overexpression of ddx43 mediates mek inhibitor resistance … · that ic 50 were at least 10-fold...

Cancer Biology and Signal Transduction

Overexpression of DDX43 Mediates MEK InhibitorResistance through RAS Upregulation in UvealMelanoma Cells

Grazia Ambrosini1, Raya Khanin2, Richard D. Carvajal1, and Gary K. Schwartz3

AbstractThe majority of uveal melanomas carry oncogenic mutations in the G proteins GNAQ and GNA11, with

consequent activation of the MAPK pathway. Selective MEK inhibitors, such as selumetinib, have shown

clinical benefit in uveal melanoma. However, mechanisms of drug resistance limit their efficacy in some

patients. Analysis of MEK inhibitor–resistant uveal melanoma cell lines revealed the induction of RAS protein

expression and activity. This effect was mediated by the RNA helicase DDX43, which was remarkably

overexpressed in these cells. Depletion of DDX43 in MEK inhibitor–resistant cells decreased RAS proteins

and inhibited ERK and AKT pathways. On the contrary, ectopic expression of DDX43 in parental uveal

melanoma cells induced RAS protein levels and rendered cells resistant to MEK inhibition. Similar to DDX43

depletion, downregulation of KRAS, HRAS, and NRAS inhibited downstream pathways in the resistant cells,

overcomingmutantGNAQsignaling.Wealso analyzed the expression ofDDX43 in livermetastases of patients

with uveal melanoma by RT-PCR, and found a significant overexpression of DDX43 in patients who did not

benefit from selumetinib therapy. In conclusion, DDX43 induces RAS protein expression and signaling,

mediating a novel mechanism of MEK inhibitor resistance. The detection of DDX43 in patients with uveal

melanoma could lead to more targeted therapies for this disease. Mol Cancer Ther; 1–8. �2014 AACR.

IntroductionUveal melanoma accounts for approximately 5% of all

melanomas.Most arise frommelanocyteswithin theuvealtract, which consists of the iris, ciliary body, and choroidof the eye (1). This type of melanoma has a very hightendency to metastasize to the liver (2). As opposed tocutaneusmelanoma, BRAF andRASmutations are absentor extremely rare in uveal melanoma (3). Rather, activat-ingmutations inG-proteina-subunits, GNAQorGNA11,have been detected in nearly 80% of primary uveal mel-anomas (4–6), and mediate the activation of the MAPKpathway (4). The prognosis of patients with metastaticuveal melanoma is poor with a median 1-year survivalrate of less than 30% (7, 8). Nearly all patients with uvealmelanoma die due to metastatic disease and there arelimited therapeutic options (9, 10). We have reported that

the small-moleculeMEK inhibitor, selumetinib, can inhib-it pERK and cyclin D1, resulting in decreased viability ofuveal melanoma cell lines (11). Furthermore, in patientswith uvealmelanoma, selumetinib treatment can result intumor shrinkage, and the sustained inhibition of pERKmay be predictive of benefit (12). Recently, our groupreported the results of a randomized phase II clinical trialof selumetinib versus temozolomide in patients withmetastatic uveal melanoma (13). These results indicatedan improvement in the response rate and a significantmedian progression-free survival. Thus, selumetinib isthe first drug in medical oncology to ever show a clinicalbenefit in patients with this disease. Despite these prom-ising results, "de novo" and "acquired" resistance to thisdrug and other MEK inhibitors remains a major clinicalproblem (14). For example, resistance to selumetinib hasbeen described in colorectal cancer cells carrying BRAFand RAS mutations, where resistance is mediated by theamplification of the driving oncogene (15, 16). KRASoverexpression has also been implicated in resistance toMEK inhibition in colon carcinoma (17). RAS activationhas been described as amechanism of acquired resistanceto BRAF inhibitors in cutaneus melanoma with BRAFmutation, due to receptor tyrosine kinase (RTK) activation(18) or NF1 loss (19). In uveal melanoma with GNAQ/GNA11 mutations, the mechanisms of resistance to MEKinhibitors are unknown and understanding this processwill prove critical to develop more effective therapies forthe treatment of this disease.

Authors' Affiliations: 1Jennifer Goodman Linn Laboratory of New DrugDevelopment, Department of Medicine; 2Department of Bioinformatics,Memorial Sloan-Kettering Cancer Center; and 3Department of Medicine,Columbia University Medical Center, New York, New York

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

Corresponding Author: Grazia Ambrosini, Columbia University, The Her-bert Irving Comprehensive Cancer Center, 1130 St. Nicholas Ave, Room207, New York, NY 10032. Phone: 212-851-4905; Fax: 212-851-4906;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-14-0095

�2014 American Association for Cancer Research.

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DDX43, also called HAGE, is a member of the DEAD-box family of ATP-dependent RNA helicases. It wasfirst identified as a cancer/testis antigen, and it is highlyexpressed in many tumor types compared with normaltissues (20). In particular, DDX43 is overexpressed in50% of acute myeloid leukemias (21), and its expressionwas associated with advanced disease and poor prog-nosis (22). These helicases browse RNA molecules andpromote the dissociation of the RNA from ribonucleo-proteins (23). In this way, they support processes suchas gene transcription and posttranscriptional regula-tion, including pre-mRNA splicing, translation initia-tion/elongation, and RNA degradation (24–26). Theiraltered expression levels have been also implicated intumor initiation, progression, and maintenance (27).Recently, DDX43 was found highly expressed in mela-noma (28), and it promoted protein expression of NRASby unwinding of mRNA secondary structures, andenhancing NRAS translation (29). Here, we report thatthe expression of DDX43 is elevated in uveal melanomacells with acquired resistance to selumetinib and inpatients with metastatic uveal melanoma who did notbenefit from selumetinib treatment. Furthermore, wedemonstrated that DDX43 regulates RAS proteinexpression and AKT activation. Selumetinib-resistantcells became sensitive to AKT inhibition, suggestingalternative strategies for the treatment of patients withuveal melanoma with high DDX43 expression and resis-tance to MEK inhibitors.

Materials and MethodsCell lines and reagents

The GNAQ (Q209P)-mutant cell lines, Omm1.3 andMel270, were kindly provided by Dr. Bruce Ksander(HarvardMedical School, Boston, MA) in 2009. The cellshave been sequenced for the presence of mutations incodons 209 (exon 5) and 183 (exon 4) of GNAQ andGNA11. A karyotype test was also performed for thecell line in 2012, confirming the authenticity of the celllines as reported previously (30). The resistant cell lines,Rs-Omm13 and Rs-Mel270, were derived from the cor-responding cell lines after at least 3 weeks of exposure toescalating doses up to 1 mmol/L of selumetinib. Cellswere cultured in RPMI medium supplemented with10% FBS, 100 U/mL penicillin and 100 mg/mL strepto-mycin, and maintained at 37�C in 5% CO2. Cells weretreated with selumetinib (AZD6244, ARRY-142866,AstraZeneca) and MK2206 (supplied by Merck; ref. 31),PD0325901 (32), and GSK1120212 (Selleck Chemicals;ref. 33).

Cell viability assaysCells were plated in 96-well plates, and treatedwith the

indicated concentrations of drugs. Viability was assessedafter 4 days of treatment using the Cell Counting Kit 8from Dojindo Molecular Technologies according to themanufacturer’s instructions. Cell viability is expressed asa percentage of untreated cells.

Immunoblotting and RAS-GTP pull-down assaysThe cells and the biopsy tissues were lysed in RIPA

buffer supplemented with protease inhibitor cocktailtablets (Roche Diagnostics) and 1 mmol/L NaVO3. Totalprotein concentration of the lysateswasmeasuredbyBCAassay (Bio-Rad), and equal amounts of protein wereloaded on 4% to 12% PAGE gels (Invitrogen). Polyviny-lidene difluoride membranes were blocked with 5% non-fat dried milk in PBS buffer containing 0.1% Tween-20(PBST) and probed with antibody for pERK, ERK, pAKT,pan-AKT, pRB, c-Jun, cyclin D1, tubulin (Cell SignalingTechnology), DDX43 (Abcam), KRAS, HRAS (Abnova),and NRAS (Santa Cruz Biotechnology). GTP-bound RASwasmeasured using the RAFRAS-binding domain (RBD)pull-down and Detection Kit (Thermo Scientific), asinstructed by the manufacturers.

RNAi-mediated gene knockdownDDX43-1 and -2 siRNA were from OriGene. siRNA

against GNAQ (sc-35429), KRAS (sc-35731), NRAS (sc-36004), HRAS (sc-29340), and control siRNA (sc-37007)were purchased from Santa Cruz Biotechnology. Theywere transfected using Lipofectamine RNAiMAX reagent(Life Technologies). The human DDX43 cDNA clone inpCMV6-XL5 was obtained from OriGene.

Tissue sample collection and qRT-PCRMatched tumor biopsies were collected from patients

with metastatic uveal melanoma (clinicaltrials.gov, #NCT01143402). The protocol was approved by the Institu-tional Review Board of Memorial Sloan-Kettering CancerCenter (New York, NY), and all patients signed informedconsent forms. Total RNAwas extracted from flash-frozenspecimens and cell lines using TRIzol (Life Technologies)followed by DNase digestion and RNeasy (Qiagen) col-umn purification following the manufacturer’s protocol.The RNA integrity was verified using an Agilent Bioana-lyzer 2100 (Agilent). Of note, 1 mg of total RNAwas reversetranscribed using the Thermoscript RT-PCR system (LifeTechnologies). The resultant cDNA was used in RT-PCRreactions using an iCycler (Bio-Rad) with predesignedTaqMan Gene expression Assays for DDX43 and GAPDHgenes (Life Technologies). Triplicate Ct values were aver-aged, the targets were interpolated from the standardcurves and normalized to GAPDH. The results are shownas DDX43/GAPDH ratio and analyzed with theWilcoxonMann–Whitney test for two-sided P value.

ResultsGNAQ-mutant cell lines with acquired resistance toselumetinib display differential expression of RASand DDX43 and deregulated MAPK/AKT pathways

To explore mechanisms of drug resistance, we gener-ated MEK inhibitor–resistant uveal melanoma cell lines.TheGNAQ-mutant cell lines, Omm1.3 andMel270,whichare sensitive to selumetinib treatment (34), were chroni-cally exposed to increasing doses of selumetinib. Theresulting cell lines, Rs-Omm1.3 and Rs-Mel270, showed

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that IC50 were at least 10-fold higher than their parentalcells, Omm1.3 and Mel270, in cell viability assays (Fig.1A). The cells also showed cross resistance to the MEKinhibitors PD0325901 and GSK1120212 (SupplementaryFig. S1). We then examined the expression levels andphosphorylation status of different components of theERK pathway after MEK inhibition. In parental cellsOmm1.3 and Mel270, increasing doses of selumetinib for24 hours caused a decrease in pERK, cyclin D1, and pRb(Fig. 1B), whereas c-Jun and pAKT were slightlyincreased. In Rs-Omm1.3 and Rs-Mel270, pERK was alsoinhibited by the treatments (Fig. 1B), even though thesignal seems to rebound over time (Supplementary Fig.S2). Furthermore, the downstream signaling was deregu-lated in these cells, with sustained expression of cyclinD1,pRb, and high expression of c-Jun and pAKT, indepen-dently of treatments (Fig. 1B).

We next analyzed the expression of RAS in all the celllines. The parental cells expressed low levels of RAS,which slightly increasedwithhigher doses of selumetinib.In contrast, RAS protein levels were elevated in the resis-tant cells regardless of treatments (Fig. 1B).

To test whether RAS protein expression correspondedto increased activity, we used RAF-RBD pull-downassays. In the parental Omm1.3 and Mel270 cells, basalRAS expression and activity were relatively low andtemporarily induced by selumetinib without changes inRAS expression (Fig. 1C). This activation of RAS is pos-sibly mediated by the downregulation of the Sproutyproteins by ERK inhibition, as reported (35). We havealso shown that GNAQ-mutant cells have elevatedexpression of Spry2 that is downregulated by selumetinib(34). In contrast, both RAS expression and activity wereelevated in Rs-Omm1.3 and Rs-Mel270 cell lines, and

Figure 1. Selumetinib-resistant cells exhibit RAS and DDX43 upregulation and increased ERK/AKT pathway activation. A, after continuous exposure toselumetinib, the cell lines Rs-Omm1.3 and Rs-Mel270 became resistant to the drug compared with their parental cells, Omm1.3 and Mel270. Cellviability on day 4was calculated as percentage of untreated controls. Each point is amean�SD. B, immunoblot analysis of parental and selumetinib-resistantOmm1.3 (left) andMel270 (right) cells treatedwith increasing concentrations of selumetinib for 24 hours. Both selumetinib-resistant cell lines Rs-Omm1.3 andRs-Mel270 showed sustained expression of cyclin D1, pRB, c-Jun, pAKT, and RAS independently of treatment. C, whole-cell lysates were subjectedto pull-down assays with GST-bound CRAF RAS-binding domain (RBD) for active RAS. The cells were treated with selumetinib for 24 hours or treatedand washed afterwards and kept in culture for 24 hours (ND, no drug). The active RAS pull-down (top bands) and total lysates (bottom bands) wereimmunoblotted with a pan-RAS antibody. D, parental and selumetinib-resistant cells were analyzed for DDX43 expression by immunoblotting, beforeand after selumetinib treatments. E, real-time PCR was performed using gene-specific primers and the values were normalized to GAPDH ashousekeeping gene. Relative mRNA levels of DDX43 in parental and selumetinib-resistant cell lines are shown. Each experiment was performed intriplicate. Bars, � SD.

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remained active even when selumetinib was removed for24 hours (Fig. 1C). These results show that the selumeti-nib-resistant cells have sustained activation of the RAS,ERK, and AKT pathways.

To determine whether the increase of RAS proteincorresponded to higher RAS mRNA levels, we per-formed RT-PCR from parental andMEK inhibitor–resis-tant cells with or without treatment. We did not find anydifference in RNA expression for KRAS or NRAS (datanot shown). Rs-Omm1.3 and Rs-Mel270 cells weresequenced for MEK, KRAS, NRAS, HRAS mutationsand these were negative. FISH analysis was performedto exclude gene amplification of the oncogene GNAQ orRAS proteins. Assays to detect activation of RTK werealso negative.

It has been reported that the DEAD-box RNA heli-case antigen DDX43 regulates NRAS protein expres-sion through unwinding of duplex RNA in melanomacells (29). Thus, we analyzed the expression of DDX43

in uveal melanoma cells, and found a remarkableupregulation of DDX43 in the MEK inhibitor–resistantcells, compared with low or no expression in theirparental cells (Fig. 1D). Treatments with selumetinibdid not further increase DDX43 expression either withincreasing doses (Fig. 1D) or over time (SupplementaryFig. S2). The mRNA expression of DDX43 was analyzedby RT-PCR in all the cell lines, showing a 9-fold and7.6-fold increase in Rs-Omm1.3 and Rs-Mel270 cells,compared with the respective parental cell lines(Fig. 1E).

DDX43 mediates the expression of RAS andresistance to selumetinib

To determine whether DDX43 was involved in MEKinhibitor resistance, DDX43 was overexpressed in theselumetinib-sensitive uveal melanoma parental cell lines,Omm1.3 and Mel270. In these cells, DDX43 induced theprotein levels of KRAS, HRAS, NRAS, and increased

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Figure 2. DDX43 mediates the expression of RAS and resistance to selumetinib. A, parental uveal melanoma (UM) cell lines Omm1.3 and Mel270 weretransfected with DDX43 cDNA or an empty vector (V). Cell lysates were subjected to Western blot analysis for expression of DDX43, KRAS, HRAS,NRAS, pERK, and pAKT. B, the cell line Omm1.3 and Mel270 overexpressing DDX43 are less sensitive to increasing doses of selumetinib, after 4 days oftreatments. Graph reports the mean � SD of three independent experiments. C, DDX43 downregulation decreases RAS protein expression/activity andrestores MEK sensitivity. siRNA-mediated knockdown of DDX43 (þ) and control siRNA (�) in the MEK inhibitor–resistant cell lines decreases KRAS,HRAS, NRAS and downstream signaling molecules pERK, pAKT, and c-Jun. D, pull-down assays for RAS activity in cells after DDX43 downregulation.Total RAS, DDX43 levels, and tubulin are also shown. E, cell viability of Rs-Omm1.3 and Rs-Mel270 cells was measured after DDX43 knockdown and3 days of selumetinib treatments. Bars are the mean of three experiments, expressed as percentage of untreated cells,� SD. �, P < 0.001 and #, P < 0.002 forcomparison of DDX43 siRNA with and without selumetinib treatment.

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pAKT (Fig. 2A). Surprisingly, the levels of pERK did notchange, possibly because the pathway is regulated bymutant GNAQ through PKC activation, as reported(36). Nevertheless, higher expression of RAS has impor-tant implications as it regulates multiple pathways,including the PI3K/AKT pathway (37, 38). Next, wetested whether DDX43 affected the response to selume-tinib.DDX43-expressing uvealmelanoma cellsweremoreresistant to selumetinib treatment compared with cellstransfected with an empty vector (V) in viability assays(Fig. 2B), suggesting that DDX43 may mediate mechan-isms of resistance to MEK inhibition through RAS andAKT activation.We also evaluated the effect of DDX43 depletion in the

MEK inhibitor–resistant cells by using two siRNAsequences (siDDX43-1, Fig. 2C; and siDDX43-2, Supple-mentary Fig. S3). Silencing of DDX43 corresponded todecreased protein expression of KRAS, NRAS, HRAS andthe downstream effectors pERK, pAKT, and c-Jun in bothresistant cell lines (Fig. 2C). DDX43 depletion did notchange KRAS or NRAS mRNA levels (SupplementaryFig. S4) as previously reported (29), suggesting thatDDX43 regulates RAS posttranscriptionally. The decreasein RAS protein levels induced by DDX43 depletion cor-

responded to a reduction in RAS activity in both MEKinhibitor–resistant cell lines (Fig. 2D). Most importantly,the depletion of DDX43 caused inhibition of cell viabilityof the MEK inhibitor–resistant cells, and this effect wasfurther enhanced by the treatment with selumetinib(Fig. 2E), suggesting that DDX43 is required for MEKinhibitor resistance.

To confirm that the effects of DDX43 depletion aremediated by RAS downregulation, each RAS proteinwas silenced by gene-specific siRNA. KRAS and HRASknockdown caused downregulation of pMEK, pERK,and pAKT in both cell lines (Fig. 3A), whereas NRASdid not. c-Jun was downregulated by KRAS and NRAS,whereas HRAS regulated expression of c-Jun only in Rs-Mel270. We have reported that GNAQ silencing inhibitspERK and pAKT, and decreases cell viability of theparental GNAQ-mutant uveal melanoma cells (31). Wealso tested the contribution of mutant GNAQ on down-stream signaling pathways in the resistant cells. GNAQdownregulation induced minimal effects on down-stream pathways (Fig. 3B). Similar to DDX43 down-regulation, KRAS and HRAS depletion decreased cellviability of the selumetinib-resistant cell lines, whereasNRAS had partial effects (Fig. 3C). GNAQ depletion did

Figure 3. RAS proteins regulate cell signaling in MEK inhibitor–resistant cells. A, selumetinib-resistant Rs-Omm1.3 (left) and Rs-Mel270 (right) cells weretransfectedwith siRNA for KRAS, HRAS, andNRAS and analyzed for downstreamsignaling, that is, pMEK, pERK, pAKT, and c-Jun by immunoblotting. B, thecellswere also transfectedwithGNAQsiRNAandanalyzed for downstreamsignaling.C,KRASandHRASare necessary for survival ofMEK inhibitor–resistantuvealmelanoma (UM) cells. Rs-Omm1.3 andRs-Mel270cells depletedof eachRASprotein orGNAQwere tested in cell viability assays after 72hours of siRNAtransfection. �, P < 0.0001 and ��, P < 0.001 for comparison of cells transfected with siKRAS and siHRAS versus control siRNA, for both cell lines.






not affect cell viability of Rs-Omm1.3 and Rs-Mel270cells (Fig. 3C), further demonstrating that RAS proteinsbecome the dominant mediators of cell signaling inthese cells.

AKT inhibition inhibits cell viability of selumetinib-resistant GNAQ-mutant cells

Elevated DDX43 and RAS expression mediated theactivation of downstream survival pathways, especiallypAKT (Figs. 1B and 2A). Thus, we assessed the effects ofAKT inhibition in uveal melanoma cell lines. The AKTinhibitor MK2206 (AKTi) inhibited AKT phosphoryla-tion in parental and MEK inhibitor–resistant cells (Fig.4A). In cell proliferation assays, AKT inhibition hadminimal effects in the parental cells, Omm1.3 andMel270 (Fig. 4B), while it greatly inhibited cell viabilityof the resistant cell lines, Rs-Omm1.3 and Rs-Mel270(Fig. 4B), suggesting that in these cells the DDX43/RAS-mediated activation of AKT is necessary for cell surviv-al, and its inhibition may be a means of overcomingMEK inhibitor resistance.

DDX43 is highly expressed in liver metastasis ofpatients with uveal melanoma

To determine the relevance of DDX43 expression inpatients with uveal melanoma, we assessed the expressionof DDX43 by RT-PCR in liver metastasis of patients withuveal melanoma carrying GNAQ or GNA11 mutations

who were treated on the phase II study of selumetinib.We analyzed biopsies from 14 patients, 6 of whom hadpartial response or stable disease for�16weeks, defined as"responders" (R) according to RECIST, and 8 patients whohad disease progression before 16 weeks with no evidenceof radiological response, defined as "nonresponders" (NR).The "responder" patients had low expression of DDX43(Fig. 5A). In contrast, DDX43 was highly expressed in the"nonresponder" group, and there was a statistically signif-icant associationwithpooroutcome (P¼ 0.045). The tissuesfrom six representative patients were also analyzed byimmunoblotting for protein expression. The patients whoresponded to therapy had complete inhibition of pERKwith selumetinib treatment, whereas the "nonresponders"showed none or minimal inhibition (Fig. 5B). The proteinlevels ofDDX43 inbiopsy tissueswere low in "responders,"whereas it was highly expressed in "nonresponders." Sim-ilarly, levels of total KRAS and NRASwere elevated in the"nonresponder" group, while HRAS was not detectable inthese specimens.

DiscussionThe RAS/RAF/MEK/ERK pathway is often activated

by genetic alterations in upstream signaling molecules,such as RAS and BRAF in cutaneous melanoma, andGNAQ/11 in uveal melanoma (4, 5). Hyperactivation of

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Figure 4. Inhibition of AKT decreases cell viability of MEK inhibitor–resistant cells. A, parental and selumetinib-resistant uveal melanoma(UM) cellswere treatedwith 1.5mmol/L ofMK2206 (AKTi) for 24 hours andanalyzed by immunoblotting for target inhibition of pAKT. B, parental andresistant cells were exposed to increasing concentrations of AKTi (0–5mmol/L) and analyzed in viability assays after 4 days of treatment. Graphshows the mean of three independent experiments � SD.

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Figure 5. DDX43 mRNA and protein levels are higher in liver biopsies of"responder" versus "nonresponder" patients. A, DDX43 expression wasanalyzed by RT-PCR in biopsies of 14 patients (6 "responders" and 8"nonresponders"). Triplicate values were normalized with GAPDH ashousekeeping gene, and reported as a box plot showing significantassociation with poor outcome in patients with uveal melanoma treatedwith selumetinib. B, liver biopsies from six representative patients (P)before (�) and after (þ) selumetinib treatment were analyzed byimmunoblotting with antibodies for pERK, DDX43, KRAS, NRAS,and tubulin.

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this signaling cascade results in dysregulated cell prolif-eration and malignant transformation. Targeting thispathway with MEK inhibitors has shown activity in dif-ferent types of cancers (39, 40), including uvealmelanoma(13). However, resistance to MEK inhibitors is commonand undermines the efficacy of these treatments (41, 42).Resistance to MEK inhibitors has been extensivelydescribed in cutaneus melanoma with BRAF and RASmutations (43). Also in colon carcinoma, MEK inhibitionleads to gene amplification of RAS and activation ofdownstream pathways (16). Activated RAS has beenlinked to MEK inhibitor resistance as it drives the activa-tion of both the PI3K-AKT and MEK-ERK pathways (37).Here, we found that in uveal melanoma cell lines, thesustained inhibition ofMEK leads to increased expressionof DDX43, and consequent induction of RAS and down-stream pathways. Furthermore, using RT-PCR, we foundthat DDX43 is overexpressed in patients who did notrespond to selumetinib treatment, confirming the in vitrofindings. Altered expression of DDX43 has been reportedin human cancers (27, 44). In particular, DDX43 wasrequired for cell proliferation of malignant melanoma-initiating cells by promoting the expression of NRASthrough RNA unwinding (29). However, this is the firsttime that DDX43 has been linked to MEK inhibitor resis-tance through the regulation of RAS. Interestingly, it wasreported that MEK inhibitor–resistant colon carcinomacells showed increased KRAS expression, which repre-sented a potential mechanism ofMEK inhibitor resistance(17). However, the modalities of KRAS protein inductionremained to be established.Recently, Lito and colleagues have reported that RAF

inhibitors cause relief of negative feedback with activationof RAS and rebound of ERK activity, without changes inRASexpression (35).Wehave showna similar activation ofRAS,whichwas inducedbyMEK inhibition in theparentalcells. However, this effect was sustained in uveal melano-ma selumetinib-resistant cells, where the expression ofboth DDX43 and RAS was elevated. DDX43 silencingdecreased RAS proteins and downstream signaling, i.e.,pERK and pAKT, making DDX43 a mediator of MEKresistance that could represent a class effect to all MEKinhibitors. Little and colleagues reported an increase in c-Jun in colorectal cancer cells with acquired resistance toselumetinib (16). Indeed, c-Jun was highly expressed inMEK inhibitor–resistant cells, and it could be downregu-lated by knockdown of DDX43 and RAS, but not GNAQ,further confirming the importance of RAS signaling inthese cells.In our cells, DDX43 mediated "acquired" resistance to

the MEK inhibitor, as its expression increased after

long-term treatments. However, DDX43 seemed tomediate intrinsic resistance to selumetinib in patientswith uveal melanoma with elevated expression at base-line. It was reported that DDX43 is highly expressed inchronic myeloid leukemia due to gene demethylation(22), and correlated with poorer prognosis in terms ofcytogenetic response to drug treatments. Similarchanges in DDX43 gene methylation may occur in uvealmelanoma.

DDX43 has been also identified as a tumor-specific geneexpressed in sarcoma (20), and it has been associatedwithpoor clinical outcome in patients with breast cancer (45).Recently, several studies have provided new insights onRNA helicases and their emerging roles in cell signaling.For example, DDX3, another member of this family, wasfound to regulate the Wnt–b-catenin network in agenome-wide siRNA screen in human embryonic kidneycells (46). Another report showed DDX5 as a regulator ofalternative splicing of HRAS (47).

Our results provide evidence that DDX43 increased theexpression and activation of RAS in uveal melanoma cellswith GNAQ mutation, and mediated resistance to MEKinhibitors.

Finally, these findings would indicate that DDX43 mayhave predictive value in determining who will mostbenefit from a MEK inhibitor therapy in this disease.

Disclosure of Potential Conflicts of InterestR.D. Carvajal is a consultant/advisory boardmember for AstraZeneca.

No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: G. Ambrosini, R.D. Carvajal, G.K. SchwartzDevelopment of methodology: G. Ambrosini, G.K. SchwartzAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.D. Carvajal, G.K. SchwartzAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): G. Ambrosini, R. Khanin, R.D. Carvajal, G.K. SchwartzWriting, review, and/or revision of the manuscript: G. Ambrosini, R.D.Carvajal, G.K. SchwartzAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): G. Ambrosini, R.D. Carvajal, G.K.SchwartzStudy supervision: G. Ambrosini, R.D. Carvajal, G.K. Schwartz

Grant SupportG. Ambrosini, Cycle for Survival (Philanthropy).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 February 3, 2014; revised May 5, 2014; accepted May 16, 2014;published OnlineFirst June 4, 2014.

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Ambrosini et al.




Published OnlineFirst June 4, 2014.Mol Cancer Ther Grazia Ambrosini, Raya Khanin, Richard D. Carvajal, et al. CellsResistance through RAS Upregulation in Uveal Melanoma Overexpression of DDX43 Mediates MEK Inhibitor

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