PPARγ inhibition regulates the cell cycle, proliferation and motility of bladder cancer cells

Abstract Peroxisome proliferator‐activated receptor gamma (PPARγ) is a member of the nuclear receptor family of ligand‐activated transcription factors and plays an important role in regulating cell proliferation, inflammation and lipid and glucose homeostasis. Our results revealed that PPARγ was up‐regulated in human bladder cancer (BCa) tissues both at transcriptional and translational levels. Moreover, down‐regulation of PPARγ mRNA or inhibition of PPARγ function (using GW9662, antagonist of PPARγ) could significantly suppress the proliferation of BCa cells. Furthermore, the cell cycle arrested in G0/G1 phase was also induced by the down‐regulated PPARγ possibly through AKT‐mediated up‐regulation of p21/p27, whereas no significant transformation of apoptosis was observed. In addition, knockdown or inhibition of PPARγ might reduce the invasion and migration of BCa cells by affecting epithelial‐mesenchymal transition‐related proteins through AKT/GSK3β signalling pathway. Additionally, in vivo studies showed that BCa cell proliferation was significantly suppressed by GW9662. In conclusion, our results indicated that PPARγ might be crucial for BCa tumorigenesis by interfering with the motility and viability of BCa cells.

urothelial layers of the bladder. 2 Currently, the preferred therapeutic method for NMIBC is transurethral resection of the bladder tumour, followed by intravesical instillations of chemotherapy or immunotherapy. 3 According to an analysis of 2596 patients with NMIBC, the 5-year recurrence rate reached 31% to 78% and the risk of progression to muscle-invasive BCa (MIBC) ranged from 1% to 45%. 4 The gold standard therapeutic method for MIBC is radical cystectomy, which is still associated with many unfavourable outcomes. 5,6 Furthermore, even after receiving radical cystectomy, the BCa recurrence and distant metastasis rate remain at approximately to 50%, and the 5-year survival rate is only 50%-66%. 7,8 Therefore, more effective therapeutic strategies are needed for BCa treatment.
Peroxisome proliferator-activated receptor gamma (PPARγ), a member of the nuclear receptor family of ligand-activated transcription factors that regulates gene expression via the formation of protein heterodimers with the retinoic X receptor (RXR), is a key nuclear receptor regulating adipocyte differentiation and glucose homeostasis. 10 Peroxisome proliferator-activated receptor gamma has been reported to interact with multiple signalling pathways, including BCL2, NFκB, p53, p21, STAT, cyclooxygenase-2 (COX-2) and Cyclin D1. 11 Additionally, PPARγ is highly expressed in certain cancer cell. However, its function in tumour progression remains controversial. 10,12 Some previous studies have suggested that the activation of PPARγ can prevent tumours in tissues such as the lung, colon, prostate, breast and others, 13,14 while others have revealed that activated PPARγ is oncogenic. 18,19 Therefore, the role of PPARγ in tumorigenesis needs to be further clarified. In this study, we aimed to identify the effects of PPARγ in BCa.
Ligands of PPARγ contain endogenetic fatty acids and thiazolidinedione (TZD) classes of anti-diabetic drugs such as pioglitazone, which has weak PPARα activity, and rosiglitazone, which is highly selective for PPARγ. 22 Some clinical trials have shown that PPARγ agonists are associated with increased risks of bone fractures 23,24 heart failure 25 and BCa. 26 For instance, an increased incidence of BCa has been observed, as a side effect in rodent toxicity studies using PPAR agonists to examine anti-diabetic effects, 18 whereas other studies indicated no increased risk. 27,28 Recently, a comprehensive retrospective study showed an increased hazard ratio with long-term, high-dose treatment with pioglitazone in BCa. 29 In addition, some compounds that antagonize PPARγ have been reported to inhibit the proliferation or invasiveness of human tumour cell lines, such as colon carcinoma, renal cell carcinoma, oesophageal carcinoma, hepatocellular carcinoma and others 20,21,30 Therefore, in our study, we chose GW9662, the selective and irreversible PPARγ antagonist, 31 to treat the BCa cells and detected the alterations in viability and motility.
Our group has focused on novel biomarkers and pathways for the long-term prevention of BCa progression. Our previous studies have suggested that HJURP may regulate apoptosis and proliferation in BCa cells through the PPARγ-SIRT1 feedback loop 32 and that simvastatin can induce cell cycle arrest in G1/G0 phase and inhibit proliferation in BCa cells via the PPARγ signalling pathway. 33 In this study, we aimed to investigate the function of PPARγ gene and its effects on BCa tumorigenesis.

| Ethical statement for human bladder tissues
As described by Wang et al 33

| Total RNA isolation from bladder cells and tissues
Total RNA was extracted from BCa cells and bladder tissues using the Qiagen RNeasy Mini Kit (Cat. #74101; Qiagen, Hilden, Germany) and QIAshredder from Qiagen (Cat. #79654, Qiagen) according to the manufacturer's instructions. Quantity control of the isolated RNA was assessed using a NanoDrop ® ND-2000 UV-Vis spectrophotometer (Thermo Scientific, Madison, WI, USA).

| Reverse transcription and quantitative realtime PCR
The cDNA was synthesized from 1 μg of total RNA using the RevertAid Ace quantitative real-time PCR (qPCR RT) kit (Toyobo, Shanghai, China).

| Knockdown of PPARγ in BCa cells
Negative control small interfering RNA (siRNA) and PPARγ tar-

| Clonogenic survival assay
To six-well plates were added 800 UM-UC-3 cells/well, 1000 T24 cells/well and 3000 5637 cells/well for growth into colonies for 7-10 days. After removing the medium, fixing the cells with 4% PFA, and staining with crystal violet for 30 minutes, imaging and counting were performed.

| Isolation of total protein and Western blot analysis
Bladder cancer cells were lysed and sonicated on ice in radioimmunoprecipitation assay buffer including phosphatase inhibitor and protease inhibitor (Sigma-Aldrich) for approximately 30 minutes. They were then centrifuged at 12 000 g for 15 minutes, and the supernatant was collected. The Bradford protein assay (Bio-Rad, Munich, Germany) was used to determine the protein concentration using TA B L E 1 List of primers for quantitative real-time PCR bovine serum albumin (BSA) as the standard. Protein was separated using 7.5%-10% SDS-PAGE gels and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), which was then blocked in 5% fat-free milk (BD biosciences) at room temperature for 2 hours and incubated with primary antibodies overnight (Table 2) and secondary antibodies for 2 hours (Table 3). Bands were detected using an enhanced chemiluminescence (ECL) kit (Bio-Rad, Hercules, CA, USA) and visualized with Biomax MR films (Kodak, Rochester, NY).  Tables 2 and 3).

| Haematoxylin and eosin staining
All the tissue paraffin sections from xenotransplanted cancer samples were stained with haematoxylin and eosin (H&E). They were then serially deparaffinized and rehydrated in xylene, 100% ethanol, 96% ethanol, 80% ethanol, 70% ethanol and H 2 O. The sections were subsequently stained with 10% haematoxylin (Sigma-Aldrich) for 7 minutes, and 1% eosin (Sigma-Aldrich) with 0.2% glacial acetic acid was used to stain the cytoplasm after washing to reveal the nuclei. The tissue sections were washed and dehydrated successively in 70%, 80%, 96% and 100% ethanol, followed by 10 minutes in xylene. Images of the sections were obtained using an inverted phase contrast microscope (Cat. #DMI 1; Leica, Wetzlar, Germany).

| Xenograft mouse model
Male BALB/c nude mice aged 3 weeks were obtained from Beijing HFK Bioscience Co., Ltd. in Beijing, China. After a 1-week adaptation period, the mice that were maintained in a temperature and humidity-controlled and specific pathogen-free environment in the laboratory animal facility of Zhongnan Hospital of Wuhan University, were injected into the right flank with 3 × 10 6 T24 cells dispersed in 0.2 mL PBS. Ten days later, mice with transplanted tumours that were approximately 3 × 3 mm were randomly assigned to one of two groups (n = 5). GW9662 (1 mg/kg body weight) and vehicle were injected intraperitoneally every other day for 2 weeks. Tumour size was measured every 2 days using a caliper, and the values were

| Statistical analyses
All experiments were performed at least three times, and representative data were from three iterations. One-way ANOVA and two-tailed Student's T test were used to evaluate the statistical significance of differences between subgroups. All the statistical analyses were conducted with spss 16.0, and the cut-off level was set at probability values of P < 0.05.

| Up-regulation of PPARγ in BCa tissues compared with normal and paracancerous tissues
First, we searched in the Oncomine database, which showed that the expression of PPARγ was significantly up-regulated in BCa tissues at the transcriptional level ( Figure 1A). Our qRT-PCR results also showed increasing expression of PPARγ in BCa in comparison to paracancerous BCa tissues (n = 9, Figure 1B) Figure 1D; Table S1). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that PPARγ was related to cell cycle, focal adhesion, apoptosis, PI3K-AKT signalling pathway and so on ( Figure 1E; Table S2).

| Knockdown of PPARγ impaired the viability of BCa cells
To  Figure 2D; Figure S1D). The clonogenic survival results showed a distinct reduction in colony-forming efficiency ( Figure 2E; Figure   S1C), which was statistically significant ( Figure 2F). Moreover, Ki-67, an important marker of cell proliferation, was significantly reduced in the siRNA-treated group compared with the NC group ( Figure 2C).

| Down-regulation of PPARγ inhibited cell motility with alterations in AKT/GSK3β/EMTrelated proteins
Metastasis was the greatest challenge in clinical management of tumours, which was associated with cell motility. Our transwell invasion and migration assay revealed that knockdown of PPARγ could decrease the cell invasion and migration rate of BCa cells ( Figure 3A; Figure S1E), which was confirmed by the statistical analysis ( Figure 3B). In addition, proteins related to EMT were analysed by Western blot and IF staining, which showed that E-cadherin, a marker of the epithelium, was up-regulated, and the mesenchymal markers N-cadherin and vimentin exhibited an especially strongly down-regulation in the PPARγ-siRNA group ( Figure 3C,D; Figure S1H). Moreover, consistent with our KEGG TA B L E 3 List of secondary antibodies and counterstaining of nuclei pathway enrichment, the proteins associated with the AKT/ GSK3β signalling pathway were altered, for example, p-GSK3β was up-regulated and p-AKT was down-regulated ( Figure 3C).
Furthermore, double IF staining showed that the fluorescence of p-AKT (red) counterstained with PPARγ (green) was intensely decreased in the PPARγ-siRNA group compared with NC group ( Figure S1F).

| Knockdown of PPARγ induced cell cycle arrest in G0/G1 phase but had no significant effect on apoptosis
Pathway enrichment analysis revealed that PPARγ was related to cell cycle, apoptosis and PI3K-AKT signalling pathway ( Figure 1E). Therefore, we used flow cytometry to assess the effect of PPARγ  Figure 4C; Figure S1I). We also noted that the upstream indicators of the cell cycle pathway, such as p21 and p27, were up-regulated, and phosphorylated/total AKT was down-regulated ( Figure 4C; Figure S1I). However, as shown by the flow cytometry analysis, PPARγ deficiency could not induce significant apoptosis in BCa cells ( Figure 4A,B; Figure S1G).

| The PPARγ antagonist GW9662 inhibited the motility and proliferation of BCa cells
To further study the effect of drug inhibitor of PPARγ protein on  Figure 5E).

| GW9662 suppressed BCa cell growth in vivo
We established a mouse model by transplanting T24 cells ( Figure 6A) to further evaluate the effect of PPARγ on BCa cell growth in vivo. The results suggested a significantly suppressed tumour growth in the GW9662 (1 mg/kg) injection group compared with the vehicle group ( Figure 6B). The cancer tissues dissected from mice were stained with H&E ( Figure 6C). Moreover, the IF results indicated that the positive staining for Ki-67, which is an important marker of cell proliferation, 37 was considerably lower in the GW9662-injected group in comparison to the vehicle group ( Figure 6D).

| D ISCUSS I ON
Our group has revealed a variety of potential biomarkers 32,38,39 based on transcriptome data of normal bladder tissues vs BCa which has been demonstrated to play a crucial role in cancer cell invasion and migration. 52 The association of vimentin and cytokeratin was also revealed in the transitional cell carcinoma genesis of urinary bladder patients, and thus could be a favourable marker in the early diagnosis of transitional BCa. 53 Consistently, in this study, the potential BCa marker vimentin was reduced in BCa T24 cells treated with PPARγ deficiency. The phosphorylation of AKT at Ser9 could inhibit the activity of GSK-3β. 54 Consistently, in our study, we noted that phosphorylated/total AKT was down-regulated and phosphorylated/total GSK3β was up-regulated, which indicated that the alteration of EMT might be regulated via the AKT/GSK3β signalling pathway.
To better understand the effect of PPARγ on BCa, GW9662 was used for subsequent pharmacologic inhibition, a potent and pure antagonist of PPARγ. 31 Recent studies have revealed that GW9662, with or without PPARγ ligands, could be a potential therapeutic strategy targeting glioblastoma stem cells. 55,56 In accordance with the knockdown assays, our results revealed that GW9662 could in- All values are presented as the mean ± SD from at least three independent research results. *P < 0.05, **P < 0.01, ns means no significance as in PPARγ knockdown assay. The reason might be that GW9662 treatment could cause multiple alterations of pathway-related proteins, which could be caused by the drug effect that was different from PPARγ knockdown. And the deep mechanism still needs to be further studied. Interestingly, we found that GW9662 did not mod- BCa and GW9662 has the potential to become a targeted therapeutic strategy against BCa.
F I G U R E 5 GW9662 inhibited the viability and motility of BCa cells. A, To determine the appropriate concentrations of GW9662, the proliferation of UM-UC-3 and T24 cells treated with GW9662 at different concentrations (0, 0.1, 1, 10, 20 and 40 μmol/L) were detected using the methyl thiazolyl tetrazolium assay. The results after treatment for 48 h are shown. B, Alterations of cell survival were detected using the clonogenic forming assay after treatment with GW9662. The colony number in each well was counted and statistically analysed. Cell types and drug concentrations are indicated. C, The migration assay showed a decreased number of migrated T24 and UM-UC-3 cells after treatment with GW9662, as confirmed by the statistical analysis. D, GW9662 treatment with different concentrations inhibited the invasion rate of BCa UM-UC-3 and T24 cells. The scale bar is indicated. E, Western blot analysis of vimentin, slug, MMP2 and MMP9 showed a considerable decrease after GW9662 treatment. GAPDH was used as internal reference. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

ACK N OWLED G EM ENTS
The excellent technical assistance of Yuan Zhu, Xi Tong, Shanshan Zhang and Danni Shan is gratefully acknowledged.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests. F I G U R E 6 GW9662 inhibited BCa growth in vivo. A, BALB/c nude mice were subcutaneously injected with T24 cells and allowed to grow for 10 d. Then GW9662 (1 mg/kg) and vehicle were then intraperitoneally injected every other day for additional 14 d. After the mice were killed, tumour tissues were dissected. B, The tumour volume was calculated before each injection, and statistical analysis was carried out with the t test. The days after transplantation of T24 cells and the tumour size are indicated. *P < 0.05, **P < 0.01. C, Representative haematoxylin and eosin staining of tumour tissues dissected from the tumour-bearing mice. The scale bar is indicated. D, The proliferation of T24 cells treated with GW9662 and vehicle in vivo was measured by immunofluorescence staining of Ki-67 (green). Nuclei were stained with 4',6-diamidino-2phenylindole (DAPI; blue), and the scale bar is indicated