ProteinKinaseCInhibitorAEB071TargetsOcularMelanoma...

Therapeutic Discovery

Protein Kinase C Inhibitor AEB071 Targets Ocular MelanomaHarboring GNAQ Mutations via Effects on the PKC/Erk1/2and PKC/NF-kB Pathways

Xinqi Wu1,2, Jingjing Li1,2, Meijun Zhu3, Jonathan A. Fletcher3, and F. Stephen Hodi1,2

AbstractSomatic GNAQ mutations at codon 209 have been identified in approximately 50% of uveal melanomas

and have been reported to be oncogenic through activating PLCb/PKC/Erk1/2 pathways. We hypoth-

esized that protein kinase C (PKC) may provide new opportunities for therapeutic targeting of uveal

melanoma carrying GNAQ mutations. To test this hypothesis, uveal melanoma cells harboring wild-type

or mutant GNAQ were treated with the PKC inhibitor AEB071 (sotrastaurin) or infected with lentivirus-

expressing short hairpin RNAs (shRNA) targeting PKC isoforms. Notably, AEB071 at low micromolar

concentrations significantly inhibited the growth of uveal melanoma cells harboring GNAQ mutations

through induction of G1 arrest and apoptosis. However, AEB071 had little effect on uveal melanoma cells

carrying wild-type GNAQ. AEB071-mediated cell inhibition in the GNAQ-mutated uveal melanoma was

accompanied by inhibition of extracellular signal–regulated kinase (Erk)1/2 phosphorylation, NF-kB,decreased expression of cyclin D1, survivin, Bcl-xL, and XIAP, and increased expression of cyclin-

dependent kinase inhibitor p27Kip1. AEB071 suppressed the expression of PKC a, b, d, e, and q in

GNAQ-mutated uveal melanoma cells. Our findings from shRNA-mediated knockdown studies revealed

that these PKC isoforms are functionally important for uveal melanoma cells harboring GNAQ mutations.

Furthermore, inhibitors of Erk1/2 and NF-kB pathways reduced viability of uveal melanoma cells.

Together, our findings show that AEB071 exerts antitumor action on uveal melanoma cells carrying

GNAQ mutations via targeting PKC/Erk1/2 and PKC/NF-kB pathways. Targeted PKC inhibition with

drugs such as AEB071 offers novel therapeutic potential for uveal melanoma harboring GNAQ mutations.

Mol Cancer Ther; 11(9); 1905–14. �2012 AACR.

IntroductionUveal melanoma is the most common primary intraoc-

ular malignant tumor in adults, with an incidence of 7cases per million annually (1). Approximately half ofpatients with uveal melanoma develop metastases to theliver within 15 years of initial diagnosis. With no effectivetreatment modality available the median survival time ofpatients with uveal melanomawithmetastasis is less than6 months (2).The etiology of uveal melanoma has not been fully

understood. Mutations in the GNAQ gene have been

identified in approximately 50% of uveal melanoma and83% blue nevi (3–5). The GNAQ gene encodes for the asubunit of q class of heterotrimeric GTP-binding proteins(G proteins) that are composed of 3 subunits (Ga, Gb,and Gg) and transduce signals from 7-transmembraneG-protein–coupled receptors (GPCR) to intracellular cas-cades (6). Activation ofGPCRs results in exchange ofGDPfor GTP on the Ga subunit, resulting in the dissociation ofthe GTP bound form of Ga from Gbg . Both Ga and Gbgcan then activate downstream cellular signaling path-ways. The signal is terminated when GTP is hydrolyzedto GDP by the intrinsic GTPase activity of the Ga subunit.The majority of GNAQ mutations occur at codon 209within the GTPase catalytic domain, resulting in loss ofthe intrinsic GTPase activity and constitutively activationof GNAQ. Expression of mutated GNAQ results in mela-nocyte transformation and increased extracellular signal–regulated kinase (Erk)1/2 phosphorylation, indicatingthat mutant GNAQ behaves as a dominant acting onco-gene (4, 5). Currently, there are no available therapiestargeting GNAQ.

Theprotein kinaseC (PKC) family is awidely expressedgroup of serine/threonine kinases comprising multiple

Authors' Affiliations: 1Department of Medical Oncology, 2MelanomaDiseaseCenter, Dana-FarberCancer Institute andHarvardMedical School;and 3Department of Pathology, Brigham and Women's Hospital andHarvard Medical School, Boston, Massachusetts

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

Corresponding Author: F. Stephen Hodi, Dana-Farber Cancer Institute,450 Brookline Avenue, Boston, MA 02215. Phone: 617-632-5053; Fax:617-582-7992; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-0121

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on May 22, 2018. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst May 31, 2012; DOI: 10.1158/1535-7163.MCT-12-0121

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isoforms that can be divided into 3 structurally andfunctionally distinct subgroups (7, 8). These are theconventional PKCs (PKCa, PKCb, and PKCg), whichare activated by diacylglycerol (DAG) and phospholip-id and are Ca2þ dependent; the novel PKCs (PKCd,PKCe, PKCq, and PKCh), which are also activatedby DAG and phospholipids, but are not Ca2þ depen-dent; and the atypical PKCs, which do not require DAGor Ca2þ for activation. PKCs regulate key biologicprocesses including cell proliferation, apoptosis, differ-entiation, angiogenesis, tumor development, andchemoresistance (7, 9–15). PKCs are involved inGNAQ-mediated activation of the MAPK/Erk1/2 path-ways (6, 16). It has been known that GNAQ transducessignals from GPCRs to phospholipase Cb (PLCb; ref. 6).PLCb enzymes catalyze the hydrolysis of phosphatidy-linositol biphosphate to release inositol trisphosphateand DAG that function as second messengers propa-gating and amplifying the Ga-mediated signal throughactivation of PKCs. Active PKCs further activate Erk1/2through the RAF/MAPK/Erk1/2 pathway (16). Usingshort hairpin RNA (shRNA)-mediated downregulationof PKC isoforms b, e, and qwe have recently shown thatthese isoforms are functionally important for GNAQ-mutated uveal melanoma cells (17). The oncogenicproperties of mutant GNAQ and the importantPKC roles in GNAQ-mediated Erk1/2 activation andGNAQ-mutated uveal melanoma cells (4, 16) suggestedthat PKC may provide new opportunities for therapeu-tic intervention of uveal melanoma carrying GNAQmutations. To test this hypothesis, uveal melanoma cellscarrying wild-type GNAQ or GNAQ mutated at codon209 were treated with the PKC inhibitor AEB071 (sotras-taurin), a PKC inhibitor that has potent activity againstclassical and novel PKC isotypes (18). AEB071 selec-tively inhibited the growth of uveal melanoma cellsharboring GNAQ mutations by targeting PKC/Erk1/2and PKC/NF-kB pathways.

Materials and MethodsCell lines

The sources and GNAQ mutational status of uvealmelanoma cell lines C918, Ocm1, Ocm3, Mel285, Mel202,92.1, and Omm1.3 have been described previously (19).Uveal melanoma cells were cultured in RPMI-1640 con-taining 10% FBS, 50 mg/mL penicillin, and 100 mg/mLstreptomycin at 37�C and 5% CO2. These cell lines wererecently authenticated by short tandem repeat PCR anal-ysis at Biosynthesis Inc. Human epidermal melanocyteswere purchased fromLifeline Cell Technology andgrownin the medium provided by the company.

Viability assayCells were seeded in 96-well plates at 2 � 103 cells per

well and incubated over night followed by treatmentwithAEB071 (provided by Novartis) for 3 days. Cell viabilitywas measured as previously described (17).

Cell-cycle analysisCells were collected by trypsinization and fixed in cold

ethanol. After incubation with RNase A and PI, cell-cycledistribution was determined by flow cytometric analysis(fluorescence-activated cell sorting, FACS).

Analysis of apoptosisApoptotic cellswere detectedbyAnnexinV-fluorescein

isothiocyanate staining and FACS as described pre-viously (17). Compensation for AEB071 autofluorescencewas conducted.

Knockdown of PKC isotypes by shRNAThe constructs (pLKO.1-puro) containing shRNA tar-

get sequences for PKCa, PKCd, or GFP were providedby Dana-Farber Cancer Institute shRNA Core Facility.Lentivirus expressing PKC shRNA was produced asdescribe previously (17). Cells were infected with virusfor 3 days and cell viability was determined using MTSassay.

ImmunoblottingPreparation of whole-cell lysates and immunoblotting

have been described previously (19). Antibodies againstPKC isoforms were: PKCa (Cell Signaling #2056), PKCd(Cell Signaling #2058), PKCq (BD Biosciences #610090);PKCq Thr538 (Cell Signaling #9377), PKCq/d Ser643/676(Cell Signaling #9376), and PKCe (BD Biosciences#610085). Antibodies against Akt, phospho-Akt, Erk1/2,phospho-Erk1/2, cyclin D1, Bcl-xL, XIAP, survivin,HADC1, and glyceraldehyde-3-phosphate dehydroge-nase (GAPDH)were purchased fromCell Signaling Tech-nology. Antibodies against PKCbII, p27Kip1, and RelA(p65) were purchased from Santa Cruz Biotechnology.Actin antibody was purchased from Sigma-Aldrich. Pro-tein signal intensity was measured using NIH ImageJsoftware and normalized to that of actin.

Nuclear extract preparation and electrophoresismobility shift assay

Nuclear extraction and NF-kB electrophoresis mobilityshift assay (EMSA) kits purchased from Signosis wereused to isolate nuclear extracts fromuvealmelanoma cellsand conduct EMSA. The instructions provided by themanufacturer were followed.

Measurement of interleukin-6 in cell culturemediumCellswere treatedwith dimethyl sulfoxide (DMSO) or 5

mmol/L AEB071 for 72 hours in 6-well plates. Mediumwas collected and centrifuged to remove cells. Interleukin(IL)-6 in the supernatant was determined using theHuman IL-6 Quantikine ELISA Kit purchased from R&DSystems.

Statistical analysisData are presented as mean� SD. Differences between

treated and control groups was analyzed using Student ttest and considered significant with P � 0.05.

Wu et al.

Mol Cancer Ther; 11(9) September 2012 Molecular Cancer Therapeutics1906




ResultsThe PKC inhibitor AEB071 selectively inhibitsgrowth of uveal melanoma cells harboring GNAQmutationsWe first evaluated the effect of AEB071 (Fig. 1A)

on viability of a panel of 7 uveal melanoma cell lines(Table 1). Sequencing analysis confirmed that GNAQ iswild-type in cell lines C918, Ocm1, Ocm3, and Mel285,whereas codon 209 of GNAQ is mutated from CAA(glutamine) to CTA (leucine) in cell lines Mel202 and92.1 and to CCA (proline) in cell line Omm1.3. Cell linesOcm1 and Ocm3 have BRAF V600E mutation (19, 20).AEB071 at low micromolar concentrations significantlydecreased viability of all 3 GNAQ-mutated cell lines(Fig. 1B). The IC50 value of AEB071 was approximately0.8, 3, and 4 mmol/L for 92.1, Omm1.3, and Mel202cells respectively. AEB071 had little effect on viabilityof 3 GNAQ wild-type cells up to 10 mmol/L (Fig. 1Band Supplementary Fig. S1). In addition, the viability ofhuman epidermal melanocytes was not reduced byAEB071 (Fig. 1B) and may have been modestlyincreased by the inhibitor. Along with decreased via-bility, microscopic examination found morphologicalterations in AEB071-treated cells harboring GNAQmutations but not wild-type GNAQ or normal melano-cytes (Fig. 1C). Treated Mel202, 92.1, and Omm1.3 cellslost spindle shape and became flattened with increasedsize or round floating dead cells with condensedcytoplasm.

AEB071 induces G1 arrest in GNAQ-mutated uvealmelanoma cells

To better understand growth inhibitory effect ofAEB071 on GNAQ-mutated cells, we investigated wheth-er cell-cycle progression was altered by drug exposure.AEB071 markedly increased the G1 phase populationwhereas decreasing the S-phase population in uveal mel-anoma cells harboring GNAQmutations (Fig. 2A and B).There was no significant change in the cell-cycle patternfor cell lines carrying wild-type GNAQ (C918, Mel285,and Ocm3). A decrease in the S-phase population with aconcomitant increase in the G2–M phase population wasobserved in Ocm1 cells. In agreement with this G1 arrest,AEB071 also significantly increased the accumulation ofp27Kip1,while decreasing the expression of cylinD1 in all 3GNAQ-mutated cell lines tested (Fig. 2C). In comparison,the expression of p27Kip1 and cyclin D1 was not signifi-cantly altered by AEB071 in GNAQwild-type cells. Thesefindings suggest that AEB071 selectively induced G1

arrest in GNAQ-mutated cells through altering theexpression of regulators critical for the G1 to S transition.

AEB071 induces apoptosis in GNAQ-mutated uvealmelanoma cells

We next examined whether AEB071 promotedapoptosis in uveal melanoma cells. Treatment with 2 and5 mmol/L AEB071 for 72 hours significantly increasedAnnexin V–positive (apoptotic) populations in GNAQ-mutated 92.1 and Omm1.3 cells (Fig. 3A). Further

Figure 1. Effect of AEB071 onGNAQ wild-type and mutated uvealmelanoma cells. A, molecularstructure of AEB071 (sotrastaurin;adapted from ref. 43). B, AEB071selectively reduced viability of uvealmelanoma cells harboring GNAQmutations. Cells were treated withvarying amount of AEB071 for 72hours. Data are presented asmean � SD of 4 or 5 independentexperiments. NM, normalmelanocytes; WT, GNAQ wild-type;MT, GNAQ mutation. C, AEB071caused morphologic alterations inGNAQ-mutated cells. After AEB071treatment for 3 days, Mel202, 92.1,and Omm1.3 cells lost spindle shapeand became flattened with increasedsize or round floating dead cells withcondensed cytoplasm. Themorphology of GNAQ wild-typeuvealmelanomacellswas not alteredby AEB071.

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Protein Kinase C Inhibition in Uveal Melanoma

www.aacrjournals.org Mol Cancer Ther; 11(9) September 2012 1907




demonstration of apoptosis was observed with caspase-3cleavage in treated cells (Fig. 3B). For Mel202 cells, only aminimal increase in the Annexin V–positive cell popula-tion was observed after AEB071 treatment (Fig. 3A), butsignificant caspase-3 cleavage was induced (Fig. 3B). Incontrast, AEB071 did not increaseAnnexinV–positive cellpopulations or caspase-3 cleavage in uveal melanomacells harboring wild-type GNAQ (Fig. 3A and B). AEB071further inhibited the expression of the antiapoptotic pro-teins survivin, Bcl-xL and XIAP, in a dose-dependentmanner in Mel202, 92.1, and Omm1.3 cells (Fig. 3C).Under the same conditions, survivin, Bcl-xL, and XIAPwere not affected or were modestly increased in GNAQwild-type uveal melanoma cells (Fig. 3C). These resultsindicate that AEB071 selectively induced apoptosis inuveal melanoma cells harboring GNAQ mutations.

AEB071 inhibits expression and phosphorylation ofPKC isoforms in uveal melanoma cells

To better understand differential responses of uvealmelanoma cells to AEB071 based on GNAQ mutational

status, we examined inhibition of PKC isoforms byAEB071 in uveal melanoma cells. Immunoblottingshowed that treatment with AEB071 for 6 hours in theabsence of serum resulted in decreased PKCd/qSer643/676

phosphorylation in Mel202, 92.1, and C918 cells andPKCqThr538 phosphorylation in Mel202 cells (Fig. 4A). Ithas been reported that expression of PKC isoforms can bedownregulated byPKC inhibition (21).We thus examinedthe expression of PKC isoforms after prolonged AEB071treatment (5 mmol/L for 24 hours) in the presence of 10%FBS.Uvealmelanoma cell lines showedvarying reductioninPKCexpression afterAEB071 treatment (Fig. 4B). PKCawas reduced in some wild-type (C918 and Ocm1) and inGNAQ-mutated (92.1 and Omm1.3) cells, whereas PKCbwas reduced only in mutated cells (Mel202 and 92.1).PKCq was decreased in all 3 GNAQ mutated but only inone wild-type (Ocm3) cells. PKCe was downregulated inall 3 GNAQ wild-type cell lines and 2 mutated cell lines(Mel202 and 92.1). These findings suggest that AEB071may have greater overall inhibitory effect on multiplePKC isoforms in uveal melanoma cells with mutatedGNAQ: PKCa, b, d, e, and/or q expression was sup-pressed by AEB071 in GNAQ-mutated cells whereasPKCa, and PKCd and PKCe expression was affected inGNAQ wild-type cells.

PKC isoforms are functionally important for uvealmelanoma cells harboring GNAQ mutations

As the expression of PKCa and d was suppressed byAEB071 in both GNAQ wild-type and mutated uvealmelanoma cells, we next investigated whether theseisoforms were of functional importance for uveal

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Figure 2. AEB071 induces G1 arrestin uveal melanoma cells harboringGNAQ mutations. A, uvealmelanoma cells were treated withDMSO or 5 mmol/L AEB071 for 24hours and subjected to cell-cycleanalysis. B, bar graphs ofpercentages of G1, S, and G2–Mpopulations. C, AEB071 selectivelyincreased p27 and decreasedcyclin D1 expression in GNAQmutant uvealmelanomacells. Cellswere treated with 0, 2, or 5 mmol/LAEB071 for 72 hours and analyzedby immunoblot. WT, GNAQ wild-type; MT, GNAQ mutation.

Table 1. Uveal melanoma cell lines used andtheir GNAQ mutational status

GNAQ Codon 209 Cell lines

Wild-type CAA (Q) C918, Mel285,Ocm1, Ocm3

Mutation CTA (L) Mel202, 92.1CCA (P) Omm1.3

Wu et al.





melanomacells by shRNA-mediated knockdown to betterunderstand the mechanisms for the differential AEB071response in GNAQ wild-type and mutated cells. PKCaand PKCd expression was significantly downregulatedby their shRNA in C918 and Mel202 cells (Fig. 4C). Inter-estingly, knockdown of PKCa or PKCd significantlydecreasedviability ofMel202 andOmm1.3 cells, but failedto significantly decrease the viability of C918 cells(Fig. 4D), indicating that PKCa and PKCd are functionallyimportant in uveal melanoma cells harboring GNAQmutations. We have previously found that PKC isoformsb, e, and q are functionally critical for GNAQ-mutateduvealmelanomacells (17).Thesefindings togethersuggestthat AEB071 suppressed growth of GNAQ-mutated uvealmelanoma cells via inhibition of multiple PKC isoforms.

AEB071 selectively decreases Erk1/2phosphorylation in uveal melanoma cellsharboring GNAQ mutationsTo further define the molecular mechanisms underly-

ing the antiproliferative action of AEB071, we nextassessed downstream effectors of PKC-mediated path-ways thatwere potentially affected byAEB071. It has beenpreviously reported that PKC can activate the phosphoi-nositide 3-kinase (PI3K)/Akt and mitogen-activated pro-tein kinase (MAPK) pathways as well as GSK3bSer9 phos-phorylation. The PKC inhibitor enzastaurin inhibits Aktand GSK3bSer9 phosphorylation in some types of cancercells (21–26). Similarly, AEB071 decreased GSK3bSer9

phsophorylation in all uveal melanoma cell lines studiedhere (Supplementary Fig. S2). However, AEB071 onlysignificantly inhibited Erk1/2 phosphorylation in GNAQ

mutant cells whereas it had minimal effect on Akt phos-phorylation in both GNAQ wild-type and mutated cells(Fig. 5). Total Akt and Erk1/2 levelswere not significantlyaltered byAEB071 in any of the cell lines examined. Thesefindings showAEB071-induced selective inhibition of thePKC/MAPK pathway in uveal melanoma cells carryingGNAQ mutations. We have found that Erk1/2 inhibitiondecreased uveal melanoma cell viability (19). Therefore,AEB071 may exert its antiproliferative effects in partthrough suppression of Erk1/2 activation in GNAQ-mutated uveal melanoma cells.

AEB071 selectively inhibitsNF-kBactivity inGNAQ-mutated uveal melanoma cells

PKC isoforms are involved in GPCR-mediated NF-kBactivation (27–30). In most cells, NF-kB is present as alatent, inactive, IkB-bound complex in the cytoplasm. Onactivation, IkB is phosphorylated and targeted to degra-dation. The released NF-kB translocates into the nucleuswhere it drives the expression of its target genes (29). It iswell known that aberrantNF-kB activity promotes tumor-igenesis and metastasis (31). We next examined whetherAEB071 alteredNF-kBactivity inuvealmelanomacells. Inthe absence of AEB071, nuclear p65 (RelA) levels aremore or less similar among wild-type and mutant celllines (Fig. 6A). In contrast, in the presence of AEB071,nuclear p65 levelswere significantly decreased inGNAQ-mutated uveal melanoma cells whereas they were mini-mally altered in wild-type cells (Fig. 6A). Consistent withthis, reduced DNA binding activity of NF-kB wasdetected only in the nuclear extracts of GNAQ-mutatedcells exposed to AEB071 (Fig. 6B). In agreement with

Figure 3. AEB071 inducedapoptosis in uveal melanoma cellsharboring GNAQ mutations. A, cellswere treated with DMSO or 5 mmol/LAEB071 for 72 hours. Apoptotic cellswere detected using Annexin V-FITCstaining and FACS analysis. B, cellswere treated with DMSO or 5 mmol/LAEB071 for 72 hours and subjectedto immunoblot analysis of cleavedcaspase-3. Increased caspase-3cleavage was observed in GNAQ-mutated uveal melanoma cells. C,AEB071 decreased the expression ofantiapoptotic proteins Bcl-xL, XIAP,and survivin in GNAQ-mutated cells.WT, GNAQ wild-type; MT, GNAQmutation.

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decreased NF-kB activity, elevated IkBa levels weredetected in AEB071-treated GNAQ-mutated cells (Fig.6C). Besides, the secretion of IL-6, one of the target genesof NF-kB, was substantially inhibited by AEB071 inGNAQ-mutated Omm1.3 cells but not GNAQ wild-typeC918 cells (Fig. 6D). These findings together indicate thatAEB071 selectively inhibited NF-kB activation in uvealmelanoma cells harboring GNAQmutations. This notionis also in agreement with the selective downregulation of

NF-kB target genes including survivin, Bcl-xL, XIAP, andcyclin D1, by AEB071 in GNAQ-mutated cells (Figs. 2Cand 3C).

NF-kB activity is functionally important for uvealmelanoma cells

To determine whether NF-kB activity is functionallyimportant for uveal melanoma cells, both GNAQ wild-type andmutated uvealmelanoma cellswere treatedwithan inhibitor of IkB kinase (IKK)1/2 inhibitor that pos-sesses potent NF-kB inhibition activity (32). This treat-ment resulted in the dramatic decrease in viability of bothGNAQ wild-type and mutant uveal melanoma cell lines(Supplementary Fig. S3). These findings suggest that NF-kB activity is critically important for uvealmelanoma cellsand its suppression contributes to AEB071-inducedgrowth inhibition of uveal melanoma cells harboringGNAQ mutations.

DiscussionThere are currently no drugs available for targeting the

oncogenic GNAQ mutations that occur frequently in

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Figure 4. PKCa and PKCd are functionally important for uveal melanoma cells harboring GNAQ mutations. A, effect of AEB071 on phosphorylationof PKC isoforms in uveal melanoma cells. Cells were treated with 5 mmol/L AEB071 in the absence of serum and analyzed for PKC phosphorylation. B,AEB071 inhibitedPKCexpression in uvealmelanomacells. Cellswere treatedwith 5mmol/LAEB071 for 24hours in thepresenceof 10%FBSandsubjected toimmunoblot analysis. PKCd, e, and q levels in AEB-treated cells relative to control (DMSO-treated) cells are also shown. Relative levels (fold changes)are presented as mean � SD of 3 to 4 experiments. Except PKCq in C918 and Ocm1 cells and PKCe in Omm1.3 cells the difference between AEB-treatedand control cells was statistically significant (Student t test, P < 0.05 or 0.01). C, PKCa and PKCd were downregulated by shRNA-mediatedknockdown. C918 and Mel202 cells were infected with lentivirus expressing shRNA for PKCa or PKCd for 4 days and protein levels of these isoforms weredetermined by immunoblot analyses. D, knockdown of PKCa and PKCd significantly reduced viability of Mel202 and Omm1.3 cells. Cell viability wasdetermined 4 days after infection with lentivirus expressing PKC shRNA using MTS assay. Results are presented as mean � SD of percent viabilityfrom 3 independent experiments. #, P < 0.01 versus cells expressing control GFP shRNA.

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Figure 5. AEB071 selectively inhibited Erk1/2 phosphorylation in GNAQ-mutated uveal melanoma cells. Cells were treated with AEB071 for 72hours and analyzed for the expression of Erk1/2 and Akt and theirphosphorylation by immunoblot analyses. WT, GNAQ wild-type; MT,GNAQ mutation.

Wu et al.





primary and metastatic uveal melanoma. In the presentstudy, we describe the first small-molecule inhibitor thatselectively exhibits antiproliferative activity of uveal mel-anoma cells harboring GNAQ mutations: the novel PKCinhibitor AEB071 reduced viability of GNAQ-mutateduveal melanoma cell lines, but had little effect on thosecarryingwild-typeGNAQ.AEB071-induced growth inhi-bition is associated with reduced expression of PKC iso-forms a, b, d, e, and/or q, accompanied by inhibition ofErk1/2 phosphorylation, and NF-kB activation. We havepreviously shown that PKCq, PKCb, and PKCe are func-tionally important for GNAQ-mutated uveal melanomacells and that inhibition of Erk1/2 by MAP–ERK kinase(MEK)1/2 inhibitors reduced uveal melanoma cell via-bility (17). Here, we show that PKCa, PKCd, and NF-kBare also functionally important for GNAQ-mutated uvealmelanoma cells. Together, our findings suggest thatAEB071may selectively exert antiproliferative activity onGNAQ-mutated uveal melanoma cells via targeting thePKC/Erk1/2 and PKC/NF-kB pathways.AEB071-inducedgrowth suppression ofGNAQmutant

cells is associated with pronounced G1 arrest and induc-tion of apoptosis. Themolecularmechanisms forAEB071-induced G1 arrest involve altered expression of positiveand negative regulators of transition through G1 phase ofcell cycle, including cyclin D1 and p27Kip1 (33, 34).AEB071-induced apoptosis is associated with decreasedexpression of antiapoptotic proteins, yet the underlyingmolecular mechanisms remain to be revealed.

The PI3K/Akt and MAPK pathways are frequentlyactivated in malignant tumors and are critical for cancercell survival and proliferation (35–37). Erk1/2 activationis common in uveal melanoma and has been reportedto play a crucial role in uveal melanoma development(38, 39). AEB071 inhibited Erk1/2 phosphorylation inGNAQ mutated but not GNAQ wild-type uveal melano-ma cells, and hadminimal impact onAkt phophorylation.Similarly, we have recently found that PKC inhibitorenzastaurin inhibits phosphorylation of Erk1/2 but notAkt in GNAQ-mutated cell lines (17), although enzas-taurin has been reported to inhibit Akt phosphorylationbut not Erk1/2 phosphorylation in other types of cancercells (24, 26, 40). The inhibition of Erk1/2phosphorylationis therefore likely a common mechanism for the antipro-liferative action of PKC inhibitors in GNAQ-mutateduveal melanoma cells. Further studies are needed toidentify the PKC isoform(s) that are most crucial forErk1/2 phosphorylation in GNAQmutant uveal melano-ma, and whose inhibition by AEB071 leads to decreasedErk1/2 phosphorylation. PKCbII and PKCq are amongthe candidates, because activation of these isoformstriggers several signaling pathways including MAPK(9, 13, 17, 41), and siRNA downregulation of PKCbIIdecreased Erk1/2 phosphorylation in metastatic hepato-cellular carcinoma cells (42).

In addition to the PKC/Erk1/2 pathway, activation ofmany GPCRs can trigger the PKC/IKK/NF-kB pathwaythrough G proteins, including GNAQ (27–30, 43).

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Figure 6. AEB071 selectively inhibited NF-kB activation in uveal melanoma cells harboring GNAQ mutations. A, immunoblot analysis of nuclear RelA (p65)levels of uveal melanoma cells treated with 0 or 5 mmol/L AEB071 for 40 hours. HDAC1 (histone deacetylase 1) was used as loading control for nuclearextracts (56). B, EMSA analysis of NF-kB DNA-binding activity in the same nuclear extracts used in A. NF-kB/DNA complexes were determined asdescribed inMaterials andMethods. C, AEB071 increased IkBa protein levels in uveal melanoma cells with mutated GNAQ. Cells were treated with DMSO or5 mmol/L AEB071 for 20 hours and subjected to immunoblot analysis of IkBa. D, AEB071 reduced IL-6 secretion from Omm1.3 cells. Cells weretreated with DMSO or 5 mmol/L AEB071 for 72 hours. Medium was collected for ELISA analysis of IL-6. Data are presented as mean � SD of percentsecretion of 3 independent experiments. �, P < 0.05.






Overexpression of mutant GNAQ(Q209L) leads to con-stitutive activation of NF-kB, which is mediated by PKCdand to a lesser extent PKCa and PKCe in human umbilicalvein endothelial cell (HUVEC; refs. 27, 28). It is not knownwhether the same PKC isoforms have comparable roles inuveal melanoma cells. Importantly, we show that AEB071selectively repressedNF-kBactivation inuvealmelanomacells harboringGNAQmutations and this is accompaniedby downregulation of multiple PKC isoforms, in partic-ular PKCa, PKCd, and PKCe. The downregulation ofthese PKC isoforms may contribute to AEB071 suppres-sion of NF-kB activity. Comparison studies using an IKKinhibitor corroborate that the NF-kB pathway has criticalfunctional roles inuvealmelanoma cells.NF-kB inhibitionmight therefore be another mechanism for the antiproli-ferative action of AEB071 on uveal melanoma cells har-boring GNAQ mutations. The association between PKCinhibition and decreasedNF-kB activity in GNAQ-mutat-ed uveal melanoma cells suggests that these cells mightrely on GNAQ/PKC/NF-kB pathways for NF-kB activa-tion. Other pathways presumably regulate NF-kB activa-tion in GNAQ wild-type cells. For example, Ocm1 andOcm3 cells have been shown to carry the common V600EBRAF mutation that constitutively activates the MAPKand NF-kB pathways (19, 20, 44–46). High c-Met expres-sion has been found in C918 and Mel285 cells (47) and ithas been reported that NF-kB can be activated by hepa-tocyte growth factor (HGF)/c-Met signaling (48, 49).

Our data indicate thatmultiple PKC isoforms includinga, b, d, e, and q are suppressed by AEB071 in GNAQ-mutated uveal melanoma cells whereas only PKCa andPKCd were affected in GNAQ wild-type cells. The find-ings from shRNA knockdown studies confirmed thefunctional importance of PKCa and PKCd in GNAQ-mutated uveal melanoma cells. Knockdown of these 2PKC isoforms had no (PKCa) or little (PKCd) affect onviability of GNAQwild-type cells, suggesting that these 2PKC isoforms are less important in GNAQwild-type cellsthan in GNAQ-mutated cells. Similarly, our previousshRNA-mediated knockdown studies have found thatPKCb, PKCe, and PKCq are functionally more importantfor GNAQ-mutated than wild-type uveal melanoma cells(17). Together, these findings suggest that GNAQ-mutat-ed uveal melanoma cells are more dependent on thesePKC isoforms than GNAQ wild-type cells and thatAEB071 exerts antiproliferative action onGNAQ-mutatedcells via suppression of these PKC isoforms. These find-ings also provide a plausible explanation for the differ-ential response/sensitivity of GNAQ wild-type andmutated uveal melanoma cells to AEB071.

GPCRs have a pivotal role in many physiologic func-tions and in multiple diseases, including tumorigenesisand metastasis of cancers (50). Frequent somatic muta-tions in GPCRs have been found in melanoma and muta-tions in GRM3, which is a glutamate receptor and amember of the metabolic GPCRs, activate the MAPK/ERK pathway, and promote growth and migration ofmelanoma cells (51). Furthermore, overexpression of

diverse GPCRs were found in number of primary andmetastatic cancers including melanoma, breast, prostate,non–small cell lung cancer, gastric tumors, head and necksquamous cell carcinoma, and diffused large B-cell lym-phoma (52, 53). GPCRs can also influence cancer progres-sion through cross-talk with growth factor receptors suchas EGFR and insulin-like growth factor-1R, G-proteins(such as Gaq/11 and Ga12/13), chemokines, HedgehogandWNT signaling pathways, regulation of the apoptoticresponse and viral factors. Various GPCRs can beinvolved in cancer through activating NF-kB (50). Asmore data linking GPCRs to cancer emerge, these recep-tors are attractive potential targets for tumor therapy. Inparticular, the GPCR/ERK and GPCR/NF-kB pathwayscould be valuable targets for innovative anticancer drugdiscovery.

We show that PKC inhibitor AEB071 (sotrastaurin) atlowmicromolar concentrations exerts significant antipro-liferative effect on GNAQ-mutated uveal melanoma cellsthrough targeting the PKC/MAPK and PKC/NF-kBpathways. Our findings support PKCs as important tar-gets for therapeutic intervention of uveal melanoma har-boring GNAQ mutations. Clinical studies have shownthat a blood concentration of 4 mmol/L was achievedwithin 5 hours after an oral dose of 500 mg AEB071(18). AEB071 could thus be of therapeutic potential foruveal melanoma with GNAQ mutations. A clinical trialwithAEB071 inpatientswithuvealmelanoma is currentlyenrolling patients. AEB071 has primarily been studied asan anti-inflammatory agent for the treatment of diseasessuch as psoriasis and in preventing solid organ rejectionfollowing transplant (54, 55). How the immunosuppres-sive effects of this drug may influence the clinical efficacywill have to be determined, and monitoring of changes inantitumor immune responses as a function of treatmentshould be helpful to discern such changes. Given thatpreclinical models for uveal melanoma are difficult andmostly comprise xenograft studies, much of these ques-tions will likely be answered by clinical investigation.There may be future opportunities to combine AEB071or other PKC inhibitors with other rational small-mole-cule inhibitors (e.g., MEK or PI3K) or immune therapysuch as ipilimumab to improve the efficacy and durabilityof any clinical activity. It would be also of great interest toinvestigate antitumor effects of other specific PKC inhi-bitors in melanoma and other cancers with GPCR muta-tions that may activate MAPK/ERK and/or NF-kBpathways.

Disclosure of Potential Conflicts of InterestF.S. Hodi has served as a nonpaid consultant to Novartis and received

clinical trial support from Novartis. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: X. Wu, F.S. HodiDevelopment of methodology: X. Wu, J.A. Fletcher, F.S. HodiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Wu, J. Li, M. Zhu, J.A. Fletcher, F.S. Hodi

Wu et al.





Analysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): X. Wu, M. Zhu, J.A. Fletcher, F.S. HodiWriting, review, and/or revision of the manuscript: X. Wu, M. Zhu, J.A.Fletcher, F.S. HodiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J.A. Fletcher, F.S. HodiStudy supervision: F.S. Hodi

Grant SupportThis work was supported in part by Sharon Crowley Martin

Memorial Fund for Melanoma Research (F.S. Hodi), the Malcolm

and Emily Mac Naught Fund for Melanoma Research (F.S. Hodi) atDana-Farber Cancer Institute, NIH 1P50CA127003-04 (J.A. Fletcher),and the Virginia and Daniel K. Ludwig Trust for Cancer Research (J.A.Fletcher).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 7, 2012; revised April 18, 2012; acceptedMay 7, 2012;published OnlineFirst May 31, 2012.

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2012;11:1905-1914. Published OnlineFirst May 31, 2012.Mol Cancer Ther Xinqi Wu, Jingjing Li, Meijun Zhu, et al.

B PathwaysκPKC/NF-Harboring GNAQ Mutations via Effects on the PKC/Erk1/2 and Protein Kinase C Inhibitor AEB071 Targets Ocular Melanoma

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