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Cancer Therapy: Preclinical

Identification of Unique MEK-Dependent Genes in GNAQMutant Uveal Melanoma Involved in Cell Growth, Tumor CellInvasion, and MEK Resistance

Grazia Ambrosini1, Christine A. Pratilas2, Li-Xuan Qin3, Madhavi Tadi2, Oliver Surriga1,Richard D. Carvajal1, and Gary K. Schwartz1

AbstractPurpose: Metastatic uveal melanoma represents the most common intraocular malignancy with very

poor prognosis and no effective treatments. Oncogenicmutations in the G-proteina-subunit q and 11 havebeen described in about 85% of uveal melanomas and confer constitutive activation. Multiple signaling

pathways are induced as a consequence of GNAQ/11 activation, which include the MEK/ERK kinase

cascade.Weanalyzed the transcriptional profile of cell lines treatedwith amitogen-activated protein (MAP)/

extracellular signal–regulated (ERK) kinase (MEK) inhibitor to identify gene targets of activated GNAQ and

to evaluate the biologic importance of these genes in uveal melanoma.

Experimental Design: We conducted microarray analysis of uveal melanoma cell lines with GNAQ

mutations treated with the MEK inhibitor selumetinib. For comparison, we used cells carrying BRAFV600E

and cells without either mutation. Changes in the expression of selected genes were then confirmed by

quantitative real-time PCR and immunoblotting.

Results:We found that GNAQmutant cells have aMEK-dependent transcriptional output and identified

a unique set of genes that are downregulated byMEK inhibition, including the RNAhelicaseDDX21 and the

cyclin-dependent kinase regulator CDK5R1whereas Junwas induced.We provide evidence that these genes

are involved in cell proliferation, tumor cell invasion, and drug resistance, respectively. Furthermore, we

show that selumetinib treatment regulates the expression of these genes in tumor tissues of patients with

metastatic GNAQ/11 mutant uveal melanoma.

Conclusions:Our findings define a subset of transcriptionally regulated genes by selumetinib in GNAQ

mutant cells and provide new insights into understanding the biologic effect of MEK inhibition in this

disease. Clin Cancer Res; 18(13); 3552–61. �2012 AACR.

IntroductionThe RAS/RAF/MEK/ERK pathway is often activated by

genetic alterations in upstream signalingmolecules, such asRAS and BRAF. Hyperactivation of this signaling cascaderesults in dysregulated cell proliferation and malignanttransformationof anumber of human tumors (1). Recently,the presence of somatic activating mutations in the hetero-trimericG-proteina-subunit q (GNAQ) and11 (GNA11) in46% and 30% of primary uveal melanomas, respectively,has been shown (2–4). Thesemutations occur in codon 209

in the RAS-like domain, resulting in loss of GTPase activityand constitutive activation. Approximately 85% of ocularmelanomas arise from melanocytes within the uveal tract,which consists of the iris, ciliary body, and choroid of theeye (5), and account for a significant rate of deaths. Uvealmelanoma is biologically distinct from cutaneous melano-ma, as recurrent chromosomal abnormalities have beendetected in chromosomes 3 and 8 (6), as well as a very hightendency to metastasize to the liver (7). NRAS mutationshave not been detected in uveal melanoma (8), and BRAFmutations are considered rare (9, 10); however, activationof themitogen-activatedprotein kinase (MAPK)pathwaybymutant GNAQ/11 appears to be critical for the develop-ment of this disease (2). G-protein a-subunits transduceexternal signals from7-transmembrane domainG-protein–coupled receptor (GPCR) to intracellular signaling path-ways. The canonical target of GNAQ/11 stimulation isphospholipase C-b, which, in turn, stimulates inositol lipidsignaling [i.e., calcium and protein kinase C (PKC)], inresponse to mitogenic GPCR agonists (11, 12). The consti-tutively active mutant GNAQ and GNA11 have beenreported to activate the extracellular signal–regulated (ERK)

Authors' Affiliations: 1Laboratory of New Drug Development and Depart-ment of Medicine, and Departments of 2Molecular Pharmacology andChemistry and 3Epidemiology and Biostatistics, Memorial Sloan-KetteringCancer Center, New York

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

CorrespondingAuthor:Grazia Ambrosini,Memorial Sloan-KetteringCan-cer Center, 1275 York Avenue, Box 128, NewYork, NY 10065. Phone: 646-888-2184; Fax: 646-422-0631; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-3086

�2012 American Association for Cancer Research.

ClinicalCancer

Research

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pathway (2, 4), and knockdown of mutant GNAQ in uvealmelanoma cells resulted in MAPK inhibition, reducedgrowth, and induced apoptosis (2). However, the transcrip-tional output of ERK signaling downstream of mutant G-proteins is not well characterized. Targeting MAP/ERKkinase (MEK) with allosteric small-molecule inhibitors hasbeen reported to be effective in suppressing cell growth and,in some cells, inducing apoptosis. In particular, melanoma,thyroid, and non–small cell lung cancer with mutantBRAFV600E have been shown to be sensitive to MEK inhi-bitors (13–15). In a large number of tumor types treatedwith the MEK inhibitor selumetinib (AZD6244), Dry andcolleagues have reported transcriptional signatures thatpredict MEK addiction or drug resistance (16). In anotherstudy, using microarray analysis of cells treated with a MEKinhibitor, Pratilas and colleagues have found genes, includ-ing members of the dual specificity phosphatase and spro-uty gene families, that were differentially regulated by MEKinhibition in BRAFV600E cells but not in receptor tyrosinekinase–driven tumor cells with similarly elevated levels ofpERK (17). They also showed that BRAFV600E cells haveelevated ERK-dependent transcriptional output and dis-abled feedback inhibition of RAF/MEK signaling. Here, wereport that uvealmelanoma cells withGNAQmutations arehighly sensitive to MEK inhibition with selumetinib.Expressionmicroarray analysis identified aMEK-dependenttranscriptional profile that is, in part, similar to that ofBRAFV600E melanoma cells. In addition, we identified sev-eral genes unique to GNAQ mutant cells, which areinvolved in proliferation and tumor cell invasion. Further-more, pre- and posttreatment tumor biopsies from an

ongoing clinical trial of selumetinib in patients with uvealmelanoma indicate that these genes are transcriptionallyregulated and may correlate with clinical benefit.

Materials and MethodsCell culture

Omm1.3, Mel202, and Mel270 have been kindly pro-vided by Dr. Bruce Ksander (Harvard Medical School,Boston, MA). OCM1A and 92.1 were from Dr. WilliamHarbour (Washington University, St. Louis, MO). OCM3,Mel290, and C918 were from Robert Folberg (University ofIllinois, Chicago, IL). Uveal melanoma cell lines have beensequenced for the presence of activating mutations incodons 209 (exon 5) and 183 (exon 4) of GNAQ andGNA11. Two cell lines had Q209L mutation (92.1,Mel202), whereas Omm1.3 and Mel270 had Q209P muta-tion. None had GNA11 mutations. Cells were cultured inRPMI medium supplemented with 10% FBS, 100 units/mLpenicillin, and 100 mg/mL streptomycin and maintained at37�C in 5% CO2. Cells were treated with selumetinib(AZD6244, graciously supplied by AstraZeneca).

Cell viability assaysCells were plated in 96-well plates and treated with the

indicated concentrations of selumetinib, PD0325901, ordimethyl sulfoxide (DMSO) in triplicates. Viability wasassessed after 4 days of treatment using the Cell CountingKit 8 (CCK8) fromDojindoMolecular Technologies accord-ing to the manufacturer’s instructions. Survival is expressedas a percentage of untreated cells. The calculations ofcombination index values were conducted using the Com-puSyn software (ComboSyn; ref. 18).

Microarray data analysisCells were treated in triplicate with 250 nmol/L selu-

metinib, or 0.01% DMSO as control, for 8 hours. Fol-lowing RNA extraction with TRIzol reagent (Invitrogen),cDNA was synthesized in the presence of oligo(dT)24-T7from Genset Corp. cRNA was prepared using biotinylatedUTP and CTP and was hybridized to Human HT-12oligonucleotide Illumina arrays in triplicates. We useda single array slide for each cell line to minimize the effectof experimental artifact. Differential expression analysiswas conducted to identify genes whose expression isaffected by the treatment by comparing the posttreatmentexpression versus the pretreatment expression. The arraydata were log2-transformed and quantile-normalized. Foreach cell line, gene expression was compared betweentime points using the empirical Bayesian method and theR LIMMA package. An empirical Bayes t test was appliedto each gene (19). Linear models and empirical Bayesmethods were used for assessing differential expression inmicroarray experiments and a P value cutoff of 0.0001was used to select differentially expressed genes. There areabout 47,000 markers on the Illumina array and 5 genesare expected by chance to have a P value

ImmunoblottingCells were lysed in radioimmunoprecipitation assay

(RIPA) buffer supplemented with protease inhibitor cock-tail tablets (Roche Diagnostics) and 1 mmol/L Na3VO4.Equal amounts of protein were loaded on 4% to 12%PAGEgels (Invitrogen). Polyvinylidene difluoride (PVDF) mem-branes were blockedwith 5%nonfat driedmilk and probedwith pERK, ERK, cyclin D1, CDK5R1 (p35), c-Jun, anda-tubulin (Cell Signaling); SPRY2, DUSP6, ETV5, andDDX21 (Abcam).

Quantitative real-time PCRReverse transcription of 1 mg of RNA was done using the

SuperScript III First-Strand Synthesis System (Invitrogen).Quantitative real-time PCR (qRT-PCR) assays were done onthe 7300 Real Time PCR System (Applied Biosystems).TaqMangene expression assays,which include gene-specificprobe primer sets (Applied Biosystems), were used to detectthe indicated genes and glyceraldehyde-3-phosphate dehy-drogenase (GAPDH)/hypoxanthine phosphoribosyltrans-ferase (HPRT) mRNA. The relative expression of each genewas calculated by the DDCT method.

RNA interference–mediated gene knockdownThe list of siRNA is reported in the Supplementary Meth-

ods. siRNAs were transfected using Lipofectamine RNAi-MAX reagent (Invitrogen). After transfections, cells werecounted using a Nexcelom cell counter and plated in 96-well plates for cell viability assays. The statistical significanceof the experimental results was determined by the 2-sidedt test.

Tissue sample collection and analysisMatched tumor biopsies were collected from patients

with metastatic uveal melanoma (clinicaltrials.gov; no.NCT01143402) before and after 14 days of selumetinibtreatment. Flash-frozen specimens were then lysed inRIPA lysis buffer and analyzed by immunoblotting.The protocol was approved by the Institutional ReviewBoard of Memorial Sloan-Kettering Cancer Center (NewYork, NY), and all patients signed informed consentforms.

ResultsThe MEK inhibitor selumetinib inhibits cell viability ofGNAQ mutant uveal melanoma

Using a panel of uveal melanoma cell lines expressingGNAQQ209L/P, mutant BRAFV600E or wild-type (WT) forboth, we investigated the effects of MEK inhibition on cellviability with selumetinib. As shown in Fig. 1A, theGNAQQ209L/P cells exhibited dose-dependent decreasein cell viability at nanomolar concentrations (IC50< 0.1 mmol/L). The BRAFV600E cells were the most sensi-tive, with IC50 < 0.05 mmol/L, whereas cells without eitherBRAF or GNAQmutations exhibited little sensitivity, withIC50 > 1 mmol/L. This corresponded to inhibition of pERKwith selumetinib in the sensitive mutant cells (Fig. 1B;

Supplementary Fig. S1A), whereas induction of pMEK isconsistent with relief of negative feedback (20). The effectof MEK inhibition on GNAQ mutant uveal melanomashowed an accumulation in the G1 phase of the cell cycle(Supplementary Fig. S1B).

GNAQ downregulation by siRNA induced a decrease inexpression of pERK in the GNAQQ209L/P cells (Supplemen-tary Fig. S2A). This corresponded to a decrease in cellviability which was not appreciably increased by selumeti-nib (Supplementary Fig. S2B). Suppression of GNAQ inWTand BRAFV600E cells did not inhibit pERK (SupplementaryFig. S2A) and did not affect cell viability with or withoutselumetinib (Supplementary Fig. S2B). In addition, over-expression of a GNAQQ209L plasmid in a WT cell line(Supplementary Fig. S2C) sensitized the cells to selumeti-nib, as compared with cells with an empty vector (Supple-mentary Fig. S2D). These results confirm that GNAQQ209L/P

signals to MEK and specifically renders mutant cells sus-ceptible to MEK inhibition, as reported by Van Raamsdonkand colleagues (2).

Expression profile of GNAQQ209L/P cells compared withBRAFV600E MEK signature

To identify the transcriptional profile of MEK inhibitionin GNAQQ209L/P cells and potential novel targets ofGNAQQ209L/P signaling, we used microarray gene expres-sion analysis. Cells from each genetically defined subgroupof uveal melanoma (3 cell lines with GNAQQ209L/P, 1 linewith BRAFV600E, and 1 cell line WT for both) were treatedwith 250 nmol/L selumetinib or vehicle for 8 hours. Geneswhose change upon MEK inhibition exceeded defined sta-tistical thresholds for all 3 GNAQQ209L/P cell lines wereconsidered significant and defined the ERK-dependent tran-scriptional output ofGNAQQ209L/P uvealmelanoma cells. Atotal number of 387 genes met the significance level of P �0.0001 in the GNAQQ209L/P cells. Identical parameters wereused to define the set of genes regulated differentially byMEK inhibitor in the BRAFV600E and WT cell lines, and adirect comparison was conducted to evaluate commongenes among the genetic subgroups. Of the 387 genesdetermined to have changed significantly in response toselumetinib, 308 were differentially expressed only inGNAQQ209L/P cells, whereas 29 overlapped with BRAFV600E

MEK-dependent genes, 42 overlapped with genes meetingsignificance in WT cells, and 8 genes were common to all 3groups (Fig. 1B; Venn diagram). The top 19 genes withhighest significance and fold change common to allGNAQQ209L/P cells before and after treatment with selume-tinib are displayed in a heatmap (Fig. 1D). A list of P valuesand fold change of representative genes for each group(GNAQQ209L/P-only and overlap groups) are shownin Table 1. Of note, several of the genes that were sharedbetween GNAQQ209L/P and BRAFV600E cells were previouslydescribed ERK targets, such as CCND1, transcription factorsETV5, MYC, and genes involved in the feedback inhibitionof MEK/ERK signaling, that is, dual specificity phosphatase6 (DUSP6), and sprouty family members SPRY2, SPRY4.These genes are key components of "MEK signatures"

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Clin Cancer Res; 18(13) July 1, 2012 Clinical Cancer Research3554




described in other tumor cells (16, 17), and we confirmedtheir decline in expression (mRNA and protein levels) afterselumetinib treatment, using immunoblotting and qRT-PCR (Fig. 2). Following exposure to selumetinib for up to24 hours, pERK levels were determined by immunoblotting(Fig. 2A). In the less sensitiveWT cell line, pERKwas slightlydownregulated at 2 hours and quickly rebounded at latertime points, whereas its inhibition was more complete incells with BRAFV600E and GNAQQ209L/P mutations. Basalexpression levels of ETV5,DUSP6, and SPRY2 proteinswerealmost undetectable in the WT cell line, whereas they werehighly expressed in the mutant cells (both BRAFV600E andGNAQQ209L/P), confirming elevated transcriptional outputof MEK signaling in these cells (17). Cyclin D1 was durablydownregulated in BRAFV600E cells, whereas its expressionrebounded in GNAQQ209L/P and WT cells at later time

points. The induction of pMEK did not occur in BRAFV600E

cells, in accordancewith the reported abrogationofnegativefeedback between ERK and RAF proteins by BRAFV600E

(17, 20). The microarray results of these MEK-dependentgenes were also confirmed by qRT-PCR in 3 GNAQQ209L/P

cell lines, where these genes were suppressed (Fig. 2B–D).The high basal expression of these transcripts and theirdownregulation by selumetinib confirmed that the MEK/ERK pathway is active in GNAQQ209L/P cells and that thetranscriptional events described in GNAQQ209L/P are atleast, in part, similar to reported MEK functional activa-tion signatures. For example, Pratilas and colleaguesreported a signature of 52 MEK-dependent genes inBRAFV600E cells (17), of which 19 are represented amongour 345 genes (5.5%) and are significantly overenriched(P < 0.0001).

Figure 1. Selumetinib inhibits cell viability of BRAFV600E and GNAQQ209L/P cell lines. A, cells with wild-type GNAQ/BRAF (C918, Mel290), mutant GNAQ(92.1, Omm1.3, Mel270, Mel202), and BRAFV600E (OCM3, OCM1A) were treated with increasing concentrations of selumetinib (0, 25, 50, 100, 250, 500,1,000 nmol/L) and analyzed for cell viability on day 5, calculated as percentage of untreated controls. Data are representative of 3 independent experiments.Bars, mean � SD. B, immunoblots of pERK and pMEK in response to increasing concentration of selumetinib in cells with different mutational status.Microarray results: C, Venn diagram summarizing differentially expressed genes in selumetinib-treated GNAQQ209L/P cell lines (green circle), BRAFV600E

(red circle), andWTcells (blue circle), with corresponding overlapping genes as indicated. D, heatmap representation of top 19 genes identified asmeeting thestatistical threshold (see Materials and Methods) for significant change in expression in all the cell lines and with fold change > 2 in GNAQQ209L/P cell lines, inresponse to 250 nmol/L selumetinib (gray bar), or DMSO as control for 8 hours, in triplicate. One replicate of the Mel290 cell line was excluded as itdid not cluster with the other 2 replicates. Cell lines with individual replicates are columns, genes are rows.

MEK Regulated Genes in Uveal Melanoma with GNAQ Mutation

www.aacrjournals.org Clin Cancer Res; 18(13) July 1, 2012 3555




MEK inhibition regulates unique genes in GNAQQ209L/P

cellsThe gene profile of MEK inhibition in GNAQQ209L/P cell

lines also comprised uniquely regulated genes (Table 1).From this list, we selected 3 genes, 2 downregulated(DDX21, CDK5R1) and 1 upregulated (Jun) for validationby immunoblotting and qRT-PCR (Fig. 3). Immunoblotdetection showed that DDX21 and CDK5R1 protein levelswere downregulated in GNAQQ209L/P cells after MEK inhi-bition over time but not in the BRAFV600E and WT cell lines(Fig. 3A). Jun increased exclusively in the GNAQQ209L/P

cells, whereas it was quickly downregulated in BRAFV600E

cells. In contrast, Jun protein expression was high inWT cells and did not change upon treatment (Fig. 3A).Selumetinib also reduced DDX21 and CDK5R1 mRNAlevels in GNAQQ209L/P cells, whereas they did not signifi-cantly change in WT cells (Fig. 3B and C). In the BRAFV600E

cells there was a decrease in mRNA levels but generally to alesser extent than the GNAQQ209L/P cells and this wasassociated with relatively minor changes in the proteinlevels of DDX21 and CDK5R1 (Fig. 3A–C). Also, Jun wasinduced upon treatment in GNAQQ209L/P cells, whereas

it was profoundly decreased in BRAFV600E (Fig. 3D), inaccordance with the protein levels. There was also a slightdecrease in Jun mRNA in WT cells that did not affect theprotein levels (Fig. 3D).

To prove specificity of MEK-dependent signaling down-stream of GNAQ and to exclude drug-unrelated effects, theexpression levels of these genes were analyzed in cells afterGNAQ knockdown. Phospho-MEK was markedly down-regulated in the GNAQQ209L/P cells after GNAQ depletion(Fig. 3E). A slight decrease was seen in WT cell line possiblydue to the disruption of WT GNAQ signaling. Consistentwith the effects of selumetinib, CDK5R1 and DDX21 weredownregulated in theGNAQQ209L/P cells, whereas c-Junwasincreased, although less than was observed with drug alone(Fig. 3E). To determinewhether the effects onGNAQQ209L/P

cells were restricted to selumetinib, we treated uveal mel-anoma cells with a different MEK inhibitor, PD0325901.Both theGNAQQ209L/P andBRAFV600E cellswere sensitive toincreasing concentrations of the drug (Supplementary Fig.S3B). In terms of protein expression, the change in c-Jun(induction inGNAQQ209L/P, suppression in BRAFV600E, andno change in WT) was similar to selumetinib, but

Table 1. List of differentially expressed genes in response to selumetinib

Genotype Probe ID Gene name P values Log2 FC

GANQQ209L/P ILMN_1730928 CDK5R1

suppression of CDK5R1 and DDX21 was observed in bothGNAQQ209L/P and BRAFV600E cell lines (Supplementary Fig.S3A). Interestingly, PD0325901 inhibited pERK in the WTcell line but this resulted in essentially no reduction in cellviability, nor a decrease in cyclin D1 expression. Overall,these results suggest that all the effects of selumetinib ontranscriptional expression in uveal melanoma cellsmay notbe shared by other MEK inhibitors. This will require furtherinvestigation.

MEK-dependent GNAQQ209L/P-specific genes mediatecell proliferation and cell migrationOf the genes uniquely downregulated by selumetinib in

GNAQQ209L/P cells, we focused on DDX21 and CDKR51.DDX21 is amember of RNAhelicase family, which containsa motif (DExD/H) highly conserved from bacteria tohumans (21). They are multifunctional proteins involvedin RNA unwinding and play a role in transcription regula-tion (22). To test whether DDX21 is involved in the anti-proliferative effects of selumetinib, we carried out geneknocdown experiments. Three GNAQQ209L/P mutant celllines, as well as BRAFV600E and WT cells, were transientlytransfected with 2 different DDX21-specific or control siR-NAs (Fig. 4A, bottom; Supplementary Fig. S4A). Depletionof DDX21 reduced cell viability of all the cell lines, indi-cating a role for DDX21 in cell proliferation that is inde-pendent of the GNAQ status. In fact, the most affected wasthe WT cell line with 60% inhibition of proliferation. TheGNAQQ209L/P cells showed about 40% inhibition relative tocontrol siRNA–transfected cells (Fig. 4A, top; Supplemen-tary Fig. S4B). The addition of selumetinib decreased via-bility of the GNAQQ209L/P cell lines by only an additional10% to20%(Supplementary Fig. S5C–S5E), suggesting that

DDX21 downregulation contributes to the antiproliferativeeffects of selumetinib in these cells. Furthermore, DDX21downregulation did not affect migration of the cell lines(data not shown).

CDK5R1 encodes p35, a specific activator of the serine/threonine kinase CDK5,which plays a crucial role in centralnervous system development andmaintenance (23). A rolefor CDK5 and p35 in cell migration has also been reported(24, 25). Seeking to establish a possible functional roleunderlying the observed differences in CDK5R1 expression,we assessed the impact of knockdown of this protein inuveal melanoma cells using 2 different siRNAs. CDK5R1siRNA did not significantly inhibit cell viability (Supple-mentary Fig. 4C). All 3 GNAQQ209L/P cell lines depleted ofCDK5R1 (Fig. 4B, bottom) showed a decrease in cell migra-tion (Fig. 4B, top) compared with cells with control siRNA.The WT cells (Fig. 4B), as well as the BRAFV600E (Supple-mentary Fig. 6), showed no inhibition of migration withsiRNA to CDK5R1, whereas GNAQ siRNA inhibited migra-tion of theGNAQQ209L/P cell lineOmm1.3 (SupplementaryFig. 6). Interestingly, selumetinib inhibited migration ofboth GNAQQ209L/P and BRAFV600E, but not the WT cells(Supplementary Fig. 6), suggesting that selumetinib mayhave effects on the cellmigration of BRAFV600E cells, but thiseffect is independent of CDK5R1.

Jun expression is related to sensitivity to MEKinhibition

c-Jun is a component of the AP-1 transcription complexregulated by c-jun-NH2-kinase (JNK), and it is involved ina number of cell responses, such as cell proliferation andcell death (26). c-Jun was upregulated in GNAQQ209L/P

cells after selumetinib treatment, showing a differential

Figure 2. Validation of MEK-regulated genes in GNAQQ209L/P

cells. These genes wererepresentative of those sharedbetween GNAQQ209L/P andBRAFV600E MEK–dependentsignature. A, immunoblot analysis ofcells treated with 250 nmol/Lselumetinib over the time, usingantibodies for the indicatedproteins.Each blot is representative of at least2 experiments showing sameresults. B–D, relativemRNA levels ofselected ERK output genes beforeand after selumetinib in threeGNAQQ209L/P cell lines. Cells weretreated with selumetinib for 8 hoursand RT-PCR was carried out usinggene-specific primers for DUSP6,ETV5, SPRY2, and cyclin D1. Valueswere normalized with GADPH andHPRT as housekeeping genes usingthe DDCT method. Each experimentwas carried out in triplicates. Bars,�SD.






regulation compared with cells with BRAFV600E. Knock-down of c-Jun by 2 different siRNAs (Fig. 4C; Supplemen-tary Fig. S7A) significantly increased the antiproliferativeeffects of selumetinib in GNAQQ209L/P cell lines (Fig. 4D;Supplementary Fig. S7B), suggesting that c-Jun inductionmay be involved in mechanisms of resistance to MEKinhibition. In contrast, c-Jun did not seem to play a roleinWTandBRAFV600E cells as its knockdowndidnot alter thesensitivity to the selumetinib when compared with controlsiRNA (Fig. 4D; Supplementary Fig. S7B).

ValidationofGNAQQ209L/P-specific ERK transcriptionaloutput in tumor tissues

To assess the clinical efficacy of MEK inhibition onuveal melanoma, we are conducting a phase II clinicaltrial of selumetinib versus temozolomide in patientswith metastatic uveal melanoma (clinicaltrials.gov; no.NCT01143402). Matched tumor biopsies are collected toexamine target modulation between baseline and day 14from patients with GNAQ/11 mutations receiving selume-tinib. Results from 3 representative biopsy pairs are shown(Fig. 5A). The results of all the patients in the trial will bereported separately. Sustained inhibition of pERK and sup-pression of cyclin D1 were observed in patients A and B onday 14, but not in patient C. This correlated to best clinicalresponse by Response Evaluation Criteria in Solid Tumors

(RECIST; ref. 27): partial response in liver metastases inpatient A (Fig. 5B), stable disease in patient B, and progres-sion of disease in patient C. Furthermore, DDX21 wasdownregulated by selumetinib in patients A and C,CDK5R1 decreased in patient A and B, whereas they couldnot be detected in patients B and C, respectively. c-Junexpression increased in patient C (Fig. 5A). A similar trendhas been seen in other patients treated on this clinical trial.These preliminary results are consistent with our in vitrostudies indicating the existence of unique subset of genes inGNAQQ209L/P cells that are regulated by selumetinib and theexpression of which could have an impact on clinicaloutcome.

DiscussionUveal melanoma represents the most common intraoc-

ular malignancy. However, there are no effective treatmentsfor this aggressive disease. Selumetinib is the only MEKinhibitor in clinical trials currently in the United States forpatients with uveal melanoma. Here, we report that cellswith GNAQQ209L/P mutations are sensitive to MEK inhibi-tion by selumetinib, and sensitizationwas associated with aMEK-dependent gene expression profile. Some features ofthis profile are overlapping with that elicited in BRAFV600E

uveal melanoma cells and other cell types (16), which

Figure 3. Confirmation of MEK-dependent genes differentiallyregulated by selumetinib inGNAQQ209L/P cells. A, immunoblotanalysis of DDX21, CDK5R1, andJun expression after treatment with250 nmol/L selumetinib over thetime in cell lines with differentmutational status. Total RNA wasextracted from cells after 8 hours oftreatment and qRT-PCR wascarried out using gene-specificprimers for DDX21 (B), CDK5R1 (C),and Jun (D). Values are relative tomRNA levels of untreated cells.Each experiment was carried out 2or 3 times in triplicates.E, Western blot analysis of uvealmelanoma cells after GNAQknockdown by siRNA using theindicated antibodies.

Ambrosini et al.





supports the MEK dependence of GNAQQ209L/P cells. Thisgene profile includes the dual-specificity phosphatases(DUSP4/6), the sprouty homologues (SPRY1/2/4), whichare known transcriptional targets of the ERK pathwayinvolved in negative feedback regulation of ERK. The Etsvariant transcription factor ETV5was also regulated byMEKinhibition, along with cell-cycle division–associated pro-tein 7 (CDCA7), the proto-oncogene MYC, and the solutecarrier family 16, member 6 (SLC16A6).Additional features of the MEK profile were identified

as specific for GNAQQ209L/P cells. A number of genessuppressed in GNAQQ209L/P cells by selumetinib, suchas LYAR, NOP58, GNL3, and PPAT, were reported asnuclear proteins involved in cell growth and tumorigen-esis (28–31). DDX21 was recently identified as a novelbiomarker for colorectal cancer (32), whereas CDK5R1was involved in metastasis (24, 33) and associated withmeningioma progression (34). Interestingly, it has beenreported that mutant K-RAS regulates expression/stability

of CDK5 and CDK5R1 (p35) to increase malignant pro-gression and invasion of pancreatic cancer cells (35). It isplausible that mutant GNAQ acts similarly to mutant K-RAS, as CDK5R1 expression was in fact elevated in theGNAQQ209L/P cells compared with cells with other geneticbackgrounds. Furthermore, it has been reported thatCDK5 negatively regulates JNK3 activity and its targetc-Jun, to prevent apoptosis in developing neurons (36).This implicates a possible interaction between these pro-teins in promoting survival of uveal melanoma cells.CDK5R1 and DDX21 were also downregulated by selu-metinib in vivo in tissues of patients enrolled in a phase IIclinical trial we are conducting.

Jun was upregulated after selumetinib treatment in theGNAQQ209L/P cells only. Depending on the cell type anddrug treatment, c-Jun and Jun kinase have been implicatedin both pro- and antiapoptotic responses (37). In cutaneousmelanoma cells, active ERK induces c-Jun expression(38). In contrast, c-Jun was induced by MEK inhibition

Figure 4. GNAQQ209L/P MEK–dependent genes play a role in cell proliferation and metastasis. A, siRNA-mediated knockdown of DDX21 (þ) in 5 cell lines(bottom); (�), control siRNA. Cell viability wasmeasured after 4 days (top). B, migration assays of WT and GNAQQ209L/P cell lines transfected with CDK5R1 orcontrol siRNA (bottom). Cells migrated through the membrane pores were stained and counted under the microscope. Representative fields of �200magnification are shown. C, uveal melanoma cell lines were transfected with control or c-Jun siRNA and tested in proliferation assays (D) after 4 days of 250nmol/L selumetinib treatment. Experiments were repeated 3 times in triplicates. �, P < 0.0001; ��, P < 0.001; ��, P < 0.005 for comparison of c-Jun versuscontrol siRNA with selumetinib treatment.






in GNAQQ209L/P uveal melanoma cells, suggesting a differ-ential regulation of the ERK/JNK pathway. G-protein–mediated signaling is complex and involvesmultiple down-stream binding partners and various regulatory scaffolding/adaptor and effector proteins (12). For example, PKC is atarget of GNAQ activation, and it might be involved infeedback regulation of c-Jun when ERK is inhibited. Theupregulation of c-Jun could represent an alternative route tocell proliferation, which would explain the relative lowersensitivity to selumetinib of GNAQQ209L/P cells as com-pared with BRAFV600E cells. Interestingly, increased expres-sionof c-Junhas also been reported in colorectal cancer cellswith K-RAS or BRAF mutations after acquired resistance toselumetinib (39). We showed that the antiproliferativeeffect of selumetinib can be enhanced by suppressing c-Junin the GNAQQ209L/P cells. This would suggest that targetingc-Jun in the presence of MEK inhibition would result inenhanced antitumor effects and may prevent selumetinibresistance. In conclusion, our findings define a uniquemolecular profile ofMEK inhibition by selumetinib in uvealmelanoma cells with mutant GNAQ and point to a set oftranscriptionally modified genes that could have an impacton the activity of this agent in this disease.

Disclosure of Potential Conflicts of InterestC.A. Pratilas is a consultant/advisory board member for Roche. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: G. Ambrosini, C.A. Pratilas, G.K. SchwartzDevelopment of methodology: G. Ambrosini, G.K. SchwartzAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): O. Surriga, R.D. Carvajal, G.K. SchwartzAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): C.A. Pratilas, L.-X. Qin, M. Tadi, G.K.SchwartzWriting, review, and/or revision of the manuscript: G. Ambrosini, C.A.Pratilas, L.-X. Qin, R.D. Carvajal, G.K. SchwartzAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): G. Ambrosini, M. Tadi, G.K.SchwartzStudy supervision: G. Ambrosini, G.K. Schwartz

Grant SupportThis work was supported by Melanoma Research Alliance and Cycle for

Survival funds. C.A. Pratilas was supported by the NIH grant # K08-127350.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 2, 2011; revised April 18, 2012; accepted April 26,2012; published OnlineFirst May 1, 2012.

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2012;18:3552-3561. Published OnlineFirst May 1, 2012.Clin Cancer Res Grazia Ambrosini, Christine A. Pratilas, Li-Xuan Qin, et al. MEK ResistanceUveal Melanoma Involved in Cell Growth, Tumor Cell Invasion, and Identification of Unique MEK-Dependent Genes in GNAQ Mutant

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