Arkadia Regulates Tumor Metastasis by Modulation of the TGF-b

Molecular and Cellular Pathobiology

Arkadia Regulates Tumor Metastasis by Modulation of theTGF-b Pathway

Marco A. Briones-Orta1, Laurence Levy3, Chris D. Madsen2, Debipriya Das1, Yigit Erker3,Erik Sahai2, and Caroline S. Hill1

AbstractTGF-b can act as a tumor suppressor at early stages of cancer progression and as a tumor promoter at later

stages. The E3 ubiquitin ligase Arkadia (RNF111) is a critical component of the TGF-b signaling pathway, beingrequired for a subset of responses, those mediated by Smad3–Smad4 complexes. It acts by mediating ligand-induced degradation of Ski and SnoN (SKIL), which are 2 potent transcriptional repressors. Here, we investigatethe role of Arkadia in cancer using model systems to address both potential tumor-suppressive and tumor-promoting roles. Stable reexpression of Arkadia in lung carcinoma NCI-H460 cells, which we show contain ahemizygous nonsense mutation in the Arkadia/RNF111 gene, efficiently restored TGF-b–induced Smad3-dependent transcription, and substantially decreased the ability of these cells to grow in soft agar in vitro.However, it had no effect on tumor growth in vivo in mouse models. Moreover, loss of Arkadia in cancer cell linesand human tumors is rare, arguing against a prominent tumor-suppressive role. In contrast, we have uncovered apotent tumor-promoting function for Arkadia. Using 3 different cancer cell lines whose tumorigenic propertiesare driven by TGF-b signaling, we show that loss of Arkadia function, either by overexpression of dominantnegative Arkadia or by siRNA-induced knockdown, substantially inhibited lung colonization in tail vein injectionexperiments in immunodeficient mice. Our findings indicate that Arkadia is not critical for regulating tumorgrowth per se, but is required for the early stages of cancer cell colonization at the sites of metastasis. Cancer Res;73(6); 1800–10. �2012 AACR.

IntroductionThe TGF-b signaling pathway has a dual role in tumorigen-

esis (1, 2). It can function as a tumor suppressor by inhibitingcell growth, inducing apoptosis, promoting differentiation, aswell as acting on the stroma to suppress inflammation and theproduction of mitogens (2). Conversely, TGF-b can supporttumor development by inhibiting immune responses and byregulating processes necessary for the colonization of distanttissues, such as angiogenesis, cancer cell migration, and inva-sion (2, 3). At later stages of tumorigenesis, the TGF-b signal is amajor contributor to the transcriptional regulation of genesnecessary for cancer cell migration and invasion, as well asmicroenvironment remodeling (3, 4).

TGF-b binds and activates complexes of serine/threoninekinase receptors comprising TbRII and TbRI (ALK5) at the cellsurface. This leads to phosphorylation of receptor-regulatedSmads, of which the best understood are Smad2 and Smad3 (5).These activated Smads complex with Smad4 and accumulatein the nucleus where they directly regulate the transcription oftarget genes (6). Ski and SnoN are potent transcriptionalcorepressors that inhibit the transcription of a subset ofTGF-b–responsive genes (7). In the absence of TGF-b, Ski andSnoN bind Smad-binding elements (SBE) in the promoters/enhancers of target genes together with Smad4, forming atranscriptional repressor complex with histone deacetylases tosilence basal transcription (8–15). The particular elementsrecognized by Ski and SnoN are those recognized bySmad3–Smad4 complexes, and also complexes of Smad4 witha splice formof Smad2, Smad2Dexon3 (12). In response toTGF-b, Ski and SnoN are rapidly degraded via the ubiquitin–proteasome pathway (8). This degradation allows the phos-phorylated Smad3/Smad2Dexon3-containing complexes tobind SBEs in the promoters/enhancers of target genes (7, 8, 12).

While several ubiquitin ligases, namely Smurf2 and theanaphase-promoting complex, were originally proposed to beresponsible for regulating Ski and SnoN levels, (16–18), severalyears ago, we and others established that the E3 ubiquitinligase Arkadia (encoded by the RNF111 gene) was requiredfor TGF-b–induced SnoN and Ski degradation (12, 19, 20). Weshowed that in response to TGF-b, Arkadia interacts withSnoN and phosphorylated Smad2/Smad3, and this is necessary

Authors' Affiliations: 1Laboratory of Developmental Signalling and2Tumour Cell Biology Laboratory, Cancer Research UK London ResearchInstitute, Lincoln's Inn Fields Laboratories, London, United Kingdom; and3INSERM UMR S 938, Hopital St-Antoine, Paris, France

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

M.A. Briones-Orta and L. Levy contributed equally to this work.

Corresponding Author: Caroline S. Hill, Cancer Research UK LondonResearch Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's InnFields, London WC2A 3LY, United Kingdom. Phone: 44-20-7269-2941;Fax: 44-20-7269-3093; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-1916

�2012 American Association for Cancer Research.

CancerResearch

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for SnoN degradation (12). As a result, Arkadia is essential for asubset of TGF-b–induced transcriptional responses, thosemediated via Smad3/Smad2Dexon3.Like the TGF-b pathway itself, SnoN also plays a dual role in

cancer (21). Because Arkadia is a critical modulator of Ski andSnoN levels, deregulation of Arkadia function might be pre-dicted to influence tumor development and/or dissemination.We previously described a lung carcinoma cell line, NCI-H460(originally wrongly classified as the esophageal carcinoma cellline SEG-1; ref. 22) that lacked functional Arkadia, and hencedid not exhibit TGF-b–induced SnoN degradation, and wasdeficient in Smad3-dependent transcriptional responses (12).We hypothesized that Arkadia might be a novel tumor sup-pressor, with specific loss of the Smad3/Smad2Dexon3-depen-dent arm of the TGF-b pathway through loss of Arkadiaallowing cells to evade the tumor suppressive effects ofTGF-b, while maintaining TGF-b's tumor-promoting activities(12). Consistent with this, Arkadiaþ/� heterozygous mice aremore susceptible to developing tumors in a colorectal tumormodel after exposure to carcinogen, compared with wild-typemice (23). However, there was no evidence that the other alleleof Arkadiawas lost in these tumors, as might be expected for aclassical tumor suppressor. Moreover, although a number ofmutations in Arkadiawere found in primary colorectal tumorsfrom human patients, only one of them clearly resulted in anonfunctional protein (23). An alternative possibility to theidea of the 2 arms of the TGF-b pathway having differentfunctions in cancer is that the pathway as a whole may haveboth tumor-suppressive and tumor-promoting functions, butwhich predominates depends on the context. If this were thecase, thenArkadia, like SnoNand Smad4 (2, 21, 24, 25),might beexpected to exhibit a dual role in cancer.Here, we dissect the role of Arkadia in tumorigenesis, using 2

model systems designed to examine both potential tumor-suppressor and tumor-promoting activities. Our data do notsupport a prominent tumor-suppressive role. Instead, we showthat Arkadia is required for metastasis, probably at the level ofextravasation.

Materials and MethodsPlasmidsThe following plasmids were previously described: HA-

SnoN, HA-Smad3, FLAG-Arkadia, CAGA12-Luciferase andTK-Renilla (12), and HA-Ski (9). To make the stable cell lines,wild-type Arkadia and Arkadia C937A (12) were subcloned intothe 3 � Flag pBICEP-CMV2 vector (Sigma). FLAG-Arkadia 1–440 was generated by introducing a stop codon at amino acid441 in the FLAG-Arkadia construct (12).

Cell lines and cell treatmentsHaCaT, MDA-MB-231, 293T, B16, CACO-2, and HT-29

cells were cultured in Dulbecco's Modified Eagle's Medium(DMEM) containing 2 mmol/L glutamine and 10% fetal calfserum (FCS). NCI-H460 and COLO-205 cells were cultured inRPMI-1640 supplemented with 2 mmol/L glutamine and 10%FCS.MTLN3E cells were cultured in AlphaMinimum EssentialMedium containing 2 mmol/L glutamine and 10% FCS. HT-55cells were cultured in a 1:1 mix of DMEM and RPMI containing

2 mmol/L glutamine and 10% FCS. Primary human umbilicalvein endothelial cells (HUVEC; CC-2517, Lonza,) were grownin collagen-precoated flasks (BD Biocoat) in EGM-2 Bullet-Kit media with supplements (CC-3162, Lonza) at 5% CO2.Stable cell lines were obtained by transfecting NCI-H460 orMDA-MB-231 cells with either pBICEP-CMV2-FLAG-Arkadiaor pBICEP-CMV2-FLAG-Arkadia-C937A, respectively, andselecting clones with G418 (Gibco). MDA-MB-231 cells expres-sing GFP or mCherry were generated by transfecting pEGFP-N1 and pCherry-N1 plasmids (Clontech) into MDA-MB-231cells, selecting with 500 mg/mL G418 (Gibco) and by fluores-cence-activated cell sorting (FACS). The B16 cells were labeledwith Cherry or enhanced GFP (EGFP) in the same way. TheMTLN3E cells were labeled using lentivirus containing eithermyr-GFP or myr-Cherry and were FACS sorted. The MDA-MB-231 Arkadia C937A clones were labeled with a membrane-associated GFP (GFP-CAAX) using the lentivirus system andwere selected with blasticidin.

Cells were stimulated with 2 ng/mL of TGF-b (Peprotech ECLtd.) for the specified times. The ALK5 inhibitor SB-431542(Tocris) was used at 10 mmol/L. For proteasome inhibition,cells were treated with 50 mmol/L ofMG132 (Tocris) for 4 hours.

Immunoprecipitations, Western blot analyses,antibodies, and luciferase assays

Whole-cell extracts were prepared either using radioim-munoprecipitation assay buffer (12) or as described (26).Western blot analyses were conducted following standardprocedures. For transmembrane prostate androgen-induced(TMEPAI) blots, extracts were treated with PNGase asdescribed (27). Antibodies are listed in the SupplementaryMethods. Immunoprecipitations and luciferase assays wereas described (12). For luciferase assays, TGF-b induction wasfor 8 hours.

Xenografts and tail vein injection assaysFor xenografts, cells were trypsinized and 5� 106 cells were

resuspended in 100 mL PBS and injected subcutaneously intothe right and left flanks of approximately 6-week-old female,Balb/c nu/nu mice (4 mice per condition). Tumor growthwas measured with external calipers every 2 or 3 days for amaximum of 6 weeks.

For tail vein injections with unlabeled cells, the cells weretrypsinized and 1 � 106 cells were injected into the tail vein ofBalb/c nu/nu mice (4 or 5 mice per condition). Lungs wereremoved at 20 or 30 days postinjection and fixed in neutral-buffered formalin. Three sections corresponding to differentlevels of the lungs were obtained, which were stained withhematoxylin and eosin. The number of tumors in each slidewas determined by a pathologist. For the tail vein injectionswith fluorescent cells, 1 � 106 cells of a 1:1 mixture GFP- andmCherry-expressing cells (where either both were controls orone an experimental sample and the other a control) wereinjected into the tail vein of 6-week-old female, ICRF nu/numice (MDA-MB-231 and B16) or Balb/c nu/nu (MTLN3E).Additional controls for the ratio of mCherry and GFP cellswere conducted by seeding 10 mL of the cell suspension into aglass-bottom dish coated with poly-lysine (Sigma); after 2

The Role of Arkadia in Tumorigenesis

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hours, cells were fixed in 4% paraformaldehyde and imagedwith a Zeiss LSM 780 confocal microscope using a Plan-Neofluar 10 � 0.3 objective. Forty-eight hours after injection,the mice were culled, lungs extracted, and representativeimages of the tumor distribution were analyzed by confocalmicroscopy. The area occupied by fluorescent tumor cells wascalculated using Volocity software (PerkinElmer) and the GFP:mCherry ratio calculated using the total area of the green andthe red cells and was normalized using the GFP:mCherry ratioobserved in the control plates.

Cell line origin and authenticationThe parental MDA-MB-231 cells were obtained from the

ECACC/HPA culture collection and were banked at CancerResearch UK Cell Services. The MDA-MB-231 cells expressingArkadia C937A were derived from these cells. These cell lineswere authenticated using the short tandem repeat profilingand isoenzyme analysis. The NCI-H460 cells were obtainedfrom the American Type Culture Collection and were bankedat Cancer Research UK Cell Services. The Arkadia-expressingclones were derived from these cells. The MTLN3E cells wereobtained from John Condeelis (Albert Einstein College ofMedicine, Bronx, NY). We have proven that they are syngeneicwith Fisher 344 rats in agreement with their published history(28). The B16 cells were obtained from Cancer Research UKCell Services. We have proven that they are syngeneic withC57BL/6 mice and produce pigment consistent with theirpublished history (29). All cell lines were frequently tested formycoplasma and were negative.

For genomic sequencing, RNA-seq, quantitative PCR(qPCR), soft agar, and cell-cycle analyses, cell spreading andcell adhesion assays, and lists of primers, antibodies, andsiRNAs, see Supplementary Methods.

ResultsNCI-H460 cells express a truncated version of Arkadiathat is catalytically inactive and are deficient in TGF-b–induced Smad3-dependent transcription

To investigate whether Arkadia might be a novel tumorsuppressor gene, we used a lung cancer cell line, NCI-H460, thatwe previously showed had lost expression of full-length Arka-dia (12). Although NCI-H460 cells express Arkadia mRNA(Fig. 1A), they do not express full-length Arkadia protein.However, a faster migrating band was observed in extractsfrom these cells, that was absent in HaCaT extracts, suggestingthat they might express a truncated version of Arkadia (Fig.1B). This was confirmed using an siRNA SMARTpool againsthuman Arkadia (Supplementary Fig. S1A). Genomic sequenc-ing of the Arkadia/RNF111 gene in NCI-H460 cells and HaCaTcells revealed a hemizygous single nucleotide deletion in theArkadia/RNF111 gene in the NCI-H460 cells that creates a stopcodon at amino acid 441 (Supplementary Fig. S1B). Thus, NCI-H460 cells express an Arkadia protein lacking the C-terminalcatalytic RING domain.

To investigate the biological relevance of this mutation,we tested the ability of this truncated Arkadia to interactwith Ski, SnoN, and Smad3 (12, 19, 20). In immunoprecipita-tions, Arkadia 1–440 did not interact with SnoN, and only

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very weakly interacted with Ski. However, it still retained itsability to interact with Smad3 (Fig. 1C). To assay theactivity of Arkadia 1–440, we compared its ability to rescueCAGA12-luciferase activity in the NCI-H460 cells with that ofwild-type Arkadia and a dominant negative Arkadia, whichhas a point mutation in the RING domain (C937A; ref. 12).Arkadia 1–440 was substantially less active than wild-typeArkadia, showing only residual activity comparable with thatof Arkadia C937A (Fig. 1D). In 293T cells, which containendogenous Arkadia, Arkadia 1–440 had very little activity,compared with wild-type Arkadia (Fig. 1D). However, unlikeArkadia C937A, it did not exhibit dominant negative activity,probably as a result of losing its ability to interact with Skiand SnoN.

Reintroduction of functional Arkadia in NCI-H460 cellsrestores specific TGF-b responses and inhibitsanchorage-independent growth, but has no effect ontumor growth in vivo

To determine whether loss of Arkadia in NCI-H460s might beresponsible for their tumorigenicphenotype,we investigated theeffect of restoring Arkadia function. We generated 2 cell lines(WT Ark 1 and WT Ark 11) that stably express FLAG-taggedArkadia (Fig. 2A). Treatment of cells with the proteosomeinhibitor MG132 increased protein levels of overexpressedArkadia in these NCI-H460 cell lines and also endogenousArkadia in control MDA-MB-231 cells, indicating that Arkadiais normally unstable, probably due to autoubiquitination (Fig.2A and Fig. 3A; ref. 12). In both WT Ark 1 and WT Ark 11 cells,

Figure 2. Reintroduction of Arkadiain NCI-H460 cells restoresTGF-b-dependent Ski/SnoNdegradation, transcriptionalresponses, and inhibits anchorageindependent growth, but has noeffect on their tumorigenicproperties in vivo. A, Western blotanalysis showing Arkadia levels in 2NCI-H460–derived cell linesexpressing FLAG-tagged Arkadia.Cells were treated for 4 hours �MG132. Actin is a loading control. B,whole-cell extracts fromcells treated� TGF-b Western blotted using theantibodies indicated. C, luciferaseassays were conducted as in Fig. 1.D, whole-cell extracts prepared fromthe cell lines indicated and Westernblotted using antibodies shown.E, NCI-H460 parental cells or theclones expressing FLAG-taggedArkadia were grown in soft agar for10 days. The numbers of colonieswere quantified and are shownin the bar graph (right). F, thecell lines shown were injectedsubcutaneously in both flanks ofBalbc nu/nu mice. The data arethe averages of the tumor volumesfor 4 mice (8 tumors in total) percell line.

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reintroduction of Arkadia restored TGF-b–induced SnoN andSki degradation (Fig. 2B) and Smad3-dependent transcription(Fig. 2C). Induction of the TGF-b–dependent gene, transmem-brane prostate androgen-induced RNA (TMEPAI; ref. 30), wasalso recovered after reintroduction of Arkadia (Fig. 2D).

Tumor-suppressive effects of the TGF-b pathway arethought to be at least partly mediated by effects on cell growth(2). However, we found no effect of Arkadia reintroduction intoNCI-H460 cells on the rate of cell proliferation in vitro in thepresence or absence of TGF-b (Supplementary Fig. S2). Wenext determined whether restoring Arkadia altered the trans-formed phenotype of NCI-H460 cells in vitro by conductingsoft-agar assays, which measure anchorage-independentgrowth. NCI-H460 cells formed colonies in soft agar (Fig.2E). This growth depends on autocrine TGF-b signaling, as itwas abolished by SB-431542, a specific inhibitor of the TGF-btype I receptor (data not shown; ref. 31). Exogenous TGF-bcould not further promote colony formation, suggesting thatthe autocrine TGF-b activity in these cells is sufficient. Becausethe cells lack Arkadia activity, this growth is Arkadia indepen-

dent. Strikingly, neither of the NCI-H460 clones in whichArkadia has been reexpressed, were able to form a substantialnumber of colonies (Fig. 2E). Thus, anchorage-independentgrowth of NCI-H460 cells is inhibited by Arkadia.

We went on to test whether restoring Arkadia activity inNCI-H460 cells had any effect on their tumorigenic propertiesin vivo. In xenograft assays in immunodeficient mice, primarytumor growth was not affected by Arkadia (Fig 2F). We alsoinvestigatedwhether the ability of cells to colonize and developtumors in distant tissues was affected by Arkadia activity byconducting tail vein injections in immunodeficient mice. How-ever, no significant differences in the number of lung tumorsderived from parental cells or from the clones expressing wild-type Arkadia were observed, either at 20 or 30 days postinjec-tion (Supplementary Table S1). We conclude, therefore, thatalthough restoration of Arkadia activity in NCI-H460 cells atleast partly reversed the transformed phenotype in vitro, it doesnot affect tumorigenicity in vivo. Thismight be explained by thegain of additional driving mutations after acquisition of theArkadia mutation.

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Figure 3. Expression of ArkadiaC937A abolishes Arkadia functionin MDA-MB-231 cells and alterstheir adhesion and spreadingproperties. A–D, NCI-H460 cells,parental MDA-MB-231 cells, and astable MDA-MB-231 cell lineexpressing FLAG-tagged ArkadiaC937A (clone 1) were treated �MG132 for 4 hours (A) or with TGF-b for the times indicated (B–D).Whole-cell extracts were Westernblotted using the antibodiesindicated (A, B, and D). C, levels ofmRNA for the genes shown wereanalyzed either by RNA-seq or byqPCR, which was normalized toglyceraldehyde-3-phosphatedehydrogenase (GAPDH). E, GFP-labeled parental MDA-MB-231cells and Arkadia C937A cells(clone 1) were mixed with an equalnumber of parental cellsexpressing mCherry, seeded on amonolayer of HUVEC cells, andincubated for 1 hour, after whichthey were washed, fixed, andcounted. The mean ratio of GFP:mCherry cells andSDs from6wellsis plotted. F, GFP-labeled parentalMDA-MB-231 cells or ArkadiaC937A cells (clone 1) were seededon a layer of HUVEC cells, andmovies were recorded. Cellspreading was measured byquantification of changes in thearea of the cells over time using theImaris software. The graphs showthe means of the average cell areafor 4 independent wells for eachcell type with SEs. Similar resultswere obtained in 3 independentexperiments.

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Mutations in Arkadia in human cancer are rareTo obtain a more comprehensive view of Arkadia mutation

frequency in human cancer, we analyzed Arkadia protein levelsand TGF-b–induced SnoN degradation in a number of cancercell lines of different tissue origin, focusing particularly onthose identified from the Sanger Centre Cancer GenomeProject (CGP) LOH and copy number analysis that displayedLOH at the 15q22.1 locus containing the Arkadia/RNF111gene. We were unable to find another cancer cell line in whichArkadia was deleted or that contained a loss-of-functionmutation in Arkadia (data not shown). Interestingly, weobserved a direct correlation between loss of TGF-b–inducedSnoN degradation and loss of Smad4 activity. Examples are theCACO-2 cell line, which contains a point mutation in Smad4that renders it unable to form complexes with R-Smads (32),and the Colo-205 and HT-29 cell lines that are deleted forSmad4 (Supplementary Fig. S3). Thus mutation or deletion ofSmad4, which is common in certain tumors, has the sameinhibitory effect on SnoN degradation as loss of Arkadia (seeDiscussion).

Inhibition of Arkadia activity inMDA-MB-231 cells alterstheir adherence and ability to spread on endothelial cellsThe evidence presented above does not support the idea that

Arkadia is primarily a tumor suppressor. Moreover, cancer cell

lines that exhibit LOH at the Arkadia locus do not lose oracquire mutations in the other allele, suggesting the possibilitythat Arkadiamight be important formediating TGF-b's tumor-promoting functions (2). To address this, we chose a well-characterized breast cancer cell line MDA-MB-231 thatrequires TGF-b signaling for metastasis (24, 33–35) and inves-tigated how loss of Arkadia activity affected its tumorigenicproperties.

Overexpression of Arkadia C937A acts dominant negativelyto suppress the activity of an endogenous Arkadia (Fig. 1C;ref. 12). We therefore used this construct to inactivate Arkadiain MDA-MB-231 cells (Fig. 3A). Arkadia C937A prevented TGF-b–induced Ski and SnoN degradation in 3 independent clones,(Fig. 3B and Supplementary Fig. S4A). To determine the effectof dominant negative Arkadia on TGF-b–regulated targetgenes at the genomic scale, we conducted RNA-seq at 1 hourand 24 hours after TGF-b stimulation. Supplementary Fig. S5shows the filtered datasets presented as heatmaps and Sup-plementary Fig. S6 shows qPCR validations for selected genes.The similarity between the qPCR data and the RNA-seq datagave us confidence in the RNA-seq dataset as a whole. Weidentified 36 genes that are significantly (more than 2-fold) upor downregulated by TGF-b at 1 hour, 103 genes that areupregulated byTGF-b at 24 hours, and 70 genes downregulatedby TGF-b at 24 hours (Supplementary Fig. S5). Consistent with

Table 1. Gene enrichment analysis of Arkadia-dependent TGF-b regulated genes

Networks Totala PNo. genesin data Genes from active datab

Cell adhesion and cell–matrix interaction 213 7.096 � 10�4 8 TGFB1; ADAM-TS14; Collagen V; MMP-8;COL5A1; COL1A2; LAMC2; SPOCK1

Proteolysis, ECM remodeling 85 1.07 � 10�3 5 LRP1; ADAM-TS14; PAI1/Serpine1; MMP-8;SPOCK1

Proteolysis, connective tissue degradation 119 4.69 � 10�3 5 LRP1; ADAM-TS14; PAI1/Serpine1; MMP-8;SPOCK1

Development, EMT 232 5.31 � 10�3 7 AP-1; KRT14; PAI1/Serpine1; Lysyl Oxidase;SNAIL1; PDGF-B; COL1A2

Cytoskeleton and intermediate filaments 81 6.49 � 10�3 4 PPL (periplakin); KRT14; KRT17; KRT7Cardiac development, role of NADPHoxidase and ROS

133 7.47 � 10�3 5 MYH15; SNAIL1; NCF2; Gli-2; ID1

Reproduction, gonadotrophin regulation 199 9.82 � 10�3 6 Ga-specific class A orphan/other GPCRs; AP-1;FosB; JunB; VIPR1

Proliferation 221 1.59 � 10�2 6 AP-1; Ga(i)-specific amine GPCRs; CSF1R;VIPR1; HTR1D; TACSTD2

Cardiac development: TGF-b andBMP signaling

117 2.26 � 10�2 4 MYH15; SNAIL1; Gli-2; ID1

Development, neurogenesis, andsynaptogenesis

180 2.49 � 10�2 5 LRP1; Syntrophin B; KIF17; Synaptotagmin II;Synaptotagmin

NOTE: A MetaCore analysis of genes that significantly change in their TGF-b regulation (false discovery rate less than 0.05 and foldchange more than 2) between the parental cells and Arkadia C937A-expressing cells. They are ordered with respect to P value.Abbreviation: ROS, reactive oxygen species.aTotal refers to the number of MetaCore entries associated with a particular network.bIn most cases, these are individual genes. In some cases, however, multiple members of one family have been mapped to the sameMetaCore database entry for example, Lysyl oxidase or AP-1.






our previous data showing that Arkadia is required only forTGF-b responses that are dependent on Smad3/Smad2Dexon3(12), we found that a subset of TGF-b–responsive genes wasstrongly affected by dominant negative Arkadia, whereasother genes were only weakly affected, or not affected at all(Supplementary Fig. S5). Examples of strongly affected genesare the 2 well-characterized TGF-b targets, PAI-1 and TMEPAI(Fig. 3C). This was corroborated at the protein level (Fig. 3Dand Supplementary Fig. S4B). We conclude that expressionof Arkadia C937A efficiently inhibits endogenous Arkadiafunction.

MDA-MB-231 cells are resistant to TGF-b–induced growtharrest (21), and we noted an absence of genes involved in TGF-b–induced cell-cycle arrest in the MDA-MB-231 cells in theRNA-seq analysis (Supplementary Fig. S5). Inactivation of

Arkadia is therefore unlikely to affect cell growth. Indeed, wefound no difference in the growth rate of parental or ArkadiaC937A-expressing cells in vitro on plastic, in soft agar, or on thegrowth of these cells in xenograft assays in immunodeficientmice (data not shown), consistent with other studies showingthat TGF-b signaling does not have a tumor-suppressive effectin MDA-MB-231 cells (34).

To gain insight into the TGF-b–driven processes for whichArkadia is likely to be required, we conducted a MetaCoreanalysis of genes that significantly change in their TGF-bregulation between the parental and Arkadia C937A-expres-sing cells. This indicated an enrichment of genes involved incell adhesion, cell matrix interactions, epithelial-to-mesenchy-mal transition (EMT) and ECM remodeling (Table 1), andprocesses involved in tumor cell dissemination from primary

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Figure 4. Inhibition of Arkadiaactivity in MDA-MB-231 byexpression of Arkadia C937Ainhibits lung colonization. A,parental MDA-MB-231 and 3independent clones of ArkadiaC937A-expressing cells wereinjected into the tail vein ofimmunodeficient mice. After 20days, lungs were analyzed fornumber of tumors. B and C, GFP-labeled parental MDA-MB-231cells and the 3 independent clonesof Arkadia C937A-expressing cellswere mixed with an equal numberof parental cells expressingmCherry and injected into the tailvein of immunodeficient mice.After 2 days, mice were culled andlungsdirectly analyzedbyconfocalmicroscopy. Representativepictures of tumor cell distributionfor parental and one clone ofArkadia C937A-expressing cellswere taken (B, left). As a control,aliquots of the mixed GFP/mCherry cells were plated beforeinjection (B, right). Themagnification scale barcorresponds to 100 mm. The ratiobetween the GFP/mCherry cells inthe lungs from multiple mouseexperiments is plotted (C). In A andC, each point represents data froma single mouse, and means andSDs are plotted.

Briones-Orta et al.





tumors to sites of metastasis (36). During metastasis, tumorcells enter the blood or lymphatic circulation (intravasation)and then extravasate at the site of metastasis (37). Becauseboth of these processes involve invasion through a layer ofendothelial cells, we attempted to mimic this in vitro byassessing cell adhesion and ability to spread on a confluentlayer of endothelial cells (HUVEC). To visualize the cells, wefluorescently labeled them with GFP and, in the case of theparental cells, also mCherry. Equal numbers of GFP- (parentalor Arkadia C937A-expressing) and mCherry-labeled parentalcells were plated onto a layer of HUVECs. We found that theArkadia C937A-expressing cells adhered more strongly to theHUVEC cells than the parental MDA-MB-231 cells (Fig. 3E).When the GFP-labeled cells were plated onto confluent layersof HUVEC cells and filmed over a period of hours to assess cellspreading (Supplemental Movies S1 and S2; ref. 38), we con-sistently observed an inhibition in the ability to spread of theArkadia C937A-expressing cells compared with parental cells(Fig. 3F). Thus, cells inhibited in Arkadia function are moreadherent to endothelial cells, but have defects in spreading,possibly indicating a defect in remodeling of adhesions.

Inhibition of Arkadia activity in MDA-MB-231 cellsinhibits colonization of lungs of immunodeficient miceThe reduced ability of cells lacking Arkadia activity to spread

on endothelial cells suggested that Arkadia could play a role inmetastasis. We tested this directly, and observed a robustinhibition of lung colonization of the 3 individual clones ofMDA-MB-231 cells expressing Arkadia C937A, compared withparental cells in tail vein injection assays conducted over 20days (Fig. 4A). To determine whether Arkadia activity isimportant for early stages of lung colonization, we conductedthese assays over a period of just 48 hours, using the fluores-cently labeled cells described above. Mice were injected with a1:1 ratio of GFP- and mCherry-labeled cells as described in theMaterials and Methods, and after 48 hours, lung colonizationwas assessed. A dramatic decrease in the number of ArkadiaC937A-expressing cells compared with the control mCherry-labeled parental cells was observed (Fig. 4B; Fig. 4C). Becausethe effects of dominant negative Arkadia were evident just 48hours after tail vein injection, we concluded that Arkadia isrequired for early stage colonization. Taken together with thein vitro cell spreading and adhesion data, it is likely thatArkadiais required for extravasation.Arkadia C937A is catalytically inactive, but retains its ability

to interactwith partners such as SnoNand Smad2/3 (12). Itwastherefore important to exclude the possibility that the decreasein the efficiency of lung colonization by cells overexpressingArkadia C937A was due to titration of some of Arkadia'spartners. We therefore downregulated Arkadia in parentalMDA-MB-231 cells using 2 different siRNAs and investigatedthe effect on short-term lung colonization. Knockdown ofArkadia was efficient for both siRNAs, and TGF-b–inducedSnoN degradation was inhibited (Fig. 5A), as was Smad3-dependent transcription (Fig. 5B). In lung colonizationassays, we observed a significant decrease (20%–30%) for thecells in which Arkadia was downregulated compared withcontrol cells (Fig. 5C).

Finally, to confirm that Arkadia acts as a tumor promoter,we extended our analysis to 2 further cell lines for whichmetastasis is known to be driven by TGF-b: the rat mammarycarcinoma cell line MTLN3E (39) and the mouse B16 melano-ma cell line (29). In both cases, knockdown of Arkadia resultedin loss of TGF-b–induced Ski and SnoN degradation, reductionof Smad3-dependent transcription (Supplementary Fig. S7),and most importantly, substantial inhibition (between 66%and 78%) of lung colonization (Fig. 6).

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Figure 5. The ability of MDA-MB-231 cells to colonize lungs requiresArkadia function. A–C, GFP- and mCherry-labeled MDA-MB-231 cellswere transfected with a control siRNA (NT), and GFP-labeled MDA-MB-231 cells were transfectedwith 2 different siRNAs against Arkadia (Ark #1and #2). Twenty-four hours later, cells were split for immunoblots,CAGA12-Luciferase reporter assays, andmice experiments. A,whole-cellextracts were prepared from the GFP-labeled cells treated � TGF-b andWestern blotted using the antibodies indicated. B, the GFP-labeled cellswere transfected with the CAGA12-Luciferase and TK-Renilla reporters,then treated � TGF-b. Coupled Luciferase/Renilla assays were asin Fig. 1. C, equal numbers of NTmCherry cells weremixedwith either NTGFP cells or Ark #1 andArk #2 siRNAGFP cells andwere injected into thetail vein of immunodeficient mice. Analysis and quantitation of theexperiment were as in Fig. 4.






DiscussionA role for Arkadia in tumorigenesis had been hypothesized

since it was first described as the ubiquitin-ligase controllingthe cellular levels of Ski and SnoN (12, 19, 20). Here, weinvestigated a potential tumor suppressor function for Arkadiaby restoring its activity in the NCI-H460 cell line that contains ahemizygous nonsense mutation. Although reexpression ofArkadia decreased the transformed phenotype of these cellsin vitro, we found no effect on growth of tumors in xenograftassays, or in lung colonization assays. These results couldindicate that the inactivating mutation we identified in Arka-dia is not a cancer driver mutation (40). However, it is alsopossible that loss of Arkadia constitutes an early priming eventfor tumorigenesis, and that acquisition of subsequent muta-tions in this cell line prevents the reexpression of Arkadiareversing the tumorigenicity of these cells in vivo. In supportof this, another recent study concluded that Arkadia has tu-mor suppressive activity in colorectal cancer (23). Interest-ingly, the Arkadiaþ/� mice used in that study were onlysusceptible to cancer when treated with a carcinogen (23),suggesting that loss of Arkadia is not sufficient for tumorigen-esis, but may sensitize cells to other oncogenic signals. More-over, unlike classical tumor suppressors, there was no tenden-cy for the tumor cells in the Arkadiaþ/� mice to lose the otherallele. Consistent with this, complete loss of Arkadia seems tobe very rare in both tumor samples and cancer cell lines. In

this study, we identified a cell line that exhibits a hemizigousnonsense mutation (S441�), but were unable to find other celllines containing mutations in Arkadia, even in cell linesshowing LOH at 15q22.1. Interestingly a small number ofnonsense mutations: E389�, E561�, R598�, Q605�, Q722�, andQ899� (of unknown zygosity), that would similarly delete theRING domain of Arkadia, have been found in tumors of theupper aerodigestive tract, large intestine, and hematopoeticand lymphoid tissue sequenced by the CGP at the SangerInstitute (reported in the COSMIC database) and in a colo-rectal tumor (23). In addition, 4 missense mutations have alsobeen reported in the COSMIC database, but how these muta-tions affect Arkadia function is unknown. These findingsindicate that Arkadia mutations do occur in human cancer,but are rare.

It is well established that different components of the TGF-bpathway are mutated in cancer at different rates. Whereasinactivating mutations and deletions in Smad4 and TGFBR2are common in certain cancers,mutations inALK5, Smad2, andSmad3 are relatively rare (41–43). Arkadia seems to be in thislatter class. In our search for cell lines containing mutations inArkadia, we made the unexpected discovery that deletion ofSmad4, or acquisition of Smad4mutations that abolish Smad–Smad interactions also abolished TGF-b–induced degradationof SnoN, that is, it confers the same effect on Ski and SnoNlevels as would loss of Arkadia. We therefore speculate that

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Figure 6. MTLN3E and B16 cells require Arkadia to colonize lungs. A and B, GFP and mCherry-labeled MTLN3E cells or B16 cells were transfected with acontrol siRNA (NT), and GFP-labeled MTLN3E and B16 cells were transfected with siRNAs against Arkadia as indicated. Twenty-four hours later cells weresplit for immunoblots, qPCR for Arkadia/GAPDH, and 48-hour lung colonization mouse experiments, which were as described in Fig. 4.

Briones-Orta et al.





cancer cells may lose Smad4 in preference to Arkadia toachieve stabilization of Ski and SnoN.The observation that cancer cells do not preferentially lose

all Arkadia function led us to investigate whether it might playadditional roles at later stages of tumorigenesis. To explicitlyaddress this, we have used 3 different tumor cell lines thatmetastasize in a TGF-b–dependent manner (29, 34, 39). Ourresults clearly show that Arkadia has a potent tumor-promot-ing activity. Arkadia inactivation has no effect on primarytumor growth, in agreement with previous work showing thatmanipulation of the TGF-b pathway in these cells had no effecton mammary tumor growth (34). Instead, we show a dramaticeffect of loss of Arkadia activity on lung colonization in tail veininjection experiments in immunodeficient mice. The fact thatwe can detect these effects within 48 hours, coupled with ourobservation that MDA-MB-231 cells expressing dominant neg-ative Arkadia adhere more strongly than parental cells to aconfluent layer of HUVEC cells, which mimics the capillarywall, and do not spread as efficiently, leads us to conclude thatArkadia is likely important for extravasation rather thangrowth and survival of the cells in the lungs.Our RNA-seq analysis uncovered a large group of genes

whose TGF-b regulation was perturbed by loss of Arkadiaactivity. Importantly, this list contained genes previouslyimplicated in lung metastasis of MDA-MB-231 cells, such asANGPTL4, Id1, LOX, and SNAI1 (refs. 33, 34, 44, 45; Supplemen-tary Figs S5 and S6). We have used gene enrichment analysisto further define classes of genes that might be responsible forlung colonization and identified genes involved in cell adhe-sion, cell matrix interactions, EMT, and ECM remodeling asparticularly affected by loss of Arkadia activity. Specific com-binations of these genes are likely to be responsible for drivingmetastasis in this tumor model.Our data indicate that Arkadia regulates metastasis in

mouse models of breast cancer and melanoma. TGF-b signal-ing has both tumor-suppressive and tumor-promoting roles,and it is therefore difficult to target this pathway for cancertherapy (46). Because Arkadia is an enzyme that is required for

only a subset of TGF-b responses, it might be amenable toinhibition by small molecules, and thus represents a possibletherapeutic target for cancer.

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

Authors' ContributionsConception and design: M.A. Briones-Orta, L. Levy, C.D. Madsen, C.S. HillDevelopment of methodology: M.A. Briones-Orta, L. Levy, C.D. Madsen, E.SahaiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.A. Briones-Orta, L. Levy, C.D. Madsen, D. Das,Y. ErkerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.A. Briones-Orta, L. Levy, C.D. Madsen, C.S. HillWriting, review, and/or revision of the manuscript: M.A. Briones-Orta, E.Sahai, C.S. HillAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M.A. Briones-Orta

AcknowledgmentsThe authors thank the Biological Resources Unit for help with the mouse

experiments and Emma Nye, Bradley Spencer-Dene, and Gordon Stamp fromExperimental Histopathology for the analysis of the tumors, Florence Tatin andTaija Makinen for the GFP-CAAX plasmid, Nik Matthews and the advancedsequencing facility for the RNA-seq analysis, and RichardMitter for data analysis.The authors also thank the Light Microscopy Facility, Equipment Park, MikeHowell and the High Throughput Screening Facility, FACS facility at the LondonResearch Institute for the technical support, the Hill lab and Ilaria Malanchi fordiscussions and comments on the manuscript, and Becky Randall for help withthe preparation of the manuscript.

Grant SupportThis study was funded by Cancer Research United Kingdom, a postdoc-

toral fellowship from CONACYT and a Marie Curie International IncomingFellowship PIIF-GA-2009-235980 (M.A. Briones-Orta), and a FEBS long-termfellowship (C.D. Madsen). L. Levy received funding from Marie Curie Actions(ERG FP7), la Ligue Nationale Contre le Cancer, and l'Association pour laRecherche sur le Cancer. Y. Erker was supported by the Minist�ere de laRecherche et Technologie.

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

Received May 18, 2012; revised October 29, 2012; accepted November 21, 2012;published OnlineFirst March 6, 2013.

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2013;73:1800-1810. Published OnlineFirst March 6, 2013.Cancer Res Marco A. Briones-Orta, Laurence Levy, Chris D. Madsen, et al. Pathway

βArkadia Regulates Tumor Metastasis by Modulation of the TGF-

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