small molecule agonists of ppar-g exert therapeutic...

Therapeutics, Targets, and Chemical Biology

Small Molecule Agonists of PPAR-g Exert TherapeuticEffects in Esophageal Cancer

Hiroshi Sawayama1, Takatsugu Ishimoto1, Masayuki Watanabe1, Naoya Yoshida1, Hidetaka Sugihara1,Junji Kurashige1, Kotaro Hirashima1, Masaaki Iwatsuki1, Yoshifumi Baba1, Eiji Oki2, Masaru Morita2,Yoshinobu Shiose3, and Hideo Baba1

AbstractThe transcription factor PPAR-g plays various roles in lipid metabolism, inflammation, cellular differen-

tiation, and apoptosis. PPAR-g agonists used to treat diabetes may have utility in cancer treatment.Efatutazone is a novel later generation PPAR-g agonist that selectively activates PPAR-g target genes andhas antiproliferative effects in a range of malignancies. In this study, we investigated PPAR-g status inesophageal squamous cell carcinoma (ESCC) and investigated the antiproliferative effects of efatutazone.PPAR-g was expressed heterogeneously in ESCC, in which it exhibited an inverse relationship with Ki-67expression. PPAR-g expression was associated independently with good prognosis in ESCC. Efatutazone, butnot the conventional PPAR-g agonist troglitazone, inhibited ESCC cell proliferation in vitro and in vivo.Mechanistic investigations suggested that efatutazone acted by upregulating p21Cip1 protein in the nucleusthrough inactivation of the Akt pathway and dephosphorylation of p21Cip1 at Thr145 without affecting thetranscriptional activity of p21Cip1. We also found that treatment with efatutazone led to phosphorylation ofthe EGF receptor and activation of the mitogen-activated protein kinase (MAPK) pathway. Accordingly, thecombination of efatutazone with the antiepithelial growth factor receptor antibody cetuximab synergized tonegatively regulate the phosphoinositide 3-kinase–Akt and MAPK pathways. Together, our results suggestthat efatutazone, alone or in combination with cetuximab, may offer therapeutic effects in ESCC. Cancer Res;74(2); 575–85. �2013 AACR.

IntroductionPPAR-g is a member of the nuclear hormone receptor

superfamily of ligand-activated transcription factors (1).PPAR-g plays a variety of roles in adipose cell differentiation,modulation of metabolism, and the inflammatory response (2,3). The protein interacts with and/or regulates multiple sig-naling pathways, including those associatedwith p21Cip1 (4, 5)and p27 (6, 7) to regulate the cell cycle, with nuclear factorkappa b (8) to reduce the expression of cytokines such asinterleukin-6, and interleukin-8, and with cyclooxygenase-2and prostaglandin E2 to suppress inflammation (9, 10). Geneticstudies have indicated that PPAR-g functions as a tumor

suppressor in a variety of tissues, including the breast (11),prostate (12), and colon (13).

Ligands for PPAR-g include endogenous ligands such as fattyacids, D12,15 prostaglandin J2 (14), and exogenous ligands, suchas the thiazolidinedione class of antidiabetic drugs includingtroglitazone, rosiglitazone, and pioglitazone (15, 16). Numer-ous recent studies have demonstrated that PPAR-g ligandsexert antiproliferative effects. The first-generation thiazolidi-nedione troglitazone showed antiproliferative effects in hepa-tocellular carcinoma (7), colorectal cancer (17, 18), and breastcancer (19). However, these effects were limited, and smallclinical trials showednobeneficial effects of these conventionalPPAR-g ligands (20, 21).

Efatutazone is a novel third-generation thiazolidinedionePPAR-g agonist, which is at least 500-fold more potent thantroglitazone in terms of PPAR response–element activationand inhibition of cancer cell growth, rather than inducingapoptosis (4). Furthermore, efatutazone demonstrated antitu-mor effects in patients with advanced malignancies (22). Thisagent therefore shows great potential for practical cancertherapy.

Combination therapy using efatutazone with antitumoragents represents an important means of achieving significanttumor regression (22). PPAR-g agonists have shown activityboth in vitro and in vivo in combination with conventionalanticancer drugs including platinum-based drugs (23), taxanes(4), and irinotecan (24). However, the pathways affected by

Authors' Affiliations: 1Department of Gastroenterological Surgery, Grad-uate School of Medical Sciences, Kumamoto University, Kumamoto;2Department of Surgery and Science, Graduate School of MedicalSciences, Kyushu University, Fukuoka; and 3Research and DevelopmentDivision, Daiichi Sankyo Co., Ltd., Tokyo, Japan

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

H. Sawayama and T. Ishimoto contributed equally to this work.

Corresponding Author: H. Baba, Department of GastroenterologicalSurgery, Graduate School of Medical Sciences, Kumamoto University,1-1-1 Honjo, Kumamoto 860-8556, Japan. Phone: 81-96-373-5587; Fax:81-96-373-4378; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-1836

�2013 American Association for Cancer Research.

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Published OnlineFirst November 22, 2013; DOI: 10.1158/0008-5472.CAN-13-1836

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treatment with efatutazone, and the most suitable combina-tion therapy (in terms of molecular-targeted agents) based ontheoretical signaling mechanisms remain unclear.

Esophageal carcinoma affects more than 450,000 peopleworldwide, and its incidence is increasing rapidly. SCC is thepredominant form of esophageal carcinoma worldwide (25).Furthermore, esophageal carcinoma is an aggressive diseasewith a propensity to spread both locoregionally and distally,and multidisciplinary therapy has therefore been tested foresophageal squamous cell carcinoma (ESCC; refs. 26, 27). Theantiepithelial growth factor receptor (EGFR) antibody, cetux-imab, was approved for head and neck SCC (28, 29), and newmolecular-targeted therapies have been expected for the treat-ment of ESCC (30).

The effects of PPAR-g are tissue and cancer specific. Thesignificance of PPAR-g expression and antiproliferative effectsof PPAR-g agonists in ESCC have been reported in a fewstudies, however these studies were limited because they onlyevaluated the mRNA levels in small number of samples (31), inadenocarcinomas (32), or using conventional PPAR-g agonists,such as troglitazone (33). Therefore the expression of thePPAR-g protein in ESCC and the antiproliferative effects ofthe new generation PPAR-g agonist remained unclear. In thisstudy, we investigated the relationship between the expressionlevels of PPAR-g and Ki-67 in patients with ESCC, and exam-ined the antiproliferative effects and underlying mechanism ofefatutazone monotherapy both in vitro and in vivo. We alsoinvestigated the effects of combination therapy using efatuta-zone with molecular-targeted agents, based on signaling anal-ysis. The results of this study may provide a novel therapeuticstrategy for ESCC, and suggest that efatutazone, both alone andin combination with an anti-EGFR antibody, may improve theoutcomes of patients with ESCC.

Materials and MethodsChemicals

Efatutazone was kindly provided by Daiichi Sankyo, Inc..Troglitazone, U0126,MK-2206 dihydrochloride, and cetuximabwere purchased from Cayman, Wako, Selleck Bio., and Kuma-motoUniversityHospital, respectively. For the in vitro analyses,efatutazonewas prepared in dimethyl sulfoxide (DMSO) beforeaddition to cell cultures. Antibody information is provided inthe Supplementary Materials and Methods.

PatientsThis study involved 145 consecutive patients who under-

went surgical resection of ESCC at Kumamoto UniversityHospital from January 2000 to December 2008 (SupplementaryFig. S1).

Cell linesESCC cell lines (TE series) and HT-29 were obtained from

Cell Resource Center for Biomedical Research Institute ofDevelopment, Aging and Cancer, Tohoku University, the RikenBioResource Center Cell Bank, and Keio University. The celllines have been tested and authenticated by Cell ID System inOctober 2013. Cell lines (TE series and HT-29) were cultured in5% CO2 at 37�C in RPMI 1640 supplemented with 10% FBS.

Xenograft modelSix-week-old nude mice (Balb-nu/nu slc) were inoculated

subcutaneously in the right flank with 5 � 106 TE-4 cells in200 mL PBS containing 50% Matrigel (BD, Becton, Dickinsonand Company). When the tumors reached approximately 80mm3 in diameter, the mice were randomized into treatmentgroups. This subcutaneous xenograft model was used toassess the therapeutic effects of efatutazone (10 mg/kg) andtroglitazone (10 mg/kg) as single agents. Control animalsreceived 0.5 w/v% methylcellulose solution (vehicle). Efatu-tazone and troglitazone were suspended in 0.5 w/v% meth-ylcellulose solution and administered to the animals daily byoral gavage in a volume of 0.1 mL/10 g body weight, using ananimal-feeding needle. The anti-EGFR antibody, cetuximab(1 mg/injection) or placebo (PBS) was injected intraperito-neally twice a week to assess the effects of combinationtherapy with efatutazone. Caliper measurements were madetwice a week using digital calipers, and the tumor volumeswere estimated using the following formula: V ¼ L � W � D� p/6, where V is the tumor volume, L is the length, W is thewidth, and D is the depth (30, 34).

Statistical analysisComparisons between treatment groupsweremade using 2-

tailed paired or unpaired Student t tests, as appropriate, basedon the results of F tests. TheMann–Whitney U test was used inthe event of a nonnormal distribution. The log-rank test wasused for survival analysis, and the Kaplan–Meier method wasused to assess survival time distribution. For analyses ofesophageal cancer-specific mortality, deaths as a result ofcauses other than ESCC were censored. Univariate Cox regres-sion analysis was also performed. The independent effect ofPPAR-g on mortality was assessed by performing multivariateCox regression analysis. Statistical significance was defined asa P value <0.05. All data were processed and analyzed using thePASW Statistics 18 software program.

Study approvalAll animal procedures and care were approved by the

Animal Care and Use Committee of Kumamoto University(approval number H24347). Written informed consent wasobtained from the human subjects.

ResultsPPAR-g expression in the normal epithelium and ESCC

We investigated PPAR-g expression in normal esophagealepithelial samples and tumor lesions from 145 patients withresectable ESCC using immunohistochemical analysis (Sup-plementary Table S1). PPAR-g was ubiquitously expressed inthe squamous layer of the normal esophageal epithelium in all145 patients, and there was no variation (Fig. 1A). The positivestaining of PPAR-g was detected in 36 (24.8%) tumors in thepatients with ESCC. PPAR-g expression exhibited a significantinverse relationship with Ki-67 expression (P < 0.001, Mann–Whitney U test; Fig. 1B–D).

A recent study reported on the relationship between PPAR-gexpression and prognosis in colorectal cancer (35), and PPAR-gmRNA expression has demonstrated prognostic value in ESCC

Sawayama et al.

Cancer Res; 74(2) January 15, 2014 Cancer Research576




(n¼ 55; ref. 31). However, the relation between PPAR-g proteinexpression and prognosis in ESCC remains unclear. Accordingto this study, PPAR-g expression was associated with a goodprognosis in terms of disease-free survival (log-rank P ¼0.035; Fig. 1E) and esophageal cancer-specific survival (log-rank P¼ 0.033; Fig. 1F) in the 145 patients with ESCC accordingto univariate and multivariate Cox regression analysis (Sup-plementary Table S2).We therefore hypothesized that PPAR-g was associated with

tumor-suppressive effects against ESCC, and that efatutazone,a novel third-generation PPAR-g agonist, could be a potentiallyuseful anticancer agent in patients with ESCC.

PPAR-g expression in ESCC cell lines andantiproliferative effects of efatutazonePPAR-g expressionwas examined in 9 humanESCC cell lines

and in the colon cancer cell line HT-29, which was used as apositive control for PPAR-g expression (36). Various levels ofPPAR-g expression were observed in the ESCC cell lines (Fig.2A). However, efatutazone demonstrated antiproliferativeeffects in all 9 ESCC cell lines (Fig. 2B). We investigatedantiproliferating effects and underlying mechanism of efatu-tazone using TE-4, TE-8, and TE-11, TE-6 cells, which expresshigh, medium and low levels of PPAR-g , respectively.

We used 3 siRNAs for PPARG to confirm that the antitumoreffects of efatutazone occurred in a PPAR-g–dependent man-ner. We selected TE-8 cells, because the PPAR-g expression ofTE-8 cells was strongly suppressed at both the mRNA andprotein levels using siRNAs for PPARG (Fig. 2C and D; Sup-plementary Fig. S2A and S2B). TE-8 ESCC cells were trans-fected with PPARG siRNA for 48 hours, followed by treatmentwith control (DMSO) or 50 mmol/L efatutazone for 48 hours. Inthe presence of the scrambled siRNA (negative control), efa-tutazone enhanced the expression of pyruvate dehydrogenasekinase isozyme-4 (PDK4), which is an established marker forthe activity of PPAR-g as a transcriptional factor (37, 38), by 2.5-fold compared with the control level, but this effect wasblocked in the presence of PPARG siRNA (Fig. 2E). Theinhibition of proliferation by efatutazone was related to thePDK4 mRNA level (Fig. 2F). Efatutazone inhibited the prolif-eration of the ESCC cell lines in a time-dependentmanner (Fig.2G), and PDK4 levels were also increased in a similarly time-dependent manner and correlated with antiproliferative effectof efatutazone (Fig. 2H). The mRNA level of PDK4 aftertreatment with 25 mmol/L efatutazone was increased morethan after treatmentwith 25 mmol/L troglitazone (Supplemen-tary Fig. S3A). No significant antiproliferative effects of treat-ment with troglitazone were detected compared with the

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Figure 1. Expression of PPAR-g and Ki-67 in tissue samples from 145 patients with ESCC. A, immunohistochemistry of PPAR-g and Ki-67 in normalesophageal squamous epithelium. B, PPAR-g–positive and Ki-67 negative in an ESCC specimen. C, PPAR-g–negative and Ki-67 positive in an ESCCspecimen. Scale bar, 50 mm. D, relationship between the expression levels of PPAR-g and Ki-67 (n ¼ 145). The horizontal bars show median 25th and 75thpercentiles. E and F, Kaplan–Meier curves according to PPAR-g status. ��, P < 0.001 (Mann–Whitney U test).

Tumor-Suppressive Effects of PPAR-g Agonist in ESCC

www.aacrjournals.org Cancer Res; 74(2) January 15, 2014 577




control, however, the proliferation of both TE-4 and TE-8 cellswas inhibited by the treatment with efatutazone (Supplemen-tary Fig. S3B). Because the antiproliferative effects of efatuta-zone were observed in various ESCC cell lines, we investigatedthe mechanism underlying the antiproliferative effects ofefatutazone using TE-4 cells, which highly express PPAR-g .

In vitro antiproliferative effects of efatutazone in ESCCcells and dependence on Akt-p21Cip1

Efatutazone was previously reported to inhibit cell cycleprogression via p21Cip1 in anaplastic thyroid carcinoma (4,5). In order to clarify the effects of efatutazone on the cellcycle in ESCC, we subjected TE-4 and TE-11 cells to cell cycleanalysis by flow cytometry. Accumulation of cells in the G1

phase, and reductions in the S and G2/M phases occurredafter treatment with efatutazone for 48 hours (Fig. 3A,Supplementary Fig. S4A). In addition, PDK4 mRNA levelswere upregulated by treatment with efatutazone for 48hours, whereas p21Cip1 mRNA levels remained unaffectedby the treatment (Fig. 3). However, p21Cip1 protein levelswere significantly upregulated in Western blot analysis (Fig.

3C). We therefore focused on the posttranslational modifi-cation of the p21 protein (39).

Activation of Akt, which associates with p21Cip1 and phos-phorylates it at threonine 145 (Thr145), results in increasedcytoplasmic localization of p21Cip1 in breast cancer (40). Wetherefore stimulated the phosphoinositide 3-kinase (PI3K)–Akt pathway with EGF (100 ng/mL) to investigate thesesignaling changes in the ESCC cell lines. We found that Aktwas activated by treatment with EGF, and p21Cip1 was thengradually phosphorylated at Thr145 in ESCC cells (Fig. 3D).

We treated TE-4 cells with efatutazone for 48 hours, followedby stimulation with or without EGF for 10 minutes to deter-mine if efatutazone was associated with inactivation of Akt.Efatutazone reduced the phosphorylation of bothAkt at Ser473and p21 at Thr145 (Fig. 3E). Localization of p21Cip1 wasconfirmed by confocal microscopy. Some p21Cip1 protein wasdetected in the cytoplasm in control cells, but strong stainingwas detected in the nucleus following treatment with efatu-tazone for 48 hours (Fig. 3F).

The PPAR-g expression of TE-6 cells was lower (Fig. 2A) andthe antiproliferative effects of treatmentwith efatutazonewere

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Figure 2. PPAR-g expression in ESCC lines and in vitro antiproliferative effects of efatutazone. A,Western blot analysis for PPAR-g . B, cell proliferation assay inESCC cell lines treated with 25 mmol/L efatutazone for 3 days. C, Western blot analysis for PPAR-g in TE-8 cells after transfection with a scrambled siRNAor 3 kinds of siRNA for PPARG. D, PPARG mRNA levels in TE-8 cells were examined by real-time RT-PCR. E, PDK4 mRNA levels in TE-8 cells aftertreatment with 50 mmol/L efatutazone for 48 hours were compared with control (DMSO) cells. F, proliferation of TE-8 cells after treatment with 50 mmol/Lefatutazone for 48 hours was compared with that of control (DMSO) cells. G, cell proliferation assay in TE-4 cells treated with 25 mmol/L efatutazone.H. PDK4 mRNA levels in TE4 cells after treatment with 25 mmol/L efatutazone were compared with those of control cells. n.s., not significant; �, P < 0.05;��, P < 0.01 (Student t test).

Sawayama et al.





lower than those observed in the other ESCC cell lines (Fig.2B). We showed that the dephosphorylation of both AktSer473 and p21 Thr145 in the TE-6 cells was lower than thatobserved in TE4 cells (Fig. 3E and Supplementary Fig. S4B).Treatment with efatutazone could not dephosphorylate Aktat Ser473 in TE-8 cells after treatment with siRNA for PPARG(Supplementary Fig. 4C). These data indicated that inacti-vating the Akt pathway was one of the major targeted effectsof efatutazone.Furthermore we used the Akt inhibitor MK-2206 to detect

the association between the antiproliferative effects of efatu-tazone and inactivation of Akt. The antiproliferative effects ofefatutazone compared with the control decreased withincreasing dose of MK-2206 (Fig. 3G). The antiproliferativeeffects were correlated with p21 protein levels and wereinversely related to phosphorylation of p21 at Thr145 (Fig. 3H).These data indicate that efatutazone regulates p21Cip1

protein levels in the nucleus by inactivating Akt and depho-sphorylating p21 at Thr145, without affecting the transcriptionof p21Cip1.

In vivo antiproliferative effects of efatutazoneTo the best of our knowledge, no previous studies have

demonstrated the antiproliferative effects of conventionalPPAR-g agonists, such as troglitazone, against ESCC cell linesin vivo. We therefore compared 3 treatments in a mousexenograft model established using TE-4 ESCC cells. Animalswere divided into a control group, a troglitazone group and anefatutazone group. Tumor volume did not differ significantlybetween the control group and the troglitazone group; how-ever, a 49.6% � 13.6% (average � SD) reduction in tumorvolume was observed in the efatutazone group compared withthe control group (Fig. 4A, Supplementary Fig. S5A). ThemRNAexpression of PLIN2, one of the markers for the activity ofPPAR-g as a transcriptional factor, was doubled in the efatu-tazone group compared with the control group. The mRNAexpression levels of p21Cip1 and p27 were not increased byefatutazone treatment (Fig. 4B, Supplementary Fig. S5B), butp21Cip1 protein levels were significantly upregulated (Fig. 4C).

We therefore investigated the phosphorylation status ofAkt at Ser473 and of p21 at Thr145. Inactivation of Akt and

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Figure 3. Antiproliferative effects of efatutazone in ESCC cells occur via p-Akt-p21Cip1. A, cell cycle analysis in TE-4 cells after treatment with efatutazone for48 hours. B, PDK4 and p21Cip1 mRNA levels after treatment with efatutazone for 48 hours. C, Western blot analysis for p21 in TE-4 cells treated with25 mMefatutazone for 48 hours.Whole cell lysates were examined in three independent experiments and the average and standard division of the p21 proteinlevel was calculated. D, Western blot analysis for EGFR (p-EGFR), Akt (p-Akt), and p21 (p-p21) in TE-4 cells treated with EGF (100 ng/mL) for various times. E,TE-4 cells treated with 25 mmol/L efatutazone, followed by stimulation with or without EGF (100 ng/mL). Whole cell lysates were analyzed by Western blotanalysis. F, localization of p21Cip1 after treatment with 25 mmol/L efatutazone for 48 hours was examined by confocal microscopy. Originalmagnification, �600. G, antiproliferative effects of efatutazone or control, in combination with various concentrations of MK-2206 for 72 hours, assessed inTE-4 cells. H, Western blot analysis for p21 (p-p21) in TE-4 cells treated with control, efatutazone alone, MK-2206, or efatutazone combined withMK-2206 for 48 hours. n.s., not significant; �, P < 0.05; ��, P < 0.01 (Student t test).






dephosphorylation of p21 at Thr145 were detected morefrequently in the efatutazone group than the control group(Fig. 4C). Immunohistochemical staining also demonstrateddephosphorylation of Akt at Ser473 and p21 at Thr145 in thecytoplasm in the efatutazone group (Fig. 4D). We immuno-histochemically examined the expression levels of bothp21Cip1 and Ki-67 in the nucleus and found that the rate ofpositive staining for p21Cip1 was significantly increased (Fig.4D and E), whereas that for Ki-67 was significantly decreased(Fig. 4D and F) by efatutazone compared with both the controland troglitazone groups.

Efatutazone activates EGFR/MAPK signalingThe antiproliferative effects of efatutazone were associ-

ated with dephosphorylation of Akt at Ser473. Because Akt isdownstream of EGFR signaling via the PI3K–Akt pathway,we investigated the phosphorylation of EGFR at tyrosine1068 (Tyr1068). We confirmed that efatutazone inactivatedAkt pathway within 3 hours, whereas efatutazone graduallyphosphorylated EGFR at Tyr1068 for 24 hours in TE-4 cells invitro (Fig. 5A). Furthermore, using conditioned medium(Supplementary Fig. S6), we found that phosphorylationof EGFR was not caused by efatutazone directly, but was

induced by medium from efatutazone-treated in TE-4 cells(Fig. 5B). These data indicated that secreted factors aftertreatment with efatutazone stimulated phosphorylation ofEGFR at Tyr1068 in ESCC cells. We confirmed that activatedEGFR was detected in 3 ESCC cell lines, TE-4, TE-8, and TE-11 cells after treatment with efatutazone for 48 hours in vitro(Fig. 5C). Tyr1068 of EGFR was significantly phosphorylatedfollowing treatment with efatutazone, and extracellular sig-nal–regulated kinase (ERK) 1/2 was phosphorylated atThr202/Tyr204 in TE-4 xenograft tumor lysates, comparedwith both control and troglitazone-treated tumors (Supple-mentary Fig. S7A and S7B). EGFR phosphorylation atTyr1068 and ERK 1/2 phosphorylation at Thr202/Tyr204were also detected in xenograft tumor lysates from all 3cell lines (Fig. 5D). Immunohistochemical staining showedactivated EGFR at the membrane in TE-4 xenografts aftertreatment with efatutazone (Fig. 5E).

In vitro effects of combination of efatutazone and MEKinhibitor or anti-EGFR antibody

Importantly, the antiproliferative effects of efatutazonewereassociated with inactivation of Akt, whereas EGFR/MAPK(mitogen-activated protein kinase) signaling was activated.

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Figure 4. Antitumor effects of efatutazone in human tumor xenograft models. A, growth of TE-4 xenografts treated with control, troglitazone, or efatutazonedaily. B,mRNAexpression levels of PLIN2 andp21Cip1 in TE-4 cells. C,Western blot analysis for Akt (p-Akt), p21 (p-p21), andPPAR-g in TE-4 xenografts. Thep21 protein level was examined in three independent TE-4 xenografts and the average and standard division of the p21 protein level was calculated. D,immunohistochemical analysis for p-AKT, p-p21, p21, andKi-67 in TE-4 xenografts. Scale bar, 50mm.E and F, the rate of positive staining for p21 andKi-67 inthe nucleus of TE-4 xenografts. n.s., not significant; ��, P < 0.01 (Student t test).

Sawayama et al.





We therefore used molecular-targeted agents in combinationwith efatutazone to suppress MAPK or EGFR signaling.The MEK inhibitor U0126 (Supplementary Fig. S8A) was

used in combination with efatutazone. Dephosphorylation ofERK 1/2 was detected after treatment with efatutazone com-bined with U0126. However, EGFR/Akt signaling was activatedby combined treatment, compared with treatment with efa-tutazone alone (Fig. 5F). The growth inhibitory effects ofU0126, efatutazone, and the combination of the 2 agents were37.3 � 5.0, 31.1 � 3.6, and 51.6 � 3.9, respectively, comparedwith the control (Fig. 5G, Supplementary Fig. S8B).The anti-EGFR antibody cetuximab (Supplementary Fig.

S8C and S8D) was also investigated in combination withefatutazone. Significantly, phosphorylation of EGFR atTyr1068 caused by treatment with efatutazone was notdetected after treatment with the combination of efatuta-zone and cetuximab. Furthermore, both the PI3K–Akt andMAPK pathways were inactivated by this combination ther-

apy (Fig. 5F). The growth inhibitory effects of cetuximab,efatutazone, and efatutazone combined with cetuximabwere 19.7 � 6.1, 31.1 � 3.6, and 44.9 � 0.5, respectively,compared with the control (Fig. 5H, Supplementary Fig.S8E). Interestingly, treatment with efatutazone-conditionedmedium for 10 minutes activated EGFR, however EGFR wasnot phosphorylated at Tyr1068 when TE-4 cells were pre-treated with cetuximab for 6 hours (Fig. 5I).

In vivo effects of combination therapy with efatutazoneand cetuximab

Cetuximab exhibited better efficacy than U0126 when usedin combination with efatutazone in vitro. We therefore inves-tigated the antiproliferative effects of efatutazone combinedwith cetuximab in a mouse TE-4 cell xenograft model. Micewere divided into 4 treatment groups: control group, efatuta-zone group, cetuximab group, and combination group (Fig.6A). The tumor inhibitory effects after treatments were

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Figure 5. Treatment with efatutazone led to phosphorylation of EGFR and activation of MAPK pathway. A, Western blot analysis for EGFR (p-EGFR) andAkt (p-Akt) in TE-4 cells for various times after treatment with efatutazone. B, TE-4 cells were treated with control (lane 1) or efatutazone (lane 2) for 48 hours,and the medium was collected. Conditioned medium (cond. med.) control and cond. med. efatutazone were adjusted to have the same density of bothefatutazoneandDMSO.TE-4cellswere treatedwith theseconditionedmedia (lanes3, 4) for 10minutes, andEGFR (p-EGFR)was thendetermined.C,Westernblot analysis for EGFR (p-EGFR) after treatment with 25 mmol/L efatutazone for 48 hours in vitro. D, Western blot analysis for EGFR (p-EGFR) and ERK(p-ERK) using whole cell lysates of xenografts. E, immunohistochemical analysis of p-EGFR in TE-4 xenografts. Scale bar, 50 mm. F,Western blot analysis forEGFR (p-EGFR), ERK (p-ERK), and Akt (p-Akt) in TE-4 cells treated for 24 hours. G, percentage of inhibiting effect of TE-4 cells after treatment with U0126,efatutazone, and efatutazone combined with U0126 compared with control. H, percentage of inhibiting effect of TE-4 cells after treatment with cetuximab,efatutazone, and efatutazone combined with cetuximab compared with control. Representative experiments, n ¼ 3, mean � SE. I, TE-4 cells werepretreated with or without cetuximab (300 mg/mL) for 6 hours, followed by stimulation with the conditioned media.






17.6% � 11.0%, 41.0% � 8.5%, and 71.7% � 5.4% (average �SD) in the cetuximab group, efatutazone group, and combi-nation group, respectively, compared with the control groupafter 38 days (Fig. 6B).

EGFR was more dephosphorylated in the combination-treatment group than in the control group, and inactivationof both the PI3K–Akt and MAPK pathways was demonstrat-ed (Fig. 6C). Activation of EGFR at the membrane in TE-4xenografts after treatment with efatutazone was suppressedin tumors of animals treated with efatutazone plus cetux-imab, as demonstrated by immunohistochemical staining(Fig. 6D).

The body weights of the mice were measured twice a week.The mice treated with efatutazone alone tended to gain moreweight than control mice, but the body weights of mice treatedwith efatutazone combined with cetuximab showed no signif-icant difference from the controls (Fig. 6E).

DiscussionPPAR-g expression is tissue and cancer specific, and the

expression of PPAR-g protein in patients with ESCC and theantiproliferative effects and mechanism of action of the newgeneration PPAR-g agonists in ESCC remain unclear. Wetherefore investigated the expression of PPAR-g in 145 patientswith ESCC, and clearly demonstrated the antiproliferativeeffects and mechanism of action of the novel PPAR agonist,efatutazone.

We demonstrated that PPAR-g was expressed in normalesophageal squamous epithelium, and was expressed heteroge-neously in ESCC tumors. It exhibited an inverse relationshipwithKi-67 expression, as determinedby immunohistochemistry.

To the best of our knowledge, efatutazone is the first PPAR-gagonist shown to inhibit the proliferation of ESCC cell linesboth in vitro and in vivo. Importantly, the results of this studysuggest that the novel antiproliferative mechanism of efatu-tazone involves regulation of p21Cip1 protein levels in thenucleus by inactivating Akt signaling and dephosphorylatingp21 at Thr145, without affecting the transcriptional activity ofp21Cip1.

The EGFR/MAPK pathway was activated by efatutazone,and combined treatment with efatutazone and cetuximabsuppressed both the PI3K–Akt and MAPK pathways, leadingto synergistic antiproliferative effects (Fig. 7).

PPAR-g is affected by endogenous ligands such as D12,15

prostaglandin J2, and serves as a transcriptional factor in vivo(14). However, the current investigation demonstrated thatPPAR-g expression seems to be heterogeneous during carci-nogenesis. In addition, PPAR-g expression had an inverserelationship with Ki-67 expression, and may be associatedwith tumor-suppressive effects. We therefore investigated ifactivation of PPAR-g by a high-affinity PPAR-g ligand inducedantitumor effects in ESCC.

The conventional PPAR-g agonist troglitazone partiallyinhibited ESCC proliferation in vitro in previous studies (41).However, our results showed no significant antiproliferative

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Figure 6. In vivo effects of combination therapy using efatutazonewith cetuximab. A, growth of TE-4 tumors implanted in nudemice. B, percentage of inhibitingeffect after treatment with cetuximab, efatutazone alone, or efatutazone combined with cetuximab compared with control. C, Western blot analysis for EGFR(p-EGFR), ERK (p-ERK), and Akt (p-Akt) in TE-4 cells after treatment. D, immunohistochemical analysis of p-EGFR in TE-4 xenografts after treatment. Scalebar, 50 mm. E, body weights of mice. n.s., not significant; �, P < 0.05; ��, P < 0.01 (Student t test).

Sawayama et al.





effect of troglitazone in ESCC cell lines, whereas efatutazonecaused a 49.6% reduction in proliferation of xenografted ESCCcells, and the tumor-reduction rate was similar to the maxi-mumeffect of efatutazone against HT-29 colon cancer cells in aprevious xenograft study (24). This result suggests that efatu-tazone represents a promising antitumor agent for patientswith ESCC. However, the mechanisms underlying the antipro-liferative effects of efatutazone are poorly understood.This study demonstrated that efatutazone upregulated

p21Cip1 protein levels in the nucleuswithout affecting p21Cip1mRNA. A previous study showed that Akt activation wasassociated with phosphorylation of p21 at Thr145, which ledto p21Cip1 translocation from the nucleus to the cytoplasm(40). Cytoplasmic p21Cip1 has also been associated withsurvival pathways (42) and a poor prognosis in breast cancer(43). Interestingly, Akt activation by EGF stimulation also led tophosphorylation of p21 at Thr145 in ESCC, whereas Aktinactivation induced by efatutazone led to dephosphorylationof p21 at Thr145, resulting in upregulation of p21Cip1 proteinlevels in the nucleus in this study.Significant tumor regression may be typical for treatments

combining PPAR-g agonists with conventional cytotoxic anti-cancer agents (4, 23, 24). We found that additional antiproli-ferative effects were detected in ESCC cell lines when efatu-tazone was used in combination with 5-fluorouracil, cisplatinor docetaxel in vitro (data not shown); however, the antipro-liferative effects and mechanism of action of efatutazone incombination with molecular-targeted agents are unclear.Importantly, EGFR/MAPK signaling was activated by efatuta-zone. EGFR was gradually activated after treatment withefatutazone and the cause of the phosphorylation of EGFRTyr1068 was a secreted protein (factors) produced by theefatutazone-treated cells. Activation of these pathways maylimit the antiproliferative effects of efatutazone, and the addi-tion of molecular-targeted agents to efatutazone may help to

suppress these growth pathways. We used U0126 in combina-tion with efatutazone, but the additive effect was small. Recentstudies reported ERK-dependent negative feedback in severalcancers (44, 45). This ERK-dependent negative feedback waslost after treatment with a MEK inhibitor, and the ability ofreceptor tyrosine kinase ligands to activate growth signalingwas markedly enhanced (44, 46). Similar processes may havebeen responsible for the effects observed when efatutazonewas combined with U0126. ERK-dependent negative feedbackwas lost after treatment with U0126, and the secreted factorsproduced by the efatutazone-treated cells stimulated EGFR,followed by the remarkable activation of Akt pathway.

The activation of EGFR induced by the secreted factorsafter treatment with efatutazone was not detected afterpretreating ESCC cells with cetuximab. We therefore usedcetuximab in combination with efatutazone, and demon-strated a notable synergistic effect based on signal analysis.This combination therapy may have other benefits. Althoughthe tolerability of efatutazone has been demonstrated in aclinical study, more than half of the patients (51.6%) sufferedfrom peripheral edema, which is a recognized adverse effectof efatutazone treatment (22). In our study, mice treatedwith efatutazone alone also tended to gain more weight thancontrol mice, although this adverse weight gain was reducedin mice treated with efatutazone combined with cetuximab.A recent study showed that PPAR-g agonists rapidly stim-ulated sodium-coupled bicarbonate absorption from therenal proximal tubule, followed by plasma volume expan-sion. The PPAR-g agonist–induced transport stimulationwas dependent on PPAR-g–Src–EGFR–ERK (47). In ourstudy, inactivation of MAPK pathway by efatutazone com-bined with cetuximab may have suppressed the sodium-coupled bicarbonate absorption from the renal proximaltubule and led to a reduction in the adverse weight gaincaused by treatment with efatutazone alone.

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Figure 7. Effects of treatment with efatutazone alone and in combination with cetuximab. A, no treatment. EGFR is phosphorylated by EGF stimulation,and both the PI3K–Akt and MAPK pathways are activated. Activated Akt phosphorylates p21 Thr145 and leads to p21Cip1 translocation from thenucleus to the cytoplasm. Cytoplasmic localization of p21Cip1 is associated with survival pathways. B, efatutazone inactivates Akt, and p21 Thr145 isdephosphorylated. This leads to an increase in the p21Cip1 protein level in the nucleus and G1 arrest. However, the EGFR/MAPK pathways are activated.C, the combination of efatutazone with cetuximab suppresses both the PI3K–Akt and MAPK pathways and leads to synergistic antiproliferative effects.






In conclusion, this study demonstrated the tumor-suppres-sive effects and major underlying mechanism of efatutazoneinvolving inactivation of the Akt pathway. Treatment withefatutazone combined with cetuximab produced a synergisticeffect by negatively regulating both the PI3K–Akt and MAPKpathways. These results suggest that efatutazone could be usedboth alone and in combination with cetuximab as a potentialtherapeutic approach for ESCC.

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

Authors' ContributionsConception and design: H. Sawayama, T. Ishimoto, J. Kurashige, Y. Shiose,Hideo BabaDevelopment of methodology: H. Sawayama, T. Ishimoto, J. Kurashige, K.HirashimaAcquisition of data (provided animals, acquired and managed pati-ents, provided facilities, etc.): H. Sawayama, T. Ishimoto, M. Watanabe,N. YoshidaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Sawayama, T. Ishimoto, M. Watanabe

Writing, review, and/or revision of the manuscript: H. Sawayama,T. Ishimoto, Y. ShioseAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): H. Sugihara, Y. ShioseStudy supervision:M.Watanabe, M. Iwatsuki, Y. Baba, E. Oki, M. Morita, HideoBaba

AcknowledgmentsThe authors thank Drs. Youhei Tanaka, Hironobu Shigaki, Hirohisa Okabe,

Koichi Kinoshita, Seiya Saito, and Yukiharu Hiyoshi for their valuableresearch advice, Naomi Yokoyama, Kenichi Yamaguchi, and Keisuke Miyakefor technical assistance, and Dr. Kenichi Iyama for providing the pathologicaldiagnoses.

Grant SupportThiswork was supported in part by the scholarship for the Graduate School of

Medical Sciences, Kumamoto University, Japan.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received July 4, 2013; revised October 16, 2013; accepted November 3, 2013;published OnlineFirst November 22, 2013.

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2014;74:575-585. Published OnlineFirst November 22, 2013.Cancer Res Hiroshi Sawayama, Takatsugu Ishimoto, Masayuki Watanabe, et al. Esophageal Cancer

Exert Therapeutic Effects inγSmall Molecule Agonists of PPAR-

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