Caffeic Acid Directly Targets ERK1/2 to Attenuate …cancerpreventionresearch.aacrjournals.org/content/canprevres/7/10/...Caffeic Acid Directly Targets ERK1/2 to Attenuate Solar UV-Induced

Research Article

Caffeic Acid Directly Targets ERK1/2 to Attenuate SolarUV-Induced Skin Carcinogenesis

Ge Yang1,2,3, Yang Fu1,3, Margarita Malakhova1, Igor Kurinov4, Feng Zhu1, Ke Yao1, Haitao Li1,Hanyong Chen1, Wei Li1, Do Young Lim1, Yuqiao Sheng1,2,3, Ann M. Bode1, Ziming Dong2, and Zigang Dong1

AbstractCaffeic acid (3,4-dihydroxycinnamic acid) is a well-known phenolic phytochemical present in coffee and

reportedly has anticancer activities. However, the underlying molecular mechanisms and targeted proteins

involved in the suppression of carcinogenesis by caffeic acid are not fully understood. In this study,we report

that caffeic acid significantly inhibits colony formation of human skin cancer cells and EGF-induced

neoplastic transformation of HaCaT cells dose-dependently. Caffeic acid topically applied to dorsal mouse

skin significantly suppressed tumor incidence and volume in a solar UV (SUV)–induced skin carcinogenesis

mouse model. A substantial reduction of phosphorylation in mitogen-activated protein kinase signaling

was observed in mice treated with caffeic acid either before or after SUV exposure. Caffeic acid directly

interacted with ERK1/2 and inhibited ERK1/2 activities in vitro. Importantly, we resolved the cocrystal

structure of ERK2 complexed with caffeic acid. Caffeic acid interacted directly with ERK2 at amino acid

residues Q105, D106, and M108. Moreover, A431 cells expressing knockdown of ERK2 lost sensitivity

to caffeic acid in a skin cancer xenograft mouse model. Taken together, our results suggest that caffeic

acid exerts chemopreventive activity against SUV-induced skin carcinogenesis by targeting ERK1 and 2.

Cancer Prev Res; 7(10); 1056–66. �2014 AACR.

IntroductionCoffee is among the most widely consumed beverages in

the world. More than 50% of Americans drink coffee daily.The consumption of around 4 cups of coffee a day isassociated with a 38% lower risk of breast cancer in pre-menopausal women (1). Consumption of at least 5 cups ofcoffee a day is associated with a 40% lower risk of braintumors in humans compared with non-coffee drinkers (2).Recent results also show that daily consumption of 6 ormore cups of coffee is associated with a 30% reducedprevalence of nonmelanoma skin cancer (3). Skin canceris the most common cancer in the United States with more

than 1 million new cases reported each year, comprisingapproximately 40% of all diagnosed cancers (4). Epidemi-ologic evidence suggests that solar ultraviolet (SUV; i.e.,sunlight) irradiation is the most important risk factor forskin carcinogenesis (5, 6).

Caffeic acid (3, 4-dihydroxy cinnamic acid) is the majorphenolic compound naturally found in coffee. A 200 mLcup of coffee provides about 35 to 175 mg of caffeicacid. Caffeic acid reportedly exerts a broad spectrum ofpharmacologic activities, including anti-inflammatory,antioxidant, immunomodulatory, and neuroprotectiveactivities (7–9). Recent studies suggest that caffeic acid hasanticancer effects against human renal carcinoma (10) andcolon cancer metastasis (11). However, the underlyingmolecular mechanisms and targeted proteins involved inthe suppression of carcinogenesis by caffeic acid are notfully understood.

The mitogen-activated protein kinase (MAPK) pathwayencompasses different signaling cascades of which the Ras–Raf–MEK–extracellular signal-regulated kinase 1 and 2(ERK1/2) pathway is one of the most commonly deregu-lated in human cancer. This pathway mediates multiplecellular functions, including proliferation, growth, andsenescence (12). Abnormalities in ERKs signaling werereported in approximately 1 of 3 of all human cancers,including lung, liver, and melanoma (13). SUV irradiationactivates ERKs in human skin (14) and compared withactinic keratosis, squamous cell carcinomas show higherERKs phosphorylation (15). Hyperactivation of ERKs

1The Hormel Institute, University of Minnesota, Austin, Minnesota. 2Phys-iology andPathophysiology,BasicMedical College, ZhengzhouUniversity,ZhengZhou, China. 3The First Affiliated Hospital of Zhengzhou University,ZhengZhou, China. 4Cornell University, NE-CAT, APS, Argonne, Illinois.

Note:Supplementary data for this article are available atCancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Notes:G. Yang, Y. Fu, and M. Malakhova contributed equally to this work.

Current Address for W. Li: Central South University, Changsha, Hunan,China.

Corresponding Authors: Zigang Dong, The Hormel Institute, University ofMinnesota, 801 16th Avenue North East, Austin, MN 55912. Phone: 507-437-9600; Fax: 507-437-9606; E-mail: [email protected]; Ziming Dong,Zhengzhou University, No. 100 Kexue Road, Henan, China, 450001.Tel: þ86-0371-67781956; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-14-0141

�2014 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 7(10) October 20141056

Research. on July 7, 2018. © 2014 American Association for Cancercancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst August 7, 2014; DOI: 10.1158/1940-6207.CAPR-14-0141

http://cancerpreventionresearch.aacrjournals.org/

results in deregulated cell proliferation in several humancancers, including skin cancer (16, 17), suggesting thatinhibition of ERKs signaling represents a potentialapproach for SUV-induced skin cancer prevention.Here, we report the cocrystal structure of caffeic acid with

ERK2 and show that caffeic acid directly inhibited SUV-induced activation of ERK1/2 and suppressed ERKs signal-ing. Treatment of dorsal mouse skin with caffeic acid eitherbefore or after SUV exposure strongly reduced skin tumornumber and size in mice.

Materials and MethodsReagents and antibodiesCaffeic acid was purchased from Sigma-Aldrich. Active

ERK1 and 2 human recombinant proteins for kinase assayswere fromMillipore. Antibodies to detect total ERK1 and 2,phosphorylated Elk1 (Ser383), total Elk1, and phosphor-ylated p90RSK (Thr359/Ser363) were obtained fromCell Signaling Technology. Antibodies against phosphory-lated ERK1 and 2 (Thr202/Tyr204), total p90RSK, phos-phorylated c-Myc (Thr58/Ser62), total c-Myc and b-actinwere from Santa Cruz Biotechnology. The antibody todetect Ki-67 was purchased from Thermo Scientific.CNBr-Sepharose 4B beads were from GE Healthcare.

SUV irradiation systemThe SUV resource was comprised of UVA-340 lamps (Q-

Lab Corporation), which provide the best possible simu-lation of sunlight in the critical short wavelength regionfrom365nmdown to the solar cutoff of 295nmwith a peakemission of 340 nm. The percentage of UVA andUVB of theUVA-340 lamps was measured by a UVmeter as 94.5% and5.5%, respectively.

Cell culture and transfectionA431 skin cancer cells and HaCaT keratinocytes were

cultured with DMEM supplemented with 10% FBS andantibiotics. SK-MEL-5 and SK-MEL-28melanoma cells werecultured with minimum essential medium (MEM) supple-mented with 10% FBS and antibiotics. Cells were cytoge-netically tested and authenticated before being frozen. Eachvial of frozen cells was thawed andmaintained in culture foramaximumof 8weeks. Cells were kept at 37�C in a 5%CO2

incubator. Cells were cultured at 37�C in a 5% CO2 incu-bator. For transfection experiments, the jetPEI (Qbiogen,Inc.) transfection reagent was used following the manufac-turer’s instructions.

Primary mouse keratinocyte harvest and cultureSKH1 hairless mice were euthanized and the dorsal skin

was removed. All subcutaneous tissue was scraped from theskin until the skin was semitranslucent. The skin (hairy sideup)was spread out on a dish and cut into 0.5-� 1-cm strips.Trypsin without EDTA (20 mL, 0.25%) was added to thedish, and the dish was placed in a 37�C incubator for 1.5hours.DMEM/10%FBS (20mL) is added and the epidermisscraped off into the medium. The medium was collectedand stirred at 100 rpm on amagnetic stirrer for 20minutes.

The medium is filtered through a sterile 70 mmTeflonmeshand the filtrate centrifuged. The keratinocyte growth medi-umwas added to resuspend the cells and cells subsequentlymaintained at 37�C in a 5% CO2 incubator.

MTS assayTo estimate cytotoxicity, cells were seeded (8� 103 cells/

well) in 96-well plates and cultured overnight. Cells werethen fedwith freshmediumand treatedwith different dosesof caffeic acid. After culturing for various times, the cellswere harvested and cytotoxicity of caffeic acidwasmeasuredusing an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxy-methoxyphenyl)-2H-tetrazdium) assay kit (Promega)according to the manufacturer’s instructions.

Soft agar assayCells (8 � 103/mL) were suspended in 1 mL of 0.3%

basal medium eagle (BME) top agar containing 10% FBSwith various concentrations of caffeic acid and placedover a lower layer of solidified BME, 10% FBS, and 0.5%agar (3 mL) with the same concentrations of caffeic acid.The cultures were maintained at 37�C in a 5% CO2

incubator for 1 to 2 weeks and then colonies werecounted under a microscope using the Image-Pro Plussoftware (v.6) program (Media Cybernetics).

In silico target identificationThe PHASEmodule of the Schr€odingermolecularmodel-

ing software package (18) is a shape similarity method (19)and was used to search for biologic targets of caffeic acid onthe basis of its distinctive structure. The parameter of atom-type for volume was set to MacroModel, which meansoverlapping volumes are computed only between atomsthat have the same MacroModel atom type. The targetdatabase was obtained from the Protein Data Bank andour in-house chemical library. To provide more structureorientations for possible alignment, we set the maximumnumber of conformers per molecule in the library to begenerated at 100 while retaining up to 10 conformers perrotatable bond.We filtered out conformers with a similarityscore below 0.70. Finally, we obtained a final list of com-poundswith reported protein targets with a shape similarityscore higher than 0.70.

ERK2 purification and crystallizationPurification of the full-length (residues 1–360) human

ERK2 was performed as described previously (20). Briefly, aHis-tagged ERK2 protein was purified on HisPur Ni-NTAresin (Thermo Scientific); the His-tag removed by incuba-tion with thrombin; and the protein was repurified by FPLCon a Superdex 200 column.Untagged ERK2was crystallizedin a sitting drops plate by mixing protein with precipitantsolution comprised of 1.1 to 1.4 mol/L ammonium sulfate,2% PEG 500MME, and 0.1 mol/L HEPES-NaOH (pH 7.5).The ERK2 crystals were soaked in precipitant solution con-taining 2.5 mmol/L caffeic acid in 2.5% dimethyl sulfoxide(DMSO) for 1 to 2 days. The crystals were cryoprotectedin precipitant solution (1.8mol/L ammonium sulfate) with

Caffeic Acid Targets ERKs to Inhibit UV-Induced Skin Cancer

www.aacrjournals.org Cancer Prev Res; 7(10) October 2014 1057




the addition of 20% xylitol, and flash-cooled in liquidnitrogen. The ERK2 mutant, Q105A, was purified, crystal-lized, and soaked with caffeic acid in a manner similar towild-type (WT) ERK2. ERK2 was also cocrystallized with 2mmol/L AMP-PNP and 5 mmol/L MgCl2 under similarcrystallization conditions. Despite all attempts, electrondensity was clearly visible only for the adenosine moietyof AMP-PNP but not for any phosphate groups.

ERK2/caffeic acid cocrystal structure determinationThe high-resolution diffraction data to a resolution of 1.8

A�were collected at the Advanced Photon Source NE-CAT

beamline 24ID-E using a 30- � 50-mm beam and theQuantum 315 CCD detector. X-ray diffraction data wereintegrated and scaled using the HKL2000 package (21). Thestructure was resolved by molecular replacement using astarting model of the refined ERK2/norathyriol structure(PDB code 3SA0; ref. 20). All calculations were performedusing PHENIX (22). The coupling cycles of slow-coolingannealing, positional, restrained isotropic temperature fac-tor, and Translation/Libration/Screw (TLS) refinementswere followed by visual inspection of the electron densitymaps, including omit maps, coupled with a manual modelrebuilt using the graphics program COOT (23). The refinedelectron density clearly matched the amino acid sequenceof ERK2 with the exception of the N- and C-terminus(residues 1–5 and 358–360) and a weak electron densitywas observed for residues 23 to 34 and 336 to 340. Thevalues of the free R-factor weremonitored during the courseof the crystallographic refinement and the final value of freeR-factors did not exceed the overall R-factor by more than4%. The structures of the ERK2-mutant Q105A and ERK2 ina complex with adenosine (AMP-PNP was used for cocrys-tallization) were refined using a similar protocol (data notshown). The coordinates and structure factors for ERK2complexed with caffeic acid have been deposited in theProtein Data Bank under accession code 4N0S.

In vitro ERK1 and 2 kinase assaysThe recombinant nonphosphorylated His-tagged RSK2

(1 mg) was used as a substrate in an in vitro kinase assaywith 200 ng of active ERK1 or 2 (Millipore). Reactionswere conducted at 30�C for 60 minutes in kinase buffer(25mmol/L Tris-HClpH7.5, 5mmol/Lb-glycerophosphate,2 mmol/L DTT, 0.1 mmol/L Na3VO4, 10 mmol/L MgCl2)containing 100 mmol/L unlabeled ATP with or without10 mCi of [g-32P] ATP. Reactions were stopped by adding5�protein loadingbuffer andproteinswere resolvedby10%SDS-PAGE and visualized by autoradiography.

In vitro pull-down assayCaffeic acid–conjugated Sepharose 4B or Sepharose 4B

beads were prepared as reported (24). For the in vitro or exvivo pull-down assay, active ERK1 or 2 (200 ng) or lysatesfrom HaCaT cells (1 mg) were mixed with 100 mL of caffeicacid–conjugated Sepharose 4B or Sepharose 4B beads in400 mL of reaction buffer (50 mmol/L Tris–HCl pH 7.5, 5mmol/L EDTA, 150 mmol/L NaCl, 1 mmol/L DTT, 0.01%

NP-40, and 2 mg/mL BSA). After incubation with gentlerocking at 4�C overnight, the beads were washed five timeswithwashing buffer (50mmol/L Tris–HCl pH7.5, 5mmol/L EDTA, 150mmol/LNaCl, 1mmol/L DTT, and 0.01%NP-40), and proteins bound to the beads were analyzed byWestern blotting.

ATP and caffeic acid competition assayFor the ATP competition assay, active ERK2 (200 ng) was

incubated with different concentrations of ATP (0, 10, or100 mmol/L) in reaction buffer at 4�C for 2 hours. Caffeicacid–conjugated Sepharose 4B or Sepharose 4B beads (con-trol) were added followed by incubation at 4�C for 2 hours.After washing five times with washing buffer, the proteinsbound to the beads were analyzed by Western blotting.

Western blot analysisFor Western blotting, cells (2 � 106) were cultured in

10 cmdishes for 24 hours. The cells were cultured inDMEMwithout FBS for 24 hours to eliminate the influence of FBSon the activation of MAPKs. Cells were treated with caffeicacid (0–80 mmol/L) for 1 hour before being exposed toSUV (60 kJ UVA/m2 and 3.6 kJ UVB/m2) and harvested15minutes later. Cells were disrupted on ice for 30minutesin lysis buffer [20 mmol/L Tris pH 7.5, 150 mmol/L NaCl,1 mmol/L Na2EDTA, 1 mmol/L EGTA, 1% Triton X-100,2.5 mmol/L sodium pyrophosphate, 1 mmol/L b-glycero-phosphate, 1 mmol/L sodium vanadate, and 1 mmol/Lphenylmethylsulfony fluoride (PMSF)]. After centrifuga-tion at 20,817 � g for 15 minutes, the supernatant fractionwas harvested as the total cellular protein extract. Theprotein concentration was determined using the Bio-Radprotein assay reagent. Total cellular protein extracts wereseparated by SDSPAGE and transferred to polyvinylidenefluoride membranes in 20 mmol/L Tris-HCl (pH 8.0),containing 150 mmol/L glycine, and 20% (v/v) metha-nol. Membranes were blocked with 5% non-fat drymilk in TBS containing 0.05% Tween 20 (TBS-T) andincubated with primary antibodies against phosphorylat-ed (p)-ERK1/2, ERK1/2, p-p90RSK, p90RSK, p-Elk1, Elk1,p-c-Myc, c-Myc, or b-actin at 4�C overnight. Blots werewashed three times in TBS-T buffer, followed by incuba-tion with the appropriate horseradish peroxidase–linkedIgG. Specific proteins were visualized using an enhancedchemiluminescence detection reagent.

Xenograft mouse modelFemale BALB/c (nu/nu) mice (6-week-old) were pur-

chased from The Jackson Laboratory and maintainedunder "specific pathogen-free" conditions. All studieswere performed following guidelines approved by the Uni-versity of Minnesota Institutional Animal Care and UseCommittee (Minneapolis, MN). Mice were divided into6 groups (n¼ 8): (i) mice injected with A431 sh-mock cellsand treated with vehicle; (ii) mice injected with A431 sh-mock cells and treated with 10mg/kg caffeic acid; (iii) miceinjected with A431 sh-mock cells and treated with 100 mg/kg caffeic acid; (iv) mice injected with A431 sh-ERK2 cells

Yang et al.

Cancer Prev Res; 7(10) October 2014 Cancer Prevention Research1058




and treated with vehicle; (v) mice injected with A431 sh-ERK2 cells and treated with 10 mg/kg caffeic acid; and (vi)mice injected with A431 sh-ERK2 cells and treated with 100mg/kg caffeic acid. Mice were administered vehicle (10%DMSO and 20% polyethylene glycol 400 in PBS) or caffeicacid in vehicle i.p. every day for 3 consecutive weeks,beginning 3 days before injection of cells. A431 cells (3 �106 in 50 mL PBS with 50 mLMatrigel) were injected s.c. intothe right flank of each mouse. Tumor volume (length �width� depth� 0.52) was measured three times per week.Body weights were recorded once per week.

Mouse skin tumorigenesis studyFemale SKH-1 hairlessmicewere purchased fromCharles

Rivers Laboratories andmaintained according to guidelinesestablished by the Research Animal Resources and Institu-tional Animal Care and Use Committee, University ofMinnesota (Minneapolis, MN). SKH-1 mice (5–6-week-old; 25 g mean body weight) were divided into 6 age-

matched groups: (i) vehicle-treated (n ¼ 12); (ii) 20mmol/L caffeic acid (n ¼ 12); (iii) vehicle þ SUV (n ¼15); (iv) 10 mmol/L caffeic acid administered before SUV(n ¼ 15); (v) 20 mmol/L caffeic acid administered beforeSUV (n¼ 15); (vi) 20 mmol/L caffeic acid administered afterSUV (n¼ 15). The dose of SUV was progressively increased(10% each week) because of the ensuing hyperplasia thatcan occur with SUV irradiation of the skin. The vehicle wasacetone and SUV irradiation was administered three timesper week for 15 weeks with the dose at week 1 at 30 kJ/m2

UVA and 1.8 kJ/m2UVB. Atweek 6, the dose of SUV reached48 kJ/m2 (UVA) and 2.9 kJ/m2 (UVB) and this dose wasmaintained until 15 weeks. Mice were weighed and tumorsmeasured by caliper once a week until week 29 or tumorsreached a total volume of 1 cm3. At that point mice wereeuthanized and half of the dorsal skin was immediatelyfixed in 10% neural-buffered formalin and processed forhematoxylin and eosin (H&E) staining and IHC. The otherhalf was frozen for Western blot analysis.

Figure 1. Caffeic acid reduceshuman skin cancer cell colonyformation. A, chemical structure ofcaffeic acid. B, caffeic acid inhibitsA431, SK-MEL-5, or SK-MEL-28colony formation dose-dependently. Representativephotographs are shown and data,means � SD of values from threeindependent experiments; theasterisk (�) indicates a significant (P< 0.05) decrease in colonyformation in cells treated withcaffeic acid compared with theDMSO-treated group.






Statistical analysisAll quantitative data are expressed asmeans� SDor SE as

indicated. The Student t test or a one-way ANOVA was usedfor statistical analysis. A P value of <0.05 was used as thecriterion for statistical significance.

ResultsCaffeic acid inhibits human skin cancer cell colonyformation and EGF-induced neoplastic transformationof HaCaT cells

In the present study, we examined the effect of caffeic acid(Fig. 1A) on human skin cancer cell colony formationand EGF-induced transformation ofHaCaT cells. Treatmentof A431 skin cancer cells or SK-MEL-5 and SK-MEL-28melanoma cells with caffeic acid significantly inhibited

colony formation in soft agar in a dose-dependent manner(Fig. 1B). HaCaT cells were also treatedwith caffeic acid andwas not cytotoxic (Supplementary Fig. S1A). However,caffeic acid dramatically inhibited EGF-promoted transfor-mation dose-dependently (Supplementary Fig. S1B). Col-ony formation in soft agar is an ex vivo indicator and a keycharacteristic of the transformed cell phenotype (25) andthese results indicate that caffeic acid can inhibit colonyformation of human skin cancer cells and neoplastic trans-formation of HaCaT cells induced by EGF.

ERKs are identified by in silico screening as plausibletargets of caffeic acid

To identify potential targets of caffeic acid, we conductedin silico screening by using a smallmolecule shape similarity

Figure2. Caffeic acid inhibits ERKs kinase activities bydirectly bindingwithERK1or 2 in anATP-competitivemanner and attenuatesMAPKsignaling in primarymouse epidermal keratinocytes. A, a His-tagged–RSK2 fusion protein was used in an in vitro kinase assay with active ERK1 or 2 and results visualized byautoradiography. Coomassie blue staining serves as a loading control. B, caffeic acid directly binds with ERK1 or 2. Binding of caffeic acid with ERK1 or2 was confirmed byWestern blot analysis. C, caffeic acid binds to ERK2 competitively with ATP. Active ERK2 (0.2 mg) was incubatedwith ATP at 0, 1, 10, 100mmol/L and 100 mL of caffeic acid-Sepharose 4B or Sepharose 4B (negative control) beads in reaction buffer in a final volume of 500 mL. Pulled downproteins were detected by Western blotting. D, after starvation in serum-free medium for 24 hours, cells were treated with caffeic acid at the indicatedconcentration for 1 hour, then exposed toSUV (60 kJ/m2UVA, 3.6 kJ/m2UVB), incubatedat 37�C in a5%CO2 incubator for 15minutes and thenharvested andprotein levels determined by Western blotting.

Yang et al.





approach. Caffeic acid was screened against all the crystal-lized ligands available from the Protein Data Bank and ourin-house natural compound library. Screening resultsshowed that caffeic acid has a shape and pharmacophoresimilarity of 0.76 with ERK00071, which is a known ERK2inhibitor. Thus, ERK2 was suggested as potential proteintarget of caffeic acid.

Caffeic acid inhibits ERK1/2 in vitro kinase activity andsuppresses SUV-induced activation of ERKs signalingWe performed an in vitro kinase assay using recombi-

nant active ERK1 or 2 in the presence of various con-centrations of caffeic acid. CAY10561, a well-knowninhibitor of ERK2, was used as a positive control. Thephosphorylation of RSK2, a well-known ERKs substrate,was inhibited by caffeic acid in a concentration-depen-dent manner (Fig. 2A). We also performed an in vitrobinding assay using caffeic acid–conjugated Sepharose 4Bbeads. No obvious band was observed when the recom-binant active ERK 1 or 2 was incubated with controlSepharose 4B beads, whereas a strong band was seenwhen ERK1 or 2 was incubated with caffeic acid–conju-gated Sepharose 4B beads (Fig. 2B). An ATP-competitivepull-down assay demonstrated that caffeic acid binds toERK2 in an ATP-competitive manner (Fig. 2C). Altogeth-er, the results clearly indicate that caffeic acid binds torecombinant ERK 1 or 2 to inhibit their respective activity.

To confirm the effect of caffeic acid on ERKs signaling, weused SUV to induce the activation of various ERKs substratesusing primary mouse epidermal keratinocytes or HaCaTcells. The results show that caffeic acid inhibited SUV-induced phosphorylation of ERKs and also reduced phos-phorylation of their downstream target proteins, includingRSK2, c-Myc, and Elk1 (Fig. 2D and Supplementary Fig. S2;ref. 26).

Cocrystal structure of caffeic acid and ERK2To identify the bindingmode of ERKs and caffeic acid, we

resolved the crystal structure of ERK2 bound with caffeic

acid at 1.8 A�resolution. The details of data collection and

structure refinement for the ERK2/caffeic acid complex arepresented in Supplementary Table S1. Caffeic acid is boundat the ATP-binding site (Fig. 3A–C). The catechol moiety ofcaffeic acid occupies a position at the adenine ring of AMP-PNP that is visible in electron density as adenosine. Twohydroxyl groups of caffeic acid form hydrogen bonds withamino acid residues of ERK2 located in the hinge region,including the carboxyl groupofQ105 and themain chainofD106 and M108. To confirm the importance of the Q105residue in bindingwith caffeic acid, we performed an in vitropull-down–binding assay using caffeic acid–conjugatedSepharose 4B beads. Compared with WT ERK2, a purifiedrecombinant ERK2 mutant, Q105A, showed decreasedbinding with caffeic acid–conjugated Sepharose 4B beads

Figure 3. Crystal structure of theERK2/caffeic acid complex. A,superimposition of ERK2 boundwith caffeic acid and ERK2 boundwith adenosine. The bindinginteractions within the hinge regionare shown as dotted lines. Thecaffeic acid molecule (blue sticks)interacts with ERK2 amino acidresidues Q105, D106, and M108.The catechol moiety of caffeic acidis positioned in the same plane asthe adenine moiety of adenosine(yellow lines). The three AMP-PNPphosphate groups were invisibleon the electron density map. B,electron density map 2Fo-Fc forcaffeic acidwas contoured at 1.5s.C, surface and ribbonrepresentation of the ERK2/caffeicacid structure. Caffeic acid boundat the ATP pocket is shown as balland sticks. D, caffeic acid bindingwith a WT recombinant ERK2compared with its binding with anERK2 Q105A mutant. Proteinswere incubated with caffeic acid–conjugated Sepharose 4B beadsand analyzed by Western blotanalysis.






(Fig. 3D). We attempted to obtain the Q105A-mutantcrystal structure bound with caffeic acid. However, noelectron density of caffeic acid was observed in the refinedstructure of the Q105A mutant despite identical soakingexperiments. This result suggests that the Q105A mutantwas not able to strongly bind with caffeic acid in thecrystallized form.

Knockdownof ERK2decreases the anticancer effects ofcaffeic acid

We determined whether knocking down ERK2 expres-sion could influence the sensitivity of human skin cancercells or HaCaT cells to caffeic acid. We first determinedthe efficiency of shRNA knockdown, as well as theeffect of shRNA transfection on anchorage-independentgrowth. The expression of ERK2 in each cell line wasobviously decreased after shRNA transfection (Fig. 4A and

Supplementary Fig. S3A). Colony number dramaticallydecreased after ERK2 shRNA transfection compared withthe sh-mock group (Fig. 4B and Supplementary Fig. S3B).Next, cells transfected with ERK2 shRNA or sh-mock weretreated with caffeic acid or vehicle and subjected to a softagar assay. The results showed that caffeic acid (40 mmol/L) inhibited colony formation of each cell type trans-fected with sh-mock by more than 80%. In contrast, thesame dose of caffeic acid inhibited colony formation ofeach cell type transfected with ERK2 shRNA by no morethan 50% (Fig. 4C and Supplementary Fig. S3C). Theseresults suggested that ERK2 plays an important role in thesensitivity of human skin cancer cells or HaCaT cells tothe antiproliferative effects of caffeic acid.

Using a xenograftmodel, we found that administration ofvehicle or caffeic acid (10 or 100 mg/kg) had no effect onbody weight (Fig. 5A). More importantly, we observed that

Figure 4. Knockdown of ERK2 inhuman skin cancer cells decreasessensitivity to caffeic acid.A, efficiency of ERK2 shRNA inA431 and SK-MEL-5 cells. B,colony formation of A431 andSK-MEL-5 cells transfected withsh-mock, ERK2 shRNA#1,shRNA#2, shRNA#3 or shRNA#4.Colony number dramaticallydecreased after ERK2 shRNAtransfection compared with thesh-mock group. Data, means �SD. The asterisk (�) indicates asignificant decrease in colonynumber compared with the sh-mock group (P < 0.05). C,sensitivity of skin cancer cellstransfected with sh-mock orERK2 shRNA to treatment withcaffeic acid (40 mmol/L). Data,means � SD. The asterisk (�)indicates a significant decrease insensitivity to caffeic acid (P < 0.05).

Yang et al.





knocking down ERK2 expression decreased xenografttumor volume (Fig. 5B and C), but also decreased thesensitivity of xenograft skin cancer growth to caffeic acid(Fig. 5B and D). These results indicated that when ERK2levels were decreased, caffeic acid lost the ability to exert itspreventive effects against skin cancer development.

Caffeic acid suppresses SUV-induced skincarcinogenesis in SKH-1 hairless mice in vivoTo further study the antitumorigenic activity of caffeic

acid in vivo, we evaluated the effect of caffeic acid in an SUV-induced mouse skin tumorigenesis model. We used SUV tomimic sunlight because although UVB is a major etiologicfactor for the development of skin cancer, UVA is the mostabundant component of SUV irradiation (5). The resultsdemonstrated that topical application of caffeic acid todorsal skin–inhibited skin cancer development inmice thatwere exposed to SUV compared with mice treated withvehicle only and exposed to SUV (Fig. 6A). Caffeic acid(10 or 20 mmol/L) significantly inhibited the average num-ber (P < 0.05, Fig. 6B) or volume (P < 0.05, Fig. 6C) oftumors per mouse. Notably, application of caffeic acideither before or after UV resulted in similar observations;a significant reduction in tumor formation. Skin and tumorsamples were processed for H&E and IHC staining. Aftertreatmentwith SUV, epidermal thickness in the vehicle/SUVgroup was increased by edema and epithelial cell prolifer-ation, whereas caffeic acid–treated groups showed a smaller

increase in epidermal thickness and less inflammation (Fig.6D). IHC data showed that Ki-67, which is a well-knowncellularmarker for proliferation,was dramatically increasedin the vehicle/SUV group compared with the vehicle group.However, Ki-67 expression was decreased in the SUV–caf-feic acid–treated groups, compared with the vehicle/SUVgroup (Fig. 6D). The IHC staining for phosphorylation of c-Myc, which is downstream of ERKs, showed a significantlyreduced level of phosphorylation in mice treated withcaffeic acid (Fig. 6D). In addition, Western blotting analysisofmouse skin showed that the phosphorylation of p90RSK,c-Myc, and Elk1 induced by SUV was dramatically sup-pressed in the caffeic acid–treated groups (Fig. 6E). Overall,these results indicate that caffeic acid might serve as aneffective chemopreventive agent against SUV-mediated skincancer acting by suppressing the activity of ERKs.

DiscussionMany research groups have observed that coffee con-

sumption is linked to a reduced risk of several cancers,including liver, colorectal, mouth and throat cancer, andbreast cancer in premenopausal women (1, 11, 27–31).Caffeic acid is one of the main metabolites produced bythe hydrolyzation of chlorogenic acid, a major phenolicphytochemical found in various foods, especially coffee.Previous studies have shown that caffeic acid inhibits skintumor promotion induced by 12-O-tetradecanoylphorbol-

Figure 5. Knockdown of ERK2 decreases preventive effects of caffeic acid against skin cancer development in a xenograft mousemodel. A, caffeic acid doesnot affectmouse bodyweight. Micewere treatedwith caffeic acid or its vehicle i.p. once/day for 3weeks. Bodyweight of eachmousewasmeasured once perweek and data, means � SD. B, representative photographs of mice from each group injected with sh-mock or ERK2 shRNA#1 A431 cells. At 19 days afterinjectionof A431cells,micewere euthanizedwithCO2.C, knockdownof ERK2suppresses tumor growth in anA431 xenograftmousemodel. D, knockdownofERK2 decreases preventive effects of caffeic acid against skin cancer development. Mice either injected with A431 sh-mock or A431 ERK2 shRNA#1 cellswere given caffeic acid or vehicle as described in Materials andMethods. In all groups, tumor volume wasmeasured three times per week after injection withA431 cells. Data, means � SD and the asterisk (�) indicates a significant difference (P < 0.05) in tumor volume (mm3).






13-acetate in mouse skin (31). Also caffeic acid has beenimplicated as a protective agent against UVB-induced skindamage (32). Although accumulating evidence suggeststhat caffeic acid has the potential to inhibit skin cancerdevelopment, the actual effects and the molecular mechan-isms remain unclear. In the present study, we showed achemopreventive effect of caffeic acid against SUV-inducedskin cancer development and identified ERK1 and 2 as itspossible protein targets.

UV radiation in sunlight is the most prominentand ubiquitous physical carcinogen in our natural envi-ronment. Studies have shown that sunlight induced sig-nal transduction is an important step for developmentof both nonmelanoma skin cancer and melanoma (33).Cells respond to signals produced from UV exposure byactivating signaling cascades, including the MAPK path-way. MAPKs regulate multiple critical cellular functions,including proliferation, growth, and senescence (34).

Figure 6. Caffeic acid inhibits SUV-induced skin tumorigenesis inSKH-1 hairless mice. A, externalappearance of tumors. B, averagetumor number; and C, tumorvolume per mouse. Tumor volumewas calculated using the formula:tumor volume (mm3) ¼ (length �width � height � 0.52). Data,means � SE. The asterisk (�)indicates a significant difference (P< 0.05) between the SUV groupstreated or not treated with caffeicacid. At the end of the study, 1 of 2of the skin and tumor sampleswerefixed in 10% neutral-bufferedformalin and processed for D, H&Estaining and IHC with specificprimary antibodies to detect Ki-67and p-c-Myc. E, the other half ofthe skin and tumor samples wasanalyzed for protein expression byWestern blotting.

Yang et al.





Studies in various skin cell lines demonstrated that EGFreceptors (35), MAPKs (36), and PI3K (37) are specificsignaling molecules in UVB-induced skin carcinogenesis.Some studies showed that when HaCaT cells were irra-diated by UVB (0.2 kJ/m2), ERK1 and 2 were phosphor-ylated after 15 minutes and remained activated for 6hours (38). In our previous study, phosphorylation ofERKs and RSK2 was increased by exposure to UVB (4 kJ/m2) in HaCaT cells (39), suggesting that ERKs might be auseful anticancer target.In the present work, we showed that caffeic acid effec-

tively inhibited colony formation of human skin cancercells and EGF-induced neoplastic transformation of HaCaTcells (Fig. 1 and Supplementary Fig. S1). Long-term topicaltreatment of mouse dorsal skin with caffeic acid eitherbefore or after SUV significantly reduced skin tumor for-mation (Fig. 6). The Ki-67 protein is a known cellularmarker for proliferation (40) and our IHC results showedthat expression of Ki-67 was dramatically decreased aftertreatment with caffeic acid.We provided systemic evidence, indicating that caffeic

acid directly targets ERKs. According to our ERK2/caffeicacid cocrystal structure (Fig. 3), caffeic acid stronglybinds to the ATP-binding cleft through the formation ofhydrogen bonds between hydroxyl groups and aminoacids Q105, D106, and M108 located at the hinge loop.This is the best evidence available showing that caffeicacid can target ERKs at the molecular level. We furthershowed that caffeic acid suppressed SUV-induced ERKsphosphorylation and downstream signaling in primarymouse epidermal keratinocytes and HaCaT cells (Fig. 2Dand Supplementary Fig. S2). Caffeic acid was highlyeffective in decreasing SUV-induced skin carcinogenesis,in vivo, whether applied before or after exposure to SUV(Fig. 6A and B). Western blot analysis of mouse skintissue clearly confirmed that the SUV-induced phosphor-ylation of several of ERKs’ downstream targets, includingRSK2, Elk1, and c-Myc, was suppressed by caffeic acid(Fig. 6D). In addition, knocking down ERK2 expressiondecreased the sensitivity of human skin cancer cellsand HaCaT cells to caffeic acid treatment (Fig. 4C andSupplementary Fig. S3). Finally, we used a skin cancer

xenograft model to provide in vivo evidence showing thatlow levels of ERK2 attenuated the ability of caffeic acid toexert its preventive effects against skin cancer develop-ment (Fig. 5).

Taken together, our results clearly showed that topicalapplication of caffeic acid markedly suppressed the forma-tion of skin cancer in SKH-1 hairless mice exposed to SUV.Caffeic acid functioned as a potent inhibitor of ERK1 and 2and suppressed the activity of their downstream substrates,RSK2, Elk1, and c-Myc. Therefore, caffeic acid might be agood chemopreventive agent that is highly effective againstSUV-induced skin cancer.

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

Authors' ContributionsConception and design: G. Yang, A.M. Bode, Z. Dong, Z. DongDevelopment of methodology: G. Yang, K. Yao, Z. DongAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): G. Yang, Y. Fu, M. Malakhova, I. Kurinov, K. Yao,W. Li, Z. DongAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):G. Yang, Y. Fu,M.Malakhova, I. Kurinov,F. Zhu, H. Li, A.M. Bode, Z. DongWriting, review, and/or revision of the manuscript: G. Yang, Y. Fu,M. Malakhova, I. Kurinov, H. Chen, D.Y. Lim, A.M. Bode, Z. DongAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K. Yao, D.Y. Lim, Y. Sheng, Z. DongStudy supervision: Z. Dong

Grant SupportThe crystallography work is based upon research conducted at the

Advanced Photon Source on the Northeastern Collaborative Access Teambeamlines, which is supported by a grant from the National Institute ofGeneral Medical Sciences (P41 GM103403) from the NIH. The use of theAdvanced Photon Source, an Office of Science User Facility operated for theU.S. Department of Energy Office of Science by the Argonne NationalLaboratory, was supported by the U.S. DOE under contract no. DE-AC02-06CH11357. The remainder of the work presented was supported by TheHormel Foundation and National Institutes of Health grants (to Z. Dong)R37 CA081064, CA166011, CA172457, and ES016548.

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 May 2, 2014; revised July 25, 2014; accepted July 28, 2014;published OnlineFirst August 7, 2014.

References1. Arab L. Epidemiologic evidence on coffee and cancer. Nutr Cancer

2010;62:271–83.2. Holick CN, Smith SG, Giovannucci E, Michaud DS. Coffee, tea,

caffeine intake, and risk of adult glioma in three prospective cohortstudies. Cancer Epidemiol Biomarkers Prev 2010;19:39–47.

3. Abel EL, Hendrix SO, McNeeley SG, Johnson KC, Rosenberg CA,Mossavar-Rahmani Y, et al. Daily coffee consumption and prevalenceof nonmelanoma skin cancer in Caucasian women. European journalof cancer prevention: the official journal of the European CancerPrevention Organisation 2007;16:446–52.

4. Ricotti C, Bouzari N, Agadi A, Cockerell CJ.Malignant skin neoplasms.Med Clin North Am 2009;93:1241–64.

5. Berwick M, Lachiewicz A, Pestak C, Thomas N. Solar UV exposureand mortality from skin tumors. Adv Exp Med Biol 2008;624:117–24.

6. de Gruijl FR. Skin cancer and solar UV radiation. Eur J Cancer1999;35:2003–9.

7. Chung TW, Moon SK, Chang YC, Ko JH, Lee YC, Cho G, et al. Noveland therapeutic effect of caffeic acid and caffeic acid phenyl esteron hepatocarcinoma cells: complete regression of hepatomagrowth and metastasis by dual mechanism. FASEB J 2004;18:1670–81.

8. Tanaka T, Kojima T, Kawamori T, Wang A, Suzui M, Okamoto K, et al.Inhibition of 4-nitroquinoline-1-oxide-induced rat tongue carcinogen-esis by the naturally occurring plant phenolics caffeic, ellagic, chloro-genic, and ferulic acids. Carcinogenesis 1993;14:1321–5.

9. Sul D, Kim HS, Lee D, Joo SS, Hwang KW, Park SY. Protective effectof caffeic acid against beta-amyloid-induced neurotoxicity by theinhibition of calcium influx and tau phosphorylation. Life Sciences2009;84:257–62.






10. Jung JE, Kim HS, Lee CS, Park DH, Kim YN, LeeMJ, et al. Caffeic acidand its synthetic derivative CADPE suppress tumor angiogenesis byblockingSTAT3-mediatedVEGFexpression in human renal carcinomacells. Carcinogenesis 2007;28:1780–7.

11. Kang NJ, Lee KW, Kim BH, Bode AM, Lee H-J, Heo Y-S, et al. Coffeephenolic phytochemicals suppress colon cancermetastasis by target-ing MEK and TOPK. Carcinogenesis 2011;32:921–8.

12. McCubrey JA, Steelman LS, Chappell WH, Abrams SL, Wong EW,Chang F, et al. Roles of the Raf/MEK/ERK pathway in cell growth,malignant transformation and drug resistance. Biochim Biophys Acta2007;1773:1263–84.

13. DhillonAS,HaganS,RathO,KolchW.MAPkinasesignallingpathwaysin cancer. Oncogene 2007;26:3279–90.

14. KimHH,ChoS, LeeS, KimKH,ChoKH, EunHC, et al. Photoprotectiveand anti-skin-aging effects of eicosapentaenoic acid in human skin invivo. J Lipid Res 2006;47:921–30.

15. Einspahr JG, Calvert V, Alberts DS, Curiel-Lewandrowski C, WarnekeJ,KrouseR, et al. Functional protein pathwayactivationmapping of theprogression of normal skin to squamous cell carcinoma. Cancer PrevRes 2012;5:403–13.

16. Ohori M, Kinoshita T, Okubo M, Sato K, Yamazaki A, Arakawa H, et al.Identification of a selective ERK inhibitor and structural determinationof the inhibitor-ERK2 complex. Biochem Biophys Res Commun2005;336:357–63.

17. Russo AE, Torrisi E, Bevelacqua Y, Perrotta R, Libra M, McCubrey JA,et al. Melanoma: molecular pathogenesis and emerging target ther-apies (Review). Int J Oncol 2009;34:1481–9.

18. Schr€odinger. Schr€odinger Suite 2012. Schr€odinger, LLC, New York,NY, 2012. 2012.

19. ChenH,YaoK,Nadas J, BodeAM,MalakhovaM,OiN, et al. Predictionof molecular targets of cancer preventing flavonoid compounds usingcomputational methods. PLoS ONE 2012;7:e38261.

20. Li J, Malakhova M, Mottamal M, Reddy K, Kurinov I, Carper A,et al. Norathyriol suppresses skin cancers induced by solar ultra-violet radiation by targeting ERK kinases. Cancer Res 2012;72:260–70.

21. Otwinowski Z, Minor W. Processing of x-ray diffraction data collectedin oscillation mode. Methods Enzymol 1997;276:307–326.

22. Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ,Moriarty NW, et al. PHENIX: building new software for automatedcrystallographic structure determination. Acta Crystallogr D Biol Crys-tallogr 2002;58:1948–54.

23. Emsley P, Cowtan K. Coot: model-building tools for molecular gra-phics. Acta Crystallogr D Biol Crystallogr 2004;60:2126–32.

24. Jung SK, Lee KW, Byun S, Kang NJ, Lim SH, Heo YS, et al. Myricetinsuppresses UVB-induced skin cancer by targeting Fyn. Cancer Res2008;68:6021–9.

25. FreedmanVH,Shin SI. Cellular tumorigenicity in nudemice: correlationwith cell growth in semi-solid medium. Cell 1974;3:355–9.

26. RamanM, ChenW, CobbMH. Differential regulation and properties ofMAPKs. Oncogene 2007;26:3100–12.

27. Yu X, Bao Z, Zou J, Dong J. Coffee consumption and risk of cancers: ameta-analysis of cohort studies. BMC Cancer 2011;11:96.

28. Nkondjock A. Coffee consumption and the risk of cancer: an overview.Cancer Lett 2009;277:121–5.

29. Turati F, Galeone C, La Vecchia C, Garavello W, Tavani A. Coffee andcancers of the upper digestive and respiratory tracts:meta-analyses ofobservational studies. Ann Oncol 2011;22:536–44.

30. Tavani A, LaVecchiaC.Coffee, decaffeinated coffee, tea andcancer ofthe colon and rectum: a review of epidemiological studies, 1990–2003.Cancer Causes Control 2004;15:743–57.

31. Huang MT, Smart RC, Wong CQ, Conney AH. Inhibitory effect ofcurcumin, chlorogenic acid, caffeic acid, and ferulic acid on tumorpromotion in mouse skin by 12-O-tetradecanoylphorbol-13-acetate.Cancer Res 1988;48:5941–6.

32. Staniforth V, Chiu LT, Yang NS. Caffeic acid suppresses UVB radia-tion-induced expression of interleukin-10 and activation of mitogen-activated protein kinases inmouse. Carcinogenesis 2006;27:1803–11.

33. Bode AM, Dong Z. Mitogen-activated protein kinase activation in UV-induced signal transduction. Sci STKE 2003;2003:RE2.

34. Kohno M, Pouyssegur J. Targeting the ERK signaling pathway incancer therapy. Ann Med 2006;38:200–11.

35. Wan YS, Wang ZQ, Shao Y, Voorhees JJ, Fisher GJ. Ultravioletirradiation activates PI 3-kinase/AKT survival pathway via EGF recep-tors in human skin in vivo. Int J Oncol 2001;18:461–6.

36. ChenW, TangQ, GonzalesMS, BowdenGT. Role of p38MAP kinasesand ERK in mediating ultraviolet-B induced cyclooxygenase-2 geneexpression in human keratinocytes. Oncogene 2001;20:3921–6.

37. KabuyamaY, HamayaM, HommaY.Wavelength specific activation ofPI 3-kinase by UVB irradiation. FEBS Lett 1998;441:297–301.

38. He YY, Huang JL, Chignell CF. Delayed and sustained activation ofextracellular signal-regulated kinase in human keratinocytes byUVA: implications in carcinogenesis. J Biol Chem 2004;279:53867–74.

39. ChoYY, LeeMH, LeeCJ, YaoK, LeeHS,BodeAM, et al. RSK2as a keyregulator in human skin cancer. Carcinogenesis 2012;33:2529–37.

40. Scholzen T, Gerdes J. The Ki-67 protein: from the known and theunknown. J Cell Physiol 2000;182:311–22.


Yang et al.




2014;7:1056-1066. Published OnlineFirst August 7, 2014.Cancer Prev Res Ge Yang, Yang Fu, Margarita Malakhova, et al. Skin CarcinogenesisCaffeic Acid Directly Targets ERK1/2 to Attenuate Solar UV-Induced

Updated version

10.1158/1940-6207.CAPR-14-0141doi:

Access the most recent version of this article at:

Material

Supplementary

1

http://cancerpreventionresearch.aacrjournals.org/content/suppl/2014/08/16/1940-6207.CAPR-14-0141.DCAccess the most recent supplemental material at:

Cited articles

http://cancerpreventionresearch.aacrjournals.org/content/7/10/1056.full#ref-list-1

This article cites 39 articles, 8 of which you can access for free at:

Citing articles

http://cancerpreventionresearch.aacrjournals.org/content/7/10/1056.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerpreventionresearch.aacrjournals.org/content/7/10/1056To request permission to re-use all or part of this article, use this link



http://cancerpreventionresearch.aacrjournals.org/lookup/doi/10.1158/1940-6207.CAPR-14-0141

http://cancerpreventionresearch.aacrjournals.org/content/suppl/2014/08/16/1940-6207.CAPR-14-0141.DC1

http://cancerpreventionresearch.aacrjournals.org/content/suppl/2014/08/16/1940-6207.CAPR-14-0141.DC1

http://cancerpreventionresearch.aacrjournals.org/content/7/10/1056.full#ref-list-1

http://cancerpreventionresearch.aacrjournals.org/content/7/10/1056.full#related-urls

http://cancerpreventionresearch.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerpreventionresearch.aacrjournals.org/content/7/10/1056


Documents

Caffeic Acid Directly Targets ERK1/2 to Attenuate …cancerpreventionresearch.aacrjournals.org/content/canprevres/7/10/...Caffeic Acid Directly Targets ERK1/2 to Attenuate Solar UV-Induced