cytoplasmic cul9/parc ubiquitin ligase is a...

Molecular and Cellular Pathobiology

Cytoplasmic CUL9/PARC Ubiquitin Ligase Is a TumorSuppressor and Promotes p53-Dependent Apoptosis

Xin-Hai Pei1, Feng Bai1, Zhijun Li1, Matthew D. Smith1, Gabrielle Whitewolf2, Ran Jin1, and Yue Xiong1,3,4

AbstractA wide range of cell stresses, including DNA damage, signal to p53 through posttranslational modification of

p53. The cytoplasmic functions of p53 are emerging as an important constituent of role of p53 in tumorsuppression. Here, we report that deletion of the Cul9 (formerly Parc) gene, which encodes an E3 ubiquitin ligasethat binds to p53 and localizes in the cytoplasm, resulted in spontaneous tumor development, accelerated Em-Myc–induced lymphomagenesis, and rendered mice susceptible to carcinogenesis. Cul9-p53 double-mutantmice exhibited indistinguishable tumor phenotypes as p53 single-mutant mice, indicating that the function ofCul9 in tumor suppression is largely mediated by p53. Deletion of Cul9 had no significant effect on cell-cycleprogression, but attenuated DNA damage–induced apoptosis. Ectopic expression of wild-type CUL9, but not apoint mutant CUL9 deficient in p53 binding, promotes apoptosis. These results show CUL9 as a potential p53-activating E3 ligase in the cytoplasm. Cancer Res; 71(8); 2969–77. �2011 AACR.

Introduction

Cullins are a family of evolutionarily conserved proteinsthat bind to the small RING finger protein, ROC1 (also knownas RBX1), to constitute a large number of distinct E3 ubiquitinligases. Mammalian genomes contain 6 closely related cullingenes (CUL1, 2, 3, 4A, 4B, and 5). In addition, 3 other proteins,CUL7, CUL9 (changed from the former name of PARC follow-ing the recommendation by HUGO Nomenclature Committee;refs. 1, 2; Supplementary Fig. S1A and B), and APC2 (3, 4),contain significant sequence homology to cullins over anapproximately 180-residue region that binds with ROC1 ora homologous small RING protein, APC11. CUL7 and CUL9share extensive sequence homology and contain several addi-tional functional domains, including an N-terminal p53-bind-ing sequence (2, 5–7), a region similar to HERC2 (hect domainand rcc1 domain protein 2), a centrally located DOC1 domain,and a C-terminal sequence unique to CUL9 that contains 2RING fingers separated by a sequence known as IBR (inbetween the RING fingers).

Both the function and mechanism of CUL9 are poorlydefined. Two notable features of CUL9 (ref. 2; SupplementaryFig. S1C), which are also shared by CUL7 (5–7), are itsexclusive localization in the cytoplasm and binding withp53. The significances of both features are unclear. Althoughit was initially reported that CUL9/PARC sequesters p53 in thecytoplasm and thereby antagonizes p53-mediated apoptosis(2), this finding does not seem to be reproducible as thedeletion of mouse Cul9/Parc had no detectable effect on p53stability, target gene induction, or localization (8), and CUL9did not sequester p53 in the cytoplasm in most cells, evenwhen CUL9 or both CUL9 and p53 were overexpressed (Sup-plementary Fig. S1C). In this article, we report an extensivegenetic analysis that has identified CUL9 as a p53-dependenttumor suppressor.

Materials and Methods

Generating Cul9 mutant miceThe targeting construct was generated to delete a 5.7-kb

genomic fragment containing exons 2 to 7 encoding the N-terminal 664 amino acid residues of the mouse Cul9 gene.Briefly, a 17.8-kb mouse genomic DNA fragment spanning theCul9 locus was isolated by PCR from mouse embryonic stem(ES) cell genomic DNA. LoxP sites were introduced flankingexons 2 and 7 of Cul9 by site-directed mutagenesis (Strata-gene). A neomycin (neo) resistance gene flanked by frt siteswas inserted upstream of the loxP site upstream of mouseexon 2 as a positive selection marker for homologous recom-bination, and the thymidine kinase gene was insertedupstream of exon 1 to allow for negative selection usinggancyclovir against random integration. The linearized target-ing construct was electroporated into E14 ES cells andselected with G418 and ganglocyclovir. Doubly resistantclones were screened for homologous recombination events

Authors' Affiliations: 1Lineberger Comprehensive Cancer Center, 2Curri-culum in Genetics and Molecular Biology, 3Department of Biochemistryand Biophysics, and 4Program in Molecular Biology and Biotechnology,University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

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

Current address for X-H. Pei: Molecular Oncology Program, Department ofSurgery, University of Miami, Miami, FL 33136.

Corresponding Author: Yue Xiong, University of North Carolina, 22-012Lineberger Cancer Center, Chapel Hill, NC 27599-7295. Phone: 919-962-2142; Fax: 919-966-8799; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-4300

�2011 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 2969

Research. on July 29, 2018. © 2011 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst April 12, 2011; DOI: 10.1158/0008-5472.CAN-10-4300

http://cancerres.aacrjournals.org/

and confirmed by Southern blot analysis. After removal of theneomarker by transfection of Flp recombinase, 3 independentCul9flox/þ ES clones were injected into C57BL/6 blastocysts,and chimeric mice were crossed with C57BL/6 mice to gen-erate Cul9flox/þ heterozygotes. Germline transmitted Cul9flox/þ

mice were then crossed with EIIa-Cre transgenic mice [B6.FVB-Tg (EIIa-Cre); Jackson Laboratory] to generate Cul9þ/�

heterozygotes. Successful deletion of exons 2 to 7 in Cul9þ/�

mice was confirmed by PCR and Southern blotting (Supple-mentary Fig. S2).

Mice, histopathology, immunohistochemistry, andcarcinogenesis

Cul9 heterozygotes were backcrossed for 10 generationswith C57BL/6 mice. p53 null (p53tm1Tyj) and Em-Myc trans-genic [B6.Cg-Tg(IghMyc)22Bri/J] mice in C57BL/6 back-ground were obtained from Jackson Laboratory and bred tothe Cul9 mutant mice to create double-mutant mice. Mostanalyses, including body weight and tumor development, onCul9 and Cul9;p53 double-mutant mice were done with micebackcrossed 4 to 5 generations to C57BL/6. Analysis on Cul9;Em-Myc mice was done using Cul9 mutant mice that werebackcrossed 10 generations to C57BL/6. For the determina-tion of spontaneous tumor incidence, Cul9, p53, and Cul9;p53mutant mice with visible tumors, exhibiting morbidity, weightloss, or paralysis were sacrificed. The remaining survivingmice were sacrificed at 27 months of age (for WT, Cul9þ/�,and Cul9�/�) or at 21 months of age (for p53þ/�, Cul9þ/�;p53þ/�, and Cul9�/�; p53þ/�). For the determination of lym-phoma development in Em-Myc and Em-Myc;Cul9 mutants,mice were sacrificed when they developed visible tumors orexhibited morbidity. The remaining surviving mice weresacrificed at 5 months of age. A full autopsy was done andtissues were fixed and histologically examined by 2 patholo-gists after hematoxylin and eosin (H&E) staining. Immuno-histochemistry was done as described previously (9).

To determine the carcinogenic susceptibility of Cul9mutants, mice of different genotypes were injected intraper-itoneally at 7 weeks of age with N-nitrosodiethylamine (DEN;Sigma) at 10 mg/kg body weight. All mice were sacrificed andfull necropsies were done at 11 months of age. All tumors wereconfirmed by 2 individual pathologists. The number of lungtumors was counted on the basis of the H&E staining con-firmed lesions.

ES and MEF cellsCul9 null ES cells were generated by 2 rounds of targeting.

Briefly, the linearized targeting construct was electroporatedinto E14 ES cells and selected with G418 and ganglocyclovir.Doubly resistant clones were screened for homologous recom-bination events and confirmed by Southern blot analysis(Supplementary Fig. S2). The neo marker was removed byFlp recombinase, and clones confirmed for neo excision weresubjected to another round of targeting, selection, and screen-ing for homologous recombination. Cul9NEOflox/flox ES cloneswere expanded and subjected to both plasmid-mediated(CMV-Cre) and retroviral Cre expression. Deletion of exons2 to 7 from both alleles was confirmed by genomic PCR, RT-

PCR, and Western blotting (Supplementary Fig. S2). p53 nullmice were bred to the Cul9 mutant mice to create doubleheterozygous mice. Cul9þ/� and p53þ/�;Cul9þ/� mice wereintercrossed separately and E13.5-E14.5 embryos were isolatedto generate mouse embryonic fibroblasts (MEF) as described(10). Early passage MEFs (<passage 4) from individualembryos were plated in 100-mm plates and incubated inDulbecco's modified Eagle's medium plus 10% FBS.

Proliferation and apoptosis assaysFor bromodeoxyuridine (BrdU) labeling, ES cells or early

passage MEFs (<passage 4) from individual embryos weregrown in media containing 10 mmol/L BrdU for 1 hour. Aftertrypsinization and PBS wash, cells were fixed and permeabi-lized. Incorporated BrdUwas exposed to DNase treatment andthen stained with fluorescein isothiocyanate (FITC)–conju-gated anti-BrdU antibody. Total DNA was stained by PI(propidium iodide). For apoptosis analysis, cells were exposedto the indicated amounts of UV radiation, harvested 24 hoursafter UV treatment, stained with Annexin V–FITC, and thenanalyzed by fluorescence-activated cell sorting (FACS). FACSanalysis was done using the CYAN ADP Analyzer (Dako). Dataanalysis was done using Summit v4.3 software (Dako).

To analyze apoptosis in the thymus in vivo, mice wereirradiated with 10 Gy of g-irradiation and sacrificed after 8hours. Thymus sections were analyzed for apoptosis usingApoTag kit (Chemicon) according to the manufacturer's pro-tocol. Thymocytes derived from the irradiated mice were alsostained with either JC-1 or Annexin V–FITC and PI and thenanalyzed by FACS. JC-1 staining determines mitochondrialmembrane potential (DYm). It enters cells and gives a greenfluorescent signal and is processed to give an additional redfluorescent signal only in mitochondria with preserved DYm.

Cells with preservation of DYm are JC-1Ghigh and JC-1Rhigh,and cells with loss of DYm are JC-1Ghigh and JC-1Rlow.

Results

Cul9 mutant mice develop spontaneous tumorsTo determine the function of CUL9, we generated Cul9�/�

mice by homologous recombination, using a targeting con-struct that deletes a 5.7-kb genomic fragment containingexons 2 to 7 that encodes the N-terminal 664 amino acidresidues of Cul9 protein (Supplementary Fig. S2A–F). TheCul9�/� mice, as previously reported (8), were produced inMendelian ratio without significant developmental defect. Thebody weights of both male and female Cul9�/� mice, however,were decreased (Fig. 1A). After monitoring a cohort of 28Cul9�/�, 23 Cul9þ/�, and 12 wild-type mice for 27 months, wefound that 22 (79%) Cul9�/� mice developed tumors in multi-ple organs or tissues, including lymphoma, sarcoma, andtumors in pituitary, lung, liver, and ovary (Fig. 1B and C,and Supplementary Fig. S3). Cul9þ/� heterozygote mice alsodeveloped tumors with a similar spectrum and incidence(74%, n ¼ 23), whereas only 33% (n ¼ 12) WT mice displayedtumors at similar age. Lymphoma and sarcoma developed inCul9 mutant mice frequently infiltrated into multiple organs,including thymus, spleen, liver, pancreas, lung, and skin

Pei et al.

Cancer Res; 71(8) April 15, 2011 Cancer Research2970




(Supplementary Fig. S3). Both Cul9�/� and Cul9þ/�mice had adecreased life span compared with WT controls. The meantumor-free survival times for Cul9�/�, Cul9þ/�, and WT were19.9, 20.8, and 26.4 months, respectively (Fig. 1C). Only 2 of 16(13%) tumors developed in Cul9þ/� mice were found to havelost the remaining WT allele (Fig. 1D, and data not shown).These results reveal a haploinsufficient function of Cul9 intumor suppression.

Cul9 deficiency promotes lymphomagenesis andmetastasis in Em-Myc miceEm-Myc transgenic mice ectopically express the c-Myc

oncogene from the immunoglobulin heavy chain enhancer,and reproducibly develop and die from tumors of the Blymphocyte lineage with a mean survival time of 4 months(11, 12). To further corroborate the tumor suppression func-

tion of Cul9, we generated Cul9;Em-Myc double-mutant miceand followed lymphoma development for 5 months. No lym-phoma developed in Cul9 mutant mice in the absence ofectopic Myc expression by this age. Loss of Cul9 significantlyaccelerated the onset of lymphoma development in Em-Mycmice. The mean lymphoma-free survival times were reducedfrom 126.4 days in Cul9þ/þ;Em-Myc mice to 108.9 days inCul9þ/�;Em-Myc, and 85.1 days in Cul9�/�;Em-Myc mice,respectively (Fig. 2A and B). The earliest lymphomas weredetected in Cul9�/�;Em-Mycmice at 25 days of age. Three of 5Cul9þ/� or Cul9�/� mice younger than 2 months of agedeveloped lymphoma, but no tumor developed in Cul9þ/þ

mice by this age. Overall, 66% (6/9) of the Cul9þ/þ;Em-Mycmice developed lymphoma, whereas 80% of Cul9þ/�;Em-Mycmice (16/20) and 100% (10/10) Cul9�/�;Em-Myc mice hadlymphoma in 5 months. All tumors that developed were either

Figure 1. Cul9-deficient micedevelop spontaneous tumors. A,loss of Cul9 results in body weightdecrease. Male and female miceat the indicated ages wereweighed. Averages and SDs werecalculated from 4 to 5mice in eachtime point. B, tumor incidence andspectrum in WT and Cul9 mutantmice. C, tumor-free survival curveof WT and Cul9 mutant mice. D,genomic DNA extracted from themicrodissected samples of Cul9heterozygote mice was amplifiedby PCR to detect Cul9 alleles. mt,mutant.

CUL9/PARC Is a Tumor Suppressor

www.aacrjournals.org Cancer Res; 71(8) April 15, 2011 2971




B-cell lymphoma or pre-B lymphomas regardless of the gen-otypes (Supplementary Fig. S4B).

Haplo or complete loss of Cul9 not only accelerated thelymphoma development in Em-Mycmice but also transformedEm-Myc–induced lymphoma to become more malignant. Allbut 3 lymphomas (n ¼ 26) developed in Cul9þ/�;Em-Myc, orCul9�/�;Em-Myc mice exhibited extensive periportal invasionwith lymphoma cell clusters spreading throughout the liverparenchym and various degrees of lung and pancreas infiltra-tion (Supplementary Fig. S4A). In contrast, lymphomas devel-oped in Cul9þ/þ;Em-Myc mice displayed little nonlymphoidorgan infiltration, with only one Cul9þ/þ;Em-Myc lymphoma (n¼ 6) showing minor periportal invasion in the liver (Fig. 2B,and Supplementary Fig. S4A). No significant difference wasobserved in tumor invasion and infiltration between Cul9þ/�;Em-Myc, and Cul9�/�;Em-Myc mice, nor did we detect the lossof the remaining WT allele of Cul9 in lymphomas developed inCul9þ/�;Em-Myc mice (n ¼ 10, Fig. 2C, and data not shown).

Together, these results provide further evidence supportingCul9 as a haploinsufficient tumor suppressor.

Cul9 mutant mice are susceptible to carcinogenesisSpontaneous tumor development and acceleration of Em-

Myc–induced lymphomagesis in Cul9 mutant mice led us toexamine the susceptibility of Cul9mutant mice to tumorigen-esis after continuous low-dose exposure to a commonly usedchemical carcinogen DEN, which mainly targets the lung andliver. As shown in Supplementary Figure S5A, both Cul9þ/�

and Cul9�/� mice are more susceptible to DEN-inducedtumorigenesis. By the age of 11 months, when all mice werescarified for histologic and pathologic analysis, none of 3 WTmice had developed detectable tumor lesions. In contrast, 3 of6 Cul9þ/� heterozygote mice and 5 of 6 Cul9�/� homozygousmice developed multiple tumors in lung, with Cul9�/� micedeveloping significantly more tumors per lung than the Cul9þ/

� mice (Fig. 2D and Supplementary Fig. S5A). Most lung

Figure 2. Cul9 deficiencyaccelerates Em-Myc–inducedlymphomagenesis and increasesthe susceptibility to carcinogen. A,lymphoma-free survival curve ofCul9, Em-Myc, and Em-Myc;Cul9double-mutant mice. All micewere euthanized within 5 months,followed by pathologicexamination and FACS analysis.B, lymphoma incidence, subtype,and lymphoma numbers invadedinto liver (periportal invasion) inCul9, Em-Myc, and Em-Myc;Cul9double-mutant mice. Note:lymphoma was diagnosed andtyped by H&E staining and FACSanalysis. C, genomic DNAextracted from the representativelymphomas derived from 3 Cul9heterozygotes mice and from WTor Cul9 homozygote mouse wasamplified by PCR to detect Cul9alleles. TT, thymus tumor; NS,normal spleen; ST, spleen tumor.D, average numbers of lungtumors derived from the micetreated with DEN. mt, mutant.

Pei et al.





tumors developed in DEN-treated Cul9 mutant mice wererelatively well-differentiated adenomas (SupplementaryFig. S5B). Two lung tumors derived from Cul9�/� mice wereadenocarcinomas with high mitotic indices and invasivepotential. Two Cul9�/�mice developed lymphoma with multi-ple organ infiltration, one of which also developed hepatocel-lular carcinoma with metastasis to spleen, pancreas, andlymph node. In addition, we also observed more severehepatocyte dysplasia in Cul9 mutant livers than in wild-typecounterparts. Taken together, these results further corrobo-rate the function of Cul9 in tumor suppression.

Tumor suppression function of Cul9 is largely mediatedby p53To determine the functional interaction of Cul9 and p53, we

generated Cul9-p53 double-mutant mice. After monitoring acohort of total 73 mice of various genotypes over a period of 21months, we found that unlike accelerated lymphoma devel-opment of Em-Myc mice, neither haplo nor complete lossin Cul9 significantly affected tumor development in eitherp53þ/� heterozygote or p53�/� null mutant mice (Fig. 3A andB). The mean tumor-free survival times were comparable inboth p53þ/� (14 months for p53þ/�;Cul9�/� and 15.1 monthsfor p53þ/�;Cul9þ/� vs. 14.6 months for p53þ/� mice) and

p53�/� (4.3 months for p53�/�;Cul9�/� and 4.6 months forp53�/�;Cul9þ/� vs. 5.3 months for p53�/� mice) backgrounds,indicating that combinatorial loss of Cul9 and p53 had little orno effect on tumor-free survival as compared with animalslacking p53 alone. The lack of significant effect on tumorinitiation and progression in p53 mutant mice by Cul9 muta-tion cannot be attributed to either a lack of function of Cul9 insuppressing lymphomas or early death of p53 mutant micefrom lymphomas becoming rapidly lethal as Em-Myc micedeveloped similar types of lymphomas and died even earlier.One interpretation for the collaboration of Cul9mutation withMyc overexpression, but not with p53 mutation, to promotetumor growth is that Cul9 suppresses tumor growth via apathway that is distinct from Myc-promoted tumorigenesisbut is largely mediated by p53.

Disruption of Cul9 attenuates DNA damage–inducedapoptosis

Given the functional dependency of CUL9 on p53 in tumorsuppression, we examined 2 cellular activities—arresting cell-cycle progression and promoting apoptosis—underlying thefunction of p53 in tumor suppression. Deletion of one or bothalleles of Cul9 did not cause any significant alteration in thecell-cycle phase distribution in either asynchronously growingES (Fig. 4A) or MEFs (Fig. 4B). These results indicate thatCUL9, a relatively young gene evolved after the emergence ofvertebrates, does not have an essential function in cell-cycleprogression.

We next examined the function of Cul9 in response to 2common types of DNA damage, UV and g-irradiations, in bothin vitro cultured cells and in tissues of mutant mice. Cul9�/�

ES cells were exposed to UV irradiation for various lengths oftime, pulse labeled with BrdU, and analyzed by flow cytometry.UV irradiation quickly—within an hour—reduced the S-phase(BrdUþ) cell population by almost 50% and accumulated bothG1 and G2 populations (Fig. 4C and Supplementary Fig. S6A).Although not causing a significant change in the relativedistribution of these 3 cell populations, deletion of Cul9decreased UV-induced apoptosis. Starting at 4 hours afterUV irradiation, there was an evident accumulation of anapoptotic (sub-G1) cell population in WT ES cells that con-tinuously increased throughout the experimental duration of24 hours. Loss of function of Cul9 attenuated, but did notcompletely eliminate, the accumulation of apoptotic popula-tion (Fig. 4C). To confirm the proapoptotic function of Cul9,we analyzed UV-irradiated ES cells by Annexin V staining,which more sensitively and accurately detects early apoptoticcells. We found that deletion of Cul9 reduced Annexin V–positive cells in response to both 10 and 25 J/m2 of UV doses(Fig. 4D). Together, these results show that Cul9 plays asignificant role in promoting UV-induced apoptosis.

We next examined the function of Cul9 in cellular responseto DNA damage in vivo. Littermate mice were g-irradiated anddissected 9 hours after irradiation, and thymocytes wereisolated from thymus and analyzed by flow cytometry. JC-1staining, which detects early apoptotic cells by measuringDYm, showed that deletion of Cul9 attenuated apoptosis,reducing the percentages of thymocytes with loss of DYm

Figure 3. Functional dependency of Cul9 on p53 in tumor suppression. A,tumor incidence and spectrum in Cul9, p53 single-, and p53;Cul9 double-mutant mice. B, tumor-free survival curve of Cul9, p53 single-, and p53;Cul9 double-mutant mice.






from 44% in the WT thymus to 29% in Cul9�/� thymus(Fig. 4E). Parallel Annexin V staining of these irradiatedthymocytes confirmed decreased apoptosis from 35% inWT to 22% in Cul9�/� thymus. We also carried out a terminaldeoxynucleotidyl transferase–mediated dUTP nick end label-ing assay in g-irradiated thymus (Supplementary Fig. S6B) andspleen (data not shown) and found that deletion of Cul9substantially reduced apoptosis in both organs.

Cul9 promotes p53-dependent apoptosisTo determine how Cul9 and p53 functionally interact with

each other to promote DNA damage–induced apoptosis, weUV irradiated asynchronously growing MEFs of differentgenotypes and determined both cell-cycle progression bycoupled BrdU pulse labeling and flow cytometry and apoptosisby Annexin V staining 24 hours after UV irradiation. Unlikedeletion of Cul9, which had little effect on the cell-cycleprogression of MEF cells as measured by the BrdU incorpora-tion (24% vs. 23%), deletion of p53 increased S-phase (BrdUþ)cell population by 54% (24% in WT vs. 37% in p53�/� MEFs;Fig. 5A), consistent with an activity of p53 in constraining G1

progression. Codeletion of both Cul9 and p53 genes further

increased the S-phase cell population to 45%. Annexin Vstaining showed that singular deletion of either Cul9 or p53reduced UV-induced apoptosis when compared with the WTMEFs (53%), with p53 disruption (37%) exhibiting a morepronounced effect than Cul9 deletion (44%; Fig. 5B). Codele-tion of both Cul9 and p53 genes resulted in a slight furtherreduction of apoptosis (32%), indicating the function of Cul9in promoting apoptosis is mostly, but not entirely, dependenton p53.

To further corroborate the p53-mediated function of Cul9 inpromoting apoptosis, we examined the expression of Bax, aproapoptosis gene and a downstream target of p53, in Cul9-deficient thymus in response to g-irradiation. The steady statelevel of Bax protein was nearly undetectable in both unirra-diated WT and Cul9-deficient spleen (Fig. 5C, and data notshown). Bax levels were significantly increased in WT thymusafter g-irradiation for 2 and 8 hours, respectively, but theseincreases were substantially reduced in Cul9-deficient thymus(Fig. 5C).

We further examined the UV-induced p53 induction incultured ES and MEFs. Western blotting analyses showedthat deletion of Cul9 had little effect on the basal level of

Figure 4. Disruption of Cul9reduced DNA damage–inducedapoptosis. A, asynchronouslygrowing WT and Cul9�/� ES cellswere analyzed by FACS. B,asynchronously growing WT andCul9�/� MEFs cells were pulselabeled with 10 mmol/L BrdU for 1hour and analyzed by FACS. C,Cul9f/f and Cul9�/� ES cells wereexposed to 50 J/m2 UV irradiation,pulse labeled with 10 mmol/L BrdUfor 1 hour, and harvested atindicated time after UV andstained with anti-BrdU-FITC andPI. The distribution of cells indifferent phases of the cell cycle isdetermined by flow cytometry andshown in the table. D, Cul9f/f andCul9�/� ES cells were exposed tothe indicated dosage of UV for 24hours and then stained forAnnexin V. The percentages ofAnnexin V positive cells areindicated. E, littermate mice at4 months of age were irradiated(10 Gy); 9 hours later, freshlyisolated thymocytes were stainedwith JC-1 (left) and Annexin V(right). The percentages ofapoptotic cells, as measured byDYm (or JC-1Ghigh and JC-1Rlow),are indicated. A representative of3 independent experiments isshown.

Pei et al.





p53 in unperturbed asynchronously growing ES and MEFs(Fig. 5D, and Supplementary Fig. S2F and H, and S7A–C). Cul9deletion reduced, but did not abolish, the accumulation of p53following UV irradiation. Accordingly, deletion of Cul9 did notsignificantly affect the basal level of Bax in unperturbed ES andMEF cells, but it reduced the Bax protein accumulation

following UV irradiation. Deletion of p53, on the other hand,slightly decreased Cul9 protein level in embryos at E13. 5,E15.5, and E17.5 (Supplementary Fig. S8A and data not shown)but did not affect Cul9 expression in adult tissues (Supple-mentary Fig. S8B), indicating that p53 does not play a majorrole in the regulation of Cul9 gene expression.

Figure 5. CUL9 promotes a p53-dependent apoptosis. A and B,Cul9 collaborates with p53 inpromoting apoptosis. MEFs(passage 1) of different genotypeswere pulse labeled with 10 mmol/LBrdU for 1 hour and stained withanti–BrdU-FITC (A) or wereirradiated with UV (50 J/m2) for 24hours and stained with Annexin V(B). DNA synthesis and apoptosiswere determined by flowcytometry. The percentages ofBrdUþ and apoptotic cells areindicated. C, littermate mice at5 months of age were irradiated(10 Gy). Thymus was isolated andfixed at the indicated time andstained with an antibody againstBax and DAPI. Thymus notirradiated was used as control. IR,irradiation. D, deletion of Cul9reduces the accumulation of p53and p53 target gene, Bax, in MEFcells. Total cell lysates preparedfrom UV-irradiated MEF cells ofdifferent genotypes wereseparated by SDS-PAGE,followed by direct immunoblottingwith indicated antibodies. Thequantification is included inSupplementary Fig. S8. E,plasmids expressing differenthuman CUL9 mutants werecotransfected with p53expressing plasmid into U2OScells and CUL9-p53 binding wasdetermined by IP-Western. IP,immunoprecipitation; IB,immunoblotting. F, WT MEF cellswere infected for 2 days with thelentivirus indicated, stained withPI, and analyzed by FACS. Thepercentages of cells with less than2N (sub-G1) and greater than 4NDNA (polyploidy) contents wereindicated. The expression levels ofWT and mutant Cul9 transducedby lentivirus were confirmed byWestern blot (left).






Finally, to provide further biochemical evidence supportinga functional dependency on p53 for the proapoptotic activityof CUL9, we carried out a series of deletion and mutationanalyses and identified 2 point mutants of human CUL9,CUL9Q417A and CUL9F419A, that completely disrupted the bind-ing of CUL9 with p53 (Fig. 5E for Q417A mutant and data notshown for F419A). We then generated a lentivirus expressingboth the WT and Q417A mutant CUL9. Transduction of MEFswith WT Cul9 lentivirus increased the apoptotic (sub-G1) cellpopulation by 71%, from 14.1% to 24.1% (Fig. 5F). Remarkably,transduction of CUL9Q417A, when expressed at a similar level,did not cause any significant increase of apoptotic cells(14.0%). Instead, ectopic expression of CUL9Q417A substantiallyincreased the polyploidy cell population, resulting in anincrease of cells with more than 4N DNA content from16.5% in control cells and 14.2% in cells transduced withWT CUL9 to 24.6% in MEFs transduced with CUL9Q417A.Transduction of U2OS cells with WT CUL9 and CUL9Q417A

lentivirus also increased apoptosis and polyploidy, respec-tively, as evidenced by Annexin V and 40,6-diamidino-2-phe-nylindole (DAPI) staining (data not shown). These resultssuggest that increased CUL9 level causes a defect leadingto polyploidy cells that would normally be eliminated by a p53-mediated apoptosis pathway but become accumulated whenthe CUL9-p53 interaction is disrupted.

Discussion

In this article, we provide compelling genetic evidence thatCUL9 (formerly PARC) is a tumor suppressor; deletion of Cul9in mice resulted in spontaneous development of tumor,acceleration of Em-Myc–induced lymphomagenesis, and pre-disposition of mice to carcinogenesis. Consistent with itsbroad expression (2), tumors spontaneously developed inCul9mutant mice cross a wide spectrum of at least 9 differentorgans or tissues, with pituitary adenomas being more pre-valent than others. One notable feature of tumorigenesis inCul9 mutant mice is that both the penetrance of tumordevelopment and tumor-free survival are very similar betweenCul9 heterozygotes and homozygotes, suggesting haploinsuf-ficiency of Cul9 in tumor suppression. This notion is sup-ported by the observation that in both Cul9þ/�-single andCul9þ/�;Em-Myc–double mutants mice, the remaining WTallele of Cul9 was retained in most tumors examined. TheCUL9 gene has not been reported thus far to be frequentlymutated in human tumors. In addition to being a haploin-sufficient gene whose decreased expression may be suffi-ciently reduced by its function in tumor suppression, thefunctional dependency of CUL9 on p53 in tumor suppressionmay also explain the lack of widespread CUL9 mutations inhuman cancers. Deletion of both alleles of Cul9 did not exhibitany significant synergistic tumorigenic effect in p53 hetero-zygotes, suggesting that even complete loss of CUL9 functionwould not offer a significant selection advantage for thegrowth of human tumor cells with even haploid loss or partialreduction of p53 activity.

CUL9 joins a growing number of p53 regulators that med-iate different cellular stresses to activate p53, leading to either

cell-cycle arrest or apoptosis. There are 3 unique features ofCUL9 in signaling p53 to suppress tumorigenesis. First, CUL9signals p53 selectively toward apoptosis and does not seem tocause a cell-cycle arrest following DNA damage. In several celltypes we have examined (ES, MEFs, thymocytes, and splee-nocytes), deletion of Cul9 caused only an attenuated apoptosiswithout significantly affecting the cell-cycle phase distribu-tion. Second, CUL9 is an E3 ubiquitin ligase that may functionas a p53 activator. More than a dozen E3 ubiquitin ligases havebeen linked to p53 regulation (for a recent compilation, see ref.13), all promoting p53 degradation and thus acting function-ally as a negative regulator of p53. In contrast, the functions ofCUL9 in tumor suppression (Fig. 3) and in promoting apop-tosis (Fig. 5) are both dependent on p53. To the best of ourknowledge, CUL9 represents the first E3 ligase that bindsdirectly to and is functionally dependent on p53. Third, CUL9localizes exclusively in the cytoplasm. In addition to the well-established function of p53 as a nuclear transcription factor,emerging evidence suggests that p53 has an important role inthe cytoplasm, especially in mitochondrial-mediated apopto-sis (14). The regulation, in particular the activation, of p53 inthe cytoplasm remains poorly understood. Given its cytoplas-mic localization and functional dependency on p53, we sus-pect that CUL9 may activate p53 in the cytoplasm.

The biochemical mechanism by which CUL9 regulates p53remains unclear. Following DNA damage, the induction of p53was moderately and reproducibly reduced in Cul9-deficientorgans and cells. A reduced p53 induction is consistent withand likely underlines the attenuated apoptosis inCul9-deficientorgans in vivo and in both ES and MEF cells cultured in vitroafter DNA damage. However, unlike the overexpression ordecrease of other p53 E3 ligases, especially MDM2, we didnot detect a significant change in the steady state level andhalf-life of p53 in cells with either Cul9 deletion or overexpression.We have also carried out extensive investigation of subcellularlocalization and stress-linked phosphorylation at various sitesin WT and Cul9 null cells following DNA damage. Thus far, wehave not detected a significant change in either subcellulardistribution or phosphorylation of p53. Thus, it remains achallenge to elucidate the molecular mechanism by whichCUL9, a cytoplasmic localized E3 ligase, signals to p53.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank J. DeCaprio (Dana-Farber Cancer Institute) for providing a CUL9/PARC lentiviral plasmid and for discussions.

Grant Support

This study was supported by NIH grants CA065572 and GM067113 to Y.Xiong.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 28, 2010; revised February 9, 2011; accepted February 25,2011; published online April 12, 2011.

Pei et al.





References1. Dias DC, Dolios G,Wang R, Pan ZQ. CUL7: A DOCdomain-containing

cullin selectively binds Skp1.Fbx29 to form an SCF-like complex. ProcNatl Acad Sci U S A 2002;99:16601–6.

2. Nikolaev AY, Li M, Puskas N, Qin J, GuW. Parc: a cytoplasmic anchorfor p53. Cell 2003;112:29–40.

3. Zachariae W, Shevchenko A, Andrews PD, Ciosk R, Galova M, StarkMJR, et al. Mass spectrometric analysis of the anaphase-promotingcomplex from yeast: identification of a subunit related to cullins.Science 1998;279:1216–9.

4. Yu H, Peters J-M, King RW, Page AM, Hieter P, Kirschner MW.Identification of a cullin homology region in a subunit of the ana-phase-promoting complex. Science 1998;279:1219–22.

5. Andrews P, He YJ, Xiong Y. Cytoplasmic localized ubiquitin ligasecullin 7 binds to p53 and promotes cell growth by antagonizing p53function. Oncogene 2006;25:4534–48.

6. Kaustov L, Lukin J, Lemak A, Duan S, Ho M, Doherty R, et al. Theconserved CPH domains of Cul7 and PARC are protein-proteininteraction modules that bind the tetramerization domain of p53. JBiol Chem 2007;282:11300–7.

7. Kasper JS, Arai T, DeCaprio JA. A novel p53-binding domain in CUL7.Biochem Biophys Res Commun 2006;348:132–8.

8. Skaar JR, Arai T, DeCaprio JA. Dimerization of CUL7 and PARC is notrequired for all CUL7 functions and mouse development. Mol Cell Biol2005;25:5579–89.

9. Bai F, Pei XH, Godfrey VL, Xiong Y. Haploinsufficiency of p18INK4c

sensitizes mice to carcinogen-induced tumorigenesis. Mol Cell Biol2003;23:1269–77.

10. Pei XH, Bai F, Tsutsui T, Kiyokawa H, Xiong Y. Genetic evidence forfunctional dependency of p18Ink4c on Cdk4. Mol Cell Biol 2004;24:6653–64.

11. Adams JM, Harris AW, Pinkert CA, Corcoran LM, Alexander WS, CoryS, et al. The c-myc oncogene driven by immunoglobulin enhancersinduces lymphoid malignancy in transgenic mice. Nature 1985;318:533–8.

12. Langdon WY, Harris AW, Cory S, Adams JM. The c-myc oncogeneperturbs B lymphocyte development in E-mu-myc transgenic mice.Cell 1986;47:11–8.

13. Jain AK, Barton MC. Regulation of p53: TRIM24 enters the RING. CellCycle 2009;8:3668–74.

14. Green DR, Kroemer G. Cytoplasmic functions of the tumour suppres-sor p53. Nature 2009;458:1127–30.






2011;71:2969-2977. Published OnlineFirst April 12, 2011.Cancer Res Xin-Hai Pei, Feng Bai, Zhijun Li, et al. Suppressor and Promotes p53-Dependent Apoptosis

Ubiquitin Ligase Is a TumorCUL9/PARCCytoplasmic

Updated version

10.1158/0008-5472.CAN-10-4300doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2011/04/08/0008-5472.CAN-10-4300.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/71/8/2969.full#ref-list-1

This article cites 14 articles, 7 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/71/8/2969.full#related-urls

This article has been cited by 7 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

SubscriptionsReprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://cancerres.aacrjournals.org/content/71/8/2969To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-10-4300

http://cancerres.aacrjournals.org/content/suppl/2011/04/08/0008-5472.CAN-10-4300.DC1

http://cancerres.aacrjournals.org/content/71/8/2969.full#ref-list-1

http://cancerres.aacrjournals.org/content/71/8/2969.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/71/8/2969


cytoplasmic cul9/parc ubiquitin ligase is a...

Documents