Peroxisome Proliferator-activated Receptor γ Agonists Induce Cell Cycle Arrest through Transcriptional Regulation of Krüppel-like Factor 4 (KLF4)*

Background: PPARγ serves as a master transcriptional regulator of glucose and lipid metabolism, but it also plays an important role in carcinogenesis. Results: Up-regulation of KLF4 upon PPARγ activation is mediated through the PPRE in the KLF4 promoter. Conclusion: KLF4 partly mediates PPARγ-induced cell proliferation inhibition. Significance: This study provides further insights into the PPARγ signal transduction pathway as well as a novel cancer therapeutic strategy. Peroxisome proliferator-activated receptor γ (PPARγ), a subgroup of ligand-activated nuclear receptors, plays critical roles in cell cycle regulation, differentiation, apoptosis, and invasion. PPARγ is involved in tumorigenesis and is a potent target for cancer therapy. PPARγ transactivation of KLF4 has been demonstrated in various studies; however, how PPARγ regulates KLF4 expression is not clear. In this study, we reveal that PPARγ regulates the expression of KLF4 by binding directly to the PPAR response element (PPRE) within the KLF4 promoter. The PPRE resides at −1657 to −1669 bp upstream of the KLF4 ATG codon, which is essential for the transactivation of troglitazone-induced KLF4 expression. Furthermore, we found that stable silencing of KLF4 obviously suppressed the G1/S arrest and anti-proliferation effects induced by PPARγ ligands. Taken together, our data indicate that up-regulation of KLF4 upon PPARγ activation is mediated through the PPRE in the KLF4 promoter, thus providing further insights into the PPARγ signal transduction pathway as well as a novel cancer therapeutic strategy.

The peroxisome proliferator-activated receptors (PPARs) 3 are a subgroup of the ligand-activated nuclear receptor superfamily, and three isoforms have been identified: PPAR␣, PPAR␤, and PPAR␥ (1). Like other members of this superfamily, PPARs mediate transcriptional regulation by binding to their central DNA domain, which recognizes response ele-ments in the promoters of specific target genes (2). The most widely studied form among the three known forms of PPARs is PPAR␥ (3), which is expressed in a wide variety of cell types, including adipocytes, macrophages, and others (4,5). It has been found that PPAR␥ expression is not limited to cells of adipocytic lineage but is also in various other tissues, particularly malignant tissues such as human prostate cancer, gastric cancer, liposarcoma, etc. (6 -8). PPAR subtypes form functional heterodimers with members of the retinoid X receptor (RXR) family of nuclear receptors. The PPAR/RXR heterodimer regulates expression of target genes by binding to the PPAR response elements (PPREs) (9). 15-Deoxy-⌬ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), a metabolite of prostaglandin D 2 , is a potential endogenous ligand for PPAR␥, and thiazolidinediones such as troglitazone (TGZ), rosiglitazone (RGZ), ciglitazone, and pioglitazone (PGZ) are specific exogenous ligands for PPAR␥ (10). PPAR␥ is generally accepted as a master transcriptional regulator of glucose and lipid metabolism and also behaves as a tumor suppressor. Recent studies showed that PPAR␥ activation inhibits cell proliferation (11,12), promotes differentiation (13), induces apoptosis (14), and inhibits angiogenesis (15), leading researchers to postulate that PPAR␥ may play an important role in tumorigenesis (14). However, the molecular mechanisms that mediate anticancer effects of dual PPAR␥ agonists in either a PPAR␥-dependent or PPAR␥-independent manner are not yet clear, and further studies for the identification of a unique PPAR␥ target gene are required.
The Krüppel-like family of zinc finger transcription factors regulates numerous biological processes, including proliferation, differentiation, apoptosis, development, and inflammation. It is reported to have a dual function as a tumor suppressor or an oncogene depending on the types of tissues in which it is expressed. Increased expression of KLF4 was reported in the ductal carcinoma of breast cancer and oral squamous cell carcinomas (16,17), indicating that activation of KLF4 is one of the frequent steps toward carcinogenesis. In contrast, KLF4 is down-regulated in many human cancers, including esophageal, colorectal, bladder, and lung cancer and lymphoma (18 -22). In line with a tumor suppressor function of KLF4, it has been found that KLF4 is down-regulated by promoter hypermethylation and loss of heterozygosity in colorectal cancer, and its overexpression reduces tumorigenesis in colon cancer cells in vivo (19,23). These studies indicate that KLF4 negatively regulates cell cycle progression, but the mechanisms of by which KLF4 affects tumorigenesis in colorectal cancer have yet to be elucidated. It has been shown that some PPAR␥ agonists upregulate the expression of KLF4 in colon cancer cells (24,25). However, the mechanisms by which how PPAR␥ agonists active the expression of KLF4 are still unknown. Bearing in mind growth inhibition of KLF4, it might be interesting to increase KLF4 expression in these cancer cells by activating PPAR␥.
In this study, we demonstrated that KLF4 is a direct transcriptional target of PPAR␥. Computer-aided transcription factor-binding site analysis identified one consensus PPRE at Ϫ1657 to Ϫ1669 bp upstream of the KLF4 ATG codon. Further analysis by ChIP, EMSA, and luciferase reporter assay revealed that PPAR␥ can specifically bind to the PPRE in the promoter region of KLF4, which is required for PPAR␥ to transactivate KLF4. Furthermore, we found that stable silencing of KLF4 obviously suppressed the G 1 /S arrest and anti-proliferation effects induced by PPAR␥ ligands, providing further insights into the PPAR␥ signal transduction pathway as well as a novel cancer therapeutic strategy.

EXPERIMENTAL PROCEDURES
Reagents, Cells Transfection, and RNA Interference-TGZ, RGZ, ciglitazone, PGZ, 15d-PGJ 2 , GW9662, WY14643, and GW0742 were purchased from Cayman Chemical Co. (Ann Arbor, MI). All synthetic ligands were dissolved in dimethyl sulfoxide (DMSO). The final DMSO concentration in the culture medium in all experiments was kept constant at 0.1%. Unless stated otherwise, cells were exposed to these ligands for 24 h. HEK293, HCT116, HT29, LoVo, and HCT15 cells were maintained in DMEM and RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics. siRNAs (Shanghai Gene-Pharma Co., Ltd., Shanghai, China) targeting PPAR␥ (GCC-CTTCACTACTGTTGAC) and a control siRNA (AATTCTC-CGAACGTGTCACGT) were used at 50 nmol/liter. Stable silencing of KLF4 was achieved using the shRNA-based vector with the target sequence GGACGGCTGTGGATGGAAA. Transfections were performed using Lipofectamine 2000 (Invitrogen) for plasmids according to the manufacturer's recommendations.
Plasmid Construction-DNA fragments of the KLF4 and PPAR␥ cDNA coding regions were amplified by PCR and subcloned into the pCDE-HA vector as described previously (26). For generation of the KLF4 promoter-reporters, the KLF4 Ϫ2051/ϩ252 (designated as P1) and Ϫ1597/ϩ252 (designated as P2) sequences were PCR-amplified and subcloned into the pGL3-Basic vector.
RNA Isolation and PCR Analysis-Total RNA was isolated by using TRIzol reagent (Invitrogen), and conventional RT-PCR and RT-quantitative PCR (RT-qPCR) were done using a One- Step RT-PCR kit (Qiagen) and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), respectively. The primers used are listed in Table 1.
Reporter Assay-Luciferase assay was performed using the Dual-Luciferase reporter assay system (Promega) as described (26). HCT116 cells were transfected with pGL3-Basic (control) or KLF4 gene promoter constructs; 24 h after transfection, cells were left untreated or treated with 10 M TGZ for 18 h. Luciferase activity was then determined and normalized to an internal cytomegalovirus-Renilla luciferase control. Each experiment was done in triplicate and repeated at least three times.
EMSA-EMSA was performed using a gel shift assay system (Pierce) according to the manufacturer's protocol. Biotinlabeled and unlabeled wild-type oligonucleotide probes contained the binding motif of PPAR␥ (5Ј-ACGAGTTGTCC-TTTGACCTTTACTGG-3Ј), and unlabeled mutant probes contained a substitution of 2 bp (underlined, 5Ј-ACGAG-TTGTCCTTTGATATTTACTGG-3Ј (designated M1) and 5Ј-ATAAGTTGTCCTTTGACCTTTACTGG-3Ј (designated M2)). Briefly, 3 fmol of labeled oligonucleotides was incubated with nuclear extracts (5 g), which were obtained from HEK293 cells transfected with pcDNA3.1-PPAR␥ and pCDE-HA-RXR␣ using a nuclear extract kit (Pierce) according to the manufacturer's instructions. Competitor oligonucleotides were added at a 100-fold molar excess of the labeled probe except where indicated otherwise. In experiments involving antibodies, 2 g of the anti-PPAR␥ or anti-HA polyclonal antibody was added to the reaction except where indicated otherwise. DNAprotein complexes were resolved on 6% nondenaturing polyacrylamide gel and transferred to a nylon membrane, followed by detection of the biotin-labeled probe by the chemiluminescent nucleic acid detection module (Pierce) according to the manufacturer's instructions. The membranes were exposed to x-ray films to visualize the signal.
Chromatin Immunoprecipitation-HCT116 cells were treated with DMSO or TGZ (10 M) for 24 h and subjected to ChIP with a ChIP assay Kit (Upstate Cell Signaling Solutions, Charlottesville, VA). Briefly, formaldehyde was used to crosslink proteins with DNA, and cells were lysed in SDS lysis buffer.

RT-qPCR primers
The cell lysate was sonicated to shear the DNA. Chromatin samples were precleared with protein G-agarose for 2 h at 4°C and immunoprecipitated overnight at 4°C with normal mouse IgG or anti-PPAR␥ antibody bound to protein G-agarose. Formaldehyde cross-linking was reversed by incubation at 65°C, followed by proteinase K digestion and DNA purification. qPCR was then performed as described above using specific primers. 4 BrdU Incorporation-Proliferation was measured using a BrdU cell proliferation ELISA kit (Roche Applied Science) according to the instructions of the manufacturer with minor modifications. Briefly, after labeling the cells with BrdU for 1 h, coverslips bearing treated cells were washed with PBS, fixed with 3% paraformaldehyde in PBS, permeabilized with 0.25% Triton X-100 in PBS for 10 min, and washed with PBS. Coverslips were then treated with 4 N HCl at room temperature for 30 min to denature the DNA, neutralized with 0.1 M sodium borate at room temperature for 5 min, and washed with PBS. A BrdU antibody was added to the coverslips at 37°C for 1 h. Following washing with PBS, DAPI was added to the coverslips at room temperature for 3 min, after which they were washed with PBS, mounted, and visualized under a fluorescence microscope.
MTS Cell Proliferation Assay-Cells in DMEM containing 10% fatty acid-free BSA were seeded in 96-well plates at a density of 2 ϫ 10 3 cells/well. The cells were then incubated in the absence or presence of 10 M TGZ, which was renewed daily. CellTiter 96 AQ ueous (MTS) One Solution reagent (Promega) was added to each well, and absorbance was recorded at 570 nm using a BioTek ELx800 absorbance microplate reader. 4 Primer sequences are available upon request.

PPAR␥ Agonists Up-regulate KLF4 mRNA and Protein
Expression in HCT116 Cells-To examine whether PPAR␥ influences KLF4 activity, we first analyzed the expression of KLF4 in HCT116 cells treated with TGZ. RT-PCR, RT-qPCR, and Western blot results clearly showed that TGZ induced the expression of KLF4. The expression of KLF4 was detectably increased after TGZ treatment (Fig. 1, A and B). This increase was confirmed at the protein level by immunoblotting experiments (Fig. 1C). We subsequently examined the expression of KLF4 upon treatment with different doses of TGZ ranging from 2 to 20 M, and a dose-dependent stimulation of KLF4 expression by TGZ was observed (Fig. 1D). Additionally, other PPAR␥ agonists (RGZ, PGZ, and 15d-PGJ 2 ) were tested, and increased KLF4 expression was found as well. In contrast, WY14643 (PPAR␣ ligand) and GW0742 (PPAR␤ ligand) did not affect KLF4 expression (Fig. 1E). These results indicate that PPAR␥ agonists can specifically induce KLF4 expression, which is consistent with previous reports (25).
Induction of KLF4 by PPAR␥ Agonists Is PPAR␥-dependent-Previous studies showed that PPAR␥ agonists can alter gene expression in a PPAR␥-dependent and PPAR␥-independent manner. To clarify this, we tested KLF4 expression in wild-type PPAR␥ cell lines HT29 and LoVo and mutant PPAR␥(K422Q) cell line HCT15 after TGZ treatment. RT-qPCR analysis and Western blotting revealed that PPAR␥ activation led to an increase in KLF4 expression in HT29 and LoVo cells, which was consistent with the change in HCT116 cells, but not in HCT15 cells ( Fig. 2A). Additionally, this induction was blocked by cotreatment with the PPAR␥ antagonist GW9662 (Fig. 2B), suggesting that this induction is PPAR␥-dependent. Furthermore, we performed experiments using pCDE-HA-PPAR␥ or pCDE-HA-PPAR␥ siRNA to study KLF4 expression in HCT116 cells. As shown in Fig. 2C, PPAR␥ agonist-induced KLF4 expression was up-regulated upon overexpression of PPAR␥ and down-regulated in the absence of PPAR␥, suggesting partial regulation of KLF4 through PPAR␥ activation. These results indicate that the expression of KLF4 is regulated, at least in part, in a PPAR␥-dependent manner.
KLF4 Is Directly Induced by PPAR␥ Agonists-To determine a direct link between TGZ and KLF4 expression, we performed experiments using cycloheximide to block de novo protein translation to study the expression of KLF4 in HCT116 cells. As shown in Fig. 3A, TGZ-induced KLF4 expression was not influenced by cycloheximide treatment, suggesting that KLF4 is directly induced by TGZ and that this regulation may not  FEBRUARY 8, 2013 • VOLUME 288 • NUMBER 6 require de novo protein synthesis. To explore if the increase in KLF4 mRNA levels triggered by TGZ treatment was linked to post-transcriptional regulation, we measured the half-life of KLF4 mRNA by incubating cells with actinomycin D to block de novo gene transcription. RT-qPCR analysis revealed that the mRNA stability of KLF4 was not influenced by TGZ treatment (Fig. 3B). To further determine whether up-regulation of KLF4 induced by TGZ was transcriptional regulation, we measured KLF4 pre-mRNA and mRNA levels with or without TGZ treatment by RT-qPCR. As shown in Fig. 3C, both KLF4 pre-mRNA and mRNA levels increased after TGZ treatment, suggesting that up-regulation of KLF4 induced by TGZ is the result of transcriptional rather than post-transcriptional events.

Transcriptional Activation of KLF4 by PPAR␥
Up-regulation of KLF4 upon PPAR␥ Activation Is Mediated through the PPRE in the KLF4 Promoter-PPAR␥ regulates gene transcription via binding to PPREs located in the promoter regions of target genes. To further determine the direct regulation of KLF4 by PPAR␥, we searched for putative PPAR␥binding sites in the human KLF4 promoter. Remarkably, we identified one potential PPAR␥-binding site located at Ϫ1657 to Ϫ1669 bp upstream of the KLF4 ATG codon. Moreover, the putative PPRE site is highly conserved among different species (Fig. 4A), suggesting that KLF4 mRNA might be a direct target of PPAR␥. To determine whether the putative PPRE is involved in transactivation, we next measured the KLF4 promoter activity using KLF4 gene promoter-reporter construct P1 (with the putative PPRE) and depletion mutant reporter construct P2 (lacking the putative PPRE) (Fig. 4B). As shown in Fig. 4C (left   panel), a 2-fold increase in P1 was seen with TGZ treatment. In contrast, no significant activation of P2 was observed upon treatment with TGZ. Additionally, we performed cotransfection experiments using KLF4 promoter-reporter construct P1 and pCDE-HA-PPAR␥ or PPAR␥ siRNA; our data revealed that overexpression of PPAR␥ significantly activated P1 and that knockdown of PPAR␥ reduced the promoter activity (Fig. 4C,  right panel). These findings demonstrate unequivocally that PPAR␥ transactivates KLF4 by binding the putative PPRE. To further examine whether PPAR␥ can bind the putative PPRE of KLF4, EMSA was performed using biotinylated probes and pcDNA3.1-PPAR␥ and pCDE-HA-RXR vectors expressing PPAR␥ and HA-tagged RXR protein. The results show that PPAR␥ bound a single-strand probe containing the putative PPRE of KLF4 (Fig. 4D, lane 2) and that excess specific competitor KLF4 PPRE (with the putative PPRE) inhibited formation of the probe-PPAR␥/RXR complex (lane 3), whereas mutant competitor M1 (with the mutant PPRE) failed to inhibit the biotinylated probe binding to PPAR␥ (lane 4). In contrast, competitor M2, which contained a mutation near the PPRE, inhibited the biotinylated probe binding to PPAR␥ (Fig. 4D, lane 5). Furthermore, when anti-PPAR␥ or anti-HA antibody was applied to the probe/protein mixture, the binding was further shifted (Fig. 4D, lanes 6 and 7). These results demonstrate that the putative PPRE of KLF4 represents a bona fide site for the PPAR␥-binding site. PPAR␥ may regulate KLF4 gene transcription in vivo by its binding activity for chromatin. We performed a quantitative ChIP assay using samples with or without TGZ treatment and antibody to PPAR␥. The results show that PPAR␥ specifically bound the promoter region encompassing the PPRE of the KLF4 gene especially after TGZ treatment. In contrast, normal IgG did not precipitate detectable DNA, and the control sequence near the PPRE lost the immunoprecipitation signal (Fig. 4E). This provides additional evidence to support the active role of PPAR␥ in KLF4 gene transcription in vivo.
Stable Silencing of KLF4 Attenuates the Effect of PPAR␥ Agonists on the Modulation of KLF4 Target Genes-To determine whether the PPAR␥-induced KLF4 increase would modify the expression levels of known KLF4 target genes, we measured the expression levels of p21 Waf1 , p27 Kip1 , and cyclins D 1 and E 2 . HCT116 cells stably silenced with KLF4 or control shRNA were used to delineate the role of KLF4 as a mediator of these TGZmodulatable genes. As shown in Fig. 5A, shRNA markedly inhibited KLF4 mRNA expression following TGZ treatment. The response of the KLF4 target genes to TGZ was determined by real-time RT-qPCR and Western blotting in KLF4 or control shRNA-silenced cells. TGZ treatment resulted in an up-regulation of p21 Waf1 and p27 Kip1 but not cyclin D 1 or E 2 in the control shRNA-silenced cells (Fig. 5, B-D, and supplemental  Fig. S1). Ablation of KLF4 expression significantly muted the effect of TGZ on the expression of these genes.

Ablation of KLF4 Expression Reduces PPAR␥ Agonist-mediated G 1 /S Arrest and Anti-proliferation Effects in HCT116
Cells-In an effort to evaluate the biological significance of KLF4 up-regulation by TGZ, we assessed the response of the stable silencing of KLF4 in HCT116 cells to TGZ-mediated cell proliferation inhibition. The growth curves for control and KLF4 shRNA-silenced cells treated with or without TGZ were compared. Ablation of KLF4 expression reduced PPAR␥ agonist-mediated cell proliferation arrest (Fig. 6A) but had very subtle effects on long-term cell growth arrest both in vitro and in vivo (supplemental Figs. S2 and S3). To clarify the mechanisms underlying cell proliferation inhibition, BrdU incorporation and cell cycle analysis were conducted at 48 h post-TGZ treatment. TGZ treatment inhibited DNA synthesis by Ͼ50% in the control shRNA-silenced cells as opposed to 20% in the KLF4 shRNA-silenced cells (Fig. 6B and supplemental Fig. S4). The difference is statistically significant (p Ͻ 0.05). Additionally, as shown in Fig. 6C, TGZ-induced G 1 /S arrest was mark-edly muted as a result of KLF4 ablation. Taken together with the KLF4 silencing data, our results show the important role of KLF4 up-regulation in mediating the effect of TGZ on cell proliferation inhibition.

DISCUSSION
PPAR␥ is known as a master transcriptional regulator of glucose and lipid metabolism, but it also plays an important role in carcinogenesis. This receptor has the ability to bind a variety of small lipophilic compounds. In turn, these ligands direct cofactor recruitment to PPAR␥, regulating the transcription of genes in a variety of complex metabolic pathways. Indeed, PPAR␥ ligands such as thiazolidinediones (RGZ, TGZ, and PGZ), which are commonly used in the clinical setting as anti-diabetic medications, are potent and selective activators of PPAR␥. The role of thiazolidinediones in growth of cancer cells has been elucidated in some studies. In a phase II study of the use of TGZ in the treatment of patients with advanced breast cancer, no objective tumor response was observed (27). However, the study was incomplete. On the other hand, it is important to note that neither hormone status of the tumors nor the amount of PPAR␥ protein was assessed before patients were included in the study. In contrast, some studies have suggested that PPAR␥ ligands inhibit growth of malignant human cells; cause cell cycle arrest and apoptosis in a broad spectrum of tumor cell lines; and can be used as chemopreventive agents for colon, breast, and prostate carcinogenesis (28). Another study showed that up-regulation of p27 and p21 plays a role in the regulation of TGZ-induced G 1 arrest in colon cancer cell lines (29). However, up to now, no functional PPREs were definitively identified in the p27 Kip1 and p21 Waf1 promoter regions, which means that the regulation of p27 and p21 by PPAR␥ is indirect, involving other factors.
Previous studies showed that some PPAR␥ agonists induce KLF4 expression in a receptor-dependent manner in colorectal cancer cells (24,25). In line with this, we have shown that TGZ specifically increased KLF4 mRNA and protein levels and that this regulation is direct because the PPAR␥-induced expression of KLF4 was not influenced by co-incubation with cyclohexi-mide, suggesting that this regulation does not require de novo protein synthesis. Additionally, our data show that the mRNA stability of KLF4 was not influenced after TGZ treatment by incubating cells with actinomycin D. Furthermore, both KLF4 pre-mRNA and mRNA were induced by TGZ treatment, indicating that the up-regulation of KLF4 induced by TGZ is at the transcriptional level rather than the post-transcriptional level. In contrast, Chen and Tseng (30) concluded that 15d-PGJ 2 upregulates KLF4 expression independently of PPAR␥ through activation of the MAPK signaling pathway in HT29 colon cancer cells. The differences we have confirmed in ligand-receptor activity may provide an explanation for differences of action in different colorectal cancer cells. Thus, we have shown that TGZ-induced KLF4 expression was abolished in the presence of GW9662, a PPAR␥-specific antagonist, suggesting the PPAR␥-dependent manner of TGZ-induced KLF4 expression. Remarkably, we identified one functional PPAR␥-binding site with computer-assisted transcription factor-binding site analysis. This site is located at Ϫ1657 to Ϫ1669 bp upstream of the KLF4 ATG codon and is highly conserved among different species. The specificity of the KLF4-binding sites was also confirmed by EMSA and ChIP assay, as shown in Fig. 4 (D and E,  respectively). Additionally, our data indicate that PPAR␥ binds to its PPRE as a heterodimer with RXR. Upon binding a ligand, the conformation of PPAR␥ is altered and stabilized such that a binding cleft is created, and recruitment of transcriptional coactivators occurs. It has been reported that activation of the PPAR␥/RXR heterodimer can generate a synergistic response in inhibiting cell growth in colon cancer cell lines (31). Therefore, further work will be necessary to determine whether activation of the PPAR␥/RXR heterodimer generates a synergistic response in upon KLF4 expression. Furthermore, luciferase assay demonstrated unequivocally that PPAR␥ transactivates KLF4 by binding the putative PPRE (Fig. 4C).
Notably, the biological function of KLF4 depends on the genetic context, and many studies have shown that the expression of KLF4 is associated with both inhibition and induction of cell proliferation, affecting tumorigenesis both positively and negatively (23,32,33). However, recent studies showed that KLF4 is down-regulated during tumorigenesis of the gastrointestinal epithelium and is frequently lost in other human cancer types. Consistent with a tumor suppressor function of KLF4, its overexpression reduces tumorigenesis in colonic cancer cells. Although KLF4 may play a protumorigenic role in other cancer types (16), our data provide evidence that KLF4 has a role as a tumor suppressor in colorectal cancer. In this study, we have presented three lines of evidence to support the role of KLF4 in mediating the effect of TGZ on inhibition of colorectal cancer cell proliferation. First, TGZ treatment led to a direct induction of KLF4 expression. Second, stable silencing of KLF4 by shRNA significantly diminished the responsiveness to TGZ with respect to the expression of KLF4 target genes p21 Waf1 and p27 Kip1 . Third, ablation of KLF4 expression reduced TGZ-mediated cell proliferation arrest in HCT116 cells. Our preliminary findings indicate that knockdown of PPAR␥ resulted in down-regulation of KLF4 and increased cell growth (supplemental Fig. S5). Furthermore, we found that TGZ-induced anti-proliferation effects were markedly muted as a result of KLF4 ablation (Fig. 6, A-C). However, it is important to know that KLF4 is one of several transcription factors whose expression is known to be modulated by TGZ treatment. Thus, it is not surprising to find that silencing KLF4 alone may not completely block the effect of TGZ on cell proliferation arrest. Further work will be necessary to identify additional genes involved in this network.
In conclusion, our data clearly demonstrate for the first time that PPAR␥ regulates the expression of KLF4 by binding directly to the PPRE within the KLF4 promoter, leading to activation of a tumor suppressor network in colorectal cancer. Furthermore, our results provide a novel cancer therapeutic strategy and will help define the mechanisms by FIGURE 6. Ablation of KLF4 expression reduces PPAR␥ agonist-mediated G 1 /S arrest and anti-proliferation effects in HCT116 cells. A, HCT116 cells stably silenced with control (sh-ctr) or KLF4 (sh-KLF4) shRNA were incubated with DMSO (control) or TGZ (10 M) for the indicated times, and cell growth inhibition was detected using MTS assay. Values were the mean Ϯ S.D. of absorbance at 570 nm for three independent experiments. B, HCT116 cells stably silenced with control or KLF4 shRNA were left untreated (control (Ctr)) or treated with 10 M TGZ for 48 h and then analyzed by BrdU incorporation using a BrdU cell proliferation ELISA kit. Similar results were observed in a triplicate analysis. *, p Ͻ 0.05. C, cell cycle analysis was conducted at 48 h post-TGZ treatment in control and KLF4 shRNA-silenced HCT116 cells. The percentage of the G 1 phase cells was determined and is shown in a graph. Similar results were observed in a triplicate analysis. *, p Ͻ 0.05.