acetylsalicylic acid governs the effect of sorafenib in ras-mutant … · cancer therapy:...

Cancer Therapy: Preclinical

Acetylsalicylic Acid Governs the Effect ofSorafenib in RAS-Mutant CancersHeinz Hammerlindl1, Dinoop Ravindran Menon1, Sabrina Hammerlindl1,Abdullah Al Emran1, Joachim Torrano1, Katrin Sproesser2, Divya Thakkar1,Min Xiao2, Victoria G. Atkinson3, Brian Gabrielli4, Nikolas K. Haass5,Meenhard Herlyn2, Clemens Krepler2, and Helmut Schaider1,5

Abstract

Purpose: Identify and characterize novel combinations ofsorafenib with anti-inflammatory painkillers to target difficult-to-treat RAS-mutant cancer.

Experimental Design: The cytotoxicity of acetylsalicylic acid(aspirin) in combination with the multikinase inhibitor sorafe-nib (Nexavar) was assessed in RAS-mutant cell lines in vitro. Theunderlying mechanism for the increased cytotoxicity was inves-tigated using selective inhibitors and shRNA-mediated geneknockdown. In vitro results were confirmed in RAS-mutant xeno-graft mouse models in vivo.

Results: The addition of aspirin but not isobutylphenylpro-panoic acid (ibruprofen) or celecoxib (Celebrex) significantlyincreased the in vitro cytotoxicity of sorafenib. Mechanistically,combined exposure resulted in increased BRAF/CRAF dimer-ization and the simultaneous hyperactivation of the AMPKand ERK pathways. Combining sorafenib with other AMPK

activators, such as metformin or A769662, was not sufficientto decrease cell viability due to sole activation of the AMPKpathway. The cytotoxicity of sorafenib and aspirin was blockedby inhibition of the AMPK or ERK pathways through shRNAor via pharmacologic inhibitors of RAF (LY3009120), MEK(trametinib), or AMPK (compound C). The combination wasfound to be specific for RAS/RAF–mutant cells and had nosignificant effect in RAS/RAF–wild-type keratinocytes or mela-noma cells. In vivo treatment of human xenografts in NSG micewith sorafenib and aspirin significantly reduced tumor volumecompared with each single-agent treatment.

Conclusions: Combination sorafenib and aspirin exertscytotoxicity against RAS/RAF–mutant cells by simultaneouslyaffecting two independent pathways and represents a promis-ing novel strategy for the treatment of RAS-mutant cancers.Clin Cancer Res; 24(5); 1090–102. �2017 AACR.

IntroductionMutant neuroblastoma RAS viral (v-ras) oncogene homolog

(NRAS) was the first oncogene identified in melanoma (1) and itis now known that approximately 20% of all melanomas harbormutations inNRAS, 2% inKRAS and1% inHRAS (2).WhileKRASand HRAS only play a minor role for melanoma, KRAS inparticular is frequently mutated in other cancers, including lung,colon, and pancreatic carcinomas (3). Mechanistically, RASproteins are GTPases that activate downstream signaling path-

ways involved in proliferation and cell survival uponGTPbinding(3). Genetic mutations are located in codons 12, 13, and 61, andmore than 80% of mutant NRAS harbor a mutation at codon 61(4). Mutations result in reduced GTPase activity, which causespreferential binding of GTP and therefore constitutive activationof RAS signaling (5). In recent years, the advent of targetedtherapies has advanced melanoma treatment but focused onmutant BRAF, whereas no strategies directly targeting mutantNRAS have been approved (6, 7). Inhibitors for RAS are partic-ularly difficult to develop (8, 9) leaving low-response chemother-apy (10) or immunotherapy (11) with high toxicity rates astherapeutic strategies for patients with NRAS-mutant melanoma.Attempts to directly target RAS mutations include farnesyl trans-ferase inhibitors, which are supposed to prevent posttranslationalmodifications required for the integration of RAS into the plasmamembrane (12) thus preventing the interaction of RAS and theprenyl-binding protein PDEd (PDE6D) (13). Unfortunately, far-nesyl transferase inhibitors showed disappointing results in clin-ical settings (12) and inhibitors of PDEd binding to farnesylatedKRAS still require more development to optimize the drugs (14).Other attempts to target RAS-mutant cancers include blockingRAS downstream targets. Inhibition of MEK (MAP2K1, MAP2K2)(6, 15) or MEK in combination with PI3K (PIK3CA)/MTORinhibitors have shown promising results (10). However, inhibi-tion of MEK leads to the development of resistance, similar tostrategies in BRAF-mutant melanoma (16). Several mechanismsof resistance have been proposed for NRAS-mutant melanomaincluding PDGF receptor b signaling (PDGFRB) (17)

1Dermatology Research Centre, The University of Queensland, The University ofQueensland Diamantina Institute, Translational Research Institute, Brisbane,Australia. 2The Wistar Institute, Philadelphia, Pennsylvania. 3Division of CancerServices, Princess Alexandra Hospital, Brisbane, Australia. 4Mater MedicalResearch Institute, The University of Queensland, Translational Research Insti-tute, Brisbane, Australia. 5The University of Queensland, The University ofQueensland Diamantina Institute, Translational Research Institute, Brisbane,Australia.

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

H. Hammerlindl and D. Ravindran Menon contributed equally to this article.

Corresponding Author: Helmut Schaider, The University of Queensland Dia-mantina Institute, Translational Research Institute, The University of Queens-land, 37 Kent Street, Woolloongabba, Queensland 4102, Australia. Phone 617-3443-7395; Fax 617-3443-7799; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-2118

�2017 American Association for Cancer Research.

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emphasizing the importance of novel single or combinationtherapies for sustained treatment. Sorafenib (Nexavar, BAY43–9006; Bayer Healthcare Pharmaceuticals) is a multikinaseinhibitor that targets both CRAF (RAF1) and BRAF as well as theVEGFR family (KDR and FLT4) and platelet-derived growth factorreceptor family (PDGFRB; ref. 18), among others. Sorafenib isFDA-approved for the treatment of advanced renal cell carcinomaand patients with unresectable hepatocellular cancer (19) withmost common adverse events being skin rashes, diarrhea, andalopecia (20). In addition, the development of squamous cellcarcinomas and keratoacanthomas has been reported (21, 22).These side effects resulted in patients requiring dose reductions,interrupting or discontinuing therapy raising concerns about thetoxicity, efficacy, and safety of sorafenib (20). One strategy toovercome drug-induced toxicity is to combine sorafenib withother drugs to reduce the effective dose required to trigger atumor-specific response without inducing systemic toxicity. Sev-eral studies have explored possible anticancer effects of sorafenibin combination with other targeted inhibitors or radiation, whichshowed limited efficacy (reviewed in ref. 23).

Nonsteroidal anti-inflammatory drugs (NSAID) have beenreported to reduce overall cancer risk including prostate (24),colorectal (25), and skin cancer (26). The association betweenNSAIDs and melanoma risk is less clear, and several studiesyielded conflicting results (27). Acetylsalicylic acid (Aspirin) isan intriguing agent as it is one of the most widely used drugs.Synergistic effects of sorafenib and aspirin have already beendescribed in RAS wild-type hepatocellular carcinoma where ithas been reported to reduce the prometastatic effect of sorafenibmonotherapy (28).

Here we show that combined sorafenib and aspirin resulted insynergistic cytotoxicity in RAS/RAF–mutant cancers includingNRAS-mutant melanoma by simultaneously activating theAMP-activated protein kinase (AMPK) and mitogen-activatedprotein kinase (MAPK/ERK) pathways. The combined treatment,which is effective in vivo, allows the concentration of sorafenib tobe substantially reduced with the likelihood of less tissue toxicity.These data provide a rationale for the application of this combi-nation in RAS-mutant cancer patients.

Materials and MethodsCell culture

KRAS-mutant human lung adenocarcinoma cell lines A549 andH358 as well as RAS/RAF–wild-type immortalized human kera-tinocytes HaCaT were kindly provided Dr. Gerald Hoefler (Insti-tute of Pathology, Medical University of Graz, Graz, Austria). TheRAS/RAF-wild type humanbreast cancer cell line SkBr3was kindlyprovided by Dr Fiona Simpson (The University of QueenslandDiamantina Institute, Brisbane, Queensland, Australia). All celllines are routinely tested for mycoplasma as described previously(29, 30) and were authenticated in 2016 by the analytic facility ofthe QIMR Berghofer Medical Research Institute (Brisbane, Aus-tralia) via STR fingerprinting. All experiments were performedwithin 3 months after thawing the respective cell lines. Cells weregrown in RPMI1640 medium (Sigma-Aldrich), supplementedwith 5% FBS (Assay Matrix) and 2% L-glutamine (Life Technol-ogies) and maintained at 37�C in a humidified atmospherecontaining 5% CO2. Cells were harvested for individual experi-ments after washing with PBS (pH 7.4; Life Technologies) usingtrypsin (Life Technologies).

Viral vector transductionLentiviral vectors containing shRNA targeting AMPK1/2 (PRKAA1/

PRKAA2) (shPRKAA1, NM_006251, CloneID TRCN0000000861;shPRKAA2 NM_006252, CloneID TRCN0000002171), or BRAF(NM_004333, CloneID TRCN0000231130) were purchased fromSigma-Aldrich. Cells were prepared in 12-well plates to reachconfluence of 50%–80%, pretreated with 8 mg/mL polybrene(Sigma-Aldrich) for 2 hours followed by the addition of 25 mL ofthe viral supernatant. Transduced cells were subjected to selectionwith puromycin (Sigma-Aldrich) at a concentration of 5 mg/mL72 hours post transfection. Cells were then maintained inmediumcontaining 1.5 mg/mL.

ImmunoblottingWhole-cell lysates were generated using RIPA buffer (Sigma-

Aldrich) supplemented with 1% protease inhibitor cocktail(Active Motive). Protein lysates from frozen tissue samples weregenerated using 500-mL RIPA buffer per 10 mg of tissue andsonicated at 180watts 10�10 seconds. Theprotein concentrationwas measured using Bradford Protein Assay (Bio-Rad). Fifteenmicrograms of protein were separated on a 6%–10% SDS-polyacrylamide gel followed by transfer to a polyvinylidenedifluoridemembrane (Bio-Rad). Themembranewas then probedfor the protein of interest with the specific primary and thecorresponding peroxidase-conjugated secondary antibodies(Supplementary Table S1). Proteins were visualized using Amer-sham ECL PrimeWestern Blotting Detection Reagent (GEHealth-care) and scanned on an LI-COR C-DiGit Blot Scanner. Themembranes were stripped and reprobed using Restore Plus West-ern Blot stripping buffer (Life Technologies) as required in theindividual experiments. Immunoblots were quantified usingImageJ and the ratio of phosphorylated to total protein wascalculated and normalized to control of the same immunoblot.

ImmunoprecipitationCells were exposed to drugs for 24 hours as indicated in the

respective experiments, lysed using 1� Cell Lysis Buffer (CellSignaling Technology) and sonicated on ice three times for 5seconds. Cell lysate was subject to a precleaning step by

Translational Relevance

To date, no therapies directly targeting mutant RAS havebeen approved, leaving chemotherapy with very low responserates or immunotherapy as the only treatment options forRAS-mutant cancers. Here we report a novel strategy to targetRAS-mutant cancer, especially NRAS-mutant melanoma, bycombining the multikinase inhibitor sorafenib and the non-steroidal anti-inflammatory drug acetylsalicylic acid (aspirin),both of which are clinically approved and tested. The additionof aspirin strongly enhanced the in vitro and in vivo cytotoxicityof otherwise ineffective sorafenib dosages. The combinationofsorafenib and aspirin, but no other AMPK activators, simul-taneously induced activation of the AMPK and ERK pathways,which are both necessary for drug effectivity. This findingsuggests that combining sorafenib with aspirin could be aviable treatment strategy for RAS-mutant cancers, includingNRAS-mutant melanoma.

Combined Aspirin and Sorafenib for RAS-Mutant Cancer Therapy

www.aacrjournals.org Clin Cancer Res; 24(5) March 1, 2018 1091




incubating with Protein G Agarose Beads (Cell Signaling Tech-nology) for 1 hour at 4�C with gentle rocking followed by theaddition of 2 mL of c-Raf-1 antibody (BD Transduction Labora-tories) and incubated overnight at 4�C. Immunoprecipitationwasperformed by adding 10mL Protein G Agarose Beads (Cell Signal-ing Technology) per 100-mL cell lysate and incubated for 3 hours.Coimmunoprecipitation was then assessed by immunoblottingusing specific antibodies for CRAF and BRAF (SupplementaryTable S1)

InhibitorsSorafenib, trametinib, dabrafenib, and LY3009120 were pur-

chased from Selleck Chemicals. Dorsomorphin (compound C),A-769662, SC-560, andmetforminwere purchased fromCaymanChemical. Celecoxib and isobutylphenylpropanoic acid werepurchased from Sigma Aldrich.

Flow cytometryCaspase-3 activation was assessed using the Active Caspase 3

apoptosis kit (BD Biosciences) following the manufacturer'sprotocol. The samples were analyzed with a BD ACCURI C6PLUS from the Translational Research Institute (TRI) FACScore facility.

MTT assayA total of 1� 104 cells were seeded in 96-well culture plates for

allocated times as mentioned in the experiments. Cells weretreated with drugs depending on the experimental setup 48 hoursafter the initial seeding and subsequently incubated with MTT(3-(4, 5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide,Thermo Fisher Scientific) reagent (1/10 dilution in full growthmedium) at 37�C for 4hours. After incubation, 100mLDMSOwasadded and incubated for 10minutes in the incubator. Absorbancewas measured at 540 nm using a microtiter plate reader. Allexperiments were done in triplicate or duplicate and a finalconcentration of 1% DMSO was used as control.

Cell survival crystal violet stainingCells, which have been exposed to drugs for various time

points, were fixed with 4% paraformaldehyde, followed by30-minute incubation with 0.1% crystal violet in 4% paraformal-dehyde. The plates were washed and imaged using a Chemi DocTM XRS Universal Hood (Bio-Rad). A final concentration of 1%DMSO was used as control.

Drug synergy assessmentCell viability was assessed using the AlamarBlue assay and

synergy was assessed using the Bliss independence model asdescribed previously (31). Briefly, data were normalized to doxo-rubicin and DMSO controls and converted to fraction-affectedvalues (F). Next, the predicted inhibition values (P) were calcu-lated: [P¼ Faþ Fb – Fab 0<P > 1]. Predicted F equals the fractionaffected by compound "a" (Fa) at concentration � plus thefraction affected by compound "b" (Fb) at concentration yminusthe product of the two (Fab). The difference between the predictedadditive fraction affected and the experimentally observed frac-tion affected is the Bliss number. Positive value indicates synergy,a negative value indicates antagonism, and an overlap of pre-dicted and observed combination effects gives a Bliss number ofzero and indicates additivity. Because of the nature of the assay,wedetect a background noise level of � 15%.

In vivo studyAll animal experiments were performed in accordance with

institutional guidelines under Wistar IACUC protocol 111954 inNSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Laboratory)mice or in accordance with institutional guidelines of UQ animalethics committee ethics number: SOM/TRI/197/15/DRC inC.B-17/IcrHanHsdArcPrkdcscid mice (Animal Resources CentreCanning Vale). Animals were inoculated subcutaneously with1� 106 humanWM1366melanoma cells in a 100 mL suspensionof Matrigel (BD Matrigel)/complete media at a ratio of 1:1 orwith 2.5 � 106 human A549 lung adenocarcinoma cells sus-pended in completemedia. After tumors had reached a volume of100–200 mm3, mice were randomized into groups of 4 mice asindicated in the respective experiments: vehicle (0.75% hydroxylmethyl cellulose/25% ethanol/10% DMSO), sorafenib 30 mg/kgor 15 mg/kg, aspirin 100 mg/kg, or 200 mg/kg, metformin100 mg/kg, sorafenib þ aspirin and sorafenib þ metformin.Tumor size was assessed multiple times per week using a caliper.Animals were sacrificed at the experiment endpoint or whentumor volume exceeded the ethical limit. Tumors were thenharvested, snap frozen in liquid nitrogen, and stored at �80�Cuntil further processing.

IHCTumors were formalin-fixed, paraffin-embedded, and stained

using aDiscoveryUltra Ventana in the TRIHistologyCore Facility.Deparaffinzation and antigen retrieval was performed beforeusing prediluted antibodies (Supplementary Table S1) or rabbitIgG as negative control (Invitrogen). Hematoxylin and eosin(H&E) staining was performed according to common methods.Staining intensity was assessed and immunoreactivity was calcu-lated as described previously (32). Slides were scanned using theOlympus VS120 slide scanner (20�).

Statistical analysisRepresented data are expressed as arithmetic mean � SD of

three independent experiments. Unpaired t test has been used todetermine statistical significance. N.s. indicates a P value > 0.05; �,P, � 0.05; ��, indicates a P � 0.01; ��, P � 0.001

ResultsSorafenib and aspirin synergistically induce cell death inNRAS-mutant melanoma

The NRASQ61K-mutant melanoma cell line WM1366 was non-responsive to sorafenib at low concentrations (0.25–1 mmol/L)but showed an almost complete loss of viability at 5 mmol/L(Fig. 1A). Exposure to aspirin alone at concentrations up to2 mmol/L did not affect cell viability (Fig. 1A). The combinationof sorafenib and aspirin, however, showed a dose-dependenttoxicity, with effects of the combination observed with sorafenibconcentrations as low as 250 nmol/L and 2 mmol/L aspirin(Fig. 1B). The efficacy of this combination was confirmed byMTTassay, which showed a significant reduction of cell proliferationafter 72 hours of drug exposure compared with single exposure(Supplementary Fig. S1A). Next, we investigated the combinationof sorafenib and aspirin using the Bliss IndependenceModel (33)and found synergistic efficacy in a wide range of combinedconcentrations of both drugs (Fig. 1C). The combination ofsorafenib- and aspirin-induced apoptosis at a similar level tosorafenib 5 mmol/L, indicated by the level of active caspase-3

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(CASP3) (Fig. 1D). The efficacy of combined sorafenib andaspirin was confirmed in three other NRAS-mutant melanomacell lines (Supplementary Fig. S1B). Combining sorafenib withother NSAIDs, the specific COX2 (PTGS2) inhibitor celecoxib,the specific COX1 (PTGS1) inhibitor SC-560, or the nonspe-cific COX1 and COX2 inhibitor isobutylphenylpropanoic acid(Ibuprofen) showed no synergistic toxicity (SupplementaryFig. S1C–S1E), suggesting that the effect of sorafenib andaspirin is independent of the known aspirin targets COX1and COX2. Taken together, sorafenib at low concentrationsin combination with aspirin synergistically induces apoptosisin NRAS-mutant melanoma independently of COX1 andCOX2 inhibition.

KRAS- and BRAF-mutant but not RAS/RAF–wild-type cells aresensitive to combined sorafenib and aspirin

We expanded our investigation to cell lines harboring KRASG12

mutations, investigating the effects of combined sorafenib andaspirin treatment in the lung carcinoma cell line A549. The dose–response profile showed that A549 is insensitive to single-agentsorafenib and aspirin (Fig. 1E); however, the combination pro-duced a dose-dependent response with an effective sorafenibconcentration as low as 500 nmol/L when combined with2 mmol/L aspirin, and a strong cytotoxic effect at 1 mmol/Lsorafenib and 2 mmol/L aspirin (Fig. 1F). We confirmed theefficacy of the combination by MTT assay, which showed asignificantly reduced cell proliferation over 72 hours

Figure 1.

The combination of sorafenib and aspirin synergistically decreases cell viability in RAS-mutant cancers. A, Crystal violet staining of WM1366 treated withincreasing concentrations of sorafenib (250 nmol/L–5 mmol/L) or increasing concentrations of aspirin (500 mmol/L–2 mmol/L) for 72 hours. B, Crystalviolet staining of WM1366 treated with increasing concentrations of sorafenib (250 nmol/L–5 mmol/L) in the presence of increasing concentrations of aspirin(500 mmol/L–2 mmol/L) for 72 hours. C, Drug synergy assessed using the Bliss independence model. Green indicates synergistic, yellow indicates additiveand red indicates antagonistic effects of the drugs.D,Caspase-3 activation following indicated drug exposuresmeasured after 48 hours. �� ,P�0.001; �� ,P�0.01. E,Crystal violet staining of A549 treated with increasing concentrations of sorafenib (250 nmol/L–5 mmol/L) or increasing concentrations of aspirin(500 mmol/L–2 mmol/L) for 72 hours. F, Crystal violet staining of A549 exposed to increasing concentrations of sorafenib (250 nmol/L–5 mmol/L) in thepresence of increasing concentrations of aspirin (500 mmol/L–2 mmol/L) for 72 hours. G, Cell proliferation of WM164 and WM983B BRAF-mutant melanomacells following exposure to sorafenib (1 mmol/L), aspirin (2 mmol/L), or the combination for 72 hours. �� , P � 0.001. H, Crystal violet staining of HaCaT orSkBr3 cells exposed to increasing concentrations of sorafenib (250 nmol/L–5 mmol/L) alone or in combination with aspirin (2 mmol/L) for 72 hours.






(Supplementary Fig. S1F). The combination was also effective inthe KRASG12-mutant lung cancer cell line H358 (SupplementaryFig. S1G), suggesting that RAS-mutant cell lines in general aresusceptible to this combination. Furthermore, sorafenib/aspirinstrongly reduced cell proliferation and viability in BRAF-mutantmelanoma cell lines WM164 and WM983B as measured by MTT(Fig. 1G) and crystal violet staining (Supplementary Fig. S1H). Incontrast, RAS/RAF wild-type keratinocytes (HaCaT) or the RAS/RAF–wild-type breast cancer cell line SkBr3 only showed a minorresponse to combined sorafenib/aspirin (Fig. 1H). Similarly theBRAF/NRAS–wild-type melanoma cell line D24 was only mod-erately affected by the combination (Supplementary Fig. S1I),suggesting that the cytotoxic effects of sorafenib and aspirin arespecific for RAS/RAF–mutant cancers. The mutation status andsensitivity to combined sorafenib/aspirin of all tested cell lines issummarized in Table 1.

The combination of sorafenib and aspirin activates ERK andAMPK pathways

MutantRAS results in increased RAF/MEK/ERK signaling, whilesorafenib inhibits BRAF and CRAF among others, thereby inhibit-ing MAPK signaling (34). In addition to inhibiting COX1/2,aspirin has been shown to increase AMPK signaling (35). Wetherefore investigated these pathways in sorafenib/aspirin–exposed cells. Contrary to our expectations, the combinationtreatment resulted in the increased activation of ERK1 (MAPK3)and ERK2 (MAPK1) and the phosphorylation of the AMPKsubstrate acetyl-coenzyme A carboxylase (ACC/ACACA/ACACB)in NRAS- (Fig. 2A), KRAS- (Fig. 2B), and BRAF-mutant (Fig. 2Cand D) cells. In contrast, RAS/RAF-wild-type keratinocytes(HaCaT; Fig. 2E) and BRAF/NRAS–wild-type melanoma cells(D24; Fig. 2F), which are both less sensitive to the combinationof sorafenib and aspirin, showed no or only a subtle ERK and/orAMPK pathway activation, suggesting that these pathways couldbe crucial for the cytotoxic effects of the combination treatment.Sorafenib at a concentration of 5 mmol/L, which showed toxicityin WM1366 cells, also resulted in an increase of pERK and pACC(Fig. 2A), suggesting that single-agent–mediated toxicity affectssimilar pathways in these cells. Furthermore, activation of theAMPK andMAPK pathways was already observed after 4 hours oftreatment (Supplementary Fig. S2). Because there is a significantlack of specific treatment options for RAS-mutant–driven cancers,and RAS- and RAF-mutant cancer cells showed the same pathwayactivation pattern, we conducted all further experiments inNRAS-and KRAS-mutant cell lines. To test for the contribution of bothpathways, we blocked them using the selective MEK inhibitortrametinib and the AMPK inhibitor compound C (dorsomor-

phin).While single treatmentwith theMEK inhibitor resulted in adose-dependent increase in toxicity, the inhibition of MEKreduced sorafenib and aspirin-induced toxicity in NRAS- andKRAS-mutant cells (Fig. 3A). Similarly, compound C showed adose-dependent toxicity while rescuing the cells if combined withsorafenib and aspirin (Fig. 3B) confirming the importance ofactivated AMPK and ERK signaling for the toxicity of the combi-nation treatment. Simultaneous inhibition of AMPK and MEK incells treated with the combination therapy increased cell viabilityover inhibition of either pathway alone (Supplementary Fig. S3Aand S3C). The decreased toxicity of sorafenib and aspirin afterinhibition of MEK and/or AMPK was also confirmed by MTTassays (Supplementary Fig. S3B and S3D), even though no addi-tional benefit of combining trametinib and compound C wasdetected using this assay. The observations of the crystal violetstainings suggest that AMPK and ERK1/2 activation are indepen-dent. Indeed,we found that treatmentwith sorafenib, aspirin, andtrametinib resulted in activated AMPK signaling but blocked ERKsignaling, whereas treatment with sorafenib, aspirin and com-pound C resulted in decreased AMPK signaling but activatedERK1/2 (Fig. 3C). This indicates that AMPK and ERK pathwayactivation are independent events that synergistically mediatecytotoxicity of the combination treatment. To confirm the spec-ificity of the observations using pharmacologic inhibitors, weused sequence-specific shRNAs to silence BRAF and AMPKa1/2(PRKAA1/PRKAA2) inNRAS-mutantmelanoma cells (WM1366).BRAF-orAMPKa1/2-silenced cells showeddecreased sensitivity tosorafenib and aspirin compared with empty vector–transducedcontrol cells (Fig. 3D and E) suggesting that both of these proteinsare involved in sorafenib/aspirin–mediated cytotoxicity. It hasbeen shown that BRAF inhibitors can induce paradoxical MAPKpathway activation by promoting BRAF/CRAF dimerization in aRAS-dependent manner in RAS-mutant and RAS/RAF–wild-typecancers (36). CRAF immunoprecipitation showed that sorafenibin combination with aspirin, but not single-agent treatment,induced strong BRAF/CRAF complex formation in NRAS-mutantWM1366 (Fig. 3F) and BRAF-mutant WM164 melanoma cells(Fig. 3G). Interestingly, BRAF shows an electrophoretic mobilityshift inWM1366 that recently has been linked to phosphorylationand increased activity of BRAF as part of a high molecular weightcomplex in RAS-mutant cancer cells (37). We then tested theinvolvement of BRAF/CRAF complexes for the activation of theERK pathway by using the pan-RAF inhibitor LY3009120 that hasbeen shown to inhibit active dimers (38). Similar to trametinib,LY3009120 reduced the toxicity of sorafenib and aspirin pro-foundly in bothNRAS- and KRAS-mutant cells (Fig. 3H), suggest-ing that RAF activation is required for sorafenib- and aspirin-

Table 1. Overview of sensitivity to combined sorafenib and aspirin

Cell line Type Mutation status Sorafenib/aspirin sensitivity

WM1366 Melanoma NRAS YesWM1361A Melanoma NRAS YesWM852 Melanoma NRAS YesCJM Melanoma NRAS YesA549 Lung carcinoma KRAS YesH358 Non–small cell lung cancer KRAS YesWM164 Melanoma BRAF YesWM983B Melanoma BRAF YesHaCaT Immortalized keratinocyte RAS/RAF wild-type NoSkBr3 Breast cancer RAS/RAF wild-type NoD24 Melanoma RAS/RAF wild-type No

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Figure 2.

Sorafenib and aspirin hyperactivate ERK and AMPK signaling in RAS-mutant cells. Immunoblotting of whole-cell lysates of WM1366 (A), A549 (B), WM164(C), WM983B (D), HaCat (E), and D24 (F) exposed to sorafenib (1 mmol/L), aspirin (2 mmol/L), or the combination for 48 hours, using specific antibodies forphospho-acetyl CoA carboxylase (p-ACC), total acetyl CoA carboxylase (t-ACC), phospho-ERK1/2 (p-ERK1/2), and total ERK1/2 (t-ERK1/2). b-Actin wasused as loading control. Immunoblots were quantified using ImageJ. The ratio of phosphorylated to total protein normalized to control is shown above therespective blots.






Figure 3.

Inhibiting either AMPK or ERK activation reduces sorafenib/aspirin sensitivity. A, Crystal violet staining of WM1366 or A549 exposed to increasingconcentrations of trametinib (5 nmol/L–25 nmol/L) and in the presence of sorafenib (1 mmol/L) and aspirin (2 mmol/L). B, Crystal violet staining of WM1366or A549 exposed to increasing concentrations of compound C (1–10 mmol/L) and in the presence of sorafenib (1 mmol/L) and aspirin (2 mmol/L).C, Immunoblotting of whole-cell lysates of WM1366 exposed to trametinib, compound C, and/or sorafenib and aspirin using specific antibodies for phospho-acetylCoA carboxylase (p-ACC), total acetyl CoA carboxylase (t-ACC), phospho-mTOR (p-mTOR), total mTOR (t-mTOR), phospho-ERK1/2 (p-ERK1/2), and total ERK1/2(t-ERK1/2). b-Actin was used as loading control. D, Crystal violet staining of WM1366 transduced with shRNA targeting BRAF and treated with sorafenib and/oraspirin for 72hours. Knockdownwas confirmedby immunoblotting.E,Crystal violet staining ofWM1366 transducedwith shRNA targetingAMPKa1 andAMPKa2 andtreatedwith sorafenib and/or aspirin for 72hours. Knockdownwas confirmedby immunoblotting.F andG, Immunoprecipitation usingCRAF-specific antibodies afterexposure to sorafenib and/or aspirin at the indicated concentrations for 24 hours. Coimmunoprecipitation and total BRAF expression were assessed byimmunoblotting using specific antibodies for CRAF and BRAF. b-Actin was used as loading control. H, Crystal violet staining of WM1366 or A549 exposed toincreasing concentrations of the pan-RAF inhibitor LY3009120 (250 nmol/L–1 mmol/L) and in the presence of sorafenib (1 mmol/L) and aspirin (2 mmol/L) for72 hours. I, Immunoblotting of whole-cell lysates ofWM1366 exposed to LY3009120 (1 mmol/L) and in combinationwith sorafenib (1 mmol/L) and aspirin (2mmol/L)for 48 hours using specific antibodies for phospho-acetyl CoA carboxylase (p-ACC), total acetyl CoA carboxylase (t-ACC), phospho-ERK1/2 (p-ERK1/2), andtotal ERK1/2 (t-ERK1/2). b-Actin was used as loading control. Immunoblots were quantified using ImageJ. The ratio of phosphorylated to total protein normalizedto control is shown above the respective blots.

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induced cytotoxicity. Combining sorafenib, aspirin, andLY3009120 significantly rescued cell proliferation as determinedby MTT assays confirming previous findings (Supplementary Fig.S3E and S3F). LY3009120 alone or in combination with aspirinresulted in moderate activation of ERK signaling, which, eventhoughobserved at a higher concentration, is in linewith previousreports (38). The combination of LY3009120 with sorafenib andaspirin blocked hyperactivation of ERK signaling without inhibit-ing AMPK pathway activity (Fig. 3I), again suggesting that MAPKand AMPK pathway activation are independent from each other.Taken together, combined sorafenib and aspirin simultaneouslyhyperactivate ERK and AMPK signaling, which both contribute todecreased cell viability in RAS-mutant cancers.

The combination of sorafenib with other AMPK activatorsshows no synergistic effects

As a proof of principle, we tested the combination of aspirinwith the BRAFV600E-specific drug dabrafenib, which has beenreported to paradoxically activate ERK in NRAS-mutant melano-ma (9, 39). Combining dabrafenib at concentrations that are100–1,000 times higher than usually used for melanoma (40),with aspirin resulted in a profound decrease of cell viability inRAS-mutant cells (Fig. 4A; Supplementary Fig. S4A). Only thecombination induced hyperactivation of ERK and AMPK signal-ing, whereas dabrafenib alone, which showed no effect on cellviability, only resulted in hyperactivated ERK (Fig. 4B). Similar todabrafenib in combination with aspirin, dabrafenib combinedwith the antidiabetic drug metformin, which is known to be anAMPKactivator, also exerted cytotoxic effects (Supplementary Fig.S4B) and activated ERK and AMPK signaling (Fig. 4B). We thentested whether sorafenib in combination with other AMPK acti-vators is also effective. Interestingly, the combination of sorafenibwithmetformin showed no synergistic effect inNRAS- and KRAS-mutant cells (Fig. 4C; Supplementary Fig. S4C). On themolecularlevel, combined sorafenib andmetformin resulted in activation ofthe AMPK pathway but only modest activation of ERK signaling(Fig. 4D). We then hypothesized that the mechanistic differencesof AMPK activation of metformin and aspirin might influence thesynergistic effects. Metformin is an indirect AMPK activator as itinhibits oxidative phosphorylation (41) while aspirin interactsdirectlywithAMPK. To account for thismechanistic difference, wecombined sorafenibwith the allosteric AMPKactivator A-769662,which has been shown to activate AMPK by interacting with thesame protein region as aspirin (35). This combination also failedto decrease cell viability in RAS-mutant cancers (Fig. 4E). Likemetformin, the combination with A-769662 resulted in AMPKpathway activation while failing to trigger hyperactivated ERKsignaling (Fig. 4F). Similar to the in vitro results, the combinationof sorafenib and aspirin showed a significant synergistic effect inNRAS- and KRAS-mutant xenograft mouse models (Fig. 5A andB). In comparison, the combination of sorafenib and metforminfailed to decrease tumor growth (Supplementary Fig. S5A) con-firming the superior efficacy of the sorafenib and aspirin combi-nation. IHC analysis of the A549-derived tumors showed that themajority of the sorafenib or sorafenib/aspirin-treated tumorsconsisted of necrotic/dead cells with only the periphery of thetumors showing p-ERK positivity (Supplementary Fig. S5B–S5F).By applying an immunoreactivity score, it was found thatsorafenib/aspirin–treated tumors displayed a higher stainingintensity compared with sorafenib-treated tumors (Fig. 5C).Immunoblotting of A549-derived xenografts tumors treated with

three doses of combined sorafenib/aspirin or vehicle (n ¼ 3)further showed significant activation of AMPK signaling, indicat-ing a similar pathway activation pattern as observed in vitro(Fig. 5D). These data demonstrate that sorafenib in combinationwith aspirin is a suitable strategy to target RAS-mutant cancers,whereas other AMPK activators such as metformin or A-769662show no synergistic antitumorigenic effects in combination withsorafenib (Fig. 5E).

DiscussionThe identification of the prominent melanoma driver muta-

tion BRAFV600E was followed by the development of clinicallyeffective drugs specifically targeting this mutation. Despite theemergence of resistance to these drugs, treatment significantlyimproves patient survival and is viewed as a model for thedevelopment of oncogene-directed targeted therapies (42).Intensive research efforts have been undertaken to developdrugs specifically targeting mutant NRAS, the second-mostcommon driver mutation of melanoma, but no agents havebeen approved by the FDA to date (6, 7). Besides the lack ofeffective targeted therapies, mutantNRAS is also correlated withshorter survival after diagnosis of late-stage disease comparedwith NRAS/BRAF–wild-type or BRAF-mutant melanoma (4),highlighting the aggressive nature of melanomas driven by thismutation. Here we report the novel combination of sorafeniband aspirin to target RAS-mutant cancers. Considering that theclinically achievable sorafenib plasma concentration of approx-imately 5 mmol/L is associated with severe adverse effects (43),reducing the effective sorafenib dosage by combining it withclinically achievable aspirin concentrations (44, 45) could be apromising strategy to overcome toxicity issues. However,adverse effects triggered by aspirin and strategies to preventthese adverse effects must also be considered (46). The bestcharacterizedmechanism of action of aspirin is the inhibition ofthe cyclooxygenase enzymes COX1 and COX2, both of whichare acetylated at a serine residue in the active site of the enzyme,abolishing enzyme activity (47). Interestingly, the combinationwith other NSAIDs, SC-560, ibuprofen, or celecoxib whichinhibit COX1 and/or COX2 (48, 49), showed no synergistictoxicity suggesting a mechanism independent of COX1/2 inhi-bition. While epidemiologic studies provide compelling evi-dence that NSAIDs are associated with reduced risk of cancer(50), some reports also suggest that aspirin rather than otherNSAIDs show a protective effect in some cancer types (51).Indeed, several reports have suggested that the cancer preventiveaction of the selective COX2 inhibitor celecoxib is the result ofCOX2-independent effects (52, 53). Mechanistically, inhibitionof COX1/2 by aspirin is unlike other NSAIDs and the result of acovalent irreversible modification (49). While acetylation ofCOX1 completely blocks enzyme activity, acetylation of COX2modifies the enzyme activity leading to the generation oflipoxins (54), which inhibit cell proliferation and angiogenesisof colorectal cancer (55). While a contribution of such lipoxinsto the observed toxicity of sorafenib and aspirin cannot beexcluded, the lack of synergy of sorafenib with other NSAIDsthat target COX1/2 strongly suggests that the effect of aspirin isindependent of inhibition of cyclooxygenase enzymes. Recentlyit has been shown that aspirin-derived salicylate activatesAMPK by directly binding to and allosterically inhibitingdephosphorylation of AMPK (35). This aspect of aspirin is






Figure 4.

Activation of either AMPK or ERK signaling is not sufficient to induce synergistic toxicity in combination with sorafenib. A, C, and E, Crystal violet stainingof WM1366 or A549 exposed to the indicated drugs for 72 hours. B, D, and F, Immunoblotting of whole-cell lysates of WM1366 exposed to the indicateddrugs for 48 hours using specific antibodies for phospho-acetyl CoA carboxylase (p-ACC), total acetyl CoA carboxylase (t-ACC), phospho-ERK1/2 (p-ERK1/2),and total ERK1/2 (t-ERK1/2). Immunoblots were quantified using ImageJ. The ratio of phosphorylated to total protein normalized to control is shown abovethe respective blots.


Hammerlindl et al.




intriguing as activation of AMPK has been suggested to haveantitumorigenic effects in certain contexts (56), especially inmelanoma (57). Accordingly, constitutive activation of MAPKsignaling by mutant BRAF has been shown to inhibit LKB1(STK11) via ERK and p90Rsk (RPS6KA1), inhibiting AMPKactivation in response to energy stress and promoting melano-ma cell proliferation (58, 59). Furthermore, the combination ofAMPK activators, like metformin or phenformin, with MAPKpathway inhibitors has been reported to be an effective strategyto increase treatment response in BRAF-mutant melanoma(60). Thus, it was surprising to see that neither metformin norA-769662 showed synergistic effects in combination with sor-afenib. In our experiments, aspirin alone did not activate AMPKsignaling, which could be the direct result of LKB1-AMPKuncoupling. Sorafenib on the other hand, has been reportedto activate AMPK in an LKB1 and/or CAMKK2-dependent man-ner (61), which, together with the aspirin-mediated stabiliza-tion of the active state of AMPK could result in the strongly

increased activation of the AMPK pathway. shRNA-mediatedsilencing of AMPK profoundly reduced the cytotoxic effectsof sorafenib and aspirin cotreatment, suggesting that AMPKactivation is necessary but not sufficient to increase the cyto-toxicity of sorafenib. Inhibition of ERK signaling also reducedthe cytotoxicity of the combination, and the ability of the pan-RAF inhibitor LY3009120 and BRAF silencing to block thecytotoxicity of the combination, demonstrates the importanceof BRAF in the observed hyperactivation of ERK signaling.Considering that both dominant melanoma driver mutationsresult in constitutive activation of ERK signaling and thatsorafenib is a multikinase inhibitor that is supposed to inhibitthe MAPK pathway (62), the involvement of hyperactivatedMAPK signaling for the cytotoxicity of the combination treat-ment was unexpected. However, it is well known that sorafe-nib induces the observed BRAF/CRAF dimerization in BRAF-wild-type cells, which can lead to paradoxical activation ofthe ERK signaling pathway (36, 39, 63). We also observed

Figure 5.

Sorafenib in combination with aspirin increases treatment efficacy in vivo. A and B, A total of 1 � 106 WM1366 (A) or 2.5 � 106 A549 cells (B) wereinjected into immunocompromised mice (n ¼ 4 mice per group). Mice were treated daily with the indicated drugs. Statistical significance of results wascalculated using an unpaired t test. n.s., P > 0.05; �� , P � 0.01. C, Immunoreactivity score of phospho-ERK staining intensities of tumors generated fromA549 (Supplementary Fig. S5B–S5E; � , P � 0.05). D, Immunoblotting of A549-derived xenografts following 54 hours of treatment with either vehicle orcombined sorafenib (15 mg/kg) and aspirin (200 mg/kg) using specific antibodies for phospho-acetyl CoA carboxylase (p-ACC), total acetyl CoAcarboxylase (t-ACC), phospho-AMPK (p-AMPK), total AMPK (t-AMPK), phospho-ERK1/2 (p-ERK1/2), and total ERK1/2 (t-ERK1/2). Immunoblots werequantified using ImageJ. The ratio of phosphorylated to total protein normalized to mean of control is shown above the respective blots (right) and plottedas a graph (left). � , P � 0.05. E, Proposed model of the synergistic effects elicited by the combination of sorafenib and aspirin. The combination activatesthe MAPK and AMPK pathways, which together induce cell death in RAS-mutant cancer.






sorafenib/aspirin but not sorafenib or aspirin alone, mediatedBRAF/CRAF dimerization in BRAF-mutant cells which has notbeen described for more specific BRAF inhibitors like PLX4720(36). It is noteworthy that unlike other ATP-competitive RAFinhibitors, sorafenib binds and stabilizes RAF in a differentconformation (64). It is possible that this difference in thebinding mode is important for the observed synergy betweensorafenib and aspirin and the resulting increased BRAF/CRAFdimerization in BRAF-mutant cells. It is noteworthy that othermechanisms like dysregulation of the complex negative feed-back regulation of Dual-specificity phosphatases (DUSP;ref. 65) could also be involved in the hyperactivation of ERKsignaling and therefore contribute to sorafenib/aspirin inducedantitumorigenic effects. While the detailed downstream effectsof sorafenib/aspirin mediated hyperactivation of ERK andAMPK signaling remain elusive, it has been recently shown thathyperactivated ERK signaling induces apoptosis in BRAF–mutant cancers (66), resulting in an oncogene-induced senes-cence and negative selection of RAF and RAS double mutations(67) or inhibit melanoma growth and induce autophagy in vitroand in vivo (68). Beside the effectiveness of sorafenib in com-bination with aspirin to target NRAS-mutant melanoma, KRAS-mutant lung adenocarcinoma cell lines also proved to besensitive to this combination. Sorafenib has recently been testedin a phase II clinical trial for the treatment of non–small celllung cancer (NSCLC) with KRAS mutations, which showedrelevant but modest clinical activity in patients (69). Our resultssuggest that the use of sorafenib in combination with aspirin is apromising strategy for the treatment of KRAS-mutant NSCLC.Taken together, we identified molecular drivers mediating thecytotoxic effects of sorafenib in combination with aspirin. Thiswork provides a strong rationale to repurpose two clinicallyapproved drugs to treat cancer types that lack specific therapeu-tic options.

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

Authors' ContributionsConception and design: H. Hammerlindl, D. Ravindran Menon, D. Thakkar,H. SchaiderDevelopment of methodology: H. Hammerlindl, D. Ravindran Menon,S. Hammerlindl, D. ThakkarAcquisition of data (provided animals, acquired and managed patients, pro-vided facilities, etc.): H. Hammerlindl, D. Ravindran Menon, S. Hammerlindl,A.A. Emran, J. Torrano, K. Sproesser, D. Thakkar, M. Xiao, C. KreplerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Hammerlindl, D. Ravindran Menon, A.A. Emran,J. Torrano, K. Sproesser, D. Thakkar, V.G. Atkinson, N.K. Haass, C. Krepler,H. SchaiderWriting, review, and/or revision of the manuscript: H. Hammerlindl,D. Thakkar, V.G. Atkinson, B. Gabrielli, M. Herlyn, C. Krepler, H. SchaiderAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. Hammerlindl, K. Sproesser, N.K. Haass,H. SchaiderStudy supervision: H. Schaider

AcknowledgmentsThe authors thank Dr. Gerald Hoefler (Institute of Pathology, Medical

University of Graz, Graz, Austria) for kindly providing A549, H358, and HaCaTcells and Dr Fiona Simpson (The University of Queensland DiamantinaInstitute, Brisbane, Queensland, Australia) for kindly providing SkBr3 cells.The authors also acknowledge the help provided by the Translational ResearchInstitute (TRI) FACS, histology, and microscopy core facilities. This work wasfunded by the Epiderm Foundation (H. Schaider), the Princess AlexandraResearch Foundation (PARSS2016_NearMiss; to H. Schaider), NIH grants PO1CA114046 and P50 CA174523, and the Dr. Miriam and Sheldon G. AdelsonMedical Research Foundation (both M. Herlyn). A.A. Emran is funded by TheUniversity of Queensland International Scholarship (UQI); H. Hammerlindl isfunded by the International Postgraduate Research Scholarship (IPRS) and UQCentennial Scholarship (UQCent). N.K.Haass is funded by theNational Healthand Medical Research Council (APP1084893).

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 November 14, 2016; revised June 27, 2017; accepted October 26,2017; published OnlineFirst December 1, 2017.

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2018;24:1090-1102. Published OnlineFirst December 1, 2017.Clin Cancer Res Heinz Hammerlindl, Dinoop Ravindran Menon, Sabrina Hammerlindl, et al. Cancers

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