ebf1-mediated upregulation of ribosome assembly factor ... · and thus increased ribosome...

Molecular Cell Biology

EBF1-Mediated Upregulation of RibosomeAssembly Factor PNO1 Contributes to CancerProgression by Negatively Regulating the p53Signaling PathwayAling Shen1,2, Youqin Chen1,2,3, Liya Liu1,2,3, Yue Huang1,2, Hongwei Chen1,2, Fei Qi1,2,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 ribosomebiogenesis, but its potential role in cancer remains unknown.In this study, online data mining, cDNA, and tissue micro-arrays indicated that PNO1 expressionwas higher in colorectalcancer tissue than in noncancerous tissue, and its overexpres-sion was associated with worse patient survival. Gain-of-function and loss-of-function studies demonstrated thatPNO1 knockdown suppressed growth of colorectal cancercells in vitro and in vivo, while PNO1 overexpression promotedcolorectal cancer cell proliferation in vitro. In colorectal cancercells expressing wild-type p53, PNO1 knockdown enhancedexpression of p53 and its downstream gene p21, and reducedcell viability; these effectswere prevented byp53knockout andattenuated by the p53 inhibitor PFT-a. Moreover, PNO1knockdown in HCT116 cells decreased levels of 18S rRNA,of 40S and 60S ribosomal subunits, and of the 80S ribosome.It also reduced global protein synthesis, increasing nuclear

stress and inhibiting MDM2-mediated ubiquitination andp53 degradation. Overexpressing EBF1 suppressed PNO1 pro-moter activity and decreased PNO1 mRNA and protein, inhi-biting cell proliferation and inducing cell apoptosis throughthe p53/p21 pathway. In colorectal cancer tissues, the expres-sion of EBF1 correlated inversely with PNO1. Data mining ofonline breast and lung cancer databases showed increasedPNO1 expression and association with poor patient survival;PNO1 knockdown reduced cell viability of cultured breast andlung cancer cells. Taken together, these findings indicate thatPNO1 is overexpressed in colorectal cancer and correlates withpoor patient survival, and that PNO1 exerts oncogenic effects,at least, in part, by altering ribosome biogenesis.

Significance: This study identifies the ribosome assemblyfactor PNO1 as a potential oncogene involved in tumorgrowth and progression of colorectal cancer.

IntroductionColorectal cancer is the third most common cancer and fourth

leading cause of cancer-related death worldwide, with 1.2millionnew cases and over 600,000 deaths each year (1, 2).Despite recentprogress in treatment, outcomes in colorectal cancer remain poor.Therefore, a better understanding of themolecularmechanisms ofcolorectal cancer is urgently required, as is the discovery of new

diagnostic and prognostic biomarkers. In this study, cDNAmicro-array analysis of paired cancerous and noncancerous tissues frompatients with colorectal cancer was followed by high-contentscreening using lentivirus-delivered short hairpin (sh)RNA inter-ference in colorectal cancer cells. This led to the finding thatribosome assembly factor PNO1 was overexpressed in cancertissues, while PNO1 knockdown inhibited the growth of colo-rectal cancer cells.

The ribosome is a supramolecular ribonucleoprotein complexresponsible for translating mRNA into proteins. Ribosome bio-genesis is a complicated, well-orchestrated process that involvesthe transcription and processing of ribosomal RNAs, the produc-tion of ribosomal proteins, as well as the assembly and nuclearexport of ribosome subunits. In eukaryotes, ribosome biogenesisis facilitated by the coordinated function of over 200 assemblyfactors including helicases, ATPases, GTPases, and kinases, whichjoin and are released from preribosomal particles at differenttimes during ribosome maturation. Because ribosome biogenesisdetermines the capacity of a cell to synthesize proteins and henceplays a crucial role in cell growth and proliferation, dysregulationof this vital process is associated with many diseases includingcancer (3–5).

Cancer cells are characterized by an uncontrolled increase incell proliferation (6), which requires extensive protein synthesis

1Academy of Integrative Medicine, Fujian University of Traditional ChineseMedicine, Fuzhou, Fujian, China. 2Fujian Key Laboratory of Integrative Medicinein Geriatrics, Fujian University of Traditional Chinese Medicine, Fujian, China.3Department of Pediatrics, Rainbow Babies and Children's Hospital, CaseWestern Reserve University School of Medicine, Cleveland, Ohio.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

A. Shen and Y. Chen contributed equally to this article.

Corresponding Author: Jun Peng, Academy of Integrative Medicine, FujianUniversity of Traditional Chinese Medicine, 1 Huatuo Road, Minhou Shangjie,Fuzhou, Fujian 350122, China. Phone: 8659-1228-61303; Fax: 8659-1228-61157;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-3238

�2019 American Association for Cancer Research.

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and thus increased ribosome biogenesis (7). In several typesof human malignancies, overexpression of specific ribosomalproteins and ribosome assembly factors results in increasedribosome biogenesis and is associated with poor prognosis(8–16). If upregulated ribosome biogenesis plays an importantrole in malignant transformation by promoting cancer cell pro-liferation, then defects in ribosome assembly can cause cell-cyclearrest, inhibit cell proliferation, and induce cell apoptosis, ulti-mately suppressing tumor growth (17–21).

In this way, a promising anticancer strategy may be to suppressribosome biogenesis by targeting ribosome assembly factors. TheRNA-binding protein "partner of NOB1" (PNO1), also known asDim2 or Rrp2, is a ribosome neogenesis factor highly conservedfrom yeast to mammals. The human PNO1 gene is located onchromosome 2q14 and includes seven exons (22). The full-lengthcDNA sequence of PNO1 (1637 bp) contains a 759-bp openreading frame encoding a protein of 252 or 248 residues, whichcontains a conserved C-terminal K homolog (KH) domainresponsible for RNA binding (23, 24). Previous studies in yeasthave revealed that PNO1 participates in ribosome biogenesis.PNO1 is one of six assembly factors required for cytoplasmicmaturation of the 20S pre-rRNA to 18S rRNA; it binds to NOB1and increases NOB1 affinity for RNA, stimulating NOB1 to cleaveat the 30 endof pre-18S rRNA. Loss of PNO1 results in a decrease of18S rRNA and defective assembly of pre-40S ribosomal subu-nits (25–27).

In contrast to extensive studies of the yeast homolog of PNO1called Yor145, little is known about the functions of PNO1 inmammalian cells. In particular, a role in cancer progression hasnever been reported. Therefore in this study, we applied a com-bination of high-throughput "omics" technologies, online datamining, biochemistry, and molecular biology to evaluate PNO1expression in human cancers, its association with cancer progno-sis, as well as its potential oncogenic activity and the underlyingmechanisms.

Materials and MethodsSummary of the experimental design

A cDNA microarray was used to identify differentiallyexpressed genes (DEG) in paired cancerous and noncanceroustissues from patients with colorectal cancer. Cell-based high-content screening and shRNA-mediated knockdown led toidentification of PNO1. Prognostic value of PNO1 was ana-lyzed in both clinical colorectal cancer samples and onlinedatabases. Effects of PNO1 on tumor growth in vitro andin vivo, as well as on cell proliferation and cell apoptosis, wereassessed in colorectal cancer cells after PNO1 knockdownand/or overexpression. A cDNA microarray was used afterPNO1 knockdown to identify downstream regulatory mechan-isms. Bioinformatics analysis of DEGs identified the p53/p21pathway as one of the most enriched signaling pathways.Effects of p53/p21 signaling on oncogenic activities of PNO1were investigated in HCT116 cells that were exposed to a p53inhibitor or in which the p53 gene was deleted. The putativemechanism by which PNO1 knockdown activated p53 signal-ing was evaluated in colorectal cancer cells by examiningribosome biogenesis and protein synthesis, nuclear morpho-logy, and levels of p53 in nucleus, as well as ubiquitinationand degradation of p53. Regulation of PNO1 by EBF1 was

examined. Potential roles of PNO1 in breast and lung cancerswere also examined.

Patients and specimensFifty pairs of cancerous and matched noncancerous tissues (at

least 5-cm away) were obtained from patients with colorectalcancer who underwent surgical resection between 2013 and 2014at Fujian Provincial Hospital and the First People's HospitalAffiliated to Fujian University of Traditional Chinese Medicine(Fujian, China). Clinicopathologic characteristics of patients aresummarized in Supplementary Table S1. The specimens wereusedwithwritten informed consent from thepatients and approv-al by the local Ethics Committees. No patients received radio- orchemotherapy prior to surgery.

Sampleswere processed using routinemethods for IHC, or theywere snap-frozen and stored in liquid nitrogen for other use.Samples stained with hematoxylin and eosin were examined byexperienced pathologists using the pathologic tumor–node–metastasis (p-TNM) classification of the International UnionAgainst Cancer.

Microarray analysisMicroarray assays were performed to identify DEGs between

colorectal cancer tissues (n ¼ 14) and matched adjacent normaltissues (n¼ 14), as well as betweenHCT116 cells transduced withlentivirus encoding anti-PNO1 shRNA (n ¼ 3) or control shRNA(n¼ 3). These assays were carried out by Shanghai GeneChem orCapitalBio. Briefly, total RNA was extracted with TRIzol reagent(Thermo Fisher Scientific). Complementary DNA was synthe-sized, labeled, andhybridized to thehumanGeneChip Primeviewarray (Affymetrix). Scanning was conducted using a GeneChipScanner 3000 and analyzed using GeneChip GCOS 1.4 software(Affymetrix). Genes were classified as DEGs if their expressiondiffered at least 2-fold between the two conditions, and if thedifference was associated with P < 0.05. DEGs were identifiedusing volcano plots and hierarchical clustering plots. Kyoto Ency-clopedia of Genes and Genomes (KEGG) pathway enrichmentanalysis was used to identify pathways represented among theDEGs.

Cell cultureThe following cell lineswere obtained from theCell Bank of the

Chinese Academy of Sciences (Shanghai, China): human colo-rectal cancer lines RKO, HCT-8, HT-29, HCT116, and Caco2; lungcarcinoma cell line A549; breast cancer cell line MCF-7; andembryonic kidney HEK293T cells. Wild-type HCT116 cells(HCT116/p53þ/þ) and HCT116 cells lacking the p53 gene(HCT116/p53�/�) were a gift from Dr. Yao Lin (Fujian NormalUniversity, Fujian, China), which were originally obtained fromDr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD).RKO, HCT116, HCT-8, HCT116/p53þ/þ, and HCT116/p53�/�

cells were maintained in RPMI1640 (Thermo Fisher Scientific);HT-29 cells, in M50A medium (KeyGEN); Caco2, MCF-7, andA549 cells in DMEM (Thermo Fisher Scientific); and HEK293Tcells in MEM (Thermo Fisher Scientific). All media contained10% FBS (Thermo Fisher Scientific), 100 U/mL penicillin, and100 mg/mL streptomycin (Hyclone). Cells were cultured at 37�Cin a humidified atmosphere of 5% CO2. For long-term use ofHCT116 and RKO cells, cells were verified using short tandemrepeat genotyping and examined for Mycoplasma contaminationusing RT-PCR analysis.

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Lentiviral transductionThree shRNAs for each gene were cloned into the GV115

lentiviral vector (Shanghai GeneChem), which encodedenhanced GFP (EGFP) under control of the CMV promoter.The double-stranded shRNAs targeting each gene are summarizedin Supplementary Table S2. Lentivirus encoding shRNA againsta target gene or a nonsilencing control shRNA was added tocultured cells at a multiplicity of infection (MOI) of 10, asrecommended by the manufacturer. To generate a cell line stablyoverexpressing PNO1, cultures of HT-29 and HCT-8 cells weretransduced for 72 hours with a lentiviral vector (MOI: 10) encod-ing full-length human PNO1 (coding region of 756 bp; ShanghaiGeneChem). Transductants were selected for 2 weeks using puro-mycin (Thermo Fisher Scientific) at 1.5 mg/mL for HCT-8 cells or1mg/mL forHT-29 cells. In experiments to examine effects of EBF1on expression of PNO1 and its downstream targets, and on cellproliferation and apoptosis, RKO cells were transduced for72 hours with a lentiviral vector (MOI: 10) encoding full-lengthhuman EBF1 (coding region of 1776 bp; Shanghai GeneChem).Transductants were selected for 2 weeks using 1 mg/mL ofpuromycin.

High-content screening for cell growthGrowth of cultured cells was assessed using multiparametric

high-content screening. At 72 hours after shRNA transduction,cells were seeded into 96-well plates at a density of 2,000 cells/well in 100 mL of medium. Cell growth was monitored every dayfor 5days using theCellomics ArrayScanV(TI) high-content imageanalysis platform and analyzed using HCS Studio Cell AnalysisSoftware (Thermo Fisher Scientific).

Quantitative real-time PCR and tissue cDNA array analysisTotal RNA was extracted from the cell line samples or tissue

samples using RNAiso Plus reagent (Takara). Reverse transcrip-tion was performed using the PrimeScript RT reagent kit (Takara).The resulting cDNA, or a commercial tissue cDNAarray (ShanghaiOutdo Biotech), was used to examine levels of mRNAs encodingPNO1, CDKN1A/p21, THBS1, MAPK1, MCM2, CDC42, EBF1, orGAPDH using an ABI 7500 Fast Real-Time PCR System (AppliedBiosystems) and the SYBR Premix Ex Tag (Takara). The conditionsfor real-time PCR were as follows: predenaturation (95�C for 10minutes), denaturation (95�C for 15 seconds), annealing andextension (60�C for 60 seconds) for a total 40 cycles. GAPDHwasused as an internal control. Primer sequences are shown inSupplementary Table S3. mRNA levels are presented as: 2�DDCt

(with Ct being the cycle threshold), where DCt ¼ [Ct (target gene)� Ct (GAPDH)].

Western blot analysisProteins were extracted using RIPA lysis buffer (Thermo Fisher

Scientific), separated by SDS-PAGE, transferred onto a nitrocel-lulose membrane, and then blocked with a blocking buffer(Thermo Fisher Scientific) prior to overnight incubation at 4�Cwith a primary antibody (Supplementary Table S4). Membraneswere washed extensively and incubated with a horseradish per-oxidase–conjugated goat anti-rabbit or anti-mouse secondaryantibody. The blots were visualized using a chemiluminescencemethod (Thermo Fisher Scientific), and band intensities werequantified relative to intensity of b-actin using ImageJ software.Levels of target protein were expressed relative to levels in controlcells, defined as 1.00.

IHC stainingTissue sections were incubated with an anti-PNO1 antibody

(1:800; catalog no. LS-C179090, LSBio). Background stainingwasassessed by omitting the primary antibody. The intensity andpercentage of positive cells from five fields in each sample weredetermined independently by two experienced pathologistsblinded to the clinical and pathologic data. Staining intensitywas assessed using a 4-point scale (0, undetectable; 1, weak; 2,moderate; 3, strong). Percentage of positively stained cellswas expressed as one of four categories: 1, 0%–25% cells stained;2, 26%–50% stained; 3, 51%–75% stained; 4, 76%–100%stained (28). PNO1 expression was calculated by multiplying theintensity and percentage scores together.

Tissue microarray and survival analysisTissue microarray (TMA) slides (Shanghai Outdo Biotech)

contained 90 pairs of tissue samples and were hybridized withprimary antibody against PNO1 (1:500) using standard techni-ques (29). The 90 pairs of specimens were obtained from TaizhouHospital of Zhejiang Province (Zhejiang, China) from July 2007toOctober 2008; all subjects had reliable information on survival,and there was no data censoring prior to 7 years of follow-up.

Images were captured using a Nano Zoomer 2.0 HT slidescanner (Hamamatsu Photonics) and processed using NanoZoomer Digital Pathology View 1.6 software. IHC score wasdetermined independently by two experienced pathologistsblinded to the clinical and pathologic data. PNO1 expressionwas scored as described in the "IHC staining" section. PNO1expression was considered high for scores of 4–12 and low forscores of 0–3. The relationship between PNO1 expression (low orhigh) and patient overall survival was analyzed using Kaplan–Meier analysis and assessed for significance using the log-rank test.

Cell transfectionThree nonoverlapping anti-PNO1 siRNA oligonucleotides (si-

PNO1, 25-mer Stealth RNAi duplexes) and control siRNAs (si-Ctrl) were designed using the BLOCK-iT RNAi design program(Thermo Fisher Scientific; Supplementary Table S2). Cells weretransfected with siRNAs or si-Ctrl at a concentration of 10 nmol/Lusing Lipofectamine RNAiMax (Thermo Fisher Scientific).

Cell proliferation assayCell proliferationwas determinedusingCFDAorCCK-8 assays.

In the CFDA assay, cells were transfected with anti-PNO1 orcontrol siRNA for 24–72 hours, washed with PBS, and incubatedin fresh culturemediumuntil the indicated timepoints, when 200mLCFDA (25mmol/L; Thermo Fisher Scientific)was added to eachwell. Plates were incubated for an additional 2 hours at 37�C inthe dark. Fluorescence intensity was measured at 480 nm using amicroplate reader (Tecan). In the CCK-8 assay, cells were trans-fected with siRNA or transduced with lentivirus and cultured forthe indicated duration, when 10 mL CCK-8 (Cell Counting Kit-8,Dojindo) was added to each well. Plates were incubated foran additional 2 hours at 37�C and absorbance was measured at450 nm.

Colony-forming assayFor cell survival analysis, transduced cells were seeded into

12-well plates (500 cells/well) and incubated in humidified aircontaining 5% CO2 at 37�C for 10–14 days to allow colonyformation. Medium was replaced every 2–3 days. Cells were

PNO1 Knockdown Inhibits Colorectal Cancer by Activating p53

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washed with PBS, fixed with 4% paraformaldehyde, and stainedwith crystal violet. After staining, photographs were taken andnumbers of colonies counted. Data were normalized to results forcontrol cells.

For cell colony formation in agarose, transduced cells weresuspended in medium containing 0.35% low-melting-point aga-rose (ThermoFisher Scientific), and500 cellswere seeded inplatescontaining medium with 0.6% solidified agarose. After incuba-tion for 2–3 weeks, photographs were taken and numbers ofcolonies counted. Three independent experiments were per-formed, and data were normalized to results for control cells.

Cell-cycle analysisCells were fixed with 70% ethanol at 4�C overnight, washed

extensively, and incubated with FxCycle PI/RNase Staining Solu-tion (Thermo Fisher Scientific) for 30 minutes. DNA content wasanalyzed by FACS (FACSCalibur, Becton Dickinson). The pro-portion of DNA in different phases was analyzed using ModfitLTversion 3.0 (Verity Software House).

Apoptosis analysisCells were washed with ice-cold PBS followed by binding

buffer, and then stained for 15 minutes with Annexin V-APC(KeyGEN). The percentage of apoptosis was analyzed by FACS(FACSCalibur, Becton Dickinson).

Caspase activity assayThe activity of caspase-3 and -9 was determined by commercial

colorimetric assays (KeyGEN). Cells were lysed with lysis bufferfor 30 minutes on ice, and centrifuged at 14,000 rpm for 10minutes. Samples containing 100 mg total protein were incubatedat 37�C in the dark for 2 hours with 50 mL caspase-3 substrateAsp-Glue-Val-Asp (DEAD)-pNA, or with 50 mL caspase-9 sub-strate Leu-Glu-His-Asp (LEHD)-pNA. Absorbance was measuredat 405 nm.

In vivo experimentsAnimal care and experiments were performed in strict accor-

dance with the "Guide for the Care and Use of LaboratoryAnimals" and the "Principles for the Utilization and Care ofVertebrate Animals," and approved by the Committee of FujianUniversity of Traditional Chinese Medicine (Fujian, China). MaleBALB/c nude mice (6–8 weeks of age; 20–22 g) were obtainedfrom Shanghai SLAC Laboratory Animal Co. andmaintained in aspecific pathogen-free facility. HCT116 or RKO cells (1 � 106) in100 mL RPMI1640 medium containing 50% Matrigel wereinjected subcutaneously into the flank of nude mice (n ¼ 8).Starting on day 7 after the first injection, tumor growth wasmonitored once every other day for 19 days. Tumor volume(mm3) was calculated as (1/2) (length � width2), where lengthand width refer to the longest longitudinal and transverse dia-meters, respectively.

At the end of the experiment, mice were anesthetized withisoflurane. Tumor images were captured using an IVIS Spectrumwhole live-animal imaging system (PerkinElmer). Mice were thensacrificed for tissue collection.

Promoter activity assayHCT116 cells were transduced for 4–6 hours with lentivirus

encoding anti-PNO1 or control shRNAs. Then cells were trans-fected for 4–6 hours with a plasmid expressing luciferase reporter

under the control of a p53-driven promoter. Quantitation ofluciferase activity allowed assessment of promoter activity. Briefly,cells were transduced for 48 hours, washed twice with PBS, andharvested with 1 � Passive Lysis Buffer from the Dual-LuciferaseReporter Assay System (Promega). Cells were lysed and centri-fuged, and the supernatant was collected. Aliquots of supernatant(40 mL) were added to 96-well plates, followed by 20 mL luciferaseassay reagent (Promega) at room temperature. Luciferase activitywasmeasured immediately using a luminometer (Orion IIMicro-plate Luminometer, Berthold Detection Systems). Data werenormalized to the results obtained for the internal control Renillaluciferase.

To examine whether the 10 predicted transcription factorsindeed modulated PNO1 transcription, plasmids were con-structed encoding the full-length predicted factors in the GV141vector (Shanghai GeneChem), which contains a multiple cloningsite followed by a 3FLAG tag downstream of the CMV promoter,aswell as the neomycin genedownstreamof the SV40promoter. Aseparate plasmid was constructed from vector GV238 (ShanghaiGeneChem) encoding luciferase reporter downstream of a 2.0-kbfragment of the PNO1promoter. Plasmid integrity was confirmedby DNA sequencing. Briefly, HEK293T or RKO cells were cotrans-fected with each of 10 different overexpression plasmids orEBF1 overexpression plasmid (500 ng), Renilla luciferase reporter(20 ng), and luciferase reporter (500 ng) using Lipofectamine3000 Transfection reagent (Thermo Fisher Scientific). Promoteractivities were measured after 48-hour transfection.

Northern blot analysis of 18S rRNAExtracted total RNA (10 mg per sample) was fractionated on a

1.2% agarose–formaldehyde gel and transferred to Hybond NÞmembranes (Amersham). BIO-labeled probe (summarized inSupplementary Table S3) was purchased from Thermo FisherScientific. The membrane was hybridized overnight in hybridiza-tion buffer (Thermo Fisher Scientific), washed twice (5 minuteseach) under low-stringency conditions (2 � SSC, 0.1% SDS),blocked with blocking buffer at 50�C for 30 minutes, thenincubated at 50�C for 20 minutes with stabilized streptavidin–horseradish peroxidase conjugate (1:300). Membranes wereexposed to a phosphor storage screen and visualized using theChemi Doc XRSþ System (Bio-Rad).

Ribosome profile analysisTransfected HCT116 cells were treated with 100 mg/mL cyclo-

heximide for 15 minutes, washed with ice-cold PBS containing100 mg/mL cycloheximide, and then resuspended in hypotonicbuffer [10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 10mmol/L KCl, 0.5 mmol/L dithiothreitol (DTT), 100 mg/mL cyclo-heximide, 40 U/mL RNase inhibitor, 1� protease inhibitorcocktail]. Cells were centrifuged at 14,000 rpm for 15 minutesat 4�C. Total protein (1 mg) was loaded onto a linear sucrosegradient (5%–50%) and centrifuged for 3 hours at 38,000 rpm at4�C. Fractions (100 mL) were collected and precipitated withtrichloroacetic acid and analyzed by Western blotting.

Protein synthesis assayGlobal protein synthesis was assayed using a commercial kit

(Cayman Chemical). Transfected HCT116 cells were incubatedfor 30minutes with o-propargyl-puromycin in completemediumin 96-well plates (100 mL/well). Cells were fixed with cell-basedassay fixative (100 mL/well), stained with 5 FAM-azide (Cayman

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Chemical), and photographed under a fluorescence microscope(Accu-Scope). Images were collected using MetaMorph imageacquisition software (Molecular Devices). Fluorescence intensityin single cells was quantitated using Image Pro Plus software(Media Cybernetics).

Immunofluorescence stainingCells were fixed with 10% formalin for 15 minutes, permea-

bilized with 0.25% Triton X-100 for 5 minutes, blocked with10% goat serum and 5% BSA in PBS for 1 hour at roomtemperature, then incubated overnight at 4�C with primaryantibody diluted in PBS (anti-p53, 1:200; anti-nucleolin,1:200; Supplementary Table S4). Next, cells were further incu-bated with Alexa Fluor 647–conjugated anti-rabbit secondaryantibody (1:1,000) in 10% goat serum and 5% BSA in PBS for1 hour at room temperature in the dark. Finally, cells werestained with DAPI. Images were acquired on a confocal micro-scope (Ultraview Vox, PerkinElmer).

In vivo ubiquitination assayAfter transductionwith sh-PNO1 lentivirus or sh-Ctrl lentivirus

for 72 hours, cells were reseeded in 100-mm dishes, and thentransfected with 2 mg of Ub-HA overexpression plasmid. Aftertransfection for 72 hours, cells were treated with 10 mmol/Lproteasome inhibitor MG132 (MCE) for 6 hours and then lysedusing Western and IP lysis buffer (Beyotime Biotechnology)containing a proteinase inhibitor cocktail and phenylmethylsul-fonylfluoride. A total of 1 mg of soluble proteins was incubatedovernight at 4�C with 5 mg of anti-p53 (MBL) and Protein A&Gsepharose beads (Santa Cruz Biotechnology). Beads were washedthree times withWestern and IP lysis buffer and the proteins wereseparated by 15% SDS-PAGE and analyzed by Western blottingusing anti-HA antibody.

Bioinformatics analysisLevels of PNO1mRNAwere analyzed in various types of tumor

tissues from multiple cohorts in the Oncomine database (www.oncomine.org). Correlation between PNO1 mRNA level andsurvival in patients with breast or lung cancer was analyzed usinga Kaplan–Meier method (http://kmplot.com/analysis/). Thethreshold of significance was set at P < 0.05.

We used the web-based prediction program TFBIND (http://tfbind.hgc.jp) to search for potential transcription factors withbinding sites in the PNO1 promoter. Expression of PNO1 andEBF1 in colorectal cancer samples was analyzed using twodatasets (GEO ID: GSE3629 and GSE23878) with the aid ofthe R2 web application (http://r2.amc.nl). Correlation betweenlevels of PNO1 and EBF1 expression was analyzed using thePearson test.

Statistical analysisStatistical analysis was performed using SPSS 20.0 (IBM). Data

are presented asmean� SD.Differences between two groupswereassessed for significance using the independent Student t test, anddifferences among three or more groups were assessed using one-way ANOVA. Kaplan–Meier survival differences were assessedusing the log-rank test. Correlation between PNO1 and EBF1expression was analyzed using Pearson rank correlation. P < 0.05was considered significant.

Data availabilityThe authors declare that all data supporting the findings of this

study are available within the article, in the SupplementaryInformation Files, and from the authors upon request.

ResultsIdentification of PNO1 as a potential target in colorectal cancer

Microarray experiments comparing 14pairs of colorectal cancerprimary lesions and noncancerous surrounding tissue revealed1,868 DEGs, of which, 778 in primary lesions were upregulatedand 1,090 were downregulated (Fig. 1A and B; GEO Submission:GSE113513). To identify novel oncogenes, we focused on 16upregulated genes that have not been extensively investigated forpotential associationwith cancer, including PNO1, EFNA3, CKS2,CDCA5, NUF2, and DACH1 (Fig. 1C; Supplementary Table S5).High-content screening in which lentivirus was used to delivershRNAs 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 assemblyin oncogenesis (8–16), we focused on PNO1 in subsequentstudies.

PNO1 is highly expressed in colorectal cancer and is associatedwith poor prognosis

Quantitative PCR of an independent sample of 50 colorectalcancer cases previously collected fromour laboratory showed thatlevels of PNO1mRNAwere elevated in colorectal cancer (Fig. 2A),and these results were confirmedbyWestern blot analysis (Fig. 2B;n¼ 12) and IHC (Fig. 2C; n¼ 10). Quantitative PCR analysis of acDNA array based on a commercially available set of 80 colorectalcancer primary lesions and 15 noncancerous surrounding tissues(Shanghai Outdo Biotech) showed increased PNO1 expression incolorectal cancer (Fig. 2D), but no significant relationship wasobserved between PNO1 mRNA and patient survival. Analysis oflevels of PNO1 protein in an IHC-based tissue microarray basedon 90 colorectal cancer samples (Shanghai Outdo Biotech)showed increased PNO1 expression in colorectal cancer primarylesions (P<0.05 vs. noncancerous surrounding tissues; Fig. 2E), aswell as an association between higher PNO1 expression andpoorer overall survival (P < 0.05; Fig. 2F and G).

PNO1 promotes colorectal cancer cell proliferationIn cultured RKO and HCT116 cells, which constitutively

express relatively high levels of endogenous PNO1 (Supplemen-tary Fig. S1A and S1B), PNO1 knockdown based on lentivirus-delivered shRNA or on transfected siRNA (Fig. 3A and B; Sup-plementary Fig. S1C and S1D) decreased cell viability (Fig. 3C;Supplementary Fig. S1E) and colony formation (Fig. 3D; Sup-plementary Fig. S1F). It also increased the percentage of cells inG0–G1 phase, with a concomitant decrease in the percentage ofcells in S phase (Fig. 3E). PNO1 knockdown increased the per-centage of cells undergoing apoptosis (Fig. 3F), and it increasedactivity of caspases-3 and -9 (Fig. 3G and H).

In contrast, ectopic PNO1 expression inHT-29 andHCT-8 cells(Fig. 3I and J), which constitutively express low levels of endog-enous PNO1, increased cell viability and colony formation(Fig. 3K and L). Xenografts of wild-type HCT116 or RKO cellsgrew significantly faster in nude mice than xenografts in whichPNO1 was knocked down prior to inoculation (Fig. 4A and B).





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Figure 1.

Gene expression profile analysis and high-content screening suggest an oncogenic role for PNO1. cDNAmicroarray analysis was performed to identify DEGsbetween 14 pairs of colorectal cancer tissues (T) and adjacent normal tissues (N). High-content screening and lentivirus-delivered, shRNA-based interferencewere 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 tocompare gene expression profiles (fold change >2 or�2.0, P < 0.05). C, Heatmap of 16 selected DEGs. RKO cells were transduced with lentivirus encodingshRNAs specific against these 16 DEGs, and cell growth was measured using multiparametric high-content screening. D, Effects of PNO1, CDCA5, NUF2, andDACH1 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 RKOcells. Data were normalized to cell number on day 1 and are represented as fold change. � , P < 0.05 versus control shRNA.

Shen et al.





Oncogenic activities of PNO1 depend on p53 signalingMicroarray analysis of HCT116 cells in which PNO1 was

knocked down revealed 253 DEGs (Fig. 5A; GEO submission:GSE113514). Many of these DEGs, including CDKN1A (alsoknown as p21), THBS1, MAPK1, MCM2, and CDC42 (Fig. 5B,P < 0.05), have been reported to play essential roles in cellproliferation and apoptosis (30–34). KEGG pathway enrichmentanalysis of these DEGs indicated that p53 pathways were amongthe 10 most enriched signaling pathways (Fig. 5C); one of thegenes most strongly upregulated in response to PNO1 knock-down was the downstream effector p21 (Fig. 5B).

Microarray analysis (Fig. 5D) and quantitative PCR (Fig. 5E)showed a slight increase in p53 mRNA. PNO1 knockdown

increased luciferase activity in the p53-driven luciferase reporterassay (Fig. 5F). Western blot analysis confirmed increased expres-sion of both p53 and p21 at the protein level (Fig. 5G). Theinhibitory effects of PNO1 knockdown were blocked when p53was knocked out (Fig. 5H and I), and the inhibitory effects wereattenuated when cells were treated with the p53 inhibitor PFT-a(Fig. 5J).

PNO1 knockdown activates p53 through the ribosomal stresspathway by inhibiting MDM2-mediated ubiquitination anddegradation of p53

Consistently with studies in yeast (26, 27), we confirmed thatPNO1 knockdown in HCT116 cells significantly decreased levels

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Figure 2.

PNO1 expression is upregulated in colorectal cancer tissues and associated with poor prognosis of colorectal cancer patients. A, PNO1 mRNA levels in tissuesfrom 50 patients with colorectal cancer were analyzed using quantitative PCR. GAPDHwas used as an internal control. B, PNO1 protein expression in 12 pairs ofcolorectal cancer tissues and adjacent normal tissues was determined byWestern blotting. PNO1 bands were quantitated using ImageJ software, thennormalized to b-actin. C, PNO1 protein levels in 10 pairs of colorectal cancer tissues and adjacent normal tissues were determined by IHC. Representative imageswere 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). GAPDHwas 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 versusnormal tissue. F, Correlation between protein expression of PNO1 and survival of patients with colorectal cancer was analyzed in Kaplan–Meier plots generatedon the basis of results from the IHC-based tissue microarray containing 90 samples (� , P¼ 0.0182). G, Representative images of colorectal cancer tissuesshowing high or low PNO1 expression were taken at a magnification of�40 or�200.






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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 wasperformed to determine levels of PNO1 mRNA. GAPDHwas 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 toviability 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 ofcontrol cells. E, Cell-cycle distribution was determined by flow cytometry, and percentages of cells in G0–G1, S, or G2–M phases were determined. F, Cellapoptosis was analyzed using Annexin V-APC staining, followed by flow cytometry. Activity of caspase-3 (G) and caspase-9 (H) was determined using acolorimetric assay. Data were normalized to caspase activities in cells treated with control shRNA (sh-Ctrl). HT-29 and HCT-8 cells were transduced withlentivirus encoding PNO1. I,Quantitative PCRwas performed to determine levels of PNO1 mRNA. GAPDHwas used as an internal control. J,Western blotting wasperformed to determine levels of PNO1 protein. PNO1 bands were quantitated using ImageJ software and normalized to b-actin. K, Cell viability was measuredusing 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 formationassay 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.

Shen et al.





of 18S rRNA (Fig. 6A), 40S and 60S subunits of the 80S ribosome(Fig. 6B), and global protein synthesis (Fig. 6C). To determinewhether these effects on the nucleolus were related to nucleolarstress, we stained cells for p53 and the major nucleolar proteinnucleolin/C23. PNO1knockdownor treatmentwith actinomycinD (as a positive control) resulted in translocation of nucleolinfrom the nucleolus to the nucleoplasm (Fig. 6D, left) and anincrease in nuclear p53 immunoreactivity (Fig. 6D, right). West-ern blot analysis and coimmunoprecipitation (co-IP) assay indi-cated that PNO1 knockdown led to a reduction in degradation ofp53 (Fig. 6E) and ubiquitination of p53 (Fig. 6F).Moreover, co-IPanalysis indicated that PNO1 knockdown increased the bindingof RPL11 to MDM2 (Fig. 6G).

Transcription factor EBF1 negatively regulates PNO1expression in colorectal cancer

Using TFBIND (http://tfbind.hgc.jp/), we predicted 10potential transcription factors of PNO1: EGR3, TCF-3, MYCN,PBX1, MYC, VBP1, THRB1, RORA, TFCP2, and EBF1. Thesefactors were significantly up- or downregulated in colorectalcancer tissues relative to noncancerous tissue in our cDNAmicroarray (Fig. 7A; Supplementary Table S5). EBF1 displayedthe most potent regulatory effect on PNO1 transcription inHEK293T cells based on a luciferase reporter system (Fig. 7B),and this regulatory effect was confirmed in RKO cells (Fig. 7C).EBF1 overexpression reduced PNO1 expression at the mRNAand protein levels (Fig. 7D and E), as well as reduced cellviability (Fig. 7F) and induced cell-cycle arrest (Fig. 7G) andapoptosis (Fig. 7H). In microarray experiments with 14 pairedsamples of colorectal cancer and noncancerous tissue, PNO1expression was significantly higher in colorectal cancer than innoncancerous tissue (Fig. 1C), whereas the converse was truefor EBF1 expression (Fig. 7A); their expression exhibited asignificant inverse correlation (Fig. 7I), which was consistentwith the analyses in two colorectal cancer cohorts from the R2Bioinformatic Platform (Fig. 7A, J, and K). Therefore, decreasedexpression of the negative transcription factor EBF1 may be oneof the key reasons for PNO1 overexpression in colorectalcancer.

PNO1 displays oncogenic potential in lung and breast cancersData from the Oncomine database (http://www.oncomine.

com/) indicated that PNO1 mRNA levels are significantly upre-gulated in various types of malignancies, including colorectalcancer, lung cancer, and breast cancer (Supplementary Table S6).Analysis of Kaplan–Meier plots (http://kmplot.com/analysis/)indicated that higher PNO1 mRNA levels were associated withpoorer overall survival and relapse-free survival of patients withbreast or lung cancer, as well as shorter time to first progression(Supplementary Fig. S2A, P < 0.05). In cultures of A549 lungcancer cells and MCF-7 breast cancer cells, PNO1 knockdownusing siRNA transfection (Supplementary Fig. S2B and S2C)significantly decreased cell viability (Supplementary Fig. S2D)and cell survival (Supplementary Fig. S2E). These data suggestthat PNO1may play oncogenic roles in colorectal cancer as wellas other kinds of cancers.

DiscussionThe key finding of this report is that the ribosome assembly

factor PNO1may play a critical oncogenic role in tumor initiationandprogression.Overexpression of PNO1 significantly promotedcell proliferation, while depletion of PNO1 suppressed tumorgrowth in vivo and in vitro by inhibiting proliferation and inducingcell apoptosis. PNO1 knockdown suppressed cell proliferation ofcolorectal cancer cells in a p53-dependent manner through acti-vation of the ribosomal protein–MDM2–p53 pathway. Theseresults suggest that PNO1 plays an important role in tumorgrowth and may serve as an attractive therapeutic target forcolorectal cancer treatment. PNO1 expression may be driven bythe transcription factor EBF1, and the effects of PNO1may involvenegative regulation of the p53/p21 pathway. This leads us topropose an oncogenic EBF1/PNO1/p53 axis.

This study describes several important discoveries about thefunctional roles of PNO1 in colorectal cancer. We demonstratedincreased levels of PNO1mRNA and protein in clinical colorectalcancer samples and cell cultures using a combination of techni-ques, including mRNA and tissue microarrays, quantitative PCR,Western blotting, and IHC. These experiments suggest that PNO1

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Oncogenic functions of PNO1 in colorectal cancer cells in vivo. A xenograft nude mouse model was constructed to investigate the effects of PNO1 knockdown ontumor growth. HCT116 cells (A) or RKO cells (B) were transduced with lentivirus encoding anti-PNO1 shRNA (sh-PNO1) or control shRNA (sh-Ctrl), then injectedsubcutaneously into BALB/c nudemice. Tumor size, morphology, and tumor weight were monitored. Tumor fluorescence was imaged using a whole live-animalimaging 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.





http://tfbind.hgc.jp/

http://www.oncomine.com/

http://www.oncomine.com/

http://kmplot.com/analysis/


overexpression plays an essential role in malignant transforma-tion. Indeed, high PNO1 expression based on tissue microarrayanalysis correlated with poor survival of patients with colorectalcancer. These results highlight not only the potential oncogenicrole of PNO1, but also the possibility of using PNO1 as abiomarker for early diagnosis and prognosis of colorectal cancer.Thefindings herefirst need tobe validated in amuch larger clinicalsample.

The uncontrolled cell proliferation of cancer cells means thatthey require extensive protein synthesis; as a result, ribosome

biogenesis is one of the hallmarks of cancer cells (19). Someribosome assembly factors such as RIO1 and NOB1 are upregu-lated in various types of humanmalignancies, and their upregula-tion suggests poor prognosis. Similarly, we found that PNO1 issignificantly upregulated in colorectal cancer tissues and thatPNO1 knockdown inhibits ribosome biogenesis, suggesting thatPNO1 plays an essential role in tumorigenesis. Indeed, we dem-onstrated here that suppressing endogenous PNO1 can arrest cellsat the G1–S transition and induce cell apoptosis, inhibiting tumorgrowth in vivo and in vitro; conversely, overexpressing PNO1

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Figure 5.

Oncogenic activities of PNO1 depend on p53 signaling. A, cDNAmicroarray analysis was performed to determine DEGs in HCT116 cells transduced with lentivirusencoding 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 >2 or�2.0, P < 0.05). B, Levels of mRNAs encoding CDKN1A/p21, THBS1, MAPK1, MCM2, and CDC42 in a cDNAmicroarray of HCT116 cells after PNO1knockdown (left), which were confirmed by quantitative PCR (right). C, KEGG pathway enrichment analysis of DEGs was performed to identify functionallyrelated 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 cDNAmicroarray of HCT116 cells after PNO1 knockdown. E,Quantitative PCR was performed to determine mRNA levels of TP53. GAPDHwas used as an internalcontrol. F,A dual luciferase assay was performed to determine the effect of PNO1 knockdown on transcriptional activity of TP53 in HCT116 cells. G,Westernblotting was performed to determine levels of p53 and p21 proteins in both HCT116 and RKO cells (left). PNO1 bands were quantitated using ImageJ software andnormalized 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 ofPNO1 protein were assayed usingWestern blotting (left). Protein bands were quantitated using ImageJ software and normalized to b-actin (right). I, Cell viabilitywas 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. Cellviability 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 orsh-PNO1 cells without PFT-a treatment.

Shen et al.





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Figure 6.

Effects of PNO1 on ribosome biogenesis and nucleolar stress-induced p53 pathway.A, 18S rRNA productionwas detected in HCT116 cells after PNO1 knockdownusing Northern blotting with an 18S rRNA probe. EB, ethidium bromide. B, Ribosome profiling was performed to determine ribosome biogenesis in HCT116 cellsafter PNO1 knockdown. Formation of 40S subunits, 60S subunits, and 80S ribosomes was evaluated by measuring absorbance at 254 nm. The presence ofribosomes in each fraction was confirmed byWestern blotting against ribosomal protein S3 (PRS3). C, Newly synthesized protein was detected in HCT116 cellsafter PNO1 knockdown using a protein synthesis assay, � , P < 0.05 versus sh-Ctrl. D, After PNO1 knockdown or treatment with actinomycin D (ActD), cells wereimmunostained for nucleolin (left) or p53 (right), then counterstained with DAPI to visualize nuclei. Magnification,�600. E, Degradation of p53 was determinedbyWestern blot analysis in HCT116 cells after PNO1 knockdown, followed by cycloheximide (CHX) treatment or not (top). Amount of p53 was quantified bydensitometry and normalized to the level of b-actin (bottom). F, Ubiquitination of p53 was determined byWestern blot analysis using anti-HA in HCT116 cellsafter PNO1 knockdown, followed by transfection with HA-tag overexpressing plasmid. G, Coimmunoprecipitation (IP) analysis in HCT116 cells after PNO1knockdownwas performed to determine the binding of RPL11 to MDM2 using anti-MDM2 antibody.






significantly promoted cell proliferation. Development of selec-tive small-molecule inhibitors targeting PNO1 may provide apromising therapeutic strategy for patients with colorectal cancer.

The ability of PNO1depletion to inhibit tumorigenesis encour-aged us to begin exploring downstream pathways that maymediate the oncogenic effects of PNO1. Analysis of cDNAmicro-arrays and classification of functional pathways identified severalDEGs between HCT116 cells treated with anti-PNO1 shRNA orcontrol shRNA. TheseDEGs suggest that PNO1may participate inseveral pathways involved in cell-cycle progression and prolifer-ation, including p53/p21 signaling. Activation of the well-knowntumor suppressor p53 induces transcription of various genes,

including the p21 gene, which can lead to cell-cycle arrest andapoptosis as well as inhibit cell proliferation (35–39). Variouscellular insults can activate p53, including disruption of ribosomebiogenesis (21, 40, 41). In this study,we demonstrated that PNO1knockdown enhanced expression of p53 and its downstreamgenep21 in wild-type HCT116 cells as well as reduced cell viability.These effects were blocked by p53 knockout and attenuated by thep53 inhibitor PFT-a.

Ribosome biogenesis is a very complex process (42–44), highlyregulated bymore than 200 ribosome assembly factors, includingRIO1 and NOB1. While studies of the yeast homolog of PNO1showed that it plays an essential role in the processing of pre-18S

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Figure 7.

PNO1 expression in colorectal cancer is negatively regulated by transcription factor EBF1. A, Differences in levels of mRNAs encoding 10 potential transcriptionfactors between colorectal cancer tissues (T) and adjacent normal tissues (N) in cDNAmicroarray 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 < 0.05versus control. D, Effects of EBF1 overexpression onmRNA levels of PNO1 in RKO cells, as measured using quantitative PCR. GAPDHwas used as an internalcontrol. � , P < 0.05 versus control. E, Effect of EBF1 overexpression on levels of PNO1 protein in RKO cells, as measured usingWestern blotting (left). EBF1 proteinbands 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 percentagesof cells in G0–G1, S, or G2–M phases were determined.H, Cell apoptosis was analyzed using Annexin V-APC staining, followed by flow cytometry. I,Analysis of thepossible correlation between PNO1 and EBF1 mRNA levels in colorectal cancer tissues based on the cDNAmicroarray described in Fig. 1. Data were analyzedusing Pearson rank correlation (� , P < 0.05; R¼ 0.576). J and K,Analysis of the possible correlation between PNO1 (left) and EBF1 (middle) mRNA levels incolorectal cancer tissues based on the online dataset from the R2 Bioinformatic Platform (GEO ID: GSE3629 and GSE23878; right).

Shen et al.





rRNA, the physiologic functions of mammalian PNO1 remainunclear. Using RNA profiling and Northern blotting, we foundthat PNO1 knockdown in HCT116 cells decreased amounts of18S rRNA, 40S subunits, 60S subunits, and the 80S ribosome,leading thereby to significant inhibition of global protein syn-thesis. These results demonstrate the essential role of PNO1 inribosome biogenesis in mammalian cells, specifically in humancolorectal cancer cells. Further studies need to be done to explorefunctional correlation between PNO1 and NOB1 in mammaliancells.

Deficiency in ribosome biogenesis elicits a p53-dependentcellular stress response referred to as "nucleolar stress" or "ribo-somal stress" (21). We confirmed that the effect of PNO1 knock-down on the nucleolus is consistent with nucleolar stress andactivation of p53. Moreover, PNO1 knockdown resulted in trans-location of nucleolin from the nucleolus to the nucleoplasm,which is an indicator of increased nucleolar stress, and it resultedin an increase in levels of p53 in the nucleus. A p53-drivenluciferase reporter assay and quantitative PCR analysis indicatedthat PNO1 knockdown slightly increased luciferase activity andp53 mRNA expression. PNO1 knockdown led to a reduction inthe degradation and ubiquitination of p53. These studies suggestthat both the increase in p53 mRNA expression and decrease inMDM2-mediated degradation and ubiquitination of p53 con-tribute to the increase in p53 after PNO1 knockdown. Therefore,further study is needed to explore precisely how PNO1 knock-down activates p53. The ribosome protein RPL11 binds toMDM2and inhibits its ubiquitin ligase activity toward p53, resulting inp53 accumulation (21, 39, 45).We therefore detected the bindingof RPL11 and MDM2 in HCT116 after PNO1 knockdown andfound that this knockdown significantly increased the binding ofRPL11 to MDM2, suggesting that ribosomal stress increasesRPL11 binding to MDM2 and decreases ubiquitination anddegradation of p53; this may contribute to the accumulation ofp53. Future work should address PNO1-triggered downstreamprocesses in colorectal cancer.

In parallel with our efforts to explore downstream effectors ofPNO1 oncogenicity, we searched for upstream regulators ofPNO1. Web-based screening of transcription factor binding sitesidentified EBF1 as a potential transcription factor of PNO1. Wefound that EBF1 overexpression significantly decreased levels ofPNO1 mRNA and protein in colorectal cancer cells, while itincreased levels of p53 and p21 protein. Moreover, EBF1 over-expression in RKO cells significantly decreased cell viability,arrested the cell cycle at G0–G1 phase, and induced cell apoptosis.We also found that EFB1 expression correlated inversely with thatof PNO1 based on data from our colorectal cancer cDNA micro-array analysis as well as from twoonline colorectal cancer cohorts.These results suggest that EBF1 suppression may help drivecolorectal cancer by triggering PNO1 overexpression. This wouldbe consistent with previous studies that have proposed EBF1 as a

tumor suppressor in hematologic malignancies (46, 47). HowEBF1 regulates PNO1 expression should be investigated in furtherstudies. In B-cell development, EBF1 helps drive DNA demeth-ylation and chromatin remodeling, which controls the transcrip-tion of various genes (46, 47). Whether EBF1 regulates PNO1expression in the same way should be explored, and moregenerally the potential roles of EBF1 in solidmalignancies shouldbe clarified.

In conclusion, this is the first demonstration of the significanceof PNO1 in colorectal cancer. Our data suggest that the proteinplays a role in tumorigenesis and prognosis. We have also shown,for the first time, that mammalian PNO1 is critical for ribosomebiogenesis in cancer cells, suggesting that it works as a ribosomeassembly factor like its yeast homolog. PNO1 transcriptionappears to be regulated by the transcription factor EBF1, andPNO1 exerts its oncogenic effects, at least in part, by negativelyregulating the p53 signaling pathway. These findings justify thesearch for small-molecule inhibitors targeting PNO1 as a noveltherapeutic strategy in colorectal cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Chen, T.J. Sferra, J. PengDevelopment of methodology: A. Shen, L. Liu, F. QiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Shen, Y. Huang, H. Chen, F. Qi, Z. Shen,M. Wu, Q. Li, L. Qiu, N. YuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Liu, F. Qi, X. Wu, T.J. SferraWriting, review, and/or revision of the manuscript: A. Shen, Y. Chen,T.J. Sferra, J. PengAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Shen, L. Liu, J. Lin, X. WuStudy supervision: Y. Chen, J. Lin, T.J. Sferra, J. Peng

AcknowledgmentsWe thank Dr. Xiangfeng Wang from First People's Hospital Affiliated to

Fujian University of Traditional ChineseMedicine andDr. YaodongWang fromFujian Provincial Hospital for assistance with collection of clinical samples. Wethank Drs. Wei Lin, Weidong Zhu, Baochang He, and Guoqing Ji for helpfuladvice and discussions. This study was supported by the National NaturalScience Foundation of China (81673721, 81803882), the International Coop-erative Project of Fujian Department of Science and Technology (2017I0007),and a Chinese Government Scholarship from the China Scholarship Council[(2016) 3100].

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedOctober 24, 2018; revised January 31, 2019; acceptedMarch8, 2019;published first March 12, 2019.

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Shen et al.




2019;79:2257-2270. Published OnlineFirst March 12, 2019.Cancer Res Aling Shen, Youqin Chen, Liya Liu, et al. p53 Signaling PathwayContributes to Cancer Progression by Negatively Regulating the EBF1-Mediated Upregulation of Ribosome Assembly Factor PNO1

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