative mRNA 12345 Cell viability (fold change) sh-Ctrl sh-PNO1 expression Relative mRNA sh-Ctrl sh-PNO1 Days Days

BDPNO1 35 kDa PNO1 35 kDa

b-Actin 42 kDa b-Actin 42 kDa

1.4 1.4 120 120

1.2 1.2 100 100 1.0 1.0 80 80 0.8 0.8 60 60 0.6 0.6

Colonies (%) 40 40 0.4 0.4 Colonies (%)

0.2 0.2 20 20

0.0 0.0 0 0 Relative protein expression sh-Ctrl sh-PNO1 Relative protein expression sh-Ctrl sh-PNO1 sh-Ctrl sh-PNO1 sh-Ctrl sh-PNO1 E F 100 100 100 100 8060 8060 8060 8060 40 40 40 40 Counts Counts Counts Counts M1 M1 M1 M1 020 020 020 020 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 FLF-H FLF-H FLF-H FLF-H

70 30 10 % (sh-Ctrl) 70 % (sh-Ctrl) % (sh-PNO1) % (sh-PNO1) 60 60 25 8 50 50 20 6 40 40 15 30 30 4 10 20 20 2 5 10 10 Percentage of cells (%) Percentage of cells (%) Percentage of cells (%) 0 0 Percentage of cells (%) 0 0 GHG0–G1 SGG2–M 0–G1 S G2–M sh-Ctrl sh-PNO1 sh-Ctrl sh-PNO1

2.0 2.5 2.0 2.0

2.0 1.5 1.5 1.5 1.5 1.0 1.0 1.0 1.0 0.5

0.5 (fold change) 0.5 0.5 (fold change) (fold change) (fold change) Caspase-9 activity Caspase-9 activity Caspase-3 activity 0.0 Caspase-3 activity 0.0 0.0 0.0 sh-Ctrl sh-PNO1 sh-Ctrl sh-PNO1 sh-Ctrl sh-PNO1 sh-Ctrl sh-PNO1

I HT-29 HCT-8 K HT-29 HCT-8 30 14 10 8 Vector Control 25 12 PNO1 7 PNO1 8 10 6 20 5 8 6 15 4 6 4 3 10 4 2

Cell viability 2 Cell viability 5 2 (fold of change) 1 (fold of change) 0 0 0 0 12345 12345 Relative mRNA expression Relative mRNA Control PNO1 expression Relative mRNA Control PNO1 Days Days J L PNO1 35 kDa PNO1 35 kDa

b b-Actin 42 kDa -Actin 42 kDa

3.5 4 140 120 3.0 120 3 100 2.5 100 80 2.0 80 2 60 1.5 60

1.0 40 40 1 Colonies (%) Colonies (%) 0.5 20 20

0.0 0 0 0 Relative protein expression Control PNO1 Relative protein expression Control PNO1 Control PNO1 Control PNO1

Figure 3. Oncogenic functions of PNO1 in colorectal cancer cells in vitro. HCT116 and RKO cells were transduced with lentivirus encoding shRNA. A, Quantitative PCR was performed to determine levels of PNO1 mRNA. GAPDH was used as an internal control. B, Western blotting was performed to determine levels of PNO1 protein. PNO1 bands were quantitated using ImageJ software and normalized to b-actin. C, Cell viability was measured using the CCK-8 assay. Results were normalized to viability on day 1 and are represented as fold change. D, Cell survival was measured using a colony formation assay, and data were normalized to the survival of

control cells. E, Cell-cycle distribution was determined by ﬂow cytometry, and percentages of cells in G0–G1,S,orG2–M phases were determined. F, Cell apoptosis was analyzed using Annexin V-APC staining, followed by ﬂow cytometry. Activity of caspase-3 (G) and caspase-9 (H) was determined using a colorimetric assay. Data were normalized to caspase activities in cells treated with control shRNA (sh-Ctrl). HT-29 and HCT-8 cells were transduced with lentivirus encoding PNO1. I, Quantitative PCR was performed to determine levels of PNO1 mRNA. GAPDH was used as an internal control. J, Western blotting was performed to determine levels of PNO1 protein. PNO1 bands were quantitated using ImageJ software and normalized to b-actin. K, Cell viability was measured using the CCK-8 assay. Results were normalized to viability on day 1 and are represented as fold change. L, Cell survival was measured using a colony formation assay without agarose (left) or with agarose (right), and data were normalized to the survival of control cells. , P < 0.05 versus sh-Ctrl cells or control cells.

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A ] 2

) 1,200 sh-Ctrl

3 sh-PNO1 1.0 16 1,000 14 0.8 800 12 0.6 10 600 8 sr] [ m W/cm 400 0.4 2 6 (x10E-10) 4 200 0.2

Tumor weight (g) Tumor 2 Tumor volume (mm Tumor 0 0.0 0 0 1 3 5 7 9 11 13 15 17 19 [p/s/cm Days sh-Ctrl sh-PNO1 sh-Ctrl sh-PNO1 Avg radiant efficiency sh-Ctrl sh-PNO1

B ] 2,000 sh-Ctrl 2 2.0 5 ) sh-PNO1 3 1,500 4 1.5 3 1,000 1.0 sr] [ m W/cm

2 2

500 0.5 (x10E-10) 1 Tumor weight (g) Tumor

Tumor volume (mm Tumor 0 975310 11 1513 17 19 0.0 0 [p/s/cm Days sh-Ctrl sh-PNO1 sh-Ctrl sh-PNO1 Avg radiant efficiency sh-Ctrl sh-PNO1

Figure 4. Oncogenic functions of PNO1 in colorectal cancer cells in vivo. A xenograft nude mouse model was constructed to investigate the effects of PNO1 knockdown on tumor growth. HCT116 cells (A) or RKO cells (B) were transduced with lentivirus encoding anti-PNO1 shRNA (sh-PNO1) or control shRNA (sh-Ctrl), then injected subcutaneously into BALB/c nude mice. Tumor size, morphology, and tumor weight were monitored. Tumor fluorescence was imaged using a whole live-animal imaging system, and signal intensity was quantified as the number of photons within the region of interest per second. , P < 0.05 versus sh-Ctrl group. of 18S rRNA (Fig. 6A), 40S and 60S subunits of the 80S ribosome PNO1 displays oncogenic potential in lung and breast cancers (Fig. 6B), and global protein synthesis (Fig. 6C). To determine Data from the Oncomine database (http://www.oncomine. whether these effects on the nucleolus were related to nucleolar com/) indicated that PNO1 mRNA levels are significantly upre- stress, we stained cells for p53 and the major nucleolar protein gulated in various types of malignancies, including colorectal nucleolin/C23. PNO1 knockdown or treatment with actinomycin cancer, lung cancer, and breast cancer (Supplementary Table S6). D (as a positive control) resulted in translocation of nucleolin Analysis of Kaplan–Meier plots (http://kmplot.com/analysis/) from the nucleolus to the nucleoplasm (Fig. 6D, left) and an indicated that higher PNO1 mRNA levels were associated with increase in nuclear p53 immunoreactivity (Fig. 6D, right). West- poorer overall survival and relapse-free survival of patients with ern blot analysis and coimmunoprecipitation (co-IP) assay indi- breast or lung cancer, as well as shorter time to first progression cated that PNO1 knockdown led to a reduction in degradation of (Supplementary Fig. S2A, P < 0.05). In cultures of A549 lung p53 (Fig. 6E) and ubiquitination of p53 (Fig. 6F). Moreover, co-IP cancer cells and MCF-7 breast cancer cells, PNO1 knockdown analysis indicated that PNO1 knockdown increased the binding using siRNA transfection (Supplementary Fig. S2B and S2C) of RPL11 to MDM2 (Fig. 6G). significantly decreased cell viability (Supplementary Fig. S2D) and cell survival (Supplementary Fig. S2E). These data suggest Transcription factor EBF1 negatively regulates PNO1 that PNO1 may play oncogenic roles in colorectal cancer as well expression in colorectal cancer as other kinds of cancers. Using TFBIND (http://tfbind.hgc.jp/), we predicted 10 potential transcription factors of PNO1: EGR3, TCF-3, MYCN, PBX1, MYC, VBP1, THRB1, RORA, TFCP2, and EBF1. These Discussion factors were significantly up- or downregulated in colorectal The key finding of this report is that the ribosome assembly cancer tissues relative to noncancerous tissue in our cDNA factor PNO1 may play a critical oncogenic role in tumor initiation microarray (Fig. 7A; Supplementary Table S5). EBF1 displayed and progression. Overexpression of PNO1 significantly promoted the most potent regulatory effect on PNO1 transcription in cell proliferation, while depletion of PNO1 suppressed tumor HEK293T cells based on a luciferase reporter system (Fig. 7B), growth in vivo and in vitro by inhibiting proliferation and inducing and this regulatory effect was confirmed in RKO cells (Fig. 7C). cell apoptosis. PNO1 knockdown suppressed cell proliferation of EBF1 overexpression reduced PNO1 expression at the mRNA colorectal cancer cells in a p53-dependent manner through acti- and protein levels (Fig. 7D and E), as well as reduced cell vation of the ribosomal protein–MDM2–p53 pathway. These viability (Fig. 7F) and induced cell-cycle arrest (Fig. 7G) and results suggest that PNO1 plays an important role in tumor apoptosis (Fig. 7H). In microarray experiments with 14 paired growth and may serve as an attractive therapeutic target for samples of colorectal cancer and noncancerous tissue, PNO1 colorectal cancer treatment. PNO1 expression may be driven by expression was significantly higher in colorectal cancer than in the transcription factor EBF1, and the effects of PNO1 may involve noncancerous tissue (Fig. 1C), whereas the converse was true negative regulation of the p53/p21 pathway. This leads us to for EBF1 expression (Fig. 7A); their expression exhibited a propose an oncogenic EBF1/PNO1/p53 axis. significant inverse correlation (Fig. 7I), which was consistent This study describes several important discoveries about the with the analyses in two colorectal cancer cohorts from the R2 functional roles of PNO1 in colorectal cancer. We demonstrated Bioinformatic Platform (Fig. 7A, J, and K). Therefore, decreased increased levels of PNO1 mRNA and protein in clinical colorectal expression of the negative transcription factor EBF1 may be one cancer samples and cell cultures using a combination of techni- of the key reasons for PNO1 overexpression in colorectal ques, including mRNA and tissue microarrays, quantitative PCR, cancer. Western blotting, and IHC. These experiments suggest that PNO1

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A B

7 Microarray qPCR

6 3.0 sh-Ctrl 3.0 sh-Ctrl sh-PNO1 sh-PNO1 5 2.5 2.5 2.0 2.0

34 1.5 1.5 - Log ( P ) 2 1.0 1.0

10 0.5 0.5 0.0 0.0 Relative mRNA expression Relative mRNA 1 2 3 1 2 3 -4 -3 -2 -101234 expression Relative mRNA Log2 (fold change) sh-Ctrl sh-PNO1 PNO1 MCM2 PNO1 THBS1 MAPK1 CDC42 MCM2 C D CDKN1A E CDKN1A THBS1 MAPK1 CDC42 Pathway enrichment p53-microarray p53-qPCR - P 1.6 1.4 Log10 ( ) 0.0 0.5 1.0 1.5 2.0 2.5 1.4 1.2 Ubiquitin-mediated proteolysis 1.2 1.0 Bladder cancer 1.0 0.8 Protein processing in endoplasmic reticulum 0.8 0.6 p53 signaling pathway 0.6 0.4 Ubiquinone and other terpenoid-quinone biosynthesis 0.4 0.2 Bacterial invasion of epithelial cells 0.2 Proteoglycans in cancer 0.0 0.0 Relative mRNA expression Relative mRNA Viral carcinogenesis expression Relative mRNA sh-Ctrl sh-PNO1 sh-Ctrl sh-PNO1 ErbB signaling pathway DNA replication

F G HCT-116 RKO HCT-116 RKO 3.5 2.5 sh-Ctrl 2.0 sh-Ctrl 3.0 sh-PNO1 sh-PNO1 2.0 1.5 2.5 sh-PNO1 sh-PNO1 sh-Ctrl sh-Ctrl 2.0 1.5 1.0 p53 53 kDa 1.5 1.0 1.0 0.5 p21 21 kDa 0.5 0.5 0.0 0.0 0.0 Relative luciferase activity b sh-Ctrl sh-PNO1 -Actin 42 kDa p53 p21 p53 p21

+/+ -/- H HCT116/p53 HCT116/p53 IJ

+/+ 3.0 +/+ 2.0 sh-Ctrl sh-PNO1 sh-Ctrl sh-PNO1 3.0 HCT-116/p53 +sh-Ctrl HCT-116/p53 +sh-Ctrl sh-Ctrl+DMSO HCT-116/p53+/++sh-PNO1 HCT-116/p53+/+ +sh-PNO1 sh-Ctrl+PFT-a -/- 2.5 -/- 2.5 HCT-116/p53 +sh-Ctrl HCT-116/p53 +sh-Ctrl sh-PNO1+DMSO -/- 1.5 a HCT-116/p53 +sh-PNO1 HCT-116/p53-/- +sh-PNO1 sh-PNO1++PFT- PNO1 2.0 2.0

1.5 1.5 1.0 p53 1.0 1.0 OD Value 0.5 Cell viability 0.5 (fold change) 0.5 b -Actin 0.0 0.0 0.0 Relative protein expression 123 1 23 4 5 PNO1 p53 Days Days

Figure 5. Oncogenic activities of PNO1 depend on p53 signaling. A, cDNA microarray analysis was performed to determine DEGs in HCT116 cells transduced with lentivirus encoding either anti-PNO1 shRNA (sh-PNO1) or control shRNA (sh-Ctrl). A hierarchical clustering plot (left) and a volcano plot (right) were used to identify DEGs (fold change >2or2.0, P < 0.05). B, Levels of mRNAs encoding CDKN1A/p21, THBS1, MAPK1, MCM2, and CDC42 in a cDNA microarray of HCT116 cells after PNO1 knockdown (left), which were conﬁrmed by quantitative PCR (right). C, KEGG pathway enrichment analysis of DEGs was performed to identify functionally

related gene pathways. The top 10 enriched signaling pathways are shown and are ranked on the basis of log10(P). D, Levels of mRNA encoding TP53 in a cDNA microarray of HCT116 cells after PNO1 knockdown. E, Quantitative PCR was performed to determine mRNA levels of TP53. GAPDH was used as an internal control. F, A dual luciferase assay was performed to determine the effect of PNO1 knockdown on transcriptional activity of TP53 in HCT116 cells. G, Western blotting was performed to determine levels of p53 and p21 proteins in both HCT116 and RKO cells (left). PNO1 bands were quantitated using ImageJ software and normalized to b-actin (middle and right). H, HCT116/p53þ/þ and HCT116/p53/ cells were transduced with lentivirus encoding sh-Ctrl or sh-PNO1, then levels of PNO1 protein were assayed using Western blotting (left). Protein bands were quantitated using ImageJ software and normalized to b-actin (right). I, Cell viability was determined using the CCK-8 assay, and results were normalized to viability on day 1 and are represented as fold change. , P < 0.05 versus sh-Ctrl cells; #, P < 0.05 versus sh-Ctrl in HCT116/p53/ cells. J, HCT116 cells were transduced with lentivirus encoding sh-Ctrl or sh-PNO1 and treated or not with PFT-a. Cell viability was determined using the CCK-8 assay. Results were normalized to viability on day 1 and are represented as fold change. , #P < 0.05 versus sh-Ctrl or sh-PNO1 cells without PFT-a treatment.

overexpression plays an essential role in malignant transforma- biogenesis is one of the hallmarks of cancer cells (19). Some tion. Indeed, high PNO1 expression based on tissue microarray ribosome assembly factors such as RIO1 and NOB1 are upregu- analysis correlated with poor survival of patients with colorectal lated in various types of human malignancies, and their upregula- cancer. These results highlight not only the potential oncogenic tion suggests poor prognosis. Similarly, we found that PNO1 is role of PNO1, but also the possibility of using PNO1 as a significantly upregulated in colorectal cancer tissues and that biomarker for early diagnosis and prognosis of colorectal cancer. PNO1 knockdown inhibits ribosome biogenesis, suggesting that The findings here first need to be validated in a much larger clinical PNO1 plays an essential role in tumorigenesis. Indeed, we dem- sample. onstrated here that suppressing endogenous PNO1 can arrest cells The uncontrolled cell proliferation of cancer cells means that at the G1–S transition and induce cell apoptosis, inhibiting tumor they require extensive protein synthesis; as a result, ribosome growth in vivo and in vitro; conversely, overexpressing PNO1

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80S si-Ctrl si-PNO1 A B 0.10 si-Ctrl C si-PNO1 0.09 Polysome 60S 0.08 sh-Ctrl sh-PNO1 0.07 40S 2.0 OD254 18S 0.06 140 120 1.0 0.05 100 18S rRNA probe 01234567891011121314151617 181920 80 5.0 5% 50% 60 3.0 28S 40 2.0 18S sh-Ctrl 1.0 20 Protein synthesis RPS3 sh-PNO1 (relative to sh-Ctrl) EB staining 27 kDa 0 40S 60S 80S si-Ctrl si-PNO1

D Nuclein-red GFP-green DAPI-blue p53-red GFP-green DAPI-blue sh-Ctrl sh-Ctrl sh-PNO1 sh-PNO1 sh-Ctrl+ActD sh-Ctrl+ActD

EFGCHX (min) sh-Ctrl sh-PNO1 sh-Ctrl sh-PNO1 0 15 30 60 Mdm2 p53 53 kDa 90 kDa sh-Ctrl

b IP:MDM2 -Actin 42 kDa 20 kDa IB:HA RPL11 IP:p53 p53 53 kDa sh-Ctrl sh-PNO1

sh-PNO1 b-Actin 42 kDa PNO1 35 kDa

1.2

1.0 IB: p53 Mdm2 90 kDa 0.8

0.6 IB: PNO1 Input 20 kDa Input RPL11 0.4 sh-Ctrl 0.2 sh-PNO1 IB: b-Actin b-Actin 42 kDa 0.0 01530 60 Relative amount of p53 (%) CHX (min)

Figure 6. Effects of PNO1 on ribosome biogenesis and nucleolar stress-induced p53 pathway. A, 18S rRNA production was detected in HCT116 cells after PNO1 knockdown using Northern blotting with an 18S rRNA probe. EB, ethidium bromide. B, Ribosome profiling was performed to determine ribosome biogenesis in HCT116 cells after PNO1 knockdown. Formation of 40S subunits, 60S subunits, and 80S ribosomes was evaluated by measuring absorbance at 254 nm. The presence of ribosomes in each fraction was confirmed by Western blotting against ribosomal protein S3 (PRS3). C, Newly synthesized protein was detected in HCT116 cells after PNO1 knockdown using a protein synthesis assay, , P < 0.05 versus sh-Ctrl. D, After PNO1 knockdown or treatment with actinomycin D (ActD), cells were immunostained for nucleolin (left) or p53 (right), then counterstained with DAPI to visualize nuclei. Magnification, 600. E, Degradation of p53 was determined by Western blot analysis in HCT116 cells after PNO1 knockdown, followed by cycloheximide (CHX) treatment or not (top). Amount of p53 was quantified by densitometry and normalized to the level of b-actin (bottom). F, Ubiquitination of p53 was determined by Western blot analysis using anti-HA in HCT116 cells after PNO1 knockdown, followed by transfection with HA-tag overexpressing plasmid. G, Coimmunoprecipitation (IP) analysis in HCT116 cells after PNO1 knockdown was performed to determine the binding of RPL11 to MDM2 using anti-MDM2 antibody.

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D 100,000 A 4 B 1.2 C 1.2 80,000 Control 3 EBF1 1.0 1.0 60,000 2 40,000 1 0.8 0.8 20,000 0 0.6 0.6 1.5 -1

(T vs. N) 0.4 -2 0.4 1.0 -3 0.2 0.2 0.5 - 4 0.0 0.0 0.0 Relative luciferase activity Relative mRNA expression Relative mRNA Relative mRNA expression Relative mRNA Relative luciferase activity Control EBF1 EBF1 PNO1 MYC TCF3 EBF1 MYC VBP1 PBX1 EGR3 THRB TCF3 RORA MYCN EBF1 VBP1 PBX1 TFCP2 EGR3 THRB RORA MYCN Control TFCP2 6 1.2 10 EBF1 PNO1 F Control Control EBF1 EBF1 E 5 1.0 8 64 kDa EBF1 4 0.8 6 3 0.6 4 PNO1 35 kDa 2 0.4 Cell viability 2 1 0.2 (fold of change) 0 b-Actin 42 kDa 0 0.0

Relative protein expression 1 2 5

Relative protein expression 34 Control EBF1 Control EBF1 Days

50 4 G Control 60 I EBF1 H 40 50 2 Control Control 30 40 30 0 20 EBF1 20 R = -0.576 -2 10 EBF1 EBF1 10 P = 0.001

Percentage of cells (%) - Percentage of cells (%) 0 0 4 - - -2 0 2 4 G0 G1 S G2 M Control EBF1 PNO1 J 3 4 3

2 2 2 1 1 0 0 0

EBF1 R = -0.695 -2 -1 -1 P = 9.7e-19 -2 -4 -2 Relative mRNA expression Relative mRNA Relative mRNA expression Relative mRNA - - NT N T 4 2 0 24 PNO1 K 5 6 5 4 -0 0 2

- EBF1 - 5 5 R = -0.5333 -2 P = 1.4e-05 -10 -4 -10 Relative mRNA expression Relative mRNA NT expression Relative mRNA -4 -2 0 2 4 6 NT PNO1

Figure 7. PNO1 expression in colorectal cancer is negatively regulated by transcription factor EBF1. A, Differences in levels of mRNAs encoding 10 potential transcription factors between colorectal cancer tissues (T) and adjacent normal tissues (N) in cDNA microarray analysis, which was performed as described in Fig. 1. , P < 0.05, T versus N. B, A dual luciferase assay was performed to assess the effects of 10 potential transcription factors on PNO1 transcription in HEK293T cells. , P < 0.05 versus control. C, A dual luciferase assay was performed to assess the effects of EBF1 overexpression on PNO1 transcription in RKO cells. , P < 0.05 versus control. D, Effects of EBF1 overexpression on mRNA levels of PNO1 in RKO cells, as measured using quantitative PCR. GAPDH was used as an internal control. , P < 0.05 versus control. E, Effect of EBF1 overexpression on levels of PNO1 protein in RKO cells, as measured using Western blotting (left). EBF1 protein bands were quantitated using ImageJ software and normalized to b-actin (right). , P < 0.05 versus control. F, Cell viability was measured using the CCK-8 assay. Results were normalized to viability on day 1 and are represented as fold change. G, Cell-cycle distribution was determined by ﬂow cytometry, and percentages

of cells in G0–G1,S,orG2–M phases were determined. H, Cell apoptosis was analyzed using Annexin V-APC staining, followed by ﬂow cytometry. I, Analysis of the possible correlation between PNO1 and EBF1 mRNA levels in colorectal cancer tissues based on the cDNA microarray described in Fig. 1. Data were analyzed using Pearson rank correlation (, P < 0.05; R ¼ 0.576). J and K, Analysis of the possible correlation between PNO1 (left) and EBF1 (middle) mRNA levels in colorectal cancer tissues based on the online dataset from the R2 Bioinformatic Platform (GEO ID: GSE3629 and GSE23878; right).

significantly promoted cell proliferation. Development of selec- including the p21 gene, which can lead to cell-cycle arrest and tive small-molecule inhibitors targeting PNO1 may provide a apoptosis as well as inhibit cell proliferation (35–39). Various promising therapeutic strategy for patients with colorectal cancer. cellular insults can activate p53, including disruption of ribosome The ability of PNO1 depletion to inhibit tumorigenesis encour- biogenesis (21, 40, 41). In this study, we demonstrated that PNO1 aged us to begin exploring downstream pathways that may knockdown enhanced expression of p53 and its downstream gene mediate the oncogenic effects of PNO1. Analysis of cDNA micro- p21 in wild-type HCT116 cells as well as reduced cell viability. arrays and classification of functional pathways identified several These effects were blocked by p53 knockout and attenuated by the DEGs between HCT116 cells treated with anti-PNO1 shRNA or p53 inhibitor PFT-a. control shRNA. These DEGs suggest that PNO1 may participate in Ribosome biogenesis is a very complex process (42–44), highly several pathways involved in cell-cycle progression and prolifer- regulated by more than 200 ribosome assembly factors, including ation, including p53/p21 signaling. Activation of the well-known RIO1 and NOB1. While studies of the yeast homolog of PNO1 tumor suppressor p53 induces transcription of various genes, showed that it plays an essential role in the processing of pre-18S

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rRNA, the physiologic functions of mammalian PNO1 remain tumor suppressor in hematologic malignancies (46, 47). How unclear. Using RNA profiling and Northern blotting, we found EBF1 regulates PNO1 expression should be investigated in further that PNO1 knockdown in HCT116 cells decreased amounts of studies. In B-cell development, EBF1 helps drive DNA demeth- 18S rRNA, 40S subunits, 60S subunits, and the 80S ribosome, ylation and chromatin remodeling, which controls the transcrip- leading thereby to significant inhibition of global protein syn- tion of various genes (46, 47). Whether EBF1 regulates PNO1 thesis. These results demonstrate the essential role of PNO1 in expression in the same way should be explored, and more ribosome biogenesis in mammalian cells, specifically in human generally the potential roles of EBF1 in solid malignancies should colorectal cancer cells. Further studies need to be done to explore be clarified. functional correlation between PNO1 and NOB1 in mammalian In conclusion, this is the first demonstration of the significance cells. of PNO1 in colorectal cancer. Our data suggest that the protein Deficiency in ribosome biogenesis elicits a p53-dependent plays a role in tumorigenesis and prognosis. We have also shown, cellular stress response referred to as "nucleolar stress" or "ribo- for the first time, that mammalian PNO1 is critical for ribosome somal stress" (21). We confirmed that the effect of PNO1 knock- biogenesis in cancer cells, suggesting that it works as a ribosome down on the nucleolus is consistent with nucleolar stress and assembly factor like its yeast homolog. PNO1 transcription activation of p53. Moreover, PNO1 knockdown resulted in trans- appears to be regulated by the transcription factor EBF1, and location of nucleolin from the nucleolus to the nucleoplasm, PNO1 exerts its oncogenic effects, at least in part, by negatively which is an indicator of increased nucleolar stress, and it resulted regulating the p53 signaling pathway. These findings justify the in an increase in levels of p53 in the nucleus. A p53-driven search for small-molecule inhibitors targeting PNO1 as a novel luciferase reporter assay and quantitative PCR analysis indicated therapeutic strategy in colorectal cancer. that PNO1 knockdown slightly increased luciferase activity and p53 mRNA expression. PNO1 knockdown led to a reduction in Disclosure of Potential Conflicts of Interest the degradation and ubiquitination of p53. These studies suggest No potential conflicts of interest were disclosed. that both the increase in p53 mRNA expression and decrease in MDM2-mediated degradation and ubiquitination of p53 contribute to the increase in p53 after PNO1 knockdown. Therefore, Authors' Contributions Conception and design: Y. Chen, T.J. Sferra, J. Peng further study is needed to explore precisely how PNO1 knock- Development of methodology: A. Shen, L. Liu, F. Qi down activates p53. The ribosome protein RPL11 binds to MDM2 Acquisition of data (provided animals, acquired and managed patients, and inhibits its ubiquitin ligase activity toward p53, resulting in provided facilities, etc.): A. Shen, Y. Huang, H. Chen, F. Qi, Z. Shen, p53 accumulation (21, 39, 45). We therefore detected the binding M. Wu, Q. Li, L. Qiu, N. Yu of RPL11 and MDM2 in HCT116 after PNO1 knockdown and Analysis and interpretation of data (e.g., statistical analysis, biostatistics, found that this knockdown significantly increased the binding of computational analysis): L. Liu, F. Qi, X. Wu, T.J. Sferra Writing, review, and/or revision of the manuscript: A. Shen, Y. Chen, RPL11 to MDM2, suggesting that ribosomal stress increases T.J. Sferra, J. Peng RPL11 binding to MDM2 and decreases ubiquitination and Administrative, technical, or material support (i.e., reporting or organizing degradation of p53; this may contribute to the accumulation of data, constructing databases): A. Shen, L. Liu, J. Lin, X. Wu p53. Future work should address PNO1-triggered downstream Study supervision: Y. Chen, J. Lin, T.J. Sferra, J. Peng processes in colorectal cancer. In parallel with our efforts to explore downstream effectors of Acknowledgments PNO1 oncogenicity, we searched for upstream regulators of We thank Dr. Xiangfeng Wang from First People's Hospital Affiliated to PNO1. Web-based screening of transcription factor binding sites Fujian University of Traditional Chinese Medicine and Dr. Yaodong Wang from fi Fujian Provincial Hospital for assistance with collection of clinical samples. We identi ed EBF1 as a potential transcription factor of PNO1. We thank Drs. Wei Lin, Weidong Zhu, Baochang He, and Guoqing Ji for helpful fi found that EBF1 overexpression signi cantly decreased levels of advice and discussions. This study was supported by the National Natural PNO1 mRNA and protein in colorectal cancer cells, while it Science Foundation of China (81673721, 81803882), the International Coop- increased levels of p53 and p21 protein. Moreover, EBF1 over- erative Project of Fujian Department of Science and Technology (2017I0007), expression in RKO cells significantly decreased cell viability, and a Chinese Government Scholarship from the China Scholarship Council [(2016) 3100]. arrested the cell cycle at G0–G1 phase, and induced cell apoptosis. We also found that EFB1 expression correlated inversely with that The costs of publication of this article were defrayed in part by the payment of of PNO1 based on data from our colorectal cancer cDNA micro- page charges. This article must therefore be hereby marked advertisement in array analysis as well as from two online colorectal cancer cohorts. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. These results suggest that EBF1 suppression may help drive colorectal cancer by triggering PNO1 overexpression. This would Received October 24, 2018; revised January 31, 2019; accepted March 8, 2019; be consistent with previous studies that have proposed EBF1 as a published first March 12, 2019.

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2270 Cancer Res; 79(9) May 1, 2019 Cancer Research

EBF1-Mediated Upregulation of Ribosome Assembly Factor PNO1 Contributes to Cancer Progression by Negatively Regulating the p53 Signaling Pathway

Aling Shen, Youqin Chen, Liya Liu, et al.

Cancer Res 2019;79:2257-2270. Published OnlineFirst March 12, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-3238

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