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Human Cancer Biology

Oncogenic Function of SCCRO5/DCUN1D5 Requires ItsNeddylation E3 Activity and Nuclear Localization

Claire C. Bommelj�e1, Víola B. Weeda1, Guochang Huang1, Kushyup Shah1, Sarina Bains1, Elizabeth Buss1,Manish Shaha1, Mithat G€onen2, Ronald Ghossein3, Suresh Y. Ramanathan1, and Bhuvanesh Singh1

AbstractPurpose: To determine mechanisms by which SCCRO5 (aka DCUN1D5) promotes oncogenesis.

Experimental Design: SCCRO5mRNA and protein expression were assessed in 203 randomly selected

primary cancer tissue samples, matched histologically normal tissues, and cell lines by use of real-time PCR

and Western blot analysis. SCCRO5 overexpression was correlated with survival. The effect of SCCRO5

knockdownonviabilitywas assessed in selected cancer cell lines. Structure–function studieswere performed

to determine the SCCRO5 residues required for binding to the neddylation components, for neddylation-

promoting activity, and for transformation.

Results: In oral and lung squamous cell carcinomas, SCCRO5 mRNA levels corresponded with protein

levels and overexpression correlated with decreased disease-specific survival. Knockdown of SCCRO5 by

RNAi resulted in a selective decrease in the viability of cancer cells with high endogenous levels, suggesting

the presence of oncogene addiction. SCCRO5 promoted cullin neddylation while maintaining conserved

reaction processivity paradigms involved in ubiquitin and ubiquitin-like protein conjugation, establishing

it as a component of the neddylation E3. Neddylation activities in vitro required the potentiating of

neddylation (PONY) domain but not the nuclear localization sequence (NLS) domain. In contrast, both the

NLS domain and the PONY domain were required for transformation of NIH-3T3 cells.

Conclusions: Our data suggest that SCCRO5 has oncogenic potential that requires its function as a

component of the neddylation E3. Neddylation activity and nuclear localization of SCCRO5 are important

for its in vivo function. Clin Cancer Res; 20(2); 372–81. �2013 AACR.

IntroductionSeveral important anticancer therapies have been discov-

ered by leveraging the knowledge that changes in proteinhomeostasis drive human cancer pathogenesis. In thisregard, the ubiquitin/proteasome system is of significance,as there is a high prevalence of aberrations among thecomponents of this pathway (1, 2). In particular, the activityof cullin-RING type ubiquitination E3 ligases is commonlydysregulated in human cancers (3). Dysregulation oftenresults from aberrations in the substrate recognition adap-tors (i.e., FBX7, SPOP, Skp2, VHL), and rarely from abnor-malities in core components (e.g., Cul4A amplification in

breast cancer; refs. 4–7). It has long been established thatposttranslationalmodificationof cullins by neddylation is akey regulator of cullin-RING ligase (CRL) activity (neddyla-tion promotes assembly of the CRL complex and enhancesrecruitment of ubiquitin-charged E2, to facilitate ubiquitinconjugation; refs. 8–13). As is the case with ubiquitination,neddylation results from the sequential enzymatic activityof a dedicated E1 (APPBP1-Uba3), E2 [Ubc12 (a.k.a.Ube2M) andUbe2F], and E3 (14). The link between abnor-mal CRL activity and dysregulation of the proteins involvedin neddylation in human cancers has only recently beenappreciated (15). These observations have led to the devel-opment of a small-molecule�� inhibitor of neddylation E1,MLN4924, which has shown promise in preclinical andearly human trials (3).

SCCRO (a.k.a. DCUN1D1), a key component of the E3for neddylation, is activated by amplification in a widerange of human cancers (16, 17). The role that SCCROplays in cancer pathogenesis has been studied in both in vivoand in vitro experimental systems, establishing its functionas an oncogene (16, 18). In addition, overexpression ofSCCRO in human tumor samples has been independentlyassociated with poor survival outcomes (16). Of interest,SCCRO is a member of a protein family that contains fourother highly conserved paralogs in higher organisms. All

Authors' Affiliations: 1Department of Surgery, Laboratory of EpithelialCancer Biology and Departments of 2Epidemiology and Biostatistics and3Pathology,Memorial Sloan-KetteringCancerCenter, NewYork,NewYork

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

C.C. Bommelj�e and V.B. Weeda contributed equally to this work.

Corresponding Author: Bhuvanesh Singh, Memorial Sloan-KetteringCancer Center, 1275 York Ave New York, NY 10065. Phone: 212-639-2024; Fax: 212-717-3302; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-1252

�2013 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(2) January 15, 2014372

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Published OnlineFirst November 5, 2013; DOI: 10.1158/1078-0432.CCR-13-1252

http://clincancerres.aacrjournals.org/

family members have a conserved C-terminal potentiatingof neddylation (PONY) domain with a variable N-terminalregion. SCCRO paralogs are subdivided into three subfa-milies on the basis of the N-terminal sequence: SCCRO andSCCRO2 (a.k.a.DCUN1D2) contain a ubiquitin-associated(UBA) domain, SCCRO3 (a.k.a. DCUN1D3) contains amyristoylation sequence, and SCCRO4 (a.k.a. DCUN1D4)and SCCRO5 (a.k.a. DCUN1D5) contain a nuclear local-ization sequence (NLS). We have previously shown thatSCCRO is oncogenic and that its oncogenic functionrequires its neddylation activity (16, 17). SCCRO3 has alsobeen shown to play a role in oncogenesis (19). Recent workindicates that SCCRO5 has oncogenic activity (20). How-ever, the precise mechanisms by which the NLS-containingSCCRO paralogs promote oncogenesis remain unknown.Like SCCRO (at 3q26), its paralogs are located in chro-

mosomal loci that are recurrently amplified in humancancers (SCCRO2 at 13q34, SCCRO4 at 4q12, and SCCRO5at 11q22; refs. 21–23). Of these, 11q22 amplification is thesecond most frequent, occurring in a wide range of humancancers, with the highest prevalence in squamous cell car-cinomas (SCC)of the cervix, esophagus, head andneck, andlung (24–27). Moreover, there is an independent associa-tion between 11q22 amplification and worse clinical out-come, suggesting that this locus harbors genes that play arole in cancer pathogenesis (15, 28, 29). Although severalcandidate genes have been identified, including MMPs,Birc2 (cIAP1), Birc3 (cIAP2), and Yap, none has been clearlyestablished as a target that drives selection for 11q22 ampli-fication (29–31). Fine-mapping of 11q22.2-q22.3 in cer-vical, oral, and lung cancers has shown that SCCRO5(MGC2714) is located within the minimal common region

of amplification (27, 30, 32). SCCRO5 is upregulated inlaryngeal SCC and putatively plays a role in its pathogenesis(20). Given the established role of SCCRO in oncogenesis,we questioned whether SCCRO5 drives selection for 11q22amplification in human cancers and sought to elucidate themechanisms involved.

Here, we report the validation of SCCRO5 as a target thatdrives selection for 11q22 amplification in human cancers.We found thatSCCRO5mRNAexpression is associatedwitha corresponding increase in protein levels and is correlatedwith decreased disease-specific survival in oral and lungSCCs. Specific knockdown of SCCRO5 in cancer cell lineswith high endogenous levels of SCCRO5 expressionresulted in a more significant decrease in viability, than incells with low expression levels, suggesting the presence ofan oncogene addiction phenotype. The oncogenic potentialof SCCRO5 is underscored by its ability to transform fibro-blasts (NIH-3T3 cells) in vitro. Furthermore, we show that,like SCCRO, SCCRO5 functions as a component of the E3,promoting cullin neddylation while maintaining reactionprocessivity paradigms. Although only the PONY domainwas required for SCCRO50s neddylation function in vitro, itsoncogenic activity in vivo requires both the PONY domainand the NLS domain, suggesting that subcellular localiza-tion plays a role in its function. The precise mechanisminvolved in SCCRO50s in vivo neddylation activity remainsto be defined.

Materials and MethodsBioinformatic analyses

The ClustalW program (Conway Institute UCD, Dublin,Ireland) was used for multiple sequence alignments. Datafrom The Cancer Genome Atlas (TCGA) projects wereaccessed and analyzed using the cBio Cancer GenomicsPortal (http://www.cbioportal.org).

Human tissues, cell lines, and antibodiesA total of 203 randomly selected primary cancer tissue

and adjacent matched histologically normal tissue sampleswere obtained from patients undergoing surgical treatmentat Memorial Sloan-Kettering Cancer Center between Janu-ary 1, 2004, and October 1, 2007. All patients undergoingsurgical resection of primary cancers were offered an oppor-tunity to participate in the study. Only previously untreatedpatients with adequate tissue stored in our tissue bank wereincluded in the study. Informed consent was obtained fromall patients, in accordance with the guidelines establishedby the Institutional Review Board, and use of patient sam-pleswas approved by the institutionalHumanBiospecimenUtilizationCommittee.Histopathologic confirmation of allspecimens was established by an experienced pathologist.Tissues were frozen in liquid nitrogen immediately afterresection and stored at �80�C until use. Demographic andtumor data for the oral SCC, lung SCC, thyroid cancer, andlung neuroendocrine carcinoma patients are described inSupplementary Tables S1–S4.

Theorigins, cytogenetic characteristics,maintenance, andgrowth of the cell lines used in this study were as previously

Translational RelevanceThe importance of the ubiquitin–proteasome path-

way in cancer pathogenesis is established by the thera-peutic efficacy of agents that target pathway activity intreating human cancers. Neddylation, a major regulatorof ubiquitin–proteasome pathway activity, is itself sub-ject to regulation by SCCRO. SCCRO is commonlyactivated by amplification in many different types ofcancer and its overexpression is independently associat-ed with poor clinical outcome. Here, we show thatSCCRO5, a paralog of SCCRO, plays a role in cancerpathogenesis. SCCRO5 is commonly overexpressed inhuman cancers and its overexpression is associated withworse outcome in oral and lung squamous cell carcino-mas. Establishing its role in cancer pathogenesis, weshowoncogene addiction associatedwith SCCRO5over-expression in cancer cell lines. Moreover, we show thatSCCRO50s oncogenic activity requires both its functionas E3 in neddylation and its subcellular localization.Combined, our findings suggest that like SCCRO,SCCRO5 is a putative therapeutic target in oral and lungsquamous cell carcinoma and other types of cancer.

SCCRO5 Is an Oncogene and a Component of the E3 for Neddylation

www.aacrjournals.org Clin Cancer Res; 20(2) January 15, 2014 373




described (16, 33). The following antibodies were used inthis study: anti-SCCRO (developed and validated as previ-ously described; ref. 16), anti-SCCRO5 (developed andvalidated in our laboratory; data not shown), anti-Cul1(Invitrogen), anti-Cul2 (Abcam), anti-Cul3 (BD Bios-ciences), anti-ROC1 (Abcam), anti-Ubc12 (Rockland),anti-CAND1 (BD Biosciences), anti-a-tubulin, and anti-GAPDH (Millipore), and anti-actin and secondary antibo-dies conjugated to horseradish peroxidase (Santa CruzBiotechnology).

RNA isolation, cDNA synthesis, and quantitative real-time PCR

Total RNA was extracted from cell lines and tissue by useof TRIzol reagent (Life Technologies), repurified usingRNeasy Mini spin columns (Qiagen), and treated withDNase I to eliminate residual genomic DNA. One micro-gramof total RNAwas reverse-transcribed usingMultiScribeReverse Transcriptase (Applied Biosystems). Primers weredesigned for SCCRO5 and GAPDH (Supplementary TableS5), by use of the Primer3 program (Howard HughesMedical Institute, Chevy Chase, MD), to generate PCRproducts 75–300 bp in length and were obtained from acommercial source (Operon Technologies). To confirmspecificity for SCCRO5, the SCCRO5 primer set was testedon plasmids for all SCCRO paralogs. Quantitative real-timePCR (qRT)-PCR for SCCRO5 was performed, at least induplicate, in two separate experiments, on the 203 tumorand paired normal samples and on appropriate controls byuse of a Sequence Detector System 7900HT device (AppliedBiosystems), as previously described (16). To ensure thepresence of a single PCRproduct,melting curve analysiswasperformed after each experiment. A standard curve wasgenerated using serial dilutions of cDNA from MDA686and MDA1186 cell lines. The comparative threshold cyclemethod was used to calculate the SCCRO5 expression ratioin each sample relative to the value observed in the controlstandard curve, with GAPDH used as a control for normal-ization among samples (34).GAPDHwas determined to bethemost stable housekeeping gene in the tissues studied, onthe basis of geNorm analysis (M < 0.298; refs. 35, 36).Overexpressionwas defined as at least 2-fold higher levels ofmRNA expression in the tumor sample relative to thematched normal sample and the mean of all normal tissuesamples for each cancer type.

Mutation profilingPrimers were designed, using Primer3, to cover putative

exon regions of SCCRO5 (National Center for Biotechnol-ogy Information Human Genome Build 36.1), yieldingproducts �500 bp in length (Supplementary Table S6;ref. 37). Tumor DNA was extracted using the DNeasy kit(Qiagen) and was subjected to whole-genome amplifica-tion, as previously described (38). High-throughput bidi-rectional dideoxynucleotide sequencing of PCR-amplifiedgene products was performed using the Sanger sequencingplatform, as previously described (39). PolyPhred andPolyScan software were used to generate an initial automat-

ed report of sequence variations (39). For each respectiveexon, tumor sequences were compared against referencesequences listed in the National Center for BiotechnologyInformation (RefSeq) database. After visual inspection ofthe individual forward and reverse chromograms, for con-firmation of nonsynonymous sequence variations andinsertions or deletions (including duplications), a manualreview list of potential nucleotide changes was produced.Synonymous variants and those with corresponding entriesin the Single Nucleotide Polymorphism Database (http://www.ncbi.nlm.nih.gov/projects/SNP/) were excluded fromanalysis.

cDNA cloning, mutagenesis, and short hairpin RNAknockdown

Human SCCRO5 cDNA was obtained from ClontechLaboratories (Clontech). Mutations were generated inSCCRO5 by standard PCR mutagenesis, as previouslydescribed (17). Primers were designed using Primer3 (Sup-plementary Table S7). SCCRO5 and its mutated forms werecloned into pEGFP-N2 (Clontech), pBABE (BD Bios-ciences), and pGEX-4T-3 (GE Healthcare Life Sciences)vectors. Plasmid and protein purifications were performedas previously described (16, 17). shRNA against humanSCCRO5 (obtained from the Genome Sequencing Center atMemorial Sloan-Kettering Cancer Center) or lacZ shRNA(negative control) was transfected, along with packagingplasmids pLP1, pLP2, and pVSVG, intoHEK293 cells by useof Lipofectamine 2000, in accordance with the manufac-turer’s protocol (Invitrogen). Lentiviral particles were har-vested 48 and 72 hours after transfection and were filtered,pelleted, and resuspended inmedia for retroviral delivery inthe presence of polybrene (Millipore). Twenty-four hoursafter infection, cells were cultured in media containingpuromycin, to select for infected cells.

Molecular and biochemical assaysGlutathione S-transferase (GST)pull-downassays, in vitro

neddylation assays, in vitro thioester reactions, transfec-tions, and immunoblotting were performed as previouslydescribed (16, 17).

Proliferation and colony formation assaysMTS proliferation assays and colony formation assays

were performed as previously described (16, 17). For thesoft agar assay, colonieswere counted using ImageJ software(NIH, Bethesda,MD), to analyze images captured from four6-well plate wells per construct, from two independentexperiments.

Statistical analysisDescriptive statistics were used to summarize study day.

Continuous variables were compared across groups usingStudent t test or Mann–Whitney U test. Categorical com-parisonswere performed using the Fisher exact test. Survivalcurves were generated using the Kaplan–Meier method andcompared using the log-rank test. Time to recurrence wasdefined as the time between completion of primary

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Clin Cancer Res; 20(2) January 15, 2014 Clinical Cancer Research374




treatment of cancer and the development of recurrentcancer at any site (local, regional, or distant). Survivaloutcomes were censored for patients who died of causesother than cancer, were lost to follow-up, or survived to theend of the study. Statistical significance was defined as atwo-tailed P value less than or equal to 0.05. All statisticalanalyses were performed using state-of-the-art statisticalpackages (SPSS or SAS).

ResultsSCCRO5 binds to neddylation componentsIn silico analysis showed that SCCRO has four paralogs in

higher organisms, all with a highly conserved C-terminalPONY domain with variable N-terminal domains (Supple-mentary Fig. S1A). The established role of the PONYdomain in neddylation raised the possibility that, likeSCCRO, SCCRO’s paralogs participate in neddylation E3activity. To determine the role that SCCRO5 plays in ned-dylation, we assessed the binding of SCCRO5 to neddyla-tion components. GST pull-down assays using HeLa cellextracts, followed by Western blot analysis for neddylationcomponents, showed that, like SCCRO, SCCRO5 bindsto CAND1, Ubc12, Cul1, Cul2, Cul3, and ROC1 (Table 1;Fig. 1A). Previous biochemical and structural studies haveestablished that SCCRO binds to its neddylation compo-nents through its PONY domain, which contains fourresidues that are critical for binding interactions (Supple-mentary Fig. S1B; refs. 17, 40, 41). To map the bindingdomains,we created a series of SCCRO5deletions andpointmutants as GST fusions, including those involving the N-terminal NLS and C-terminal PONY domains, and used

them in pull-down assays on HeLa lysates. Deletion of thePONY domain (SCCRO5_D189–237) or mutation incritical residues (SCCRO5_D195N, SCCRO5_A219R,SCCRO5_D225N, SCCRO5_E226A, SCCRO5_A219R/D225N, and SCCRO5_D195N/A219R/D225N) resulted inloss of binding to CAND1 and Cul1-ROC1 (Table 1).Binding to Ubc12 was lost only with larger deletion of theC-terminal region of SCCRO5 (SCCRO5_D189–237),which is consistent with the location of the binding site ofUbc12 in SCCRO (17). Previous studies have shown thatthe UBA domain in SCCRO is not required for binding toneddylation components (17, 40, 41). SCCRO5contains anNLS in its N-terminus. We found that the NLS is functional,as transiently expressed GFP-SCCRO5 was exclusively pres-ent in the nucleus, whereas NLS-deletion mutant GFP-SCCRO5 (SCCRO5_D1–10 and SCCRO5_D1–46) had apan-cellular distribution on fluorescence microscopy (datanot shown). As with the UBA domain of SCCRO, deletionof the NLS or the entire N-terminus in SCCRO5(SCCRO5_D1–10 and SCCRO5_D1–46, respectively) hadno effect on binding to Cul1-ROC1, Ubc12, or CAND1(Table 1). These results show that binding with the neddy-lation components and residues involved in the interac-tions are conserved between SCCRO and SCCRO5.

SCCRO5 preferentially binds to Ubc12�Nedd8thioester

The mechanisms that maintain the processivity of thereactions that result in the conjugation of ubiquitin andubiquitin-like proteins are highly conserved. Central tomaintenance of processivity is a differential affinity betweenE1 and E3 for free and conjugated E2, respectively. Differ-ences in affinity ensures that an E2 must dissociate from itscognate E1 after it accepts transfer of ubiquitin or ubiquitin-like proteins and before it can bind to the E3. SCCRO hasgreater affinity for E2�Nedd8 thioester than for free E2,which is consistentwith its role as a component of the E3 forneddylation (17). To determine whether SCCRO5 alsoconforms to the established processivity paradigm forE3s, we performed GST pull-down assays on products froma Ubc12 thioester reaction, followed by Western blot anal-ysis for Ubc12. Even in the presence of molar-excess freeUbc12, SCCRO5 preferentially bound to Ubc12�Nedd8thioester (Fig. 1B; compare lanes 1 and 3). Moreover, theresults of GST pull-down assays showed that, as withSCCRO, critical residues involved in binding of SCCRO5to Cul1-ROC1 (lanes 3 and 4) and the N-terminal region(lane 5) were not involved in binding to Ubc12�Nedd8(Fig. 1C). These findings confirm that, in its interactionswith Ubc12, SCCRO5 maintains reaction processivityparadigms.

SCCRO5 promotes cullin neddylationStructural and biochemical studies have shown that

SCCRO promotes cullin neddylation by promoting com-plex assembly, nuclear translocation of neddylation E3components, enhanced E2 recruitment, and structural ori-entation to optimize the efficiency of the transfer of Nedd8

Table 1. Results from GST pull-downs:SCCRO5 interacts with components of theneddylation pathway

SCCRO5 Mutants Cul1-ROC1 CAND1 Ubc12

WT þ þ þD1–10 þ þ þD1–46 þ þ þD189–237 � � �A219R � � þD195N � � þD225N � � þE226A � � þA219R/D225N � � þD195N/A219R/D225N � � þNOTE: The table shows a summary of results frompull-downassaysusingGST-SCCRO5andselectedmutants to recom-binant Cul1-ROC1, Ubc12, and CAND1, which confirmedthat binding to Cul1-ROC1 and CAND1 requires an intactPONY domain but not the NLS domain. In contrast, bindingto Ubc12 was only lost with deletion of the C-terminus(SCCRO5_D189–237).






from Ubc12 to cullin (42, 43). To determine whetherSCCRO5 also promotes cullin neddylation, we performedin vitro neddylation reactions containing Nedd8, recom-binant APPBP1/Uba3 (E1), Ubc12 (E2), ATP, and whole-cell lysate from HeLa cells (as a source of Cullin-ROCsubstrates), with and without SCCRO5. Western blottingof reaction products for cullins showed a dose-dependent

increase in neddylation of Cul1, Cul2, and Cul3 with theaddition of SCCRO5 (Fig. 2A). A time-course in vitroneddylation reaction showed that SCCRO5 enhanced therate of cullin neddylation (Fig. 2B). To determine thedomains required for the observed effects on neddylation,we supplemented in vitro reactions with SCCRO5 orselected SCCRO5 mutants. PONY domain mutants

Figure 1. SCCRO5 interacts withcomponents of the neddylationpathway. A, Western blot analysisof the products of GST-SCCRO,GST-SCCRO5, and selected GST-SCCRO5 mutants after pull-downassays from HeLa lysates probedwith the indicated antibodies,which shows that, like SCCRO,SCCRO5 binds to CAND1, Cul1,Cul2, Cul3, and ROC1. Mutationsin the PONY domain(SCCRO5_D225N andSCCRO5_E226A), but not the N-terminal NLS (SCCRO5_D1–46),result in loss of binding. Thedividing line between lane 1 andlane 2 indicates the position ofomitted lanes from the same gel. B,Western blot analysis of Ubc12after GST and GST-SCCRO5 pull-down assays on products from athioester reaction (lane 1), whichshows preferential binding toUbc12�Nedd8, even in thepresence of excess free Ubc12. C,Western blot analysis of Ubc12after pull-downassaysonproductsof thioester reaction, which showsthat GST-SCCRO5 and PONY andNLS mutants bind equally toUbc12�Nedd8.

Figure 2. SCCRO5 augments cullin neddylation. A, Western blot analysis of the indicated cullins on products of an in vitro neddylation reaction using HeLalysates as a source for Cullin-ROC1 complexes supplemented with a gradient of SCCRO5, which shows a dose-dependent increase in neddylation of Cul1,Cul2, and Cul3 with the addition of recombinant SCCRO5. B, Western blot analysis of Cul1 on products of an in vitro neddylation reaction with (lanes 5–8) orwithout (lanes 1–4) the addition of SCCRO5, which shows enhanced efficiency of Cul1 neddylation by SCCRO5. C,Western blot analysis of Cul3 on productsof an in vitro neddylation reaction with concentration gradients of SCCRO5, SCCRO5_D1–46, SCCRO5_D225N, or SCCRO5_E226A (quantities in pmol),which shows a dose-dependent increase in neddylation of Cul3 with SCCRO5 and SCCRO5_D1–46 but not with SCCRO5_D225N or SCCRO5_E226A.

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(SCCRO5_D225N and SCCRO5_E226A) failed to aug-ment Cul3 neddylation beyond basal levels, whereas N-terminus deletion mutants (SCCRO5_D1–46) enhancedCul3 neddylation to levels similar to those observed inreactions supplemented with wild-type SCCRO5 (Fig.2C). Combined, these findings suggest that, as withSCCRO (17), the effect of SCCRO5 on cullin neddylationin vitro requires its interaction with neddylation E3 com-ponents through its PONY domain but not its NLSdomain.

SCCRO5 promotes cell proliferation and anchorage-independent cell growthWith SCCRO50s function in cullin neddylation estab-

lished, we next asked whether this activity is required fortransformation. To determine this, we transfected NIH-3T3cells with pBABE-SCCRO5, selected pBABE-SCCRO5mutants or empty vector, and developed two stably expres-sing clones for each construct by use of puromycin selec-tion. Equal expression of SCCRO5 and SCCRO5mutants inselected clones was confirmed by qRT-PCR and Westernblot analysis (Supplementary Fig. S2A and S2B). The resultsof MTS assays showed that the proliferation rate was sig-nificantly higher in SCCRO5-expressing clones than inSCCRO5_D195N/A219R/D225N, SCCRO5_D1–10, orempty vector transfected NIH-3T3 cells (Fig. 3A). NIH-3T3 clones were then subjected to soft agar colony forma-tion assay to assess transforming ability, which showed thatSCCRO5-transfected clones had significantly higheranchorage-independent growth, compared withSCCRO5_D195N/A219R/D225N, SCCRO5_D1–10, orempty vector transfected cells (Fig. 3B). Combined, thesefindings suggest that SCCRO50s oncogenic activity requiresits neddylation-promoting activity as well as its compart-mentalization to the nucleus.

SCCRO5 overexpression is associated with anoncogene addiction phenotypeConsistent with the concept of oncogene addiction, we

previously reported that cancer cell lines with amplificationand high levels of SCCRO expression are more susceptibleto apoptosis with RNAi knockdown of SCCRO, comparedwith cell lines with normal copy numbers and low levels ofSCCRO expression (16, 44). To validate the role thatSCCRO5 plays in cancer pathogenesis, we sought to deter-mine whether an oncogene addiction to SCCRO5 waspresent in cancer cell lines. Results of qRT-PCR andWesternblot analysis showed a wide range of SCCRO5 expression ina panel of head and neck cancer cell lines, with the highestlevels of expression in SCC15 andMDA1483 and the lowestlevel in MDA1386 (Fig. 3C). Representative cell lines withlow (MDA1386) and high (MDA1483) endogenousSCCRO5 expressionwere transfected with two independentanti-SCCRO5 shRNA constructs and with anti-lacZ shRNAas a control. Western blot analysis, after transfection withanti-SCCRO5 shRNA constructs, showed efficient knock-down of SCCRO5 levels, compared with those in the anti-lacZ shRNA-transfected cells (Fig. 3D). SCCRO levels were

not affected by transfection with any of the shRNAconstructs, which confirmed specificity. Results of MTSassays showed a more significant decrease in the viabilityof MDA1483 cells, compared with MDA1386 cells, afterSCCRO5 knockdown (Fig. 3E and F). These results indicatethe presence of an oncogene addiction phenotype andvalidate the role of SCCRO5 in cancer pathogenesis.

SCCRO5 overexpression is associated with anaggressive clinical course

To establish the clinical significance of SCCRO5 over-expression, we sought to identify mutations and to deter-mine the prevalence of SCCRO5 overexpression, as well asthe association of mutations and overexpression with out-come in a panel of randomly selected human tumors.Sequenome-based mutational screening of all cases did notidentify any mutations in SCCRO5 (data not shown). Toinvestigate the presence and frequency of overexpression,SCCRO5 mRNA levels were determined by qRT-PCR incancer tissue and matched histologically normal tissue foreach case. The highest prevalence of SCCRO5 mRNA over-expression was observed in cancer types known to harbor11q22 amplification, including gliomas (4/10; 40%), lungSCCs (14/30; 47%), and oral cavity SCCs (16/40; 40%).SCCRO5mRNA overexpression was less common in cancertypes with a lower prevalence of 11q22 amplification,including lung adenocarcinomas (2/27; 7%), lung neuro-endocrine carcinomas (6/54; 11%), ovarian carcinomas (1/40; 3%), and thyroid carcinomas (5/56; 9%;Fig. 4A; refs. 26,27, 30). Moreover, in the cohort of lung SCCs and oralcavity SCCs, SCCRO5 mRNA levels correlated withSCCRO5 protein levels (Fig. 4B).

To validate the prevalence of SCCRO5 dysregulation, weanalyzed interim results fromTCGAprojects,which showedthat the highest prevalence of SCCRO5 dysregulation(amplification and/or overexpression) was in head andneck SCCs (11.6%), followed by ovarian serous cystade-nocarcinomas (3%; copy number change only; expressiondata not available), cervical carcinomas (8.7%; copy num-ber changeonly; expressiondata not available), andbladderurothelial carcinomas (7%), with a significant correlationbetween copy number and mRNA expression in head andneck SCCs (Supplementary Fig. S3). The difference in theprevalence of dysregulation between our tumor cohort andthat in the TCGA likely represents differences in the sensi-tivity of the analytic techniques used to assess SCCRO5mRNA levels (cDNA array vs. qRT-PCR) and in the type ofcontrol tissue used for analysis (matched normal tissue vs.blood).

To determine clinical relevance, we performed a post hocanalysis to determine the association between SCCRO5overexpression and disease-free survival. We limited surviv-al comparisons to patients undergoing treatment for headand neck or lung SCC as these datasets had: (i) reasonablepower (more than 10 cases with SCCRO5 over expression),(ii) had sufficient events (only 2 recurrences were observedin the thyroid cancer cohorts; Supplementary Table 3), and(iii) had uniform histology (thyroid cohort included






Figure 3. Transgenic expression ofSCCRO5promotes proliferation and anchorage-independent growth. A, graph showing results fromanMTSassay onNIH-3T3 cells stably expressing SCCRO5 and indicatedmutants, which shows increased proliferation in cells expressing SCCRO5 but not NLS (SCCRO5_D1–10)or PONY (SCCRO5_D195N/A219R/D225N) domain mutants. B, results from a soft agar assay, which shows increased colony formation in NIH-3T3 cellsstably transfectedwithSCCRO5, comparedwith that in cells transfectedwith empty vector andSCCRO5NLSandPONYdomainmutants (bars represent themean� SD number of colonies per well of 6-well plates; P < 0.001). C, Western blot analysis, which shows SCCRO5 protein levels and correspondingmRNAlevels, on the basis of real-timePCR, in headand neck cancer cell lines. D,Western blot analysis of SCCRO5,withGAPDHas a loading control, on lysates fromMDA1386 (low endogenous SCCRO5) and MDA1483 (high endogenous SCCRO5), before and after transfection of two independent shRNA constructsagainst SCCRO5 or lacZ control, which shows efficient and specific knockdown of SCCRO5. E and F, graphs fromMTS assays showing amore pronounceddecrease in the viability of MDA1483 cells, compared with MDA1386 cells, with knockdown of SCCRO5.

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papillary, follicular and H€urthle cell carcinomas and lungneuroendocrine carcinomas included large cell carcinomas,small cell carcinomas, and carcinoid tumors; Supplemen-tary Table S4). All patients in the study cohort underwentuniform treatment that included primary surgery with orwithout adjuvant treatment (on the basis of the extent ofdisease), in accordance with established institutional pro-tocols. Clinicopathologic parameters, including age, sex,tumor node metastasis stage, and tobacco use, were notsignificantly different according to SCCRO5 expressionstatus for either cancer cohort (Supplementary TablesS1 and S2). We found that SCCRO5 overexpression(SCCRO5þ), which is defined in theMaterials andMethodssection, compared with normal expression of SCCRO5(SCCRO5�), negatively correlated with disease-free surviv-al for both head and neck SCCs and lung SCCs (P ¼0.05; Fig. 4C and D). Interestingly, time to survival analysisfor lung neuroendocrine carcinomas also shows worseoutcome in tumors with SCCRO5 over expression (Supple-mentary Fig. S4; P ¼ 0.05). However, the differences inoutcome in the lung neuroendocrine carcinoma need to beconsidered with caution given the limited sample size andvariable behavior of histologic subtypes of neuroendocrine

carcinomas. Moreover, limitation of sample size and num-ber of events in the study cohorts do not support multivar-iate analyses. Nonetheless, the correlation of overexpres-sion with an aggressive clinical course supports SCCRO5’sclinical relevance.

DiscussionSCCRO and its paralogs are commonly dysregulated in

human cancers, with aberrations of one or more paralogspresent in 50% of head and neck cancers, in 70% of lungSCCs, in 55% of ovarian serous cystadenocarcinomas, in34% of cervical and endometrial carcinomas, in 26% oflung adenocarcinomas, and in 15% of glioblastomas, onthe basis of analysis of interim data from respective TCGAprojects. Of the SCCRO paralogs, SCCRO and SCCRO5 arethe most commonly dysregulated and have a tendencytoward mutual exclusivity, which suggests that they actindependently in oncogenesis. It is well established thatoverexpression of SCCRO promotes its function as anoncogene (16, 18). By showing that overexpression ofSCCRO5 has transforming activity and by associating itwith an aggressive clinical course in human cancers, ourdata support the position that SCCRO5may function as an

Oral SCC

% S

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Lung Lung Oral Ovarian Thyroid

52

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Figure 4. SCCRO5 overexpression is common in human tumors and correlates with outcomes. A, box plot showing fold increase in SCCRO5 mRNAexpression, analyzed by qRT-PCR, in 10 gliomas, 30 lung SCCs, 27 lung adenocarcinomas (AC), 54 lung neuroendocrine (NEC) tumors, 40 oral SCCs, 40ovarian carcinomas, and 56 thyroid carcinomas, comparedwith matched normal tissues (boxes represent the lower through the upper quartile; themedian isshown as a horizontal line; whiskers represent minimum and maximum levels). The percentage of cases with overexpression is given below each plot. B,Western blot analysis showing SCCRO5 protein expression in representative head and neck and lung SCCs (T) and matched normal (N) samples. Thecorresponding fold change in mRNA levels, determined by real-time PCR, is noted below. C, Kaplan–Meier survival curves from post-hoc analysis showingrecurrence-free survival based onSCCRO5mRNA expression status in primary oral SCCs. D, Kaplan–Meier survival curves showing recurrence-free survivalfrom post hoc analysis based on SCCRO5 mRNA expression status in primary lung SCCs.






oncogene as well (20). Its role in cancer pathogenesis isfurther validated by our findings, which indicate the pres-ence of an oncogene addiction phenotype in cancer celllines that have high endogenous levels of SCCRO5expression.

Structure–function analyses show that, as with SCCRO,SCCRO50s conserved PONY domain is required for bindingto neddylation components, as well as for its neddylationand transforming activities (16, 17, 41, 42). Of interest,SCCRO50s N-terminal NLS was not required for neddyla-tion-promoting activity in vitro, which is similar to the casefor the UBA domain in SCCRO (41). Given that SCCRO’ssubcellular localization is important to its neddylationactivity, the direct or indirect association of the N-terminalmotifs with subcellular localization of the SCCRO paralogssuggests that they may regulate neddylation activity in vivo(43–45). Consistent with this finding, SCCRO’s UBA isinvolved in monoubiquitination by Nedd4-1, which pro-motes nuclear export, thereby inhibiting its neddylationactivity (unpublished data; refs. 43, 45). Similarly, themyristoylation sequence localizes SCCRO3 to the mem-brane, which affects its neddylation promoting activity(unpublished data; ref. 46). Our finding showing that theNLS is required for SCCRO50s transforming activity suggeststhat, as with the UBA, the NLS may modulate neddylationactivity in vivo. However, these findings raise a questionabout SCCRO50s role in neddylation. Our previous workshowed that SCCRO promotes nuclear translocation ofCullin-ROC1, which is required for neddylation in vivo.Given that SCCRO5 is present almost exclusively in thenucleus, it is unclear how cytoplasmic Cullin-ROC1 com-plexes are delivered to the nucleus to allow for interactionwith SCCRO5. Several possibilities exist, including coop-erative activity between other SCCRO paralogs and NLS-containing variants, presence of alternativemechanisms fornuclear translocationofCullin-ROC1 complexes, and selec-tive activity on cullins situated in the nucleus.

The functional effects of neddylation are mediated bydownstream targets that CRLs activate. The recent publica-tion byMonda and colleagues showing that SCCRO and itsparalogs have overlapping affinity to E2s (Ube2M andUbe2F) and cullins (42) suggests that theymay have redun-dant effects on CRL activity in vivo. This idea is supported bythedifferential requirements ofSCCRO for viability in yeast,whose genome does not contain other SCCRO paralogs,

and in higher organisms, where paralogs can compensatefor SCCRO loss (unpublished data; refs. 40, 43). AsSCCRO5 promotes neddylation of all cullins (with thepossible exception of Cul4A), it putatively can regulate theubiquitination of a myriad of proteins, resulting in diversecellular effects (42). Guo and colleagues suggest thatSCCRO5 may be involved in regulating ubiquitination ofthe proteins involved in DNA damage repair (20). Consis-tent with this suggestion, SCCRO5was found to be part of apanel of seven genes whose expression score predicts radi-ation response in patients with cervical cancer (47). Furtherwork is required to define the CRLs and protein targets thatare dependent on SCCRO5 in the DNA damage response,cellular activities, and oncogenesis.

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

Authors' ContributionsConception and design: C.C. Bommelje, S.Y. Ramanathan, B. SinghDevelopment of methodology: C.C. Bommelje, V.B. Weeda, G. Huang,S. Bains, B. SinghAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): S. Bains, M. Shaha, R. Ghoissein, B. SinghAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): C.C. Bommelje, V.B. Weeda, K. Shah,E. Buss, M. Gonen, S.Y. Ramanathan, B. SinghWriting, review, and/or revision of themanuscript:C.C. Bommelje, V.B.Weeda, G. Huang, M. Gonen, S.Y. Ramanathan, B. SinghAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): C.C. Bommelje, V.B. Weeda,K. Shah, E. Buss, M. Shaha, B. SinghStudy supervision: S.Y. Ramanathan, B. Singh

AcknowledgmentsThe authors thank Dr. Nicholas Socci, for help with analysis of gene

expression data, Dr. Adriana Heguy, for help with mutation analysis, andDavid Sewell for excellent editorial assistance.

Grant SupportThis work was supported, in part, by grants from Catherine van Tussen-

broek Fonds and the foundation Vrijvrouwe van Renswoude (to C.C.Bommelje); Fulbright, The Dutch Cancer Society, and the Prins BernhardCulture Foundation (to V.B.Weeda); and the Clinical Innovator Award fromthe Flight Attendants Medical Research Institute (to B. Singh).

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 May 7, 2013; revised October 21, 2013; accepted October 28,2013; published OnlineFirst November 5, 2013.

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2014;20:372-381. Published OnlineFirst November 5, 2013.Clin Cancer Res Claire C. Bommeljé, Víola B. Weeda, Guochang Huang, et al. Neddylation E3 Activity and Nuclear LocalizationOncogenic Function of SCCRO5/DCUN1D5 Requires Its

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