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Therapeutics, Targets, and Chemical Biology

Akt Kinase-Interacting Protein 1 Signalsthrough CREB to Drive Diffuse MalignantMesotheliomaTadaaki Yamada1,2, Joseph M. Amann1, Koji Fukuda2, Shinji Takeuchi2,Naoya Fujita3, Hisanori Uehara4, Shotaro Iwakiri5, Kazumi Itoi5,Konstantin Shilo6, Seiji Yano2, and David P. Carbone1

Abstract

Diffuse malignant mesothelioma (DMM) is a tumor ofserosal membranes with propensity for progressive local dis-ease. Because current treatment options are largely ineffective,novel therapeutic strategies based on molecular mechanismsand the disease characteristics are needed to improve the out-comes of patients with this disease. Akt kinase interactingprotein 1 (Aki1; Freud-1/CC2D1A) is a scaffold protein forthe PI3K–PDK1–Akt signaling module that helps determinereceptor signal selectivity for EGFR. Aki1 has been suggested asa therapeutic target, but its potential has yet to be evaluated in atumor setting. Here, we report evidence supporting its defini-tion as a therapeutic target in DMM. In cell-based assays, Aki1

silencing decreased cell viability and caused cell-cycle arrest ofmultiple DMM cell lines via effects on the PKA–CREB1 signal-ing pathway. Blocking CREB activity phenocopied Aki1 silenc-ing. Clinically, Aki1 was expressed in most human DMMspecimens where its expression correlated with phosphorylatedCREB1. Notably, Aki1 siRNA potently blocked tumor growth inan orthotopic implantation model of DMMwhen administereddirectly into the pleural cavity of tumor-bearing mice. Ourfindings suggest an important role for the Aki1–CREB axis inDMM pathogenesis and provide a preclinical rationale to targetAki1 by intrathoracic therapy in locally advanced tumors. CancerRes; 75(19); 4188–97. �2015 AACR.

IntroductionDiffuse malignant mesothelioma (DMM) is an aggressive

tumor arising from the mesothelial cells of serosal membranes.Several etiologic factors, including exposure to asbestos (1, 2),have been reported to be involved in the development of DMM.With introduction of strict occupational protective measures,the incidence of DMM has already reached its peak in theUnited States (3); however, it is predicted that there will beincreases in the number of DMM patients in Europe andAustralia until 2020 (3) and in Japan until 2040 (2). DMMis most likely to advance locally and the extent of the disease is

directly implicated in the prognosis of DMM patients (4). Theprognosis of DMM patients is extremely poor because conven-tional treatment regimens, such as chemotherapy and radio-therapy are inefficient in controlling locally advanced diseaseand/or metastasis.

Clinical trials using EGFR tyrosine kinase inhibitors (EGFR-TKIs) for treatment ofDMMhave haddisappointing results (5, 6).This mirrors non–small cell lung cancer (NSCLC) where efficacyof EGFR-TKIs does not correlate with EGFR expression levels andthe activating mutations in EGFR found in NSCLC are extremelyrare in DMM tumors (7, 8). In addition, previous reports haveshown that unlike EGFR-mutated lung cancer cells, multiplereceptor tyrosine kinases (RTK) are simultaneously activated inDMM cell lines (9). An increased understanding of the molecularmechanisms of DMM pathology will identify novel moleculesand signaling pathways responsible for DMM local progressionand provide new targets for therapy (4–6).

Akt kinase–interacting protein 1 (Aki1)/Freud-1/CC2D1A hasbeen shown to be a scaffold protein in the PI3K–PDK1–Aktpathway (10). This protein selectively forms a complex withnonmutated EGFR and Akt in response to EGF stimulation,mediates Akt activation by PDK1, and contributes to cell survivaland proliferation. Recently, we reported that Aki1 constitutivelyassociates with mutated EGFR, including the T790M gatekeepermutation, which is the most prevalent genetic alteration under-lying acquired resistance to EGFR inhibitors. Aki1 has an impor-tant role as a determinant of receptor-selective signaling formutant EGFR, andmediates the survival signal through activationof Akt (11). Aki1 is expressed in EGFR-mutant lung cancer (53/56cases) and pancreatic cancer (25/60 cases; refs. 11, 12). Thus, Aki1may have novel direct therapeutic potential for the treatment ofmany refractory malignancies.

1Department of Internal Medicine, The Ohio State University MedicalCenter, Columbus, Ohio. 2Division of Medical Oncology, CancerResearch Institute, Kanazawa University, Kanazawa, Japan. 3CancerChemotherapy Center, Japanese Foundation for Cancer Research,Tokyo, Japan. 4Department of Molecular and Environmental Pathol-ogy, Instituteof Health Biosciences, University of TokushimaGraduateSchool, Tokushima, Japan. 5Department of Respiratory Surgery,Hyogo Prefectural Amagasaki Hospital, Amagasaki, Japan. 6Depart-ment of Pathology, The Ohio State University Medical Center, Colum-bus, Ohio.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: David P. Carbone, The Ohio State University MedicalCenter, 460West 12th Avenue, Columbus, OH43210. Phone: 614-685-4479; Fax:614-293-4372; E-mail: [email protected]; and Seiji Yano, CancerResearch Institute, KanazawaUniversity, 13-1 Takara-machi, Kanazawa, Ishikawa920-8641, Japan. Phone: 81-76-265-2780; Fax: 81-76-234-4524; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-15-0858

�2015 American Association for Cancer Research.

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Because we do not currently have drugs inhibiting this target,decreasing its activity through RNAi or targeting downstreampathways remain options. RNAi is a phenomenon of RNA-depen-dent sequence-specific gene silencing inmammalian cells. Recent-ly, some drug delivery systems were reported for gene therapyusing nonviral vectors for delivery of DNA, siRNA, and miRNA(13). In particular, in vivo delivery of siRNAs can be highlyefficacious and of very low toxicity and several clinical trials usingsiRNA have shown promising results (13), although nonviralgene-based therapies have yet to be approved by the FDA (14).

In this study, we identify a crucial role for Aki1 in the survival ofDMM cells through the CREB signaling pathway and show thattargetingAki1orCREBwith siRNA is effective in controllingDMMin vitro and Aki1 siRNA inhibited the growth of pleural disease inin vivo orthotopicmodels. To the best of our knowledge, this is thefirst experimental report to identify the pivotal roles of Aki1 inDMM that provides grounds for targeting Aki1 as novel thera-peutic strategy.

Materials and MethodsCell cultures and reagents

We used seven human DMM cell lines, one noncancerousmesothelial cell line (Met-5A), and the HEK293T cell line. Y-MESO-8A and Y-MESO-14 were established in Aichi CancerResearch Center Institute (15). NCI-H290 were provided by Dr.Adi F. Gazdar (University of Texas Southwestern Medical Center,Dallas, TX) and Dr. Yoshitaka Sekido (Aichi Cancer ResearchCenter Institute, Aichi, Japan). Y-MESO-8A was authenticated byshort tandem repeat analysis. Y-MESO-14 andNCI-H290 authen-tications were not performed. MSTO-211H, NCI-H2052, NCI-H2373,NCI-H2452,Met-5A, andHEK293Twere obtained direct-ly from the ATCC within 6 months of the experiments reported.DMM cell lines are divided into three histologic origins, anepithelioid type (NCI-H290 and NCI-H2452), a sarcomatoidtype (NCI-H2052 and NCI-H2373), and a biphasic type(Y-MESO-8A, Y-MESO-14, and MSTO-211H; refs. 15–17). DMMcell lines were maintained in RPMI-1640 medium supplement-ed with 10% FBS, penicillin (100 U/mL), and streptomycin(50 g/mL), in a humidified CO2 incubator at 37�C. Met-5A cellswere cultured according to the instructions of theATCC.HEK293Tcells were cultured in DMEM supplemented with 10% FBS,penicillin (100 U/mL), and streptomycin (50 g/mL), in a humid-ified CO2 incubator at 37�C. All cells were passaged for less than 3months before renewal from frozen, early-passage stocks. Cellswere regularly screened for Mycoplasma, using MycoAlert Myco-plasma Detection Kits (Lonza). PKA inhibitor KT5720 and CBP–CREB interaction inhibitor were obtained from Sigma-Aldlichand Calbiochem, respectively.

Antibodies and Western blottingProtein aliquots of 25 mg each were resolved by SDS polyacryl-

amide gel (Bio-Rad) electrophoresis and transferred to polyviny-lidene difluoride membranes (Bio-Rad). After washing threetimes, the membranes were incubated with Blocking One (Naca-lai Tesque, Inc.) for 1 hour at room temperature, and thenincubated overnight at 4�C with primary antibodies to Akt,phospho-Akt (Thr308), p-CREB1, t-CREB1, p-PKAC, t-PKACa,HA, DYKDDDDK (FLAG), anti–beta-actin (13E5; 1:1,000 dilu-tion; Cell Signaling Technology), Aki1 (1:1,000 dilution; BethylLaboratories), cyclin A, or EGFR antibodies (1:1,000 dilution;

Santa Cruz Biotechnology). After washing three times, the mem-branes were incubated for 1 hour at room temperature withsecondary Ab [horseradish peroxidase (HRP)–conjugated spe-cies-specific Ab]. Immunoreactive bands were visualized withSuperSignal West Dura Extended Duration Substrate EnhancedChemiluminescent Substrate (Pierce Biotechnology). Each exper-iment was performed at least three times independently.

RNAi transfectionStealth RNAi siRNA for Aki1 (HSS123402, HSS123400; Invi-

trogen), EGFR (HSS103114, HSS103116; Invitrogen), and Silenc-er Select siRNA for CREB1 (s3489, s3490; Invitrogen) weretransfected with Lipofectamine RNAiMAX (Invitrogen) in accor-dance with the manufacturer's instructions. Stealth RNAi siRNANegative Control Low GC Duplex no.3 (Invitrogen) was used asscramble control throughout the experiment. Aki1, EGFR, andCREB1 knockdownswere confirmed byWestern blotting analysis.Each experiment was performed at least in triplicate, and threetimes independently.

Proliferation assayThe cells were reseeded at 2� 103 perwell in 96-well plates, and

incubated in antibiotic-containing RPMI-1640 with 10% FBS.After 24 hours of incubation, KT5720 (0.1 and 1 mmol/L), orCBP–CREB interaction inhibitor (0.1 and 0.3 mmol/L) was addedto eachwell, and incubationwas continued for a further 48 hours.These cells were then used for proliferation assay, which wasmeasured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium) dye reduction method. An aliquot of MTTsolution (2 mg/mL; Sigma) was added to each well followed byincubation for 2 hours at 37�C as previously described (18). Themedia were removed and the dark blue crystals in each well weredissolved in 100 mL of DMSO. Absorbance was measured with anMTP-120microplate reader (Corona Electric) at test and referencewavelengths of 550 and 630 nm, respectively. The percentage ofgrowth is shown relative to untreated controls. Each sample wasassayed in triplicate, with each experiment repeated at least threetimes independently.

Cell-cycle assayCells treated by Aki1 siRNA or scramble control siRNA were

collected by centrifugation and resuspended at 1�106 cells/mL inpropidium iodide (PI) staining buffer (0.1% sodium citrate, 0.1%Triton X-100, and 50 mg/mL PI) and were treated with 1 mg/mLRNase at room temperature for 30minutes. Cell-cycle histogramswere generated after analysis of PI-stained cells by FACSwith a BDBiosciences FACScan. For each culture, at least 1� 104 eventswererecorded. Histograms generated by FACS were analyzed by Mod-Fit cell-cycle analysis software (Verity) to determine the percent-age of cells in each phase (G0–G1, S, andG2–M). Each sample wasassayed in triplicate, with each experiment repeated at least twotimes independently.

Human phospho-kinase antibody arrayWe used the Human Phospho-Kinase Array Kit (R&D Systems)

to measure the relative level of phosphorylation for 43 kinasesand two related total proteins. The assaywas performed accordingto the manufacturer's instructions with modifications. Briefly,cells were cultured in RPMI-1640 containing 10% FBS, and thenwere lysed with array buffer before they reached confluence. Thearrays were blocked with blocking buffer and incubated with 450

Aki1 Is a Therapeutic Target in DMM

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mg of cell lysate overnight at 4�C. Arrays were washed, incubatedwith an HRP-conjugated phospho-kinase antibody and treatedwith ECL solution (GEHealthcare), before being exposed to film.Each experimentwas performed at least two times independently.

Luciferase reporter assayWe designed a dual-luciferase reporter driven by a 3x CRE

consensus–binding sequence in the promoter region in additionto a TATA box, which was inserted into a lentiviral constructupstream of luciferase as previously described (19). Cells werestably transduced to express CRE wild-type reporters and ratiosbetween the twowere compared after RNAi transfection of Aki1 orscramble control. Each sample was assayed in triplicate, with eachexperiment repeated at least two times independently.

Plasmid constructionpCF plasmid–expressing empty Vector (Vector), FLAG-tagged

human CREB (CREB-WT), or FLAG-tagged human CREB-S133A,which was CREB-kinase dead–mutated (CREB-KD) clones werepurchased from Addgene, Inc. Phosphorylation of CREB onSerine133 is essential for the transcriptional function of CREB(20). The X-tremeGene HP (Roche) transfection reagent was usedto transfect 211H cells with Vector, CREB-WT, or CREB-KDplasmid following the manufacturer's instructions. 211H andHEK293T cells were stably transfected with pHM6 plasmid–expressing HA-tagged recombinant human WT-Aki1 proteins(Aki1-WT) or empty vector as a control protein by using X-tremeGene Transfection Reagent. The plasmid was constructedbyCancerChemotherapyCenter, Japanese Foundation forCancerResearch (Tokyo, Japan) as previously described (10). The suc-cessfully transduced clones were identified by G418 (Sigma-Aldrich) selection. Whole-cell lysates (25 mg) were subjected toWestern blot analysis.

EGFP-Eluc gene transfectionThe cDNA of EGFP from pIRES-EGFP Vector (Invitrogen) and

Emerald Luc (Eluc) from Emerald Luc Vector (ELV-101,TOYOBO) were cloned into MaRXIVf Puro retrovirus vector asdescribed previously (21). All cDNA were sequenced. Transfec-tion was performed using Lipofectamine LTX and PLUS Reagent(Invitrogen), and retroviral infection into 211H cells was per-formed using the Retrovirus Packaging Kit (Ampho) according tothe manufacturer's instructions. Then, the cells were selected inPuromycin (Sigma-Aldrich).

Orthotopic model in SCID mice and in vivo RNAiWeused 5-week-old female SCIDmice (Clea) for this study. All

animal experiments were maintained under specific pathogen-free conditions throughout the study and complied with theGuidelines for the Institute for Experimental Animals, KanazawaUniversity Advanced Science Research Center (approval no. AP-081088). Anorthotopic implantationmodel of humanDMMwasestablished as described previously (22). Briefly, 1 � 106 211Htransfected the Eluc/EGFPgene cells (211H/Eluc) in100mLof PBSwere injected into the right pleural cavity of mice. On day 7 aftercell inoculation, 100 mg of either scramble or Aki1 siRNA com-plexed with invivofectamine (Invitrogen) was injected into theright pleural cavity. siRNA and invivofectamine complex wasprepared in accordance with the manufacturer's instructions(Invitrogen). Aki1 knockdown in tumor tissue was confirmed by

Western blotting analysis on day 7 after injection of siRNAcomplex. We evaluated tumor progression using in vivo imagingby the luminescence as described previously (21). The mice weresacrificed 19 days after tumor inoculation. Luminescence wasevaluated twice per week from day 2 to day 19 after cell inocu-lation. All surgeries were performed under sodium pentobarbitalanesthesia, and efforts weremade tominimize the suffering of theanimals.

PatientsA total of 68 tumor specimens were obtained from 67 DMM

patients with written informed consent at the Ohio State Univer-sity (Columbus, Ohio) and the Hyogo Prefectural AmagasakiHospital (Amagasaki, Japan) in studies with Institutional ReviewBoard approval. Of the 67 patients, 36 were epithelioid, 12 werebiphasic, 10 were sarcomatoid, seven were desmoplastic, and twopatients showed other types. Specimens from the 34 patients (35tumors) were obtained in tissue microarray (TMA) at the OhioStateUniversity, and the 33patients (33 tumors)were obtained inthe tissue at the Hyogo Prefectural Amagasaki Hospital.

Immunohistochemical studies for Aki1 and p-CREBParaffin-embedded tissue was cut at 4 mm and sections were

placed on positively charged slides. Slides were then placed in a60�C oven for 1 hour, cooled, deparaffinized, and rehydratedthrough xylene, and graded ethanol solutions to water. All slideswere quenched for 5minutes in a 3%hydrogen peroxide aqueoussolution to block for endogenous peroxidase. Antigen retrievalwas performed by Heat-Induced Epitope Retrieval, in which theslideswere placed in a 1 x solution of Target Retrieval Solution, pH6.0 (Dako, S1699) for 25 minutes at 96�C using a vegetablesteamer (Black & Decker) and cooled for 15 minutes in solution.Slides were stained with the Intellipath Autostainer Immunos-taining System. Detection system included Mach 3 Rabbit HRP(Biocare Medical; M3R531L) and Liquid DABþ Chromogen(Dako; K346811). All incubations on the Autostainer were per-formed at room temperature. Slides were counterstained inRichard Allen hematoxylin, dehydrated through graded ethanolsolutions, cleared with xylene, and coverslipped. Dilution experi-ments for immunohistochemical studies were performed inresection specimens according to themanufacturer's instructions.On the basis of the observed expression patterns, the tumor cellstaining in tissue and TMA was evaluated separately for p-CREB(1:700, clone 87G3; Cell Signaling Technology) and Aki1 (1:200;Aki1 aka CC2D1A, rabbit polyclonal; Sigma). Because p-CREBshowed only nuclear staining that varied in intensity and distri-butionwithin a tumor, its expressionwas assessed in regard to thestaining intensity as negative (0), low (1þ), intermediate (2þ),and high (3þ). Because immunohistochemical studies for Aki1showed only cytoplasmic staining evenly distributed within atumor but varied in intensity, its expression was assessed asnegative (0), low (1þ), intermediate (2þ), and high (3þ).

Statistical analysisData from MTT assay, luciferase reporter assay, and tumor

progression using in vivo imaging are expressed as means of �SD. The statistical significance of differences was analyzed by one-way ANOVA and Spearman rank correlations performed withGraphPad Prism Ver. 6.0 (GraphPad Software, Inc.). For allanalyses, a two-sided P value less than 0.05 was consideredstatistically significant.

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Cancer Res; 75(19) October 1, 2015 Cancer Research4190




ResultsSpecific downregulation of Aki1 inhibits cell viability andinduces cell arrest in DMM cells

To assess the involvement of Aki1 in EGFR-mediated signalingof DMM cells, we first examined the expression of Aki1 proteinand EGFR-related proteins (Akt and EGFR) in seven humanDMMcell lines and compared them with an immortalized humanmesothelial cell line, Met-5A, by Western blotting analysis(Fig. 1A). All of the cell lines examined expressed Aki1, Akt, andEGFR protein at various levels. However, the levels of Aki1 weregenerally higher in DMM cell lines than Met-5A. EGFR andphosphorylated Akt were also detected in all DMM and meso-thelial cell lines at various levels. These results indicate that Aki1expression is not correlated with EGFR-related proteins in DMMcells.

To determine the role of Aki1 in DMM cell lines, we usedspecific siRNA (siRNA) to knockdown Aki1 or EGFR. Treatmentwith Aki1-specific siRNA decreased the viability of DMM cells by40% or more in 5 of the 7 DMM cell lines examined (Fig. 1B andSupplementary Fig. S1). Treatment with an EGFR-specific siRNAdecreased the cell viability in H2373 dramatically and Y-meso8Ato a lesser degree; however, most DMM cell lines and the Met-5Aline were left unaffected with EGFR knockdown. The effectsof EGFR or Aki1 siRNAs in a noncancerous mesothelial cells,

Met-5A, were only marginal. These results suggest that Aki1knockdown inhibits viability of at least a subset of DMM cellsand is independent of the EGFR signaling.

Western blotting analyses showed that Aki1 knockdown in thefive most sensitive DMM cell lines, reduced cyclin A proteinexpression, consistent with the decrease in cell viability(Fig. 1C). Cyclin A is known to moderate the initiation andcompletion of DNA replication during S phase and reduction ofcyclin A with Aki1 knockdown suggests that Aki1 contributes tothe regulation of cell-cycle progression. Indeed, cell-cycle analysisconfirmed that knockdown of Aki1 decreased the number of cellsin S phase in 211H and H2052 (Fig. 1D).

Aki1 maintains the viability of DMM cells through CREB1activation

To elucidate the downstream activities regulated by Aki1 thatare involved in cell viability ofDMMcells, we performed a humanphosphokinase array to compare Aki1 containing with Aki1knockdown cells (Fig. 2A). Several kinase phosphorylation siteswere changed with Aki1 knockdown. Interestingly, the CREB1phosphorylation level at serine 133 (pS133) was decreased whencells were transfected with Aki1 siRNA. Examination of DMM celllines by Western blot analysis for CREB1 pS133 indicates that allof the DMM cell lines appear to have higher levels of pS133 than

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Figure 1.Role of Aki1 on cell viability and cell cycle in malignant pleural mesothelioma cells. A, the expression of Aki1- and EGFR-related proteins in malignant pleuralmesothelioma cell lines (211H, H290, H2052, H2373, H2452, Y-meso8A, and Y-meso14) and humanmesothelial cell lines (Met-5A). Theywere lysed and the indicatedproteins were detected by Western blotting analysis. B, cells were treated with Aki1, EGFR, or scramble control siRNA. After 72 hours incubation, cell viabilitywas determined by MTT assay. C, cells were treated with scramble control or Aki1 siRNA. After 24 hours incubation, cells were lysed and the indicated proteinswere detected by Western blotting. D, cells were treated with scramble control or Aki1 siRNA. After 48 hours incubation, cell cycle was determined with anAnnexin V–FITC Apoptosis Detection Kit I. The figure shows percentages of cell population (G2–M, S, and G0–G1).






the immortalized mesothelial cell line, Met-5A (Fig. 2B). Westernblotting analyses confirmed the phosphokinase array result show-ing that Aki1 downregulation decreases phospho-CREB1 levels inmultiple DMM cell lines (Fig. 2C). The Western blot analysis alsoshowed that the specific downregulation of CREB1 by siRNAreduced cyclin A protein expression in five DMM cell lines, whichis also seen with Aki1 knockdown. To evaluate how Aki1 lossaffects CREB activity, we transfected DMM cell lines with a CREreporter. The CRE-Luciferase reporter assay demonstrated that thetreatment with Aki1 siRNA significantly inhibits CREB1 activity in211H andH2052 cells compared with scramble control (P < 0.05by one-way ANOVA; Fig. 2D).

We next evaluated the role of CREB1 activity in maintainingDMM cell viability. DMM cells treated with a CREB1 siRNA showremarkably reduced cell viability when compared with cellstreated with the scrambled control (Fig. 2E and Supplementary

Fig. S2). Notably, the reduction in cell viability is very similar tocells treated with Aki1 siRNA. The immortalized mesothelial cellline,Met-5A, does not appear to be as affected by CREB1 and Aki1knockdown as the DMMcell lines (Supplementary Fig. S3). Thesefindings suggest that the Aki1–CREB1 axis has an important rolein maintaining the viability of DMM cells.

To further prove that CREB1 activity is important in DMM cellviability and is downstream of Aki1, we surmised that enforcedexpression of CREB1 might overcome Aki1 knockdown andmaintain cell viability. To do this, we transfected 211H cells withempty Vector, CREB-WT, and a mutated CREB that lacks tran-scriptional activity, CREB-S133A. Western blot analysis of trans-fected 211H cells clearly shows an increase in both the totalCREB1 and phosphorylated CREB1 (Fig. 2F). Treatment of thetransfected cells with Aki1 siRNA reduced the phosphorylationof CREB1 in the vector control cells as expected. Although

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Figure 2.Aki1maintains the viability ofDMMcells throughCREB1 activation. A, human kinase phosphorylation array analysis in 211H cellswith scramble control siRNA (a) orAki1siRNA (b) after 48 hours incubation. B, total and phosphorylation of CREB expressions in four DMM (211H, H290, H2052, and Y-meso14) and mesothelial(Met-5A) cells. Cellswere lysed and the indicatedproteinswere detectedbyWesternblotting. C, luciferase activity assayagainst CREB1. Cellswere stably transducedto express wild-type CRE reporters, which binds to CREB1 as a certain DNA sequences, and ratios between the two were compared after RNAi transfectionof Aki1 or scramble control; � , P < 0.05 versus scramble control by one-way ANOVA. D, cells were treated with scramble control, Aki1, or CREB1 siRNA. After24 hours incubation, cells were lysed and the indicated proteins were detected by Western blotting analysis. E, cells were treated with Aki1, CREB1 (#1, #2), orscramble control siRNA. After 24, 48, 72, and 96 hours incubation, cell viability was determined by MTT assay. F, pCF plasmid–expressing empty vector(Vector), FLAG-tagged human CREB (CREB-WT), or FLAG-tagged human CREB-kinase dead–mutated (CREB-KD) clones were transfected into 211H cells.They were lysed and the indicated proteins were detected by Western blotting analysis. G, total and phosphorylation of CREB1 expressions in 211H cells withtransfection of vector, CREB-WT, or CREB-KD clones were treated with Aki1 or scramble control siRNA. After 24 hours incubation, cells were lysed and theindicated proteins were detected by Western blotting. H, 211H cells with transfection of vector, CREB-WT, or CREB-KD clones were treated with Aki1 orscramble control siRNA. After 72 hours incubation, cell viability was determined by MTT assay; � , P < 0.05 versus cells with vector or CREB-KD clones treatedwith Aki1 siRNA by one-way ANOVA.

Yamada et al.





phosphorylated CREB1 inCREB-WT transfected cells was reducedin the Aki1 knockdown cells, it was still expressed at a relativelyhigh level compared with vector control (Fig. 2G). More impor-tantly, cells expressing wild-type CREB had a significantly higherviability than vector control cells or cells expressing the mutatedform of CREB (P < 0.05 by one-way ANOVA; Fig. 2H). Thesefindings confirm that there is indeed an Aki1–CREB axis and thatincreased CREB1 activity downstream of Aki1 is important inmaintaining cell viability of DMM cells.

Aki1 regulates CREB by modulating protein kinase Aphosphorylation and activity.

To gain a better understanding of how Aki1 regulates CREB1activity, Aki1 was overexpressed and PKA, a major regulator ofCREB phosphorylation at S133, was examined. To do this, wemade an Aki1 expression construct that was transfected into bothHEK293T and 211H cells. The exogenously expressed Aki1 pro-tein induced the phosphorylation of CREB1 and the catalyticsubunit of Protein Kinase A (PKAC) in both cell lines (Fig. 3A).Alternatively, treatment of two DMM cell lines with Aki1 siRNAreduced PKACphosphorylation (Fig. 3B).We also determined theeffect of the PKA inhibitor KT5720 on the cell viability of DMMand Met-5A cells. Treatment of DMM cells with KT5720 reduces

CREB1phosphorylation after 4 and8hours incubations (Fig. 3C).KT5720 also inhibits the growth of DMM cells in a dose-depen-dent manner with little effect on the Met-5A cells (Fig. 3D).In addition, we investigated the efficacy of the CBP–CREB inter-action inhibitor (CBP–CREB-I) on the cell growth of DMM andMet-5A cells. CBP–CREB-I inhibits theCREB1phosphorylation inDMMcells (Fig. 3E) and strongly suppresses the viability of DMMcells, withmarginal effects on theMet-5A cells (Fig. 3F). Thus, ourfindings clearly show that Aki1 mediates the activation of thePKA–CREB axis in DMM cells and may provide an attractivetherapeutic option in DMM (Supplementary Fig. S4).

Direct injection of Aki1 siRNA into the pleural cavity of a DMMorthotopic mouse model dramatically decreases tumorformation

We next examined the antitumor potential of Aki1 siRNA bydirectly injecting the siRNA into pleural cavity of a DMM ortho-topicmousemodel. TheDMM211Hcell line expressing luciferasewas implanted into the pleural cavity of immunocompromisedmice. Seven days after implantation of the DMM cells Aki1 siRNAor a control siRNA complexed with invivofectamine was injectedas a single dose. Bioimaging was used to monitor disease pro-gression. The single injection of Aki1 siRNA into the pleural cavity

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Figure 3.Aki1 regulates CREB bymodulating protein kinase A phosphorylation and activity. A, HEK293T and 211H cells were stably transfectedwith pHM6 plasmid–expressingHA-tagged recombinant human WT-Aki1 proteins (Aki1-WT) or empty vector (Vector). They were lysed and the indicated proteins were detected byWestern blotting analysis. B, cells were treated with scramble control or Aki1 siRNA. After 24 hours incubation, cells were lysed and the indicated proteinswere detected byWestern blotting. C, cells were treated with KT5720 (0.1 mmol/L). After 4 and 8 hours incubation, cells were lysed and the indicated proteins weredetected by Western blotting. D, cells were treated with KT5720 (0.1 and 1.0 mmol/L). After 72 hours incubation, cell viability was determined by MTT assay.E, cells were treated with CBP–CREB interaction inhibitor (CBP–CREB-I; 0.1 mmol/L). After 4 hours incubation, cells were lysed and the indicated proteins weredetected by Western blotting. F, cells were treated with CBP-CREB-I (0.1 and 0.3 mmol/L). After 72 hours incubation, cell viability was determined by MTT assay.






significantly inhibited tumor growth in comparison with that ofscramble control 12 days after Aki1 treatment (P < 0.05 by one-way ANOVA; Fig. 4A and B). We confirmed the knockdown ofAki1 and the inhibition of CREB1 phosphorylation in tumors byWestern blotting analysis (Fig. 4C). These results clearly indicatethe therapeutic potential of Aki1 siRNA against DMM localized tothe pleural cavity.

Aki1 and phosphorylated CREB are frequently expressed inhuman DMM tumors

We examined Aki1 expression in 68 clinical specimensobtained from 67 DMM patients (Fig. 5A and B and Supplemen-tary Fig. S5).Of 68DMMtumors, Aki1was expressed at some levelin 65 (95.6%). High cytoplasmic Aki1 protein expression wasdetected in 15 (22.1%), intermediate in 22 (32.4%), low in 28(41.2%), andnegative expression in 3 (4.4%) of 68DMM tumors.Aki1 protein expression was detected in 37 of 37 (100%) ofepithelioid DMM, 8 of 10 (80%) sarcomatoid DMM, and 12 of12 (100%) biphasic DMM (Supplementary Fig. S6A). We nextexamined phosphorylated CREB expression in 35 clinical speci-mens obtained from 34 DMM patients (Fig. 5A and B and

Supplementary Fig. S5). Of 35 DMM tumors, phosphorylatedCREB was expressed at some level in 30 tumors (85.7%). Highnuclear expression of phosphorylated CREB protein was detectedin 16 (45.7%), intermediate in 12 (34.3%), low in 2 (5.7%), andnegative expression in 5 (14.3%) of 35 DMM tumors. Phosphor-ylated CREB was detected in 20 of 21 (95%) epithelioid, in 1 of 5(20%) sarcomatoid, and in 6 of 6 (100%) biphasic DMM (Sup-plementary Fig. S6A). There was a statistically significant corre-lation between expression of Aki1 and phosphorylated CREB(Spearman rank correlations ¼ 0.521; P ¼ 0.002, N ¼ 34; Fig.5C). The epithelioid DMM demonstrated the strongest correla-tion between expression levels of Aki1 and phosphorylated CREB(Spearman rank correlations ¼ 0.561; P ¼ 0.009, N ¼ 21;Supplementary Fig. S6B). These findings suggested involvementof phosphorylated CREB in Aki1-mediating signaling in DMMtumors, especially epithelioid types.

DiscussionPrior observations where the knockdown of EGFR by siRNA

hardly affected cell viability ofDMM(23), suggested that targetingmolecules downstream of the multiple RTKs is a more effectivetherapeutic option.

Previously, we identified Aki1 as a scaffold protein critical fortransmitting the signal through the EGFR–PI3K–PDK1–Akt path-way. Downregulation of Aki1 dramatically decreased cell viabilityin EGFR-mutant lung cancer cell lines (11). These data suggestedthat Aki1 had a role in aberrant signaling through the EGFR–PI3K–Akt pathway. Our screening approach through monitoringaltered kinase phosphorylation in Aki1 knockdown DMM cellsled to the discovery that Aki1 was signaling through a differentpathway. On the basis of in vitro studies, we showed for the firsttime, that Aki1 is critical in proliferation and cell viability ofDMM.This process is independent of EGFR signaling and involvesCREB1 activation by modulating PKA phosphorylation.

The observation that phosphorylation of CREB1was decreasedby knockdown of Aki1 agrees with previous studies in nonsyn-dromicmental retardationwhere Aki1 is a regulator of the cAMP–PKA pathway in neuronal cell differentiation (24, 25). Mutantmice with a truncated Aki1 showed defective PKA activation andCREB phosphorylation in the neuronal system (25). Hence, Aki1has important roles in regulating the activity of the PKA–CREBaxis in neuronal differentiation and in brain development as wellas in malignant tumors.

CREB1 is a 43 kDa basic/leucine zipper transcription factor thatmediates signaling from extracellular stimulation and can bephosphorylated and activated by many kinases, including PKA,protein kinaseC,Ca2þ/calmodulin-dependent kinase, extracellularsignal–regulated kinases, Akt, and p90 ribosomal S6 kinase (26).Phosphorylation of CREB1 on Serine 133 has an important role inthe transcriptional activity of CREB1 by promoting the interactionwith the coactivator CREB-binding protein (CBP)/p300. CREBpromotes cell proliferation, cell-cycle progression, and survival ofmyeloid cells through induction of specific target genes (27). Inaddition, the overexpression and activation of CREB1 has beenassociated with poor prognosis in nonsmokers with NSCLC (28),melanomametastasis (29), and acutemyelogenous leukemia (30).With regard to the roles of CREB1 inDMM, CREB-induced inflam-mation is associated with cell growth and asbestosis-related DMM(31). Recent studies inDMMcells, both in vitro and in vivo, aswell asour study, demonstrate that CREB inhibition by RNAi or small

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Figure 4.Direct injection of Aki1 siRNA into the pleural cavity of a DMM orthotopicmouse model dramatically decreases tumor formation. 211H transfected theEluc/EGFP gene cells (211H/Eluc; 1 � 106 cells per 100 mL of PBS) and wereinjected into the right pleural cavity of 5-week-old male SCID mice. On day 7after cell inoculation, 100 mg of either scramble or Aki1 siRNA complexed withinvivofectamine was injected into the right pleural cavity. A, luminescencewas evaluated twice per week from day 2 to day 19 after cell inoculation. Bars,SE is shown for groups of 8 mice; �, P < 0.05 by one-way ANOVA. B, theappearance of representative mice is shown. C, the harvested tumors, whichare on day 7 after injection of siRNA complex, were examined for Aki1 andphosphorylated CREB1 by Western blotting analysis.

Yamada et al.





molecules, results in significant attenuation of proliferation,migra-tion, and drug resistance to cytotoxic chemotherapy, such asdoxorubicin (31, 32). In addition, our study demonstrates thattreatment with small-molecule inhibitors targeting PKA or CREBreduces proliferation of DMM cells in vitro.

Our data would suggest that inhibition of Aki1, or a down-stream pathway component such as PKA, would be an effectivestrategy to treatDMM.However, Aki1 is a scaffold proteinwithouta known small-molecule inhibitor. An inhibitor of PKA would beanoption, andfinding aPKA inhibitor is currently an area of activeresearch (33, 34). Phase I trials of an antisense RNA against thePKA regulatory subunit, RIa, were performed in multiple solidtumors (35). Thiswas a systemic approachwith i.v. infusion of theantisense RNA, and although it appeared that PKA activitydecreased as measured by serum extracellular PKA (ECPKA)activity, there were no objective responses to treatment. A previ-ous study showed that tumor growth of several cancer types wasblocked bymultiple i.p. injections of siRNAs for Zcchc11, Lin28A,or Lin28Busing a xenograftmodel (36). A recent article reports thepleural and peritoneal injection of a recombinant human p53-containing adenovirus to control malignant effusions (37). Thistreatment provided excellent local control of the effusion, wassafe, and could be an option for patients unable to undergostandard chemotherapeutic treatments. As for a local therapyusing pleural injection, we have demonstrated the efficacy of asingle direct injection into the pleural cavity of a siRNA to Aki1 in

an orthotopicmousemodel ofDMM.Given the unique aspects ofmesothelioma biology, and particularly its localization to thepleural cavity in most cases, direct injection of an antisense RNAcould be practical, and provide local control and reduce sideeffects associatedwith standard systemic cytotoxic chemotherapy.Phosphorylated CREB1 has been shown to be increased in meso-thelioma compared with reactive mesothelial hyperplasia andnormal lung tissue (31). The results of human tissue analysis inour study are in keepingwith that observation, but also for thefirsttime document that Aki1 is also frequently overexpressed andcorrelates with expression of phosphorylated CREB in DMM.Among DMM subtypes, the strongest correlation between theexpression levels of thesemoleculeswas seen in epithelioidDMM,although the cell-based analysis showed the high expression ofboth proteins even in sarcomatoid type. We cannot explain whythese correlations were not apparent in our TMA samples, but islikely related to the small number of sarcomatoid samples in ourTMA. Further experiments are needed to confirm these histologicdifferentiations in DMM.

In summary, we have demonstrated that Aki1 has roles in thecell growth, cell cycle, and transcription though the activation ofPKA–CREB1 signaling for DMM cells. Direct application of asingle dose of Aki1 siRNA into the pleural cavity of mice signif-icantly inhibited the growthof 211Hcellswithin thepleural cavityin an orthotropic implantation model. Furthermore, Aki1 isfrequently expressed in DMM tumors and correlated with

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positive 65 95.6 30 85.7

Figure 5.Aki1 and phosphorylated CREB arefrequently expressed in DMM. Clinicalspecimens from DMM patientsevaluated for Aki1 and phosphorylatedCREB by immunohistochemistry. A,high cytoplasmic Aki1 proteinexpression was detected in 15 (22.1%),intermediate in 22 (32.4%), low in 28(41.2%), and negative expressionin 3 (4.4%) of 68 DMM tumors. Highnuclear expression of phosphorylatedCREB protein was detected in 16(45.7%), intermediate in 12 (34.3%),low in 2 (5.7%), and negativeexpression in 5 (14.3%) of 35 DMMtumors. B, representative images ofepithelioid DMM, hematoxylin andeosin stain (H&E), showing diffuseexpression of calretinin, p-CREB, andAki1. C, the correlation of expressionscores between Aki1 andphosphorylated CREB in tumors ofDMM patients (Spearman rankcorrelations ¼ 0.521; P ¼ 0.002,N ¼ 34).






phosphorylation of CREB1, especially epithelioid types. Our dataprovide a rationale for targeting Aki1 by intrathoracic therapy inpatients with local invasive DMM tumors.

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

Authors' ContributionsConception and design: T. Yamada, J.M. Amann, D.P. CarboneDevelopment of methodology: T. YamadaiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Fukuda, K. ShiloAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Yamada, J.M. Amann, K. Fukuda, S. Takeuchi,H. Uehara, K. Shilo, D.P. CarboneWriting, review, and/or revision of the manuscript: T. Yamada, J.M. Amanni,K. Shilo, D.P. CarboneAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Fukuda, N. Fujita, S. Iwakiri, K. Itoi, S. Yano,D.P. CarboneStudy supervision: J.M. Amann, D.P. Carbone

AcknowledgmentsThe authors appreciate that NCI-H290 was provided by Dr. Adi F. Gazdar

(University of Texas Southwestern Medical Center, Dallas, TX) and Dr. Yoshi-taka Sekido (Aichi Cancer Research Center Institute, Aichi, Japan). The authorsthank Dr. Takayuki Nakagawa and Mr. Kenji Kita (Cancer Research Institute,Kanazawa University, Kanazawa, Japan) for technical advice and for technicalsupport.

Grant SupportThis studywas supported byGrants-in-Aid forCancerResearch (25860638 to

T. Yamada), Scientific Research on Innovative Areas "Integrative Research onCancer Microenvironment Network" (22112010A01 to S. Yano) from theMinistry of Education, Culture, Sports, Science, and Technology of Japan, andthe Dellapezze Family Foundation (D.P. Carbone).

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 April 1, 2015; revised June 6, 2015; accepted June 26, 2015;published OnlineFirst August 20, 2015.

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2015;75:4188-4197. Published OnlineFirst August 20, 2015.Cancer Res Tadaaki Yamada, Joseph M. Amann, Koji Fukuda, et al. Diffuse Malignant MesotheliomaAkt Kinase-Interacting Protein 1 Signals through CREB to Drive

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