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[Cancer Research 62, 3282-3288, June 1, 2002]
© 2002 American Association for Cancer Research

Molecular Biology and Genetics

Peroxisome Proliferator-activated Receptors Modulate K-Ras-mediated Transformation of Intestinal Epithelial Cells1

Jinyi Shao, Hongmiao Sheng and Raymond N. DuBois2

Departments of Medicine and Cell Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Department of Veterans Affairs Medical Center Nashville, Tennessee 37232


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of peroxisome proliferator-activated receptors (PPARs) exerts diverse effects on neoplastic cells. Recent work has shown that PPAR{delta} is up-regulated after loss of adenomatous polyposis coli tumor suppressor gene function and that transcriptional activation of the PPAR{gamma} nuclear receptor can lead to inhibition of carcinoma growth. In this study, we elucidate the regulation and functional importance of PPAR{gamma} and {delta} after K-Ras-transformation of intestinal epithelial cells. In conditionally K-Ras-transformed rat intestinal epithelial cells (IEC-iK-Ras), the level and activity of PPAR{delta} were markedly increased. PPAR{delta} up-regulation occurred due to increased mitogen-activated protein kinase activity and receptor activation required the endogenous production of prostacyclin via the cyclooxygenase-2 pathway. We also demonstrate that activation of the PPAR{gamma} nuclear receptor has antineoplastic effects in Ras-transformed cells. Activation of PPAR{gamma} resulted in a delay in transit through the G1 phase of the cell cycle that was associated with inhibition of phosphatidylinositol 3'-kinase/Akt activity and a reduction of cyclin D1 expression. Therefore, these two PPAR nuclear receptors, which are structurally related, have distinct roles during neoplastic transformation. PPAR{gamma} appears to modulate differentiation and signal growth inhibition, whereas PPAR{delta} is up-regulated by oncogenic Ras and activated by cyclooxygenase-2-derived prostaglandins.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PPARs3 are members of the nuclear receptor superfamily of ligand-activated transcription factors and were initially discovered as modulators of peroxisome proliferation (1) . Three isoforms of PPARs have been cloned in mammalian cells and are referred to as PPAR{alpha}, PPAR{gamma}, and PPAR{delta}. Acting in a similar fashion to other nuclear hormone receptors, ligand-activated PPARs can form heterodimers with the retinoid X receptor and are bound to PPREs in the promoter region of target genes (reviewed in Ref. 2 ). PPAR{alpha} is highly expressed in liver and critically involved in peroxisome proliferator-induced hepatocarcinogenesis in rodent models (reviewed in Ref. 3 ). PPAR{gamma} induces adipocyte differentiation (reviewed in Ref. 4 ) and has effects on the differentiation of cultured myelomonocytes and other cells (5) . PPAR{gamma} can be activated by a number of endogenous ligands that are bioactive lipid compounds, such as 15-deoxy {Delta}12,14-PGJ2, polyunsaturated fatty acids, and oxidized forms of linoleic acid (6, 7, 8) . Many chemical agents, including the thiazolidinediones, rosiglitazone (BRL-49653), and pioglitazone, can also bind and activate PPAR{gamma} (6 , 7 , 9, 10, 11) . Recently, PPAR{gamma} ligands were shown to inhibit the growth of a variety of transformed cells. Activation of PPAR{gamma} induces terminal differentiation in human liposarcoma cells (12) and breast cancer cells (13) . PPAR{gamma} ligands inhibit growth and induce apoptosis of human breast cancer (14) and prostate carcinoma cells (15) . Sarraf et al. (16) found that PPAR{gamma} induces differentiation and reverses the malignant phenotype in colon carcinoma cells. We reported that a selective PPAR{gamma} activator, BRL-49653, inhibits the growth of human colon cancer cells by inducing a significant delay in the G1 phase of the cell cycle (17) .

PPAR{delta} is highly expressed in the gut (18 , 19) and regulated by the APC/ß-catenin/TCF-4 pathway (20) . After induction of wild-type APC, the expression of PPAR{delta} decreases in colon cancer cells harboring a mutated APC gene. PPAR{delta} expression is increased in colorectal cancer, and its mRNA level is markedly elevated in primary human colorectal adenocarcinomas (19 , 20) . However, a recent study (21) reported that PPAR{delta} is dispensable for polyp formation in the intestine and colon of APCmin mice; however, disruption of PPAR{delta} gene appears to reduce the size of polyps, suggesting that the exact role of PPAR{delta} in colorectal neoplasm is unclear.

Ras mutations are found in a wide variety of human malignancies (22) . Oncogenic mutations in Ras result in activation of downstream signaling proteins, including Raf/MEK/ERK (23 , 24) and PI3K/protein kinase B (Akt; Refs. 25 and 26 ). A specific subset of Ras-targeted genes is subsequently modulated that results in oncogenic transformation (27, 28, 29, 30) . In the present study, we use a conditionally K-Ras-transformed intestinal epithelial cell line (IEC-iK-Ras) to evaluate the regulation and biological effects of PPARs after induction of K-RasVal12 expression. We are the first group to show that PPAR{delta} expression is increased after Ras activation and a PPAR{gamma} agonist inhibits K-Ras-mediated transformation, suggesting that different PPAR isoforms play distinct roles in carcinogenesis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
Rat intestinal epithelial (IEC-6) cells were obtained from American Type Culture Collection (Rockville, MD). An IEC-iK-Ras cell line with inducible activated K-RasVal12 was generated by using LacSwitch eukaryotic expression system (Stratagene, La Jolla, CA) and described elsewhere (31) . The K-RasVal12 cDNA is under the transcriptional control of the Lac operon. IPTG (Life Technologies, Inc., Gaithersburg, MD) at a concentration of 5 mM was used to induce the expression of mutated K-Ras. BRL-49653 was a gift of Glaxo-Smith-Kline (Research Triangle Park, NC). PD-98059 and UO-126 were purchased from Calbiochem (San Diego, CA). Celecoxib is a gift from G. D. Searle & Co. (St. Louis, MO). Rofecoxib is a gift from Merck & Co. (Whitehouse Station, NJ).

RNA Extraction and Northern Blot Analysis.
Total cellular RNA from vehicle or IPTG-treated IEC-iK-Ras cells was extracted according to the method described previously (32) . RNA samples were separated on formaldehyde-agarose gels and blotted onto nitrocellulose membranes. The blots were hybridized with cDNA probes labeled with [{alpha}-32P]dCTP by random primer extension. After hybridization and washes, the blots were then exposed to X-ray film for autoradiography. 18S rRNA signals were used as internal control to determine the integrity of RNA and equality of loading among lanes.

Immunoblot Analysis and Antibodies.
Immunoblot analysis was performed as described previously (32) . The cells were lysed for 30 min in radioimmunoprecipitation assay buffer (1 x PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 1 mM sodium orthovanadate), and then clarified cell lysates were denatured and fractionated by SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose membranes. The filters were then probed with the antibodies indicated, developed by the enhanced chemiluminescence system (Amersham, Arlington Heights, IL). The antipan Ras antibody was purchased from Calbiochem (La Jolla, CA), and the anti-Cdk4, anti-PPAR{delta}, and anti-PTEN antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antiphosphorylated Akt antibody was obtained from Cell Signaling Technology (Beverly, MA). Anticyclin D1 antibody was purchased from Upstate Biotechnology (Lake Placid, NY).

Immunofluorescence.
IEC-iK-Ras cells were grown in 35-mm tissue culture plates and treated with vehicle or IPTG for 48 h. The cells were fixed in methanol/acetone at -20°C for 10 min. Fixed cells were incubated with anti-PPAR{delta} antibody (Santa Cruz Biotechnology) for 2 h at room temperature. After washing the cells, they were incubated with Cy3-conjugated donkey antigoat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for an additional hour. The cells were then washed with PBS, mounted, and observed under fluorescent microscopy with appropriate filters.

Quantitation of Eicosanoids.
Subconfluent IEC-iK-Ras cell cultures in serum containing medium were established. The cells were treated with IPTG or IPTG plus 3 µM celecoxib for 24 h. PGI2 formation was quantified by measuring 6-keto-PGF1{alpha} using stable isotope dilution techniques using gas chromatography negative ion chemical ionization mass spectrometry. The results are expressed as ng of PGI2/ml of medium.

Flow Cytometry.
IEC-iK-Ras cells were seeded into 100-mm plates and treated with 5 mM IPTG or IPTG plus 20 µM BRL-49653 for 72 h. Cells were fixed in 70% ethanol, digested in 1 ml of 0.1% RNase (Sigma Chemical Co., St, Louis, MO), and stained with propidium iodide (Sigma Chemical Co.). The DNA was analyzed by flow cytometry. The cell cycle profile was expressed as a percentage of cells in each stage of the cell cycle.

Soft Agarose Assay.
Cells (1 x 104) were mixed with Sea plaque agarose (Hoeffer, Rockland ME) at a final concentration of 0.4% in DMEM medium with the applied treatments and overlaid onto a 0.8% agarose layer in 35-mm plates. The plates were incubated for 10 days, and then colonies were photographed by using an inverted microscope (9 fields/plate). The size of colonies was measured manually; the data plotted represent the mean ± SE of assays performed in triplicate (27 fields).

Transfection of Reporter Constructs.
PPAR activity was assayed by transient transfection with PPRE3-tk-luciferase construct as described previously (17) . Cells were plated in 24-well plates 24 h before transfection and then cotransfected with 0.5 µg of PPRE3-tk-luciferase and 0.2 µg of pRL-TK plasmid (Promega, Madison WI), using the Lipofectin procedure (Life Technologies, Inc.). After 48-h incubation, the cells were lysed with passive lysis buffer and used for both the firefly and renilla luciferase readings (Promega). Firefly luciferase values were standardized to renilla values.

Stable Transfection.
To establish the IEC:iK-Ras/PPRE-luc cell line, IEC-iK-Ras cells were cotransfected with PPRE3-tk-luciferase plasmid and pZeoSV2(+) vector. Stably transfected clones were selected with 400 µg/ml G418, 150 µg/ml hygromycin B, and zeocin (250 µg/ml) and are referred to as the IEC:iK-Ras/PPRE-luc cell line.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of PPARs in IEC-iK-Ras Cells.
PPAR{gamma} and {delta} are expressed in normal colonic mucosa and colorectal carcinomas (16 , 18 , 19) ; however, their function in the normal intestine is unknown. IEC cells were derived originally from rat intestinal crypt cells, and the expression or function of PPARs in these cells has not been evaluated previously. As demonstrated in Fig. 1ACitation , all three subtypes of PPARs are expressed at relatively low levels in nontransformed IEC-iK-Ras cells as determined by Northern blot analysis. Induction of K-Ras by IPTG led to a marked increase in the levels of PPAR{delta} but only a slight change in PPAR{alpha} (~2-fold). A 7-fold induction of PPAR{delta} mRNA was observed at 24 h after the addition of IPTG (Fig. 1A)Citation , indicating that expression of this PPAR isoform is highly regulated after Ras activation. However, the expression of PPAR{gamma} was not affected significantly by Ras activation. Western blot analysis revealed that the temporal changes in PPAR{delta} protein levels correlated closely with the induction of Ras expression (Fig. 1B)Citation . Immunofluorescent staining demonstrated that the PPAR{delta} immunoreactivity was very low in uninduced IEC-iK-Ras cells (Fig. 1CCitation , left panel). In contrast, abundant PPAR{delta} protein was detected in the nuclei of Ras-transformed IEC-iK-Ras cells (Fig. 1Citation C, right panel). To determine whether Ras-induction was associated with increased PPAR-driven transcriptional activity, we transiently transfected the IEC-iK-Ras cells with the PPRE3-tk-luciferase reporter vector and then treated the cells with vehicle or 5 mM IPTG. Induction of K-Ras resulted in a 7–10-fold increase in the renilla-normalized firefly luciferase activity (Fig. 1DCitation , left panel). To demonstrate the kinetics of the induction of PPRE activity by Ras, we stably transfected IEC-iK-Ras cells with the PPRE3-tk-luciferase reporter construct (IEC-iK-Ras/PPRE-luc cell). IPTG treatment led increased levels of oncogenic Ras and a significant increase in PPRE3-driven luciferase activity within 8 h, which was elevated by 5-fold at 24 h (Fig. 1DCitation , right panel). These results suggest that Ras-induced PPARs, predominantly the {delta} isoform, are functionally active and can drive transcription off the specific PPRE cis elements.



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  		Fig. 1. Expression of PPARs in IEC-iK-Ras cells. A, Northern analysis of PPARs. IEC-iK-Ras cells were treated with IPTG for the indicated times. Total cellular RNA was separated, blotted, and hybridized with PPAR cDNA probes. 18S RNA was used as a loading control. The results shown are representative of three separate experiments. B, Western blot analysis for the expression of PPAR{delta}. Cell lysates were denatured and fractionated by SDS-PAGE. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes. The filters were then probed with anti-PPAR{delta} antibody and developed by using the enhanced chemiluminescence system. The results shown are representative of three separate experiments. C, immunofluorescence staining for PPAR{delta}. IEC-iK-Ras cells were treated with vehicle (CTR) or 5 mM IPTG for 48 h. The cells were fixed by methanol/acetone and incubated with 10% normal donkey serum for 1 h and then with anti-PPAR{delta} antibody for 2 h at room temperature. After the cells were washed with PBS, they were incubated with CY3-conjugated donkey antigoat IgG for an additional hour. The cells were washed with PBS, mounted, and observed under a fluorescent microscope. D, PPRE activity in IEC-iK-Ras cells. IEC-iK-Ras cells were transiently (left panel) or stably (right panel) transfected with PPRE3-tk-luciferase expression vector. Transfected cells were treated with 5 mM IPTG, and cell lysates were used for the luciferase readings. The mean ± SE of assays performed in quadruplicate are plotted. For transient transfection, the firefly luciferase activity was normalized to the renilla luciferase activity. The results shown are representative of three separate experiments.

 
Activation of PPAR{delta} Requires MAP Kinase Activity and Endogenous PGI2.
Transformation by activated Ras involves a phosphorylation cascade that leads to activation of multiple signaling pathways, including the Raf/MEK/ERK pathway. PD-98059 has been shown to specifically inhibit the activation of MEK in vitro and in vivo (33 , 34) . It was of interest to determine whether the Ras-induced expression and activation of PPAR{delta} was associated with signaling via MEK/ERK. Treatment with PD-98059 (50 µM) completely blocked the Ras-induced expression of PPAR{delta} mRNA and protein (Fig. 2, A and B)Citation . Treatment with a structurally different MEK inhibitor, UO-126, confirmed that MEK/ERK activity is required for Ras induction of PPAR{delta} expression. As expected, the presence of PD-98059 or UO-126 also abolished the Ras induced PPRE activity in IEC-iK-Ras/PPRE-luc cells as determined by luciferase assays (Fig. 2C)Citation .



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  		Fig. 2. MAP kinase pathway and the expression of PPAR{delta}. A, inhibition of PPAR{delta} expression by MEK inhibitors. IEC-iK-Ras cells were treated with 5 mM IPTG in the presence or absence of MEK inhibitors (PD = 50 µM PD-98059 and UO = 10 µM UO-126) for 24 h. Total RNA and protein were extracted, and levels of PPAR{delta} were analyzed by Northern and Western blotting. B, inhibition of PPAR{delta} expression by PD-98059. IEC-iK-Ras cells were treated with 5 mM IPTG in the presence or absence of PD-98059 (PD). Cell lysates were collected at the indicated time points for analysis of PPAR{delta} protein. C, inhibition of PPRE activity by MEK inhibitors. IEC-iK-Ras/PPRE-luc cells were treated with 5 mM IPTG in the presence or absence of MEK inhibitors (PD = 50 µM PD-98059 and UO = 10 µM UO-126) for 24 h. Cell lysates were analyzed for firefly luciferase activity. The mean ± SE of assays performed in quadruplicate are plotted. The results shown are representative of three separate experiments.

 
We have demonstrated that expression of K-Ras strongly induces COX-2 expression in IEC-iK-Ras cells (31) . As a result, the level of PGI2, which is the predominant prostaglandin produced by these cells, is increased 11-fold after Ras induction (Fig. 3A)Citation . Treatment with 3 µM celecoxib completely blocked the production of PGI2 via selective inhibition of COX-2 activity. Endogenous PGI2 has been shown to activate PPAR{delta}-driven transcriptional activity in mammalian cells (19) . To determine whether Ras-induced PPAR{delta} activity involves ligand activation by PGI2, COX-2 activity was inhibited selectively by the addition of 3 µM or 10 µM celecoxib, whereas K-Ras was induced in IEC-iK-Ras cells that were transiently transfected with PPRE-tk-luc vector. Interestingly, the addition of celecoxib significantly reduced the PPRE activity (~50%, P < 0.05), and the presence of a stable PGI2 analogue (1 µM carboprostacyclin) almost completely reversed the inhibitory effect of celecoxib (Fig. 3B)Citation . A structurally different COX-2 inhibitor, rofecoxib, also inhibited the Ras-induced PPRE activity, and this too was reversed by carboprostacyclin treatment (Fig. 3C)Citation . Celecoxib and rofecoxib treatment inhibited the Ras-induced PPRE activity without affecting the regulation of PPAR{delta} expression. As shown in Fig. 3DCitation , PPAR{delta} protein levels were not altered by celecoxib or rofecoxib treatment, indicating that lack of COX-2-derived PGI2 was most likely responsible for decreased PPAR-driven transcriptional activity.



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  		Fig. 3. Role of PGI2 in PPRE activity. A, PGI2 production in IEC-iK-Ras cells. Subconfluent IEC-iK-Ras cell cultures were treated with IPTG in the presence or absence of 3 µM celecoxib (Ce) for 24 h. The PGI2 formation in medium was quantified by measuring 6-keto-PGF1{alpha} using stable isotope dilution techniques using gas chromatography negative ion chemical ionization mass spectrometry. The results are expressed as ng of PGI2/ml medium. B, PPRE activity and inhibition of PGI2 production. IEC-iK-Ras cells were transiently transfected with PPRE3-tk-luciferase vector and treated with vehicle or celecoxib (Ce) for 16 h before the addition of IPTG or IPTG plus 1 µM carboprostacyclin treatments. After cells were continuously incubated for 28 h, cell lysates were used for luciferase readings. The mean ± SE of assays performed in six wells are plotted. The results were similar in three separate experiments. C, PPRE activity and inhibition of PGI2 production. IEC-iK-Ras cells were transfected transiently with PPRE3-tk-luciferase vector and treated with vehicle or rofecoxib (Vi) for 16 h before the addition of IPTG or IPTG plus 1 µM carboprostacyclin treatment. After cells were continuously incubated for 28 h, cell lysates were evaluated for luciferase activity. The mean ± SE of assays performed in six wells are plotted. The results were similar in three separate experiments.

 
Inhibition of K-Ras Transformation by BRL-49653.
Currently, the role of PPAR{gamma} agonists in colorectal cancer is confusing (16 , 35 , 36) . Treatment with PPAR{gamma} agonists promotes adenoma formation in multiple intestinal neoplasia mice but inhibits the growth of xenografted human colonic tumors in nude mice. Given the importance of K-Ras in colorectal carcinogenesis and the controversy surrounding this area of research, determination of the effects of PPAR{gamma} agonists on Ras-mediated transformation was of great interest. IEC-iK-Ras cells displayed a nontransformed appearance (Fig. 4ACitation , panel a), and the addition of 5 mM IPTG resulted in increased Ras expression and morphological transformation of the IEC-iK-Ras cells observed between 48 and 72 h after IPTG treatment (Fig. 4ACitation , panel b; Ref. 31 ). To determine whether PPAR{gamma} agonists affect K-Ras-mediated transformation, IEC-iK-Ras cells were treated with IPTG in the presence of a specific ligand for PPAR{gamma}, rosiglitazone (BRL-49653). Treatment with 20 µM BRL-49653 clearly inhibited Ras-mediated transformation as indicated by a reduction in cell density and a partial restoration of cell-cell contact inhibition (Fig. 4A, panel cCitation ).



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  		Fig. 4. Activation of PPAR{gamma} and K-Ras transformation. A, morphology of IEC-iK-Ras cells. IEC-iK-Ras cells were grown on 60-mm tissue culture dishes and then treated with vehicle (a), 5 mM IPTG (b), and IPTG plus 20 µM BRL-49653 (c) for 4 days. The pictures were taken by using an inverted microscope (original magnification, x100). B, PPRE activity in uninduced IEC-iK-Ras cells. PPAR activity was assayed after transient transfection with PPRE3-tk-luciferase construct and pRL-TK plasmid. Transfected cells were treated with BRL-49653 for 24 h, and firefly and renilla luciferase activities were measured. Firefly luciferase values were standardized to renilla values and plotted as the mean ± SE of assays performed in quadruplicate. The results shown are representative of three separate experiments. C, activation of PPRE in Ras-transformed IEC-iK-Ras cells. IEC-iK-Ras cells were treated with IPTG for 72 h before transfection, and PPAR activity was assayed by transient transfection with PPRE3-tk-luciferase construct and pRL-TK plasmid. Transfected cells were treated with IPTG or IPTG plus BRL-49653 for 24 h, and firefly and renilla luciferase activities were measured. Firefly luciferase values were standardized to renilla values. The data are plotted as the mean ± SE of assays performed in quadruplicate, and the results shown are representative of three separate experiments.

 
To evaluate the activation of PPAR{gamma} by BRL-49653, we first transfected IEC-iK-Ras cells with PPRE3-tk-luciferase reporter and then treated the cells with a PPAR{gamma} agonist, BRL-49653. BRL-49653 did not induce PPRE activity in nontransformed IEC-iK-Ras cells (Fig. 4B)Citation . In contrast, BRL-49653 stimulated the PPRE activity in K-Ras-transformed IEC-iK-Ras cells in a concentration-dependent manner. A 3-fold increase in luciferase activity was observed after the cells were treated with 20 µM BRL-49653 for 24 h (Fig. 4C)Citation .

Growth Inhibition of K-Ras-transformed IEC Cells by BRL-49653.
We next evaluated the mechanism(s) by which BRL-49653 inhibited Ras transformation of intestinal epithelial cells. As demonstrated in Fig. 5ACitation , BRL-49653 also inhibited the growth of transformed IEC-iK-Ras cells in a concentration-dependent manner. Treatment with 10 µM BRL-49653 for 8 days reduced the cell number by 55.3%, and 20 µM BRL-49653 inhibited cell growth by 81.9%. Flow cytometric analysis indicated that treatment with BRL-49653 resulted in the accumulation of cells in the G1 phase of the cell cycle and inhibition of S phase entry of IEC-iK-Ras cells (Fig. 5B)Citation .



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  		Fig. 5. Growth inhibition by BRL-49653 in IEC-iK-Ras cells. A, demonstration of growth curves of IEC-iK-Ras cells. For the cell growth studies, 2 x 104 cells were seeded into 12-well plates. Media containing 5 mM IPTG and BRL-49653 were replaced daily. Cell numbers were counted at the indicated days, and values are mean ± SE from triplicate wells. This experiment was repeated three times. B, flow cytometric analysis. IEC-iK-Ras cells were treated with 5 mM IPTG or IPTG plus 20 µM BRL-49653 for 72 h. Cells were fixed in 70% ethanol, digested in 0.1% RNase, and stained with propidium iodide. The DNA was analyzed by flow cytometry, and the percentages of cells in G0-G1 and S phase are plotted. C, anchorage-independent growth. Cells (1 x 104) were mixed with Sea plaque agarose at a final concentration of 0.4% in DMEM medium containing vehicle or IPTG and overlaid onto a 0.8% agarose layer in 35-mm plates. The plates were incubated for 10 days, and then the colonies were photographed by using an inverted microscope (9 fields/plate). The size of colonies was measured manually, and the data plotted represent the mean ± SE of assays performed in triplicate (27 fields).

 
Anchorage-independent growth is an important phenotype associated with cell transformation. Nontransformed IEC-iK-Ras cells did not form colonies in soft agarose, whereas they did so after treatment with IPTG, which led to the induction of RasVal12 expression (31) . To determine whether the treatment with BRL-49653 impaired the anchorage-independent growth of IEC-iK-Ras cells, we added both IPTG and BRL-49653 to cells grown in soft agarose. The presence of BRL-49653 led to a slightly lower number of colonies (data not shown) but significantly reduced the size of transformed IEC-iK-Ras colonies (Fig. 5C)Citation .

Inhibition of Ras Signaling by BRL-49653.
Ras-mediated transformation of intestinal epithelial cells requires the activation of its downstream effectors. We reported recently that Ras-induced transformation of rat intestinal epithelial cells requires activation of the PI3K/protein kinase B (PI3K/Akt) pathway (37) . Interestingly, here we found that the PI3K/Akt pathway was also regulated by PPAR{gamma}. Induction of K-Ras increased the activity of Akt/PKB in IEC-iK-Ras cells. Western analysis showed that induction of K-Ras increased in the levels of the activated form of Akt/PKB (phosphorylated at serine 473) in IEC-iK-Ras cells that was blocked by BRL-49653 (Fig. 6A)Citation . Treatment with BRL-49653 resulted in a concentration-dependent reduction of the activation of Akt/PKB, and the addition of 20 µM BRL-49653 completely blocked the K-Ras-induced phosphorylation of Akt/PKB, whereas the expression of PTEN was not significantly altered by BRL-49653 treatment (Fig. 6B)Citation . Induction of mutated K-Ras modulated the expression of a number of genes, among them cyclin D1, a Ras target gene, whose expression greatly depends on PI3K/Akt activity (37, 38, 39, 40) . As demonstrated in Fig. 6Citation C, induction of K-Ras by the treatment with IPTG resulted in increased levels of cyclin D1 protein. Treatment with BRL-49653 inhibited the Ras-induced elevation of cyclin D1 levels in a concentration-dependent manner (Fig. 6D)Citation .



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  		Fig. 6. Inhibition of Akt/PKB by BRL-49653. A, K-Ras induction of Akt/PKB. IEC-iK-Ras cells were treated with IPTG or IPTG plus 20 µM BRL-49653 and were lysed at the time points indicated. The levels of phosphorylated Akt were determined by Western blot analysis. B, BRL-49653 inhibition of the phosphorylation of Akt/PKB. IEC-iK-Ras cells were treated with IPTG and indicated concentrations of BRL-49653 for 72 h. Levels of active-Akt and PTEN were determined using specific antibodies. C, induction of cyclin D1 protein by Ras. IEC-iK-Ras cells were treated with vehicle or IPTG, and cell lysates were collected at the indicated time points. The levels of cyclin D1 protein were determined by Western blot analysis. D, PPAR{gamma} inhibition of cyclin D1 expression. IEC-iK-Ras cells were treated with IPTG and BRL-49653 for 48 h. The levels of cyclin D1 were determined by Western blot analysis.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In addition to regulating lipid metabolism, PPARs have been implicated in a variety of biological processes, including tumor biology (reviewed in Ref. 41 ). Different PPAR isoforms appear to play distinct roles in tumorigenesis, and the PPAR{gamma} isoform may exert different effects on colonic tumors depending on the animal model being evaluated (16 , 35 , 36) . These findings suggest that the functional role of PPARs in tumorigenesis may depend on the type and stage of the neoplasm. PPAR{delta} is known to be a downstream target gene of APC/ß-catenin/TCF pathway. Mutations in the APC gene result in ß-catenin-mediated activation of TCF/lymphocyte enhance factor transcription factors that are believed to be involved in the majority of sporadic colorectal cancers (reviewed in Ref. 42 ). Our results show, for the first time, that PPAR{delta} is also a Ras target gene whose expression is controlled by the oncogenic Raf/MEK/ERK pathway. Because the K-Ras oncogene plays a key role during the adenoma-to-carcinoma sequence of events involved in the neoplastic transformation of colonic epithelial cells (42) , our data suggest the potential involvement of PPAR{delta} in the early stages of colorectal carcinogenesis.

IEC-iK-Ras cells provide a good model for determination of the role of endogenous PGI2 in PPAR{delta} activation. Induction of Ras expression after IPTG treatment greatly increases the production of PGI2, which is the predominant prostaglandin product in IEC-iK-Ras cells. Our data demonstrate that blocking the generation of PGI2 by selective inhibition of COX-2 significantly reduced PPRE activation, which was restored by the addition of the PGI2 agonist, carboprostacyclin, suggesting that Ras-induced activation of PPAR{delta} involves a COX-2-derived prostaglandin product.

PPAR{gamma} is expressed in normal colonic mucosa, human colon cancer cell lines, and human colonic adenocarcinomas (16) . Loss-of-function mutations in PPAR{gamma} have been reported in human colorectal cancers (43) . Ligands for PPAR{gamma} induce differentiation and inhibit the growth of colon carcinoma cells in culture and as xenografted tumors (16 , 17) . However, studies conducted in multiple intestinal neoplasia (min) mice indicate that PPAR{gamma} ligands slightly increase colonic polyp formation but had no effect on the number of polyps in the small intestine (35 , 36) . Additional studies are needed to address the mechanism(s) by which PPAR{gamma} affects cell growth and differentiation. Our results show that treatment with PPAR{gamma} ligands inhibited K-Ras-mediated transformation of intestinal epithelial cells. K-Ras mutations often occur at an early to intermediate stage during the development of colorectal neoplasia (42) . On the basis of our work, we would predict that PPAR{gamma} activation might lead to an antineoplastic effect during early stages of colon carcinogenesis. Recent data from two separate reports in preclinical animal models agree with our hypothesis showing that treatment of rats with PPAR{gamma} agonists significantly decreased the number of premalignant aberrant crypt foci, which developed in the intestine (44 , 45) .

In the present study, we investigated the mechanism underlying the inhibition of K-Ras-mediated transformation by PPAR{gamma} and focused our efforts on understanding the modulation of the Ras signaling pathway after treatment with a PPAR{gamma} ligand. PI3K/Akt is an important effector of Ras signaling and mediates the proliferative signaling in Ha-Ras-transformed RIE cells (37) . PI3K inhibitors, such as Wortmannin and LY-294002, dramatically enhance the PPAR activity (46) . The cross-talk between PPAR{gamma} and PI3K pathway is quite interesting and potentially very important. Patel et al. (47) reported that PPAR{gamma} agonists up-regulate the expression of PTEN that can inactivate PI3K. Interestingly, our results show that treatment with BRL-49653 inhibits the activation of PI3K/Akt pathway. Because BRL-49653 did not increase the expression of PTEN in K-Ras-transformed intestinal epithelial cells, inhibition of PI3K/Akt activity by BRL-49653 in IEC-iK-Ras cells may involve other mechanisms.

We reported previously that BRL-49653 treatment results in growth arrest of human colon carcinoma cells in the G1 phase of the cell cycle (17) and that expression of a number of genes are affected (48) . In the present study, we found that BRL-49653 causes G1 growth arrest in K-Ras-transformed intestinal epithelial cells. The concentration of BRL-49653 that was required for the growth-inhibitory effect was relatively high. Our results show that BRL-49653 at 3–20 µM induced the activity of PPRE, suggesting specific ligand-activation of PPAR{gamma}. The requirement of high concentration of BRL-49653 may result from the low expression of PPAR{gamma} and/or phosphorylation of PPAR{gamma} by Ras effectors, such as MAP kinase (49, 50, 51, 52) . The involvement of PPAR{gamma}-independent mechanisms is not excluded in BRL-49653-induced growth inhibition of IEC cells (53) .

Activation of the Ras pathway affects the expression of a number of cell cycle proteins, including cyclin D1. Previous studies demonstrate that Ras transformation results in acceleration of G1 progression and induction of cyclin D1 expression in a variety of cell types (54, 55, 56, 57, 58, 59) . Among Ras effectors, the PI3K/Akt pathway is extremely critical for the expression of cyclin D1 and cell cycle progression (38 , 40) . We have demonstrated recently that inhibition of PI3K/Akt activity results in down-regulation of cyclin D1 and G1 growth arrest in Ha-Ras-transformed intestinal epithelial cells (37) . Our data show that PPAR{gamma} inhibits cell cycle progression in IEC-iK-Ras cells that may result from the decreased expression of cyclin D1 protein.

In conclusion, PPAR{gamma} and {delta} appear to play distinct roles after K-Ras-mediated transformation of intestinal epithelial cells. We show for the first time that PPAR{delta} is a Ras target gene that is up-regulated by the Raf/MEK/ERK pathway. Additionally, treatment with a PPAR{gamma} ligand inhibits the activity of a major Ras effector, PI3K/Akt, which partially suppresses the K-Ras-mediated transformation of IEC cells. Although the role of each PPAR nuclear receptor ({alpha}, {gamma}, and {delta}) in carcinogenesis is not totally clear, it seems apparent that each isoform has a distinct role, and the ligands that activate PPAR{gamma} may counter some of the Ras effector pathways in transformed intestinal epithelial cells.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants DK-47297, CA-77839 (to R. N. D.), CA 68485 (Vanderbilt-Ingram Cancer Center), and Veterans Affairs Merit Grant. Back

2 To whom requests for reprints should be addressed, at Department of Medicine/GI; MCN C-2104, Vanderbilt University Medical Center, Nashville, TN 37232-2279. Phone: (615) 322-5200; Fax: (615) 343-6229; E-mail: raymond.dubois{at}mcmail.vanderbilt.edu. Back

3 The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; IPTG, isopropyl-1-thio-ß-D-galactopyranoside; APC, adenomatous polyposis coli; PPRE, peroxisome proliferator-activated receptor responsive element; TCF, T-cell factor; PGI2, prostacyclin; PTEN, phosphatase and tensin homologue deleted from chromosome 10; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; PI3K, phosphatidylinositol 3'-kinase. Back

Received 12/28/01. Accepted 3/26/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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