R1 Regulates Prostate Tumor Growth and Progression By ...characterize the contribution of R1 to prostate cancer pathogenesis. Implications: These ﬁndings provide evidence that R1

Oncogenes and Tumor Suppressors

R1 Regulates Prostate Tumor Growth andProgression By Transcriptional Suppressionof the E3 Ligase HUWE1 to Stabilize c-MycTzu-Ping Lin1,2,3,4, Jingjing Li5, Qinlong Li6,7, Xiangyan Li6, Chunyan Liu6, Ni Zeng1,Jen-Ming Huang6, Gina Chia-Yi Chu6, Chi-Hung Lin8, Haiyen E. Zhau6,Leland W.K. Chung6, Boyang Jason Wu5, and Jean C. Shih1,2,9

Abstract

Prostate cancer is a prevalent public health problem, espe-cially because noncutaneous advanced malignant forms sig-nificantly affect the lifespan and quality of life of men world-wide. New therapeutic targets and approaches are urgentlyneeded. The current study reports elevated expression of R1(CDCA7L/RAM2/JPO2), a c-Myc–interacting protein andtranscription factor, in human prostate cancer tissue speci-mens. In a clinical cohort, high R1 expression is associatedwith disease recurrence and decreased patient survival. Over-expression and knockdown of R1 in human prostate cancercells indicate that R1 induces cell proliferation and colonyformation. Moreover, silencing R1 dramatically reduces thegrowth of prostate tumor xenografts in mice. Mechanistically,R1 increases c-Myc protein stability by inhibiting ubiquitina-

tion and proteolysis through transcriptional suppression ofHUWE1, a c-Myc–targeting E3 ligase, via direct interactionwith a binding element in the promoter. Moreover, transcrip-tional repression is supported by a negative coexpressioncorrelation between R1 and HUWE1 in a prostate cancerclinical dataset. Collectively, these findings, for the first time,characterize the contribution of R1 to prostate cancerpathogenesis.

Implications: These findings provide evidence that R1 isa novel regulator of prostate tumor growth by stabilizingc-Myc protein, meriting further investigation of itstherapeutic and prognostic potential. Mol Cancer Res; 16(12);1940–51. �2018 AACR.

IntroductionProstate cancer is the second most frequently diagnosed

cancer and fifth leading cause of cancer-related death in menworldwide with the highest incidence rates found in Westerncountries (1). Despite decreasing death rates for prostate cancerin recent years, attributed mainly to improved early detectionand treatment, aggressive castration-resistant and metastaticprostate cancer are still incurable and lethal (2–4). A greaterunderstanding of the mechanisms governing prostate cancerinitiation and progression could help develop effective strate-gies for treating advanced prostate cancer.

The pathogenesis of prostate cancer is a multistep processinvolving a number of genetic alterations and epigeneticdysfunctions by which benign prostatic epithelial cells transi-tion to high-grade prostatic intraepithelial neoplasia (PIN),invasive adenocarcinoma, and distant metastasis (3, 5). Acti-vation of the proto-oncogene c-Myc, known to regulate thetranscription of numerous genes and pathways, is a molecularevent found constantly in different stages of disease progres-sion (6, 7). In addition to the frequent overexpression of c-Mycin PIN, with a stepwise increase from normal to low-grade PINto high-grade PIN, nuclear c-Myc protein overexpression wasobserved in both localized prostate cancer and metastaticdisease (6). Enforced expression of c-Myc in mouse prostatefurther revealed the role of c-Myc in driving the onset ofPIN, which progressed to invasive adenocarcinoma, albeit at

1Depatment of Pharmacology and Pharmaceutical Sciences, School ofPharmacy, University of Southern California, Los Angeles, California.2USC-Taiwan Center for Translational Research, University of SouthernCalifornia, Los Angeles, California. 3Department of Urology, Taipei VeteransGeneral Hospital, Taipei, Taiwan, Republic of China. 4Department of Urology,School of Medicine and Shu-Tien Urological Research Center, NationalYang-Ming University, Taipei, Taiwan, Republic of China. 5Department ofPharmaceutical Sciences, College of Pharmacy and Pharmaceutical Sciences,Washington State University, Spokane, Washington. 6Uro-OncologyResearch Program, Samuel Oschin Comprehensive Cancer Institute,Department of Medicine, Cedars-Sinai Medical Center, Los Angeles,California. 7Department of Pathology, Xijing Hospital, the Fourth MilitaryMedical University, Xi'an, Shaanxi, China. 8Institute of Clinical Medicine,School of Medicine, National Yang-Ming University, Taipei, Taiwan, Republicof China. 9Depatment of Cell and Neurobiology, Keck School of Medicine,University of Southern California, Los Angeles, California.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

T.-P. Lin and J. Li contributed equally to this article.

Corresponding Authors: Jean C. Shih, Department of Pharmacology andPharmaceutical Sciences, School of Pharmacy, University of SouthernCalifornia, 1985 Zonal Ave, PSC 518, Los Angeles, CA 90089. Phone: 323-442-1441; Fax: 323-442-3229; E-mail: [email protected]; and Boyang Jason Wu,Department of Pharmaceutical Sciences, College of Pharmacy and Pharma-ceutical Sciences, Washington State University, 205 E Spokane Falls Blvd,PBS 421, Spokane, WA 99210. Phone: 509-368-6691; Fax: 509-368-6561;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-16-0346

�2018 American Association for Cancer Research.

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different rates (8, 9). In addition, c-Myc may also contribute todisease progression to castration resistance by conferringandrogen-independent prostate cancer cell growth (10). Inspite of accumulated c-Myc studies in prostate cancer researchover the past 30 years, the precise regulation of c-Myc stillremains largely unclear, and merits continued exploration for abetter understanding of prostate cancer pathogenesis andprogression.

R1 (RAM2/CDCA7L/JPO2), a c-Myc–interacting protein essen-tial for cellular transformation, was first identified in medul-loblastoma cells (11). We demonstrated that R1 is a noveltranscription repressor, which directly interacts with the pro-moters of monoamine oxidase genes by competing with Spfamily transcription factors to suppress gene expression(12, 13). The role of R1 as a transcription factor was furthersupported by its chromatin-binding dynamics upon interac-tion with transcriptional coactivator LEDGF/p75 (14, 15).However, the role of R1 in prostate cancer and particularlythe regulatory relationship between R1 and c-Myc has not beenexplored. In this study, we investigated for the first time thefunctional and mechanistic roles of R1 in prostate cancerusing both cell line and tumor xenograft models, and alsoevaluated R1 expression in human prostate cancer samples andits correlation with disease prognosis and survival in clinicaldatasets.

Materials and MethodsClinical specimens

Prostate cancer tissue microarrays including a total of 93primary adenocarcinomas and 19 normal prostate tissues werepurchased from US Biomax. Specimens were stained withantibodies specific for R1 (Sigma-Aldrich) following our pub-lished protocol (16). Each sample was scored on the basis ofstaining intensity (I) and the proportion of tumor cells stainedby quantity (q) to obtain a final score defined as the productof I � q. The scoring system for I was: 0 ¼ negative, 1 ¼ low,2 ¼ moderate, and 3 ¼ intense immunostaining. The scoringsystem for q was: 0 ¼ negative, 1 ¼ 1%–25% positive, 2 ¼26%–50% positive, 3 ¼ 51%–75% positive, and 4 ¼ 76%–

100% positive cells. All scoring was performed by a pathologist.

Cells and reagentsHuman prostate cancer LNCaP, DU145, PC-3, and human

embryonic kidney 293T cell lines were obtained from theATCC. Human prostate cancer C4-2B (17), ARCaPE andARCaPM (18, 19) cell lines, and two pairs of patient-derivedhuman prostate normal epithelial (PNE) and prostate cancerepithelial (PCE) cell lines (20) were established as describedpreviously. All cell lines were cultured in RPMI1640 medium,T-medium, or DMEM (Thermo Fisher Scientific) supplementedwith 10% FBS (Atlanta Biologicals) and 1% penicillin/strepto-mycin (Thermo Fisher Scientific). Human prostate epithelialcells (PrEC) were purchased from Lonza and cultured followingthe manufacturer's instructions. Human R1 expression con-struct was generated by insertion of human R1 coding regionat EcoRI-BglII sites in p3�FLAG-CMV vector (Sigma-Aldrich)containing a neomycin-resistant gene. Human c-Myc-pcDNA3and HA-ubiquitin-pcDNA3 expression constructs were obtain-ed from Addgene. The pM4-min-tk-luc, containing 4 c-Myc–binding E-box sites, and the parental promoterless pmin-tk-luc

reporter constructs were kindly provided by Dr. BernhardLuscher (Hannover Medical School, Hannover, Germany;ref. 21). HumanHUWE1 promoter luciferase reporter constructwas purchased from GeneCopoeia. pRL-TK plasmid-expressingRenilla luciferase was purchased from Promega. Human R1,c-Myc, and nontargeting control shRNA lentiviral particles thatcarry a puromycin- or neomycin-resistant gene were purchasedfrom Sigma-Aldrich. Human R1 siRNAs were purchased fromSanta Cruz Biotechnology. Cycloheximide and MG132 werepurchased from Sigma-Aldrich.

Generation of stable overexpression and knockdown cellsR1 overexpression was achieved by transfection of FLAG-

tagged human R1 expression construct into cells using Lipo-fectamine 2000 reagent (Thermo Fisher Scientific) followingthe manufacturer's instructions, followed by 3-week G418selection (500 mg/mL) for stable clones. Stable shRNA–medi-ated R1 knockdown (KD) was achieved by infecting cells withlentiviral particles expressing a R1 shRNA TRCN0000364645(shR1 #1, mainly used in this study and usually dubbed as"shR1") or TRCN0000369291 (shR1 #2), followed by 2-weekpuromycin selection (2 mg/mL) for establishing stable celllines. A pCMV empty vector and scrambled control shRNAlentiviral particles were used as controls in stable overexpres-sion and KD cells, respectively. Overexpression and KD ofgene(s) in stable cells were validated by immunoblottinganalysis. Established stable cells were maintained in culturemedium supplemented with either G418 or puromycin at thesame doses used for selection.

Biochemical analysesTotal RNAwas isolated using the RNeasyMini Kit (Qiagen) and

reverse-transcribed to cDNA by M-MLV reverse transcriptase(Promega) as described previously (22). For immunoblots,cells were extracted with RIPA buffer in the presence of a proteaseand phosphatase inhibitor cocktail (Thermo Fisher Scientific),and blots were performed as described previously (22, 23)using primary antibodies against R1 (Sigma-Aldrich), FLAG(M2, Sigma-Aldrich), c-Myc (D84C12, Cell Signaling Technolo-gy), HUWE1 (Bethyl Laboratories), MIZ-1 (R&D Systems), p53(DO-1, NeoMarkers), or b-Actin (AC-15, Santa Cruz Biotechno-logy). Immunoblots were subjected to morphometric analysis byImageJ software (NIH, Bethesda, MD) for scanned films or ImageLab software (Bio-Rad) for digital images. To use ImageJ toquantify protein band intensity, protein bands on scannedWestern blot films were selected and defined in a rectangular boxas region of interest. The mean histogram value of each selectedbox was measured, which after subtracting the background his-togram value indicates the intensity of each band. The bandintensity of target protein was normalized to that of loadingcontrol for comparisons between groups.

Cell proliferation assaysTo determine the effect of R1 on cell proliferation, control, and

R1-overexpressing/KD cells were seeded on 6-well plates (2�104

cells/well). To determine the effect of R1 on contact inhibitionduring cell proliferation, control and R1-overexpressing cells wereseeded on 48-well plates with 3.5 � 105 cells per well, allowingconfluence to be reached prior to cell counting. Cell numbersfrom triplicate wells were counted by a hemocytometer.

R1 (CDCA7L) Promotes Prostate Cancer By Stabilizing c-Myc

www.aacrjournals.org Mol Cancer Res; 16(12) December 2018 1941




Colony formation assaysCells were suspended in culture medium containing 0.3% aga-

rose (FMC BioProducts) and placed on top of solidified 0.6%agarose in 6-well plates. The developed colonies were counted andrecorded under a microscope after a 2-week incubation.

Migration and invasion assaysAssays were performed using 6.5-mm transwell inserts (8-mm

pore size) coated with either collagen I or Growth Factor ReducedMatrigel (BD Biosciences) for migration and invasion assays,respectively. Cells were serum-starved overnight before seedingto eliminate the interference of any proliferative effect with cellmigration or invasion. Cells were seeded inside transwell insertscontaining culture medium without serum. After 16–48 hours,cells that translocated to the bottom surface of filters were fixed in4% formaldehyde. The fixed membranes were stained using 1%crystal violet. Assays were quantified by counting the number ofstained nuclei in five independent 200� fields in each transwell.

Tumor xenograft studiesMale 4-week-old athymic nude mice were purchased from

Taconic, housed in the animal research facility at University ofSouthern California, and fed a normal chow diet. 1 � 106 PC-3(shCon and shR1) cells were mixed with Matrigel (BD Bios-ciences) and injected subcutaneously into mice. Each mouse wasinjected on the right flank. Five mice were used for each group.Tumor size wasmeasured every 3 days by caliper from the time offormation of palpable tumors and tumors were dissected andweighed after 5 weeks. Tumor volume was calculated by theformula of length � width2 � 0.52 (24). Tumors were fixed in4% formaldehyde and embedded in paraffin for IHC analysis.

IHC stainingIHC analyses of clinical specimens and tumor xenograft sam-

pleswere performedusing antibodies against R1 (Sigma-Aldrich),Ki-67 (SP6, Abcam), c-Myc (D84C12,Cell Signaling Technology),or HUWE1 (Bethyl Laboratories) following our published pro-tocol (25) with minor modifications. Briefly, formalin-fixed par-affin-embedded sections (4 mm)were deparaffinized, rehydrated,and subjected to antigen retrieval. After incubation in DualEndogenous Enzyme Block solution (Dako) for 10 minutes,sections were treated with primary antibody diluted by differentfolds with Antibody Diluent solution (Dako) at 4�C overnight.The section was then washed three times in PBST (PBS containing0.2% Tween-20) for 5 minutes per washing. To detect specificstaining, each section was treated for 30minutes with EnVisionþDual Link System-HRP (Dako),which containedHRP-conjugatedgoat antibodies against mouse and rabbit IgG. Sections werewashed three times for 5 minutes each and specific stains weredevelopedwith 303-diaminobenzidine (Dako). Image acquisitionwas performed using aNikon camera and software.Magnificationwas �400 (scale bars: 20 mm).

Flow cytometric analysisTo determine the effect of R1 on cell-cycle progression,

control and R1-KD cells were fixed, stained with propidiumiodide (PI, 25 mg/mL), and analyzed by FACScan flow cytometer(BD Biosciences) on the basis of 2N and 4N DNA contentto determine the distribution of different cell-cycle phases. Todetermine the effect of R1 on cell apoptosis, cells subjected totransient or stable KD of R1 were fixed, costained with PI and

FITC Annexin V using the FITC Annexin V Apoptosis DetectionKit II (BD Biosciences) following the manufacturer's instruc-tions, and analyzed by BD Accuri C6 (BD Biosciences)to determine the apoptotic cell population characterized byPI�/Annexin Vþ cells. Quantitative analysis was conducted byFlowJo software (FlowJo).

Luciferase reporter assaysTo determine the direct effect of R1 on c-Myc transcriptional

activity, control and R1-KD PC-3 cells were transfected with thepM4-min-tk-luc construct, which contains 4 c-Myc–bindingE-box sites located upstream of the Firefly luciferase gene (21)and the pRL-TK Renilla luciferase reporter construct, with thelatter used to normalize transfection efficiency. The parentalpmin-tk-luc construct without any c-Myc–binding sites wasused as a negative control. To examine the direct effect of R1 onHUWE1 promoter, control and R1-overexpressing PC-3 cellswere transfected with the desired HUWE-1 promoter Gaussialuciferase reporter plasmids and the pRL-TK plasmid. Transfec-tions were performed with Lipofectamine 2000 reagent. Relativelight units were calculated as the ratio of Firefly or Gaussia lucif-erase activity to Renilla luciferase activity. Renilla luciferaseactivity was determined in harvested cell lysates by the Dual-Luciferase Reporter 1000 Assay System (Promega). Gaussia lucif-erase activity was determined in conditioned media by theSecrete-Pair Gaussia Luciferase Assay Kit (GeneCopoeia).

Quantitative real-time PCRqPCR was conducted using SYBR Green PCR Master Mix

and run with the Applied Biosystems 7500 Fast Real-Time PCRSystem (Applied Biosystems). PCR conditions included an initialdenaturation step of 3 minutes at 95�C, followed by 40 cycles ofPCR consisting of 30 seconds at 95�C, 30 seconds at 60�C, and30 seconds at 72�C. The PCR data were analyzed by the 2�DDCt

method (26). All primer sequences used were: R1 forward 50-CGAGGAAGAGGAAGATGAAGAA-30 and reverse 50-GAAAACAA-CTGCTCGGAAGAAC-30; c-Myc forward 50-TGCTGCCAAGAGG-GTCAAGT-30 and reverse 50-GTGTGTTCGCCTCTTGACATTC-30;HUWE1 forward 50-GCACTCTGCAATCCTCACAA-30 and reverse50-CCTCTGGCTCTACAGGCATC-30; FBW7 forward 50-CGTTG-CAGGGGCATACTAAT-30 and reverse 50-ATGCAATTCCCTGTC-TCCAC-30; SKP2 forward 50-TGCTAAGCAGCTGTTCCAGA-30 andreverse 50-AAGATTCAGCTGGGTGATGG-30; CHIP forward 50-CAATCTGCAGCGAGCTTACA-30 and reverse 50-CTGTTCCAG-CGCTTCTTCTT-30; TRUSS forward 50-AAGGACAGCACCTGC-CTAGA-30 and reverse 50-GTCCATGTAGGGCTCCTCAA-30; andb-Actin forward 50-TTGTTACAGGAAGTCCCTTGCC-30 andreverse 50-ATGCTATCACCTCCCCTGTGTG-30.

Site-directed mutational analysis of R1 andHUWE1 promotersSite-directed mutagenesis was used to delete the leucine

zipper domain in R1 expression construct and to mutate theputative R1-binding site identified in 1.3-kbHUWE1 promoter.Wild-type promoter-luciferase reporter construct was used asa template. Mutagenesis was carried out using QuickChange IIXL Site-Directed Mutagenesis Kit (Agilent Technologies) fol-lowing the manufacturer's instructions. The sequences of pri-mers used for creating R1 deletion promoter were forward50-GAGAGTTCAGATGCTAACTCGATGCCAGATTTCTTCCCAG-TACG-30 and reverse 50-ATCTGGCATCGAGTTAGCATCTGA-ACTCTCCTGGCTCTCATCC-30. The sequences of primers used

Lin et al.

Mol Cancer Res; 16(12) December 2018 Molecular Cancer Research1942




for mutagenesis of HUWE1 promoter were forward 50-GTCT-TGTGCAACATAGGGATGTGTTACAGTTTGATCTGGAGAGATT-C-30 (mutated nucleotides underlined). Deletion and mutatednucleotides were verified by DNA sequencing.

In vivo ubiquitination assays293T cells were transiently transfected with 2 mg of HA-

tagged ubiquitin, 1 mg of c-Myc and/or 1 mg of R1 expressionconstruct in 6-well plates, where pcDNA3.1 was used to keepthe amount of transfected DNA equal between groups. Trans-fections were performed with Lipofectamine 2000 reagent. Thecells were split 1:4 into 10-cm dishes 24 hours after transfectionto allow growth for another 48 hours. Cells were treated with10 mmol/L MG-132 for 4 hours prior to harvest and lysed inRIPA buffer supplemented with a protease and phosphataseinhibitor cocktail as described above. One milligram of celllysates was subjected to immunoprecipitation with anti-c-MycIgG resin (9E10, Thermo Fisher Scientific) overnight at 4�Cusing the Pierce Co-Immunoprecipitation Kit (Thermo FisherScientific) following the manufacturer's instructions, followedby immunoblotting with anti-HA antibody (12CA5, Sigma-Aldrich) to detect c-Myc protein–ubiquitin conjugates.

Chromatin immunoprecipitation analysis and qPCRChromatin immunoprecipitation (ChIP) assays were used

to determine the association of R1 protein with HUWE1 pro-moter in FLAG-tagged R1-overexpressing PC-3 cells by aSimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Tech-nology) following the manufacturer's instructions. Briefly,chromatin was cross-linked with nuclear proteins, enzymati-cally digested with micrococcal nuclease followed by sonica-tion, and immunoprecipitated with anti-FLAG antibody (M2,Sigma-Aldrich). After being pelleted with agarose beads andpurified, immunoprecipitates were subjected to qPCR with apair of primers specifically targeting the HUWE1 promoterregion that encompasses the R1-binding site. IgG includedin this kit was used as a negative control for IP. All primersequences used were: HUWE1 promoter forward 50-AACTG-CAGGGCTATGGTGAA-30 and reverse 50-CACTGGAGTTAT-GATGCTATGC-30; MAOA core promoter (serving as positivecontrol) forward 50-GTGCCTGACACTCCGCGGGGTT-30 andreverse 50-TCCTGGGTCGTAGGCACAGGAG-30; and HUWE1intron 2 (serving as negative control) forward 50CCCAGGTGG-ATTGTTGGGGG-30 and reverse 50-GGAACCCTTAAGCTCACA-CAGCA-30. Ten percent of chromatin prior to the IP step wassaved as input and data were presented as the percent of inputfrom three separate experiments.

Microarray datasetsTwo prostate cancer DNA microarray datasets, Tomlins (27)

and Taylor 3 (28), were downloaded directly from the Oncominedatabase by licensed access. These datasets are also publiclyavailable in Gene Expression Omnibus as GSE6099 andGSE21032 for the Tomlins and Taylor 3 datasets, respectively.

Statistical analysisData were presented as the mean � SEM in figure legends.

Correlations were determined by Pearson correlations. Allother comparisons were analyzed by unpaired two-tailed Stu-dent t test. A P value less than 0.05 was considered statisticallysignificant.

ResultsR1 is highly expressed in prostate cancer and associated withpoor prognosis/survival in patients with prostate cancer

To determine R1 expression in human prostate cancer, weanalyzed R1 protein levels by IHC in a tissue microarrayassembling both human normal prostatic (n ¼ 19) and tumor(n ¼ 93) samples. As shown in Fig. 1A, the intensity ofwidespread R1 staining in both nuclei and cytoplasm washigher in cancer cells than normal prostatic epithelial cellswith statistical significance demonstrated by quantitative anal-ysis (Fig. 1B). We also found increased R1 mRNA expressionassociated with biochemical recurrence and poor survival inpatients with prostate cancer in a prostate cancer clinicaldataset (Fig. 1C and D; ref. 28). Next, we assayed R1 proteinexpression in a spectrum of human prostate cancer cell lineswith different levels of aggressiveness and invasiveness,including LNCaP, C4-2B, DU145, PC-3, ARCaPE, andARCaPM, as well as normal human prostatic epithelial cells(PrEC). The R1 protein levels were elevated in all cancercell lines compared with PrEC cells (Fig. 1E). We also analyzedR1 protein expression in 2 pairs of human prostate normalepithelial (PNE) and prostate cancer epithelial (PCE) cellsderived from clinical prostate tumors and adjacent normalprostates. Similarly, these showed increased R1 proteinexpression in PCE1 and PCE2 cells relative to their corre-sponding controls (Fig. 1F). In addition, R1 mRNA expressionwas upregulated in all prostate cancer cell lines comparedwith PrEC cells (Fig. 1G), which was consistent with ourobservations of R1 protein changes. From these results inboth clinical samples and cell line models, we concluded thatR1 is highly expressed in prostate cancer and has potentialprognostic value for distinguishing aggressive from indolentprostate cancer.

R1 promotes prostate cancer cell proliferation, colonyformation, and prostate tumor growth

To study the potential functional role of R1 in prostatecancer, we stably overexpressed FLAG-tagged R1 in PC-3 cells.Enforced expression of R1 as confirmed by Western blotanalysis (Fig. 2A) increased PC-3 cell proliferation by upto40% in a 6-day observation course compared with controlcells where an empty vector was stably expressed (Fig. 2B).Both control and R1-overexpressing cells exhibited similarproliferation rates after confluence was reached, suggestingthat contact inhibition is not involved in R1-induced cellproliferation (Supplementary Fig. S1). We also knocked downR1 expression in 2 prostate cancer cell lines, PC-3 and C4-2B,using 2 shRNAs targeting separate nonoverlapping R1 codingregions followed by antibiotic selection to establish stablecell clones with successful KD confirmed by Western blotanalysis (Fig. 2C). Stable silencing of R1 significantly sup-pressed cell growth in both lines with upto 64% and 61%decreases for PC-3 and C4-2B cells, respectively, over a 7-daytime course (Fig. 2D). Interestingly, R1 KD did not affect cellapoptosis in PC-3 and C4-2B cells under both steady-state anddynamic conditions, where stable shRNA–mediated R1 KD or asiRNA-mediated stepwise decrease of R1 expression over timewas achieved, respectively (Supplementary Fig. S2). Moreover,we showed that overexpression of R1 increased the colony-forming ability of PC-3 cells with an increase of 63% in thenumber of colonies compared with control cells (Fig. 2E),






whereas R1 KD reduced the ability of PC-3 cells to formcolonies relative to control cells by an up to 55% drop incolony number (Fig. 2F). In addition, overexpression of R1also led to a significant increase in PC-3 cell migration andinvasion. In contrast, R1 KD reduced the ability of both PC-3and C4-2B cells to migrate and invade (Supplementary Fig.S3). Taken together, these results obtained from different cellline models strongly support the idea that R1 promotes pros-tate cancer cell aggressiveness and invasiveness.

To determine whether the R1 effects observed in cell linescould be recapitulated in vivo, we established PC-3 prostatetumor xenograft mouse models. After being implanted sub-cutaneously into male nude mice, R1-KD PC-3 cells showedslower tumor growth rates in comparison with control cellsexpressing a control shRNA that targets no known mamma-lian genes (Fig. 3A). Moreover, R1-KD cells formed tumorsthat were smaller, with an average tumor weight of 107 �63 mg, compared with large tumors with an average weightof 201 � 39 mg in controls (Fig. 3B). Ki-67 staining of tumorspecimens harvested at the experimental endpoint furtherrevealed a 43% decrease of Ki-67þ cells in R1-KD group(Fig. 3C and D). In addition, R1 protein staining showedreduced intensity in R1-KD tumor samples, suggesting thatshRNA–mediated silencing of R1 expression is highly effec-tive and sustainable under in vivo conditions (Fig. 3C). Theseresults in aggregate indicate the in vivo tumor-promotingfunction of R1.

R1 increases c-Myc expression by enhancing c-Myc proteinstability

To assess the effect of R1 on cell-cycle progression, whichmay underlie its proliferation-enhancing effect in prostatecancer cells, we conducted cell-cycle analysis to determine thedistribution of different cell-cycle phases in R1-KD cells. Asdemonstrated in Fig. 4A, R1 KD effectively reduced the per-centage of cells in S phase by 23% and 19% in PC-3 and C4-2Bcells, respectively, which was accompanied by slight increasesin the G0/G1 phase. Because R1 was first identified as a partnerprotein of c-Myc, a direct regulator of cell-cycle machinery(11, 29), we analyzed c-Myc expression profiles in R1-manipulated cells. Enforced expression of R1 increased c-Mycprotein expression in PC-3 cells, whereas shRNA-mediated KDof R1 resulted in a decrease of c-Myc protein levels in both PC-3and C4-2B cells (Fig. 4B). Using a luciferase reporter constructunder the control of 4 c-Myc–binding E-box sites as an indi-cator of c-Myc transcriptional activity (21), we found decreasedc-Myc activity as reflected by lower luciferase reporter activity inR1-KD cells (Fig. 4C) in line with reduced c-Myc proteinexpression in these cells, suggesting that R1 is able to affectboth c-Myc protein expression and activity. We also demon-strated dramatic loss of nuclear c-Myc protein staining in R1-KDPC-3 tumor samples compared with controls (Fig. 4D). How-ever, neither overexpression nor KD of R1 significantly affectedc-Myc mRNA expression in these cells (Fig. 4E). This suggeststhat R1-mediated upregulation of c-Myc may be due to

Figure 1.

R1 is highly expressed in prostate cancer and associated with poor clinical outcomes in patients with prostate cancer. A and B, Quantitative IHCanalysis of normal prostate and human prostate adenocarcinoma clinical samples. Representative images are shown in A. Scale bars: 20 mm. IHCstaining of R1 for all samples was assessed by both the percentage of cells stained and staining intensity (B). n ¼ 19 and 93 for normal prostateand prostate cancer samples, respectively (� , P < 0.05). C and D, Oncomine analysis of R1 mRNA levels in Taylor 3 prostate cancer dataset regardingdisease recurrence (C) and patient survival (D) status (� , P < 0.05). E and F, Western blot analysis of R1 protein expression in human normalprostate epithelial PrEC cells and a panel of human prostate cancer cell lines (E) as well as in 2 pairs of human prostate normal epithelial (PNE)and prostate cancer epithelial (PCE) cells established from clinical patient samples (F). The R1/b-Actin ratios of band intensity across different celllines are denoted in E. G, RT-qPCR analysis of R1 mRNA expression (mean � SEM, n ¼ 3) in PrEC and a panel of human prostate cancer cell lines. Theexpression of R1 as normalized to internal control b-actin in PrEC cells was set as 1.

Lin et al.





increased protein stability. To determine whether this mecha-nism is responsible for R1 upregulation of c-Myc, we performedan in vivo ubiquitination analysis of c-Myc protein with orwithout R1 expression. We found that R1 markedly inhibit-ed both endogenous and exogenous c-Myc ubiquitination(Fig. 4F), with an average 60% (n ¼ 5) inhibition of exogenousc-Myc ubiquitination achieved (Fig. 4G). To determine whetherR1/c-Myc physical interaction is a possible mechanism under-lying R1 upregulation of c-Myc, we examined the effect of a R1deletion construct deficient in the leucine zipper domainrequired for R1 binding to c-Myc on c-Myc ubiquitination(11). As shown in Supplementary Fig. S4, both wild-type anddeleted R1 suppressed c-Myc ubiquitination to a similar extent,suggesting that R1/c-Myc interaction is not involved in R1

upregulation of c-Myc. To examine the effect of R1 on c-Mycproteolysis, we subjected R1-manipulated cells to cyclohexi-mide treatment to inhibit protein synthesis. Consistent withthe inhibition of ubiquitination, transient overexpression ofR1 significantly reduced the proteolysis of c-Myc protein incells accompanied by a nearly 2-fold increase in c-Myc half-life(Fig. 4H and I, t1/2 ¼ 51 � 8 minutes and t1/2 ¼ 85.7 � 2.3minutes in control and R1-overexpressing cells, respectively,n ¼ 3 for both), which is parallel to decreased c-Myc proteinstability along with a 50% drop of c-Myc half-life in R1-KDcells (Fig. 4J and K, t1/2 ¼ 48 � 5 minutes and t1/2 ¼ 24 �2 minutes in control and R1-KD cells, respectively, n ¼ 3 forboth). In addition, we showed that R1 KD failed to suppressthe growth and colony formation of PC-3 cells that received

Figure 2.

R1 promotes prostate cancer cell proliferation and colony formation. A, Western blot analysis of transfected FLAG-tagged R1 protein expression inPC-3 cells that stably express an empty vector (Vector) or FLAG-tagged R1 (R1). B, Growth curves of PC-3 cells as established in A. Data representthe mean � SEM (n ¼ 3; � , P < 0.05). C, Western blot analysis of R1 protein expression in PC-3 and C4-2B cells expressing scrambled shRNA (shCon)or 2 distinct R1-targeting shRNAs (shR1 #1 and shR1 #2). D, Growth curves of PC-3 and C4-2B cells as established in C. Data represent the mean � SEM(n ¼ 3; �� , P < 0.01). E and F, Colony formation assays in cells as established in A and C, respectively. Both representative colony images (left) andquantitative analysis (right) are shown. Data represent the mean � SEM (n ¼ 3; �� , P < 0.01).






prior shRNA-mediated KD of c-Myc (Fig. 4L and M), indicatingthat c-Myc is a functional mediator of R1 effects.

R1 transcriptionally suppresses the E3 ligase HUWE1 thatmediates the ubiquitination and degradation of c-Myc protein

The stability of c-Myc protein has been reported to be con-trolled by a number of E3 ligases that target c-Myc for protea-some-mediated degradation (30, 31), which led us to speculateon possible alterations of these E3 ligases in response to R1elevation in prostate cancer cells. By qPCR screening the expres-sion levels of 5 E3 ligases, FBW7, SKP2, ChIP, TRUSS, andHUWE1, known to directly regulate c-Myc (32–36), we dem-onstrated that R1 KD significantly increasedHUWE1 expressionin both PC-3 and C4-2B cells with the others remainingunaffected by R1 (Fig. 5A). We also showed a 43% decreaseof HUWE1 mRNA levels in R1-overexpressing cells comparedwith control cells (Fig. 5B). Moreover, enforced expressionof R1 downregulated HUWE1 protein levels in PC-3 cells(Fig. 5C). HUWE1 is mainly expressed in the cytoplasm butalso partially in the nucleus to mediate ubiquitin-dependentdegradation of target proteins in different subcellular loca-tions (37). The subcellular localization of HUWE1 may beregulated by the modulated exposure of the nuclear localiza-tion signal of HUWE1 located in the middle of the proteindistal to the WWE domain (38). Examining the expressionpatterns of HUWE1 in PC-3 tumor samples, we furtherobserved dramatic induction of widespread cytoplasmic andpartial nuclear HUWE1 protein staining when R1 was knockeddown (Fig. 5D).

Considering the innate nature of R1 as a transcriptionrepressor competing with the Sp family of transcription factorsto downregulate target gene expression, as first demonstratedin transcriptional regulation of monoamine oxidase genes(12, 13), we next determined whether R1 directly regulatesHUWE1 at the transcription level. To explore this idea, weanalyzed 1.3-kb HUWE1 promoter sequences and identified aregion (�224/�218) that exhibits strong sequence similarityto the canonical GC-rich R1-binding site (Fig. 5E). Using a1.3-kb DNA segment located upstream of the transcription startsite of HUWE1 as a template, we generated a mutant HUWE1promoter reporter construct harboring 3 point mutations in the

center of the putative R1-binding element. Compared with thewild-type HUWE1 promoter reporter showing a R1-induced26% decrease in promoter activity, the mutated HUWE1 pro-moter was no longer repressed by overexpression of R1 in PC-3cells (Fig. 5F). To confirm direct occupancy of R1 with thesequences in the HUWE1 promoter in vivo, we performedChIP assays. We isolated chromatin-nuclear protein comp-lexes immunoprecipitated with anti-FLAG antibody fromFLAG-tagged R1-overexpressing PC-3 cells, and analyzed theseby qPCR using primers that specifically encompass the putativeR1-binding site in HUWE1 promoter. As shown in Fig. 5G,we were able to detect a physical association of R1 with theHUWE1 promoter sequences, which is parallel to the expectedbinding of R1 with the MAOA core promoter serving as apositive control. Moreover, limited signals were seen from thenegative controls, where either nonspecific IgG antibody wasused in the immunoprecipitation step or HUWE1 intron 2was probed to confirm the specificity of R1 binding to theHUWE1 promoter sequences. In addition, we demonstrated anegative coexpression correlation between R1 and HUWE1in a previously reported prostate cancer clinical dataset(Fig. 5H, P ¼ 0.0372 by Pearson correlation; ref. 27), whichis consistent with our findings in cell lines. These resultscollectively demonstrate that R1 suppresses the transcriptionof the c-Myc–targeting E3 ligase HUWE1 by directly interactingwith its promoter to upregulate c-Myc protein expression inprostate cancer cells.

In summary, our data suggest that the increased intrinsic R1expression in prostate cancer activates cell proliferation andcell-cycle progression by a mechanism that involves directtranscriptional suppression of the E3 ligase HUWE1 to preventc-Myc protein degradation, thereby activating c-Myc proteinexpression (Fig. 6). Our data further showed the key features ofthis pathway in prostate tumor xenograft samples and inclinical datasets, supporting the essential role of R1 in prostatetumor growth and progression.

DiscussionIn this study, we demonstrated for the first time the elevated

expression of R1 in human prostate cancer tissue samples

Figure 3.

R1 promotes the growth of prostate tumor xenografts. A and B, PC-3 cells that stably express scrambled shRNA (shCon) or R1-targeting shRNA (shR1) wereinjected subcutaneously into male nude mice (n ¼ 5 mice for each group) for the growth of tumor xenografts. Tumor growth was determined bymeasuring tumor volume (A) and tumor weight (B). The graphs in A show the mean (�SEM) tumor size at indicated times (� , P < 0.05). C, IHCanalysis of R1 and Ki-67 expression in tumor xenografts obtained at the experimental endpoint. Representative images are shown. Scale bars: 20 mm.D, Quantification of percent of Ki-67þ tumor cells at the experimental endpoint from each group (n ¼ 3). Data represent the mean � SEM (n ¼ 3; �� , P < 0.01).

Lin et al.





Figure 4.

R1 promotes cell-cycle progression and stabilizes c-Myc protein in prostate cancer cells. A, Cell-cycle analysis of phase distribution in control (shCon)and R1-KD (shR1) PC-3 and C4-2B cells by flow cytometry. Data represent the mean � SEM (n ¼ 3). B, Western blot analysis of c-Myc proteinexpression in PC-3 and C4-2B cells subjected to overexpression or KD of R1. C, Determination of relative luciferase activity (mean � SEM, n ¼ 3) of ac-Myc–responsive luciferase reporter, pM4-min-tk-luc, which contains 4 c-Myc–binding E-box sites and serves as an indicator of c-Myc transcriptionalactivity, in control and R1-KD PC-3 cells. The parental pmin-tk-luc construct containing no c-Myc–binding sites was used as a negative control. Thethymidine kinase promoter-driven pRL-TK construct was used to normalize transfection efficiency (�� , P < 0.01). D, IHC analysis of c-Myc and Ki-67expression in PC-3 (shCon and shR1) tumor xenografts. Representative images are shown. Scale bars, 20 mm. E, RT-qPCR analysis of c-Myc mRNA expression(mean � SEM, n ¼ 3) in cells as indicated in B. ns, not significant. F, In vivo ubiquitination assay of c-Myc in 293T cells transiently transfected with HA-taggedubiquitin (Ub) and empty vector or c-Myc, with or without R1. A representative image from five repeats is shown. G, Quantitative analysis of c-Mycubiquitination levels (mean � SEM, n ¼ 5) in response to R1 as described in F. The levels in the control group with transfection of ubiquitin but not c-Mycand R1 were set as 1 (� , P < 0.05; �� , P < 0.01). H–K, Western blot analysis of c-Myc protein expression in PC-3 cells subjected to transient overexpression(H and I) or stable KD (J and K) of R1, which were treated with 50 mg/mL of cycloheximide (CHX) and collected at different time points. c-Mycprotein levels were normalized to b-actin. The ratio at 0 hour is set as 100% in each group. L, Growth curves of PC-3 cells that were subjected tosequential shRNA-mediated KD of c-Myc and R1. Data represent the mean � SEM (n ¼ 3; �� , P < 0.01); ns, not significant. M, Colony formation assaysin cells as established in L. Data represent the mean � SEM (n ¼ 3; �� , P < 0.01; ns, not significant).






compared with normal counterparts. We also showed higherR1 expression at both protein and mRNA levels in a spectrumof human prostate cancer cell lines which exhibit different char-acteristics and behaviors compared with normal human pro-static cells. Overexpression and KD of R1 in human prostatecancer cell lines revealed that R1 induces cell proliferation, colonyformation, and migration/invasion. Moreover, silencing R1reduced the growth of PC-3 tumor xenografts in mice. AlthoughR1 was first identified as a c-Myc oncoprotein interactor, littleprogress has been made so far in understanding its role in cancerin general and in prostate cancer specifically. Previous studiesshowed that R1 enhances medulloblastoma transformationwith induced aggressive phenotypes (11, 39) and also hepato-cellular carcinoma progression (40). Our results obtained fromprostate cancer are consistent with those observations, furthersupporting a tumor-promoting effect of R1 in cancers.

R1 was shown to upregulate c-Myc protein expression andtranscriptional activity by enhancing protein stability. R1 hasbeen found to physically interact with the c-Myc NH2-terminaldomain essential for modulating c-Myc oncogenic properties, as

demonstrated in different cellular settings including medullo-blastoma and neuroblastoma cells (11, 41). This interactionresults in two possible mechanisms where R1 either acts directlyas an E3 ligase or regulates other factor(s) to indirectly modulatec-Myc protein stability. The lack of both the E6AP carboxylterminus (HECT) and the really interesting new gene (RING)domains in R1 protein sequences, two domains mediating thedirect transfer of ubiquitin from E2 to substrate, disqualifies R1from being an E3 ligase candidate (13, 42). Rather, we dem-onstrated that R1 acts as a repressor to transcriptionally suppressHUWE1 expression, a c-Myc–targeting E3 ligase, to indirectlystabilize c-Myc protein in the present model systems. Compel-ling evidence has indicated the critical role of HUWE1 as anE3 ligase regulating c-Myc protein stability, c-Myc–activatedgenes, and c-Myc–driven neoplasia and oncogenesis (refs. 34and 43–46). As reported in several independent studies bydifferent groups, manipulation of HUWE1 levels alone wassufficient to produce dramatic changes in c-Myc ubiquitination(34, 47), which to some extent supports our proposed modelwhere R1 exerts a significant impact on c-Myc protein stability

Figure 5.

R1 downregulates the c-Myc–targeting E3 ligase HUWE1 at the transcriptional level in prostate cancer cells. A, RT-qPCR analysis of mRNA expression ofE3 ligase genes that regulate c-Myc protein stability in control (shCon) and R1-KD (shR1) PC-3 and C4-2B cells. Gene expression levels in the controlgroup were set as 1 for both cell lines (� , P < 0.05; �� , P < 0.01; ns, not significant). B, RT-qPCR analysis of HUWE1 mRNA expression (mean � SEM, n ¼ 3)in control and R1-overexpressing PC-3 cells (�� , P < 0.01). C, Western blot analysis of HUWE1 protein expression in control and R1-overepressing PC-3cells. D, IHC analysis of HUWE1 protein expression in PC-3 (shCon and shR1) tumor xenografts. Representative images are shown. Scale bars, 20 mm.Red arrows indicate representative induced nuclear staining of HUWE1 in R1-KD tumor samples. E, The canonical sequence of a R1-binding site (top),a potential R1-binding site in HUWE1 promoter (middle), and introduced point mutations (bottom, italic and red) used to inactivate the potential R1-bindingsite. F, Determination of WT and mutated (Mut) HUWE1 promoter activity (mean � SEM, n ¼ 3) in control and R1-overexpressing PC-3 cells (��, P < 0.01;ns, not significant). G, ChIP analysis of FLAG-tagged R1-overexpressing PC-3 cells immunoprecipitated by anti-FLAG or IgG antibody followed byqPCR using a primer set for the R1-binding site in HUWE1 promoter. Primers targeting the MAOA core promoter sequences and HUWE1 intron 2 servedas positive and negative controls, respectively. Data represent the percent of input (mean � SEM, n ¼ 3). H, Coexpression correlation analysis of R1and HUWE1 mRNA expression in Tomlins metastatic prostate cancer dataset (n ¼ 14). P ¼ 0.0372 as determined by Pearson correlation.

Lin et al.





by modulating a single c-Myc–regulating E3 ligase. In line withthe previous observations of enhanced c-Myc–transformingactivity by R1/c-Myc interaction in medulloblastoma (11),increased R1 expression in prostate cancer also exacerbatedc-Myc function in prostate cancer cells by promotion of cell-cycle progression. HUWE1 has been previously reported tostabilize MIZ-1, another binding partner of c-Myc, and affectARF–p53-mediated apoptosis (44, 46), but we demonstratedmarginal effects of R1 on MIZ-1 and p53 in prostate cancercells (Supplementary Fig. S5), suggesting that the downstreameffect of R1-HUWE1 axis might be cell-context and/or cell-typedependent. In addition, we showed that c-Myc is a key down-stream mediator of R1's effect in prostate cancer cells as evi-denced by the abolition of R1 shRNA-induced suppression ofproliferation and colony formation of PCa cells by c-Mycsilencing. Although the current study emphasizes c-Myc proteinstability as a likely major mechanism underlying R1 regulationof c-Myc in prostate cancer cells, other possible c-Myc–centricmechanisms warrant further investigations to more comprehen-sively advance our understanding of how R1 contributes to theoncogenic activity of c-Myc and how the R1/c-Myc proteincomplex regulates prostate cancer as well as other malignancies.

Activation of the proto-oncogene c-Myc is an important stepnot only in the early phases of prostate cancer such as PIN butalso throughout the entire progression of prostate cancerincluding advanced recurrent and metastatic stages (7). Thesignificant influences of c-Myc in prostate cancer are mainlyattributed to its transcriptional regulation of numerous down-stream target genes. Several independent microarray geneexpression analyses from human prostate cancer and pros-tate-specific Hi-MYC mice have thus far identified a distinctc-Myc–driven expression signature (7, 8). On the other hand, c-Myc is also able to cooperate with other oncogenic signalingsuch as AKT to promote prostate tumorigenesis and altersensitivity to therapy (48). These accumulated gene and path-way profiles lay the mechanistic foundations for identifyingpotential R1-interacting factors, given the intimate regulatoryrelationship between R1 and c-Myc demonstrated by othergroups and by us. This idea has been coincidentally supportedby recent findings of AKT activation by R1 in medulloblastoma(39). Future studies exploring the gene and signaling networks

governed by R1 are merited to uncover potential unknownfunctions of R1 in prostate cancer.

One notable finding of our study was the discovery that R1directly interacts with an element in HUWE1 promoter tosuppress gene transcription, which was supported by clinicalevidence of a negative coexpression correlation between R1and HUWE1. According to the literature, R1 functions incancers largely through its role as a transcription factor. R1forms a ternary complex with transcription coactivatorLEDGF/p75 and chromatin in transcriptional regulation,which may contribute to the development of MLL fusion–driven acute leukemia (14, 49). In medulloblastoma, thecooperation of R1 with LEDGF/p75 also promotes cell migra-tion and metastasis by activation of AKT signaling (39).However, the direct suppressing effect of R1 by serving as atranscription repressor on MAOA gene expression, which wasfirst demonstrated in neuroblastoma cells, turns out to bemarginal in prostate cancer, leading to activation of MAOA(13, 50). These contrasting studies suggest a context-depen-dent role of R1 in the transcriptional regulation of target genesin different types of cancers. A cancer type–specific delineationof molecular states associated with R1 may advance ourunderstanding of R1-mediated transcriptional machinery indiverse cancers.

We provided clinical evidence that higher R1 expression isassociated with recurrence and decreased survival in patients withprostate cancer, which indicates the potential of R1 for predictionof disease prognosis in prostate cancer, particularly in advancedphenotypes. These clinical analyses are consistent with in vitroobservations of an increasing trend of R1 protein expression fromweak/low-metastatic to aggressive/high-metastatic cells.

In summary, we present the first study showing increasedintrinsic R1 expression in prostate cancer and its association withpoor clinical outcomes in patients. We also uncovered the under-lyingmolecular mechanisms contributing to R1-induced prostatecancer growth and progression. Our findings highlight the novelrole of R1 in prostate cancer and reveal R1 as a new potentialprognostic marker and therapeutic target for prostate cancer.

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

Figure 6.

A proposed working model forhow R1 promotes prostate tumorgrowth and progression bysuppressing the E3 ligase HUWE1 atthe transcriptional level to stabilizec-Myc protein.






Authors' ContributionsConception and design: H.E. Zhau, L.W.K. Chung, B.J. Wu, J.C. ShihDevelopment of methodology: T.-P. Lin, B.J. Wu, J.C. ShihAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Li, Q. Li, X. Li, N. Zeng, J.-M. Huang, C.-Y. Chu,B.J. Wu, J.C. ShihAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T.-P. Lin, J. Li, X. Li, N. Zeng, B.J. Wu, J.C. ShihWriting, review, and/or revision of themanuscript: T.-P. Lin, B.J. Wu, J.C. ShihAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B.J. Wu, J.C. Shih, C. Liu, Q. Li, C.-H. LinStudy supervision: B.J. Wu, J.C. Shih

AcknowledgmentsThe authors thank Bernhard Luscher (Hannover Medical School, Hann-

over, Germany) for providing pM4-min-tk-luc and pmin-tk-luc plasmids,Bin Qian (Department of Pharmacology and Pharmaceutical Sciences,

University of Southern California, Los Angeles, CA) for providing technicalassistance, and Gary Mawyer for editorial assistance.

This work was supported by the Department of Defense ProstateCancer Research Program grants W81XWH-12-1-0282 (to J.C. Shih andH.E. Zhau) and W81XWH-15-1-0493 (to J.B. Wu and H.E. Zhau); theDaniel Tsai Family Fund and the Boyd and Elsie Welin Professorship(to J.C. Shih); and NIH/NCI grant 2P01CA098912, the Board of GovernorsCancer Research Chair, and the Steven Spielberg Fund in Prostate CancerResearch (to L.W. Chung).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 11, 2016; revised March 22, 2018; accepted July 5, 2018;published first July 24, 2018.

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2018;16:1940-1951. Published OnlineFirst July 24, 2018.Mol Cancer Res Tzu-Ping Lin, Jingjing Li, Qinlong Li, et al. c-MycTranscriptional Suppression of the E3 Ligase HUWE1 to Stabilize R1 Regulates Prostate Tumor Growth and Progression By

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