Small molecule agonists of PPAR- γ exert therapeutic effects in esophageal cancer

1 The transcription factor PPAR γ plays various roles in lipid metabolism, inflammation, 2 cellular differentiation and apoptosis. PPAR γ agonists used to treat hyperlipidemia may have 3 utility in cancer treatment. Efatutazone is a novel later generation PPAR γ agonist that 4 selectively activates PPAR γ target genes and has anti-proliferative effects in a range of 5 malignancies. In this study, we investigated PPAR γ status in esophageal squamous cell 6 carcinoma (ESCC) and investigated the anti-proliferative effects of efatutazone. PPAR γ was 7 expressed heterogeneously in ESCC where it exhibited an inverse relationship with Ki-67 8 expression. PPAR γ expression was associated independently with good prognosis in ESCC. Efatutazone, but not the conventional PPAR γ agonist troglitazone, inhibited ESCC cell 10 proliferation in vitro and in vivo. Mechanistic investigations suggested that efatutazone acted 11 by upregulating p21Cip1 protein in the nucleus through inactivation of the Akt pathway and dephosphorylation of p21Cip1 at Thr145, without affecting the transcriptional activity of 13 p21Cip1. We also found that treatment with efatutazone led to phosphorylation of the EGF receptor and activation of the MAPK pathway. Accordingly, the combination of efatutazone 4 with the EGFR antibody cetuximab synergized to negatively regulate the PI3K-Akt and 1 MAPK pathways. Together, our results suggest that efatutazone, alone or in combination with 2 cetuximab, may offer therapeutic effects in ESCC

Introduction 1 is increasing rapidly. Squamous cell carcinoma is the predominant form of esophageal 1 carcinoma worldwide (25). Furthermore, esophageal carcinoma is an aggressive disease with 2 a propensity to spread both locoregionally and distally, and multidisciplinary therapy has 3 therefore been tested for ESCC (26,27). The anti-epithelial growth factor receptor (EGFR) 4 antibody, cetuximab, was approved for head and neck squamous cell carcinoma (28, 29), and 5 new molecular-targeted therapies have been expected for the treatment of ESCC (30). 6 The effects of PPARγ are tissue-and cancer-specific. The significance of PPARγ 7 expression and anti-proliferative effects of PPARγ agonists in esophageal squamous cell 8 carcinoma (ESCC) have been reported in a few studies, however these studies were limited 9 because they only evaluated the mRNA levels in small number of samples (31), in 10 adenocarcinomas (32), or using conventional PPARγ agonists, such as troglitazone (33).

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Therefore the expression of the PPARγ protein in ESCC and the anti-proliferative effects of monotherapy both in vitro and in vivo. We also investigated the effects of combination 1 therapy using efatutazone with molecular-targeted agents, based on signaling analysis. The 2 results of this study may provide a novel therapeutic strategy for ESCC, and suggest that 3 efatutazone, both alone and in combination with an anti-EGFR antibody, may improve the 4 outcomes of ESCC patients.  Troglitazone, U0126, MK-2206 dihydrochloride, and cetuximab were purchased from 9 Cayman, Wako, Selleck Bio. and Kumamoto University Hospital, respectively. For the in 10 vitro analyses, efatutazone was prepared in dimethyl sulfoxide (DMSO) before addition to 11 cell cultures. Antibody information is provided in the Supplementary Methods and Materials. and troglitazone were suspended in 0.5 w/v% methylcellulose solution and administered to 1 the animals daily by oral gavage in a volume of 0.1 ml/10 g body weight, using an 2 animal-feeding needle. The anti-EGFR antibody, cetuximab (1 mg/injection) or placebo 3 (PBS) was injected intraperitoneally twice a week to assess the effects of combination 4 therapy with efatutazone. Caliper measurements were made twice a week using digital 5 calipers, and the tumor volumes were estimated using the following formula: V=L×W×D×π/6, 6 where V is the tumor volume, L is the length, W is the width, and D is the depth (30, 34). Statistical analysis. Comparisons between treatment groups were made using two-tailed 9 paired or unpaired Student's t-tests, as appropriate, based on the results of F tests. The 10 Mann-Whitney U-test was used in the event of a non-normal distribution. The log-rank test 11 was used for survival analysis, and the Kaplan-Meier method was used to assess survival 12 time distribution. For analyses of esophageal cancer-specific mortality, deaths as a result of 13 causes other than ESCC were censored. Univariate Cox regression analysis was also 14 performed. The independent effect of PPARγ on mortality was assessed by performing multivariate Cox regression analysis. Statistical significance was defined as a P value <0.05. 1 All data were processed and analyzed using the PASW Statistics 18 software program.

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PPARγ expression in the normal epithelium and ESCC 9 We investigated PPARγ expression in normal esophageal epithelial samples and tumor 10 lesions from 145 patients with resectable ESCC using immunohistochemical analysis 11 (Supplementary Table S1). PPARγ was ubiquitously expressed in the squamous layer of the 12 normal esophageal epithelium in all 145 patients, and there was no variation (Fig. 1A). The  Table S2). 8 We therefore hypothesized that PPARγ was associated with tumor-suppressive effects 9 against ESCC, and that efatutazone, a novel third-generation PPARγ agonist, could be a 10 potentially useful anti-cancer agent in ESCC patients. of PPARγ expression were observed in the ESCC cell lines ( Fig. 2A). However, efatutazone 1 demonstrated anti-proliferative effects in all nine ESCC cell lines (Fig. 2B). We investigated 2 anti-proliferating effects and underlying mechanism of efatutazone using TE-4, TE-8 and 3 TE-11, TE-6 cells, which express high, medium and low levels of PPARγ, respectively. 4 We used three small interfering (si) RNAs for PPARG to confirm that the anti-tumor 5 effects of efatutazone occurred in a PPARγ-dependent manner. We selected TE-8 cells, 6 because the PPARγ expression of TE-8 cells was strongly suppressed at both the mRNA and 7 protein levels using siRNAs for PPARG. (Fig. 2C and D, Supplementary Fig. S2A and B).  isozyme-4 (PDK4), which is an established marker for the activity of PPARγ as a 12 transcriptional factor (37, 38), by 2.5-fold compared with the control level, but this effect was 13 blocked in the presence of PPARG siRNA (Fig. 2E). The inhibition of proliferation by 14 efatutazone was related to the PDK4 mRNA level (Fig. 2F) proliferation of the ESCC cell lines in a time-dependent manner (Fig. 2G), and PDK4 levels 1 were also increased in a similarly time-dependent manner and correlated with 2 anti-proliferative effect of efatutazone (Fig. 2H). The mRNA level of PDK4 after treatment 3 with 25μM efatutazone was increased more than after treatment with 25μM troglitazone 4 ( Supplementary Fig. S3A). No significant anti-proliferative effects of treatment with 5 troglitazone were detected compared with the control, however, the proliferation of both 6 TE-4 and TE-8 cells was inhibited by the treatment with efatutazone ( Supplementary Fig.   7 S3B). Since the anti-proliferative effects of efatutazone were observed in various ESCC cell 8 lines, we investigated the mechanism underlying the anti-proliferative effects of efatutazone 9 using TE-4 cells, which highly express PPARγ. cycle in ESCC, we subjected TE-4 and TE-11 cells to cell cycle analysis by flow cytometry.
1 Accumulation of cells in the G1 phase, and reductions in the S and G2/M phases occurred 2 after treatment with efatutazone for 48 h (Fig. 3A, Supplementary Fig. S4A). In addition, 3 PDK4 mRNA levels were upregulated by treatment with efatutazone for 48 h, while p21Cip1 4 mRNA levels remained unaffected by the treatment (Fig. 3B). However, p21Cip1 protein 5 levels were significantly upregulated in western blot analysis (Fig. 3C). We therefore focused 6 on the post-translational (post-transcriptional ?) modification of the p21 protein (39). 7 Activation of Akt, which associates with p21Cip1 and phosphorylates it at threonine 8 145 (Thr145), results in increased cytoplasmic localization of p21Cip1 in breast cancer (40). 9 We therefore stimulated the phosphoinositide 3-kinase (PI3K)-Akt pathway with epidermal 10 growth factor (EGF) (100 ng/ml) to investigate these signaling changes in the ESCC cell lines. 11 We found that Akt was activated by treatment with EGF, and p21Cip1 was then gradually 12 phosphorylated at Thr 145 in ESCC cells (Fig. 3D). 13 We treated TE-4 cells with efatutazone for 48 h, followed by stimulation with or 14 without EGF for 10 min to determine if efatutazone was associated with inactivation of Akt. Efatutazone reduced the phosphorylation of both Akt at Ser473 and p21 at Thr145 (Fig. 3E).

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Localization of p21Cip1 was confirmed by confocal microscopy. Some p21Cip1 protein was 2 detected in the cytoplasm in control cells, but strong staining was detected in the nucleus 3 following treatment with efatutazone for 48 h (Fig. 3F).

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The PPARγ expression of TE-6 cells was lower ( Fig. 2A) and the anti-proliferative 5 effects of treatment with efatutazone were lower than those observed in the other ESCC cell 6 lines (Fig. 2B). We showed that the dephosphorylation of both Akt Ser473 and p21 Thr145 in 7 the TE-6 cells was lower than that observed in TE4 cells ( Fig. 3E and Supplementary Fig.   8 S4B). Treatment with efatutazone could not dephosphorylate Akt at Ser473 in TE-8 cells 9 after treatment with siRNA for PPARG ( Supplementary Fig. 4C). These data indicated that 10 inactivating the Akt pathway was one of the major targeted effects of efatutazone.

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Furthermore we used the Akt inhibitor MK-2206 to detect the association between the  In vivo anti-proliferative effects of efatutazone 6 To the best of our knowledge, no previous studies have demonstrated the anti-proliferative 7 effects of conventional PPARγ agonists, such as troglitazone, against ESCC cell lines in vivo. 8 We therefore compared three treatments in a mouse xenograft model established using TE-4 9 ESCC cells. Animals were divided into a control group, a troglitazone group and an 10 efatutazone group. Tumor volume did not differ significantly between the control group and 11 the troglitazone group; however, a 49.6±13.6% (average ± SD) reduction in tumor volume with the control group. The mRNA expression levels of p21Cip1 and p27 were not increased 1 by efatutazone treatment (Fig. 4B, Supplementary Fig. S5B), but p21Cip1 protein levels were 2 significantly upregulated (Fig. 4C). 3 We therefore investigated the phosphorylation status of Akt at Ser473 and of p21 at  Ser473. Because Akt is downstream of EGFR signaling via the PI3K-Akt pathway, we 1 investigated the phosphorylation of EGFR at tyrosine 1068 (Tyr1068). We confirmed that 2 efatutazone inactivated Akt pathway within three hours, while efatutazone gradually 3 phosphorylated EGFR at Thy 1068 for 24 hours in TE-4 cells in vitro ( Fig 5A). Furthermore, 4 using conditioned medium ( Supplementary Fig. S6), we found that phosphorylation of EGFR   Immunohistochemical staining showed activated EGFR at the membrane in TE-4 xenografts 1 after treatment with efatutazone ( Fig. 5E). Importantly, the anti-proliferative effects of efatutazone were associated with inactivation of 6 Akt, while EGFR/MAPK signaling was activated. We therefore used molecular-targeted 7 agents in combination with efatutazone to suppress MAPK or EGFR signaling.

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EGFR was more dephosphorylated in the combination-treatment group than in the 4 control group, and inactivation of both the PI3K-Akt and MAPK pathways was demonstrated 5 (Fig. 6C). Activation of EGFR at the membrane in TE-4 xenografts after treatment with 6 efatutazone was suppressed in tumors of animals treated with efatutazone plus cetuximab, as 7 demonstrated by immunohistochemical staining (Fig. 6D). 8 The body weights of the mice were measured twice a week. The mice treated with 9 efatutazone alone tended to gain more weight than control mice, but the body weights of mice 10 treated with efatutazone combined with cetuximab showed no significant difference from the 11 controls (Fig. 6E).  We demonstrated that PPARγ was expressed in normal esophageal squamous 5 epithelium, and was expressed heterogeneously in ESCC tumors. It exhibited an inverse 6 relationship with Ki-67 expression, as determined by immunohistochemistry. 7 To the best of our knowledge, efatutazone is the first PPARγ agonist shown to inhibit 8 the proliferation of ESCC cell lines both in vitro and in vivo. Importantly, the results of the 9 current study suggest that the novel anti-proliferative mechanism of efatutazone involves 10 regulation of p21Cip1 protein levels in the nucleus by inactivating Akt signaling and 11 dephosphorylating p21 at Thr145, without affecting the transcriptional activity of p21Cip1.

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The EGFR/MAPK pathway was activated by efatutazone, and combined treatment with 13 efatutazone and cetuximab suppressed both the PI3K-Akt and MAPK pathways, leading to 14 synergistic anti-proliferative effects (Fig. 7).  efatutazone-treated cells. Activation of these pathways may limit the anti-proliferative effects 1 of efatutazone, and the addition of molecular-targeted agents to efatutazone may help to 2 suppress these growth pathways. We used U0126 in combination with efatutazone, but the tolerability of efatutazone has been demonstrated in a clinical study, more than half of the 1 patients (51.6%) suffered from peripheral edema which is a recognized adverse effect of 2 efatutazone treatment (22). In our study, mice treated with efatutazone alone also tended to 3 gain more weight than control mice, while this adverse weight gain was reduced in mice 4 treated with efatutazone combined with cetuximab. A recent study showed that PPARγ 5 agonists rapidly stimulated sodium-coupled bicarbonate absorption from the renal proximal 6 tubule, followed by plasma volume expansion. The PPARγ agonist-induced transport 7 stimulation was dependent on PPARγ-Src-EGFR-ERK (47). In our study, inactivation of 8 MAPK pathway by efatutazone combined with cetuximab may have suppressed the 9 sodium-coupled bicarbonate absorption from the renal proximal tubule and led to a reduction 10 in the adverse weight gain caused by treatment with efatutazone alone.

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In conclusion, this study demonstrated the tumor-suppressive effects and major 12 underlying mechanism of efatutazone involving inactivation of the Akt pathway. Treatment