From the
Departments of Cellular and Developmental Biology,
Medicine, and **Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee 37232, ¶Dana-Farber Cancer Institute and Department of Cancer Biology, Harvard Medical School, Boston, Massachussetts 02115, ||Psychiatric Genomics, Inc., Gaithersburg, Maryland 20878,
Human Genetics Program, Ohio State University Comprehensive Cancer Center, Columbus, Ohio 43210, and
Nuclear Receptor Discovery Research, GlaxoSmithKline, Research Triangle Park, North Carolina 27709
Received for publication, January 21, 2003 , and in revised form, February 17, 2003.
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ABSTRACT |
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INTRODUCTION |
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Peroxisome proliferator-activated receptor (PPAR
)1 is a ligand-activated transcription factor that is capable of initiating terminal differentiation pathways. PPAR
and related subtypes PPAR
and PPAR
are members of the nuclear hormone receptor gene superfamily (3) that form functional heterodimers with members of the retinoid X receptor (RXR) family of nuclear receptors (4). PPARs play fundamental roles in metabolic homeostasis, primarily as regulators of fatty acid storage and catabolism (5). Putative endogenous ligands for PPAR
include both polyunsaturated fatty acids and the eicosanoids 15-deoxy
12,14-PGJ2 (6, 7), 13-hydroxyoctadecadienoic acid, and 15-hydroxyeicosatetraenoic acid (8), but their respective roles in PPAR
signaling in vivo remains unclear. High affinity synthetic ligands that selectively activate PPAR
include the thiazolidinediones, a class of insulin sensitizing drugs currently in use for the treatment of insulin-resistant diabetes mellitus (9).
PPAR appears to play a dominant role in the differentiation of adipocytes. Early experiments established that ectopic expression of PPAR
in fibroblasts resulted in conversion of the cells to adipocytes (10). More recent studies using mice null for the PPAR
gene have confirmed this essential role in adipogenesis (11, 12). The cellular response induced by PPAR
during adipogenesis involves both cell cycle withdrawal and the expression of lipogenic-related genes such as the fatty acid-binding protein aP2 (13). The growth arrest pathway is characterized by a G1 cell cycle arrest and the induction of cyclin-dependent kinase inhibitors p18 and p21 (14). PPAR
has also been shown to restrict S phase entry by inhibiting the DNA binding activity of E2F/DP (15).
Current evidence suggests that PPAR can induce differentiation pathways beyond adipocytes. For example, activating ligands of PPAR
inhibit the proliferation rates of epithelial cells derived from breast, prostate, stomach, and lung (1619). In the colon, levels of PPAR
mRNA are nearly equivalent to that found in adipocytes (20) with the highest levels of receptor expression observed in the post-mitotic, differentiated epithelial cells facing the lumen (21). Consistent with this expression pattern, exposure of cultured human colon cancer cells to PPAR
agonists induces growth inhibition associated with a delay in the G1 phase of the cell cycle and an increase in several markers of cellular differentiation (2224).
Whether the anti-neoplastic, pro-differentiation effects of PPAR ligands in the colon operate in vivo is not clear. Agonists of the receptor will reduce pre-malignant intestinal lesions in rats treated with the carcinogen azoxymethane (25) but slightly increase colon polyps in Adenomatous polyposis coli mutant mice that are predisposed to intestinal adenomas (26, 27). However, Sarraf et al. (28) have reported that 8% of primary colorectal tumors contained a loss of function point mutation in one allele of the PPAR
gene, emphasizing that the receptor is likely to have a tumor suppressive function in the colon. Four unique mutations in PPAR
were identified in the study; one resulted in a truncated protein that lacked the entire ligand binding domain whereas the other three mutations caused defects in the binding of either synthetic or natural ligands.
Although activation of PPAR will initiate pathways leading to growth arrest in both colon epithelial and adipocyte lineages, it is unknown whether this occurs through similar or distinct mechanisms. For example, in both cell types activation of the receptor eventually leads to a G1 arrest and an increase in cell-specific differentiation markers. Does this occur through the initial regulation of identical target genes in both cell types, and does PPAR
require common co-regulator interactions in both instances?
Here we report the detection of an identical exonic mutation (K422Q) in the PPAR gene in four distinct colon cancer cell lines that are refractory to the decrease in cell growth or increase in differentiation markers normally induced by activators of PPAR
. Only introduction of the WT, but not mutant, receptor was able to restore PPAR
ligand sensitivity in the resistant colon cancer cell lines. In contrast, there was no difference in the ability of WT or K422Q receptor to induce adipocyte differentiation. Analysis of direct PPAR
target genes in colon cancer cells expressing the WT or K422Q mutant allele suggests that the mutation may be non-functional because of an inability of the apo-receptor to basally repress certain target genes. These results argue that codon 422 may be a part of a co-factor(s) interaction domain necessary for PPAR
to induce terminal differentiation in epithelial, but not adipocyte, cell lineages.
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EXPERIMENTAL PROCEDURES |
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Cell CultureThe HCT 15, COLO 205, HCT 116, HT-29, and NIH 3T3 cell lines were purchased from ATCC. 293-EBNA cells were purchased from Invitrogen. The MOSER S cell line was a gift from M. Brattain (University of Texas Health Sciences, San Antonio, TX). The MIP 101 and Clone A cell lines were a gift from L. B. Chen (Dana Farber Cancer Institute, Boston, MA). The HCA-7 cell line was obtained from S. Kirkland (University of London, London, United Kingdom). Cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Hyclone), L-glutamine (2 mmol/liter), penicillin (100 units/ml), and streptomycin (100 µg/ml) in a 5% CO2 atmosphere with constant humidity. For all experiments in which a receptor ligand was added, cells were grown in the above media except regular 10% FBS was replaced with 10% charcoal-stripped FBS (Hyclone).
PlasmidsFull-length WT PPAR was cloned into pBLUESCRIPT KS+. This was used as a template to generate PPAR
K422Q using oligonucleotide-directed in vitro mutagenesis (Muta-Gene; Bio-Rad). Both WT and K422Q PPAR
were cloned into pCDNA3.0 (Invitrogen) for use in transient transfection and EMSA experiments. HA-tagged WT and K422Q PPAR
were generated by PCR using Pfu Turbo Taq polymerase (Stratagene) and the proper non-tagged cDNA as a template. The 5' primer contained a XhoI site, the full-length HA epitope, and a partial region of PPAR
starting at codon 2. The 3' primer contained a HpaI site and a partial region of PPAR
starting at codon 479 (stop codon). Each amplicon was digested and cloned into the XhoI/HpaI site of the retroviral expression vector pMSCVpuro (Clontech). All plasmids were sequenced to avoid unwanted mutations.
Western Blot AnalysisCells were harvested in ice-cold 1x phosphate-buffered saline, and cell pellets were lysed in radioimmune precipitation assay buffer. Centrifuged lysates (50 µg) from each cell line were fractionated on a 420% gradient SDS-polyacrylamide gel and electrophoretically transferred to a polyvinlylidene difluoride membrane (PerkinElmer Life Sciences). Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween 20 and 5% powdered milk. The following primary antibodies were used: monoclonal anti-HA antibody clone HA.11 (1:1000; Babco), monoclonal anti-PPAR (1:500; Santa Cruz Biotechnology, Inc.), and monoclonal anti-keratin 18 and 20 antibodies (1:1000; NeoMarkers). This was followed by incubation with donkey anti-mouse horseradish peroxidaseconjugated secondary antibody (Jackson ImmunoResearch Laboratories) at a dilution of 1:50,000 for 1 h. Detection of immunoreactive polypeptides was accomplished using an enhanced chemiluminescence system (Amersham Biosciences).
Mutation DetectionMutations in the PPAR gene in the COLO 205, MIP 101, and Clone A cell lines were detected using a combination of denaturing gradient gel electrophoresis and direct sequencing as described previously (28, 29). PPAR
mutations in the HCT 15, MOSER S, HT-29, HCT 116, and HCA-7 cell lines were detected by automated dideoxy sequence analysis of PCR products that span the coding region of PPAR
1 using primers sets described previously (30).
Cell Growth MeasurementsThe day after initial seeding, cells were exposed to Dulbecco's modified Eagle's medium containing 10% charcoal stripped FBS and either 0.1% Me2SO or the indicated ligand. Cells were exposed to fresh medium and compound every 48 h. Cells were counted after 6 days of treatment using a Coulter counter. Each experiment was done in triplicate.
Transient Transfections and Luciferase AssaysCV-1 cells (5.0 x 105/well in 24-well plates) were transfected with a mix containing 0.66 µg/ml PPRE3-tk-luciferase (PPRE3-tk-luc) (a gift of R. Evans, Salk Institute, La Jolla, CA), 0.010 µg/ml pRL-SV40 (Promega), and 0.66 µg/ml pCNDA3.0/PPAR WT or pCDNA3.0/PPAR
K422Q in Opti-MEM (Invitrogen) for 5 h. HCT 15-pMSCV, HCT 15-PPAR
WT, or HCT 15-PPAR
K422Q cell lines were transfected with 0.66 µg/ml PPRE3-tk-luc, 0.010 µg/ml pRL-SV40, and 0.66 µg/ml pCDNA3.0. In each case, FuGENE 6 (Roche Applied Science) was added to the transfection mix at a lipid:DNA ratio of 3.5:1. The transfection mix was replaced with complete medium containing either vehicle or the indicated ligand. After 2436 h, cells were harvested in 1x luciferase lysis buffer. Relative light units from firefly luciferase activity were determined using a luminometer (MGM Instruments) and normalized to the relative light units from Renilla luciferase using the dual luciferase kit (Promega).
EMSAEMSAs were done based on methods reported by Schulman et al. (31). PPAR and RXR receptors were synthesized using a T7 Quick TNT in vitro transcription/translation kit (Promega). 1.0 µl of the PPAR receptor and 0.10, 0.50, 0.75, or 1.0 µl of RXR were added to a final reaction buffer volume of 20 µl that contained 1x binding buffer (20 mM HEPES, pH 7.5, 75 mM KCl, 2.0 mM dithiothreitol, 0.1% Nonidet P-40, 7.5% glycerol), 2.0 µg of poly(dI-dC), and 0.02 pmol of an 32P-labeled oligonucleotide containing a PPRE derived from the acyl-CoA oxidase promoter (GTCGACAGGGGACC AGGACA A AGGTCA CGTTCGGGAGT). After 20 min of incubation, the reactions were resolved on 5% nondenaturing acrylamide gels.
Viral Infection of Cell LinesPhoenix-Ampho cells (purchased from ATCC with prior approval of G. Nolan (Stanford University, Palo Alto, CA)) were transiently transfected with pMSCVpuro, pMSCV/HAPPAR WT, and pMSCV/HA-PPAR
K422Q using FuGENE 6 at a lipid:DNA ratio of 3.5:1. Approximately 72 h post-transfection, viral supernatants were collected, filtered, supplemented with 2 µg/ml of polybrene (Sigma), and used to infect exponentially growing HCT 15 or NIH 3T3 cells. After 48 h, cells were split 1:5 into media containing 4 µg/ml (HCT 15 cells) or 2 µg/ml (NIH 3T3 cells) puromycin (Sigma) to select for infected cells. After selection, all stable cell lines were grown in media containing 2 µg/ml puromycin prior to any experiments.
Flow CytometryHCT 15-pMSCV, HCT 15-PPAR WT, and HCT 15-PPAR
K422Q cell lines were treated with 0.1% Me2SO or the indicated receptor ligand for 48 h. The DNA content of nuclei was determined by staining nuclear DNA with propidium iodide (50 µg/ml) followed by measuring the relative DNA content of nuclei using a Facsort fluorescence-activated sorter (BD Biosciences). The proportion of nuclei in each phase of the cell cycle was determined using MODFIT DNA analysis software (BD Biosciences).
Tumor Growth in Athymic MiceAthymic mice (Harlan Sprague-Dawley, Inc.) were injected subcutaneously in the dorsal flanks with 5 x 106 cells of the HCT 15 cells expressing WT or K422Q PPAR in a volume of 0.10 ml of 1x phosphate-buffered saline. Dosing was started 1015 days post-injection for each cell line when the mean tumor volumes were
75 mm3. Mice were then orally gavaged five times/week with either vehicle (0.5% methylcellulose in 0.05 N HCl) or 10 mg/kg of rosiglitazone (in a total volume of 0.10 ml per mouse). Rosiglitazone was formulated daily by first dissolving the compound in 0.1 N HCL that had been pre-warmed to 40 °C followed by the addition of an equal volume of 1% methylcellulose. The size of each tumor was determined by direct measurement of tumor dimensions. The volume was calculated according to the equation (V = [L x W2] x 0.5), where V = volume, L = length, and W = width.
Adipogenesis AssayVirally infected NIH 3T3 cells were exposed at confluence to dexamethasone (1 µM) for 24 h followed by treatment with vehicle or rosiglitazone for 7 days with media changed every 48 h. Cells were then fixed and stained with Oil Red O (Sigma).
Northern Hybridization AnalysisNorthern blot analysis was performed as described previously (32). The indicated cell lines were treated with either 0.1% Me2SO or 2.0 µM rosiglitazone. Total RNA (20 µg) from each sample was fractionated on a 1.2% agarose-formaldehyde gel and transferred to a Hybond-NX nylon membrane (Amersham Biosciences). Filters were pre-hybridized for 4 h at 42 °C in Ultrahyb (Ambion). Hybridization was conducted in the same buffer in the presence of a 32P-radiolabeled cDNA fragment of the indicated gene. Blots were washed 4 x 15 min at 50 °C in 2x SSC, 0.1% SDS and once for 30 min in 1x SSC, 0.1% SDS. Membranes were then exposed to a phosphorimager, screen and images were analyzed using a Cyclone Storage Phosphor System and Optiquant software (Hewlitt-Packard).
Synthesis of cDNA Probes for Northern BlotsA partial cDNA fragment for adipophilin was generated by PCR with M13 forward and reverse primers using a sequence-validated human IMAGE cDNA clone (Research Genetics) as a template. Partial cDNA fragments for Gob-4 and TSC-22 were generated using reverse transcriptase PCR and gene-specific primers corresponding to base pairs (each from the translational start site) 283550 for Gob-4 and 1425 for TSC-22. The template for these PCR reactions was a random primed cDNA library of MOSER S colon carcinoma cells treated with either 0.1% Me2SO or 1 µM rosiglitazone. The Gob-4 PCR products were cloned into the pCR2.1-TOPO vector (Invitrogen), and the TSC-22 product was cloned into pPCR-Script (Stratagene). All plasmids were sequenced to confirm gene identity. The aP2 cDNA fragment was obtained from Youfei Guan (Vanderbilt University, Nashville, TN).
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RESULTS |
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There are a number of explanations for why a particular cell line could be resistant to activators of PPAR despite expressing robust levels of the receptor. Because somatic loss of function mutations have been identified in a subset of colorectal tumors, we sought to determine whether PPAR
ligand resistance in the four cell lines could be because of a loss of function mutation in the PPAR
gene. All four resistant lines contained a monoallelic point mutation in the PPAR
gene at codon 422 resulting in a change from lysine (Lys) to glutamine (Gln) (K422Q); this mutation was not found in the four sensitive cell lines (Table I). The correlation between the K422Q allele and lack of sensitivity to PPAR
ligands provided suggestive, but not definitive, evidence that this mutation caused the HCT 15, MIP 101, Clone A, and COLO 205 cell lines to be resistant to the growth inhibitory effects of PPAR
ligands.
Characterization of K422Q Mutant AlleleNo previous studies documenting the sequence of the PPAR gene in various malignancies or from individuals at risk for diabetes or obesity have reported mutations at codon 422 of the receptor. Lys-422 lies within the ninth
-helix (H9) of the ligand binding domain of the receptor. Crystallographic studies of PPAR
/RXR
heterodimers suggest a role for H9 in receptor dimerization (33). However, these studies found no direct role for Lys-422 in any polar interactions found at the dimer interface. X-ray crystallography of PPAR homodimers revealed Lys-422 to be located at the receptor surface and exposed to solvent, suggesting the possibility of involvement in co-factor interactions (Fig. 2A) (34). Lys-422 is conserved in the PPAR
cDNAs from all species reported in the NCBI Entrez nucleotide data base, including the six different species shown in Fig. 2B. However, Lys-422 is not conserved in either PPAR
or PPAR
, both of which encode a Gln at the homologous amino acid (Gln-413 and Gln-386, respectively) (Fig. 2B). Because codon 422 of PPAR
in the resistant cell lines is mutated to an amino acid (Gln) that is normally present in the homologous positions of WT PPAR
and PPAR
, it is unlikely that the K422Q mutation disrupts an important structural interaction common to all three PPARs. In fact, it may be that the Lys at position 422 present in WT PPAR
is responsible for an interaction unique to the
subtype.
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As no obvious function has been ascribed to Lys-422, we first characterized what effects the K422Q mutation might have on WT receptor activity. There was no difference in the DNA binding activity of WT PPAR/RXR
or K422Q PPAR
/RXR
on a PPRE from the acyl-coA oxidase promoter (Fig. 2C). Identical results were observed using RXR
and RXR
(data not shown). Transcriptional activity was assayed in cells transfected with either receptor cDNA and the PPRE3-tk-luc reporter vector that contains a luciferase cDNA downstream of three tandem repeats of the PPRE from the acyl-coA oxidase gene (35). There were no significant differences between WT and K422Q PPAR
in the ability of either a synthetic (rosiglitazone) or natural (15-deoxy
12,14-PGJ2) ligand to induce transcriptional activation (Fig. 2, D and E).
Wild-type, but Not K422Q, PPAR Can Rescue PPAR
Ligand Unresponsiveness in Resistant Colon Cancer CellsBecause WT and K422Q PPAR
showed equivalent activity in DNA binding and transactivation assays, it was not clear whether the presence of the K422Q mutant allele was the reason the resistant cells were refractory to the growth inhibitory effects of PPAR
ligands. To directly test this hypothesis, one of the resistant cell lines, the HCT 15 cells, was retrovirally transduced with HA-tagged WT or K422Q PPAR
and assayed for PPAR
ligand-induced growth inhibition and differentiation. Three different pooled stable cell lines, HCT 15-pMSCV (vector), HCT 15-PPAR
WT, and HCT 15-PPAR
K422Q, were generated. Both the HCT 15-PPAR
WT and HCT 15-PPAR
K422Q cell lines expressed equivalent levels of the WT or mutant receptor protein (Fig. 3A). As observed with the transiently transfected receptors, there were no differences in ligand-induced transactivation between WT and K422Q PPAR
in the stable cell lines (Fig. 3B).
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Exposure of HCT 15-PPAR WT, but not HCT 15-pMSCV or HCT 15-PPAR
K422Q, cells to a synthetic (rosiglitazone) or natural (15-deoxy
12,14-PGJ2) PPAR
agonist induced a dose-dependent decrease in cell number (Fig. 4, A and B). Similarly, only HCT 15 cells expressing the WT (but not mutant) receptor could undergo a partial arrest in the G1 phase of the cell cycle after extended exposure to a PPAR
agonist (Fig. 4C). Identical results were obtained in vivo using a nude mouse xenograft model of tumor growth. Mice bearing tumors consisting of HCT 15 cell expressing either WT or K422Q PPAR
were treated by oral gavage with vehicle or 10 mg/kg body weight of rosiglitazone. A significant reduction in tumor volume was seen only in HCT 15 cells transduced with the WT PPAR
allele (Fig. 5, A and B).
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Some members of the keratin and carcinoembryonic antigen (CEA) family of proteins represent markers of intestinal epithelial cell differentiation and have been shown to be induced by PPAR ligands in colon cancer cells (22, 24). Rosiglitazone was able to increase protein levels of keratin 18 or 20 or the RNA levels of the CEA cell adhesion molecule CEACAM6 (also known as nonspecific cross-reacting antigen or NCA) only in HCT 15 cells expressing WT, but not K422Q, PPAR
(Fig. 6).
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In summary, despite the fact that both WT and K422Q PPAR have comparable DNA binding and trans-activation activities, only the WT receptor could rescue the functional resistance of the parental HCT 15 cells. Cumulatively, these data suggest that the four PPAR
ligand-resistant cell lines identified in this study are resistant because of the presence of the K422Q allele.
Both WT and K422Q PPAR Can Induce Adipocyte DifferentiationThe above data indicate that codon 422 may be important in the ability of ligand occupied PPAR
to initiate terminal differentiation pathways in colon epithelial cells. To determine whether this was also true in the case of adipocyte differentiation, NIH 3T3 cells were retrovirally infected with vector (NIH 3T3-pMSCV), HA-tagged WT PPAR
(NIH 3T3-PPAR
WT), or HA-tagged K422Q PPAR
(NIH 3T3-PPAR
K422Q). Both WT and K422Q receptors were expressed at equivalent levels (Fig. 7A). Unlike the case with colon cancer cells, exposure of cells expressing either WT or K422Q PPAR
resulted in growth inhibition (Fig. 7B). Similarly, both WT and K422Q PPAR
could induce fibroblasts to differentiate into adipocytes, as indicated by ligand-induced increases in the adipocyte differentiation marker aP2 (Fig. 7C) and lipid accumulation (Fig. 7D).
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The K422Q Apo-receptor Cannot Repress the Basal Expression of Target Genes in Colon Cancer CellsThe K422Q allele could not induce colon epithelial differentiation markers. However, these are proteins that are only elevated after 57 days of treatment with PPAR agonists and are unlikely to be direct PPAR
target genes. To better understand why the presence of the K422Q mutation causes functional resistance to PPAR
agonists in colon cancer cells, we determined the effects the mutation had on the regulation of direct PPAR
target genes. Three PPAR
target genes, adipophillin, Gob-4, and TSC-22, which are all induced within 12 h of PPAR
ligand exposure in colon cancer cells, were selected for further study. We have previously identified adipophilin and Gob-4 as PPAR
target genes in a different colorectal cancer cell line (24). Adipophilin (also known as adipose differentiation-related factor) is a protein involved in fatty acid storage (36) whereas Gob-4 is a secreted protein associated with mature intestinal goblet cells (37). TSC-22 is a putative leucine zipper containing transcription factor originally identified as a TGF-
inducible gene (38). We have, in a previous microarray screen, identified and characterized TSC-22 as a direct PPAR
target gene in colorectal cancer cells.2
The expression levels of these three PPAR target genes was determined by Northern blot hybridization in HCT 15-pMSCV, HCT 15-PPAR
WT, and HCT 15-PPAR
K422Q cell lines treated with vehicle or rosiglitazone (Fig. 8). There was a 3-fold difference between WT and K422Q PPAR
in ligand-dependent induction of a target gene (i.e. adipophilin). The mutant receptor showed no defect in ligand-dependent repression of a target gene (i.e. Gob-4). However, as compared with WT apo-PPAR
, the most striking defect of the K422Q mutation was an inability of the apo-receptor to repress the basal expression of the PPAR
target gene TSC-22.
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The ability of some members of the nuclear hormone receptor superfamily, notably the RARs and thyroid hormone receptors, to actively repress gene expression in the absence of ligand has been well documented (reviewed in Refs. 39 and 40). This activity is dependent on their ability to bind to transcriptional co-regulators termed co-repressors. Two of the best characterized co-repressors are N-CoR (nuclear receptor co-repressor) (41) and SMRT (silencing mediator for RAR and thyroid hormone receptors) (42). Although prior studies have failed to demonstrate the ability of either N-CoR or SMRT to bind DNA-bound apo-PPAR, both of these proteins can bind to PPAR
in solution, suggesting their possible involvement in apo-PPAR
-mediated transcriptional repression (43). Thus, we tested whether apo-K422Q PPAR
is unable to repress basal TSC-22 expression because of a defect in co-repressor binding. However, in a mammalian two-hybrid assay, there was no difference between WT and K422Q PPAR
in their ability to bind to, or exhibit ligand-dependent release from, N-CoR or SMRT (data not shown).
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DISCUSSION |
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Loss of Normal PPAR Signaling during the Development of Colorectal CancerSarraf et al. (28) identified four of 55 primary colorectal tumors that harbored loss of function somatic mutations in either exon 3 or 5 of the PPAR
gene. Because PPAR
induces growth arrest in cultured colon cancer cells, these results suggested that some colorectal tumors undergo genetic selection for loss of PPAR
signaling during the development of colorectal cancer. Our results provide further support for this hypothesis by documenting four established colon cancer cell lines that are all functionally resistant to PPAR
ligands and that all contain a mutant PPAR
allele.
Information on the four patients from whom the resistant HCT 15, MIP 101, Clone A, and COLO 205 cell lines were derived is limited. Thus, it is unknown whether the K422Q mutation is a germline mutation, occurred in the primary colorectal tumors of these individuals, or was established only after these cells were cultured in vitro. The K422Q mutation was not found in an earlier study screening for PPAR gene mutations in 55 primary colorectal tumors. A more recent study failed to detect any PPAR
gene mutations in a large number of clinical samples including cancers derived from the breast, colon, prostate, and lung (44). The conclusion of the study was that loss of function mutations of the receptor is extremely rare in cancer. However, this study limited its screen to exons 3 and 5 of PPAR
. The K422Q mutation lies in exon 6, and our results emphasize that studies designed to screen for PPAR
gene mutations in cancers should be extended to include exons spanning the entire coding region. We are currently screening clinical samples of colorectal cancer to determine the incidence of the K422Q mutation in primary colorectal tumors. It will also be of interest to know whether the K422Q mutation is associated with metabolic syndromes such as insulin resistance or obesity for which germline PPAR
mutations have been identified previously (45, 46).
The K422Q mutation in the four resistant cell lines was only identified in one allele of the gene whereas the other allele encoded for wild-type receptor. Similarly, in the earlier report by Sarraf and colleagues (28), the four PPAR mutations found in primary colorectal cancers were all monoallelic with no evidence for loss of heterozygosity. However, it is not clear in these instances whether the remaining WT receptor is being expressed. In the HCT 15 cells, 10 independent PPAR
cDNA fragments that span exon 6 were cloned and sequenced using reverse transcriptase PCR; all 10 clones contained the K422Q mutation (data not shown). This would suggest that, at least in this cell line, the WT receptor is not expressed or is present at very low levels. In fact, in tumors that contain one mutated allele of PPAR
, the other allele might be silenced through alterative mechanisms (e.g. promoter methylation).
Nevertheless, PPAR does not appear to fit the classic Knudtson "two hit" hypothesis in which both alleles of a tumor suppressor are genetically inactivated. As opposed to studies that are limited only to analysis of DNA from primary colorectal cancers, our experiments with established cell lines has allowed us to conduct functional experiments that demonstrate that colon cancer cells with one mutant and one wild-type PPAR
allele are resistant to PPAR
ligand-induced growth inhibition. In theory, this loss of normal PPAR
signaling could occur through a dominant-negative or haploinsufficiency mechanism. It is possible that in a situation where both WT and K422Q PPAR
are co-expressed in the same cell line, the K422Q receptor could out-compete the WT receptor for a limiting number of binding sites (e.g. to RXR or to specific DNA elements) and thus inhibit the function of the WT receptor. Alternatively, it could simply be a gene dosage effect. PPAR
± embryonic stem cells have a reduced capacity to differentiate into adipocytes as compared with WT cells (11), and mice heterozygous for PPAR
have a greater incidence of colorectal cancer as compared with control animals (47); both of these experiments establish that haploinsufficiency can occur for the PPAR
locus.
Mechanisms of PPAR-mediated Differentiation in Distinct Cell TypesThe molecular mechanisms by which PPAR
initiates terminal differentiation pathways remain largely unknown. Here we have identified a PPAR
mutation that highlights a region of the receptor that appears to be essential for an interaction (and activity) necessary for receptor-induced colon epithelial, but not adipocyte, differentiation. What is the nature of this interaction? Analysis of direct genes regulated by either WT or K422Q PPAR
in the HCT 15 cells suggests that the mutation may cause a defect in the ability of the apo-receptor to repress gene expression. Thus, the mutation may disrupt interactions with an as yet unidentified co-repressor. These findings also imply that this type of target gene repression is only important in the ability of PPAR
to induce terminal differentiation in specific cell lineages (i.e. colon epithelial) and not others. It is also possible that the K422Q is defective in another unidentified receptor activity independent of repression (and that the defect in gene repression is a secondary event).
Finally, our evidence that PPAR induces colon epithelial and adipocyte differentiation through distinct mechanisms implies that selective PPAR
modulators could be developed that specially target differentiation in particular cell lineages. For example, a PPAR
ligand capable of inducing epithelial, but not adipocyte, differentiation might limit the undesirable side effects because of modulation of adipocytes in treatments primarily aimed at inducing terminal differentiation of an epithelial cancer. As a first step in pursuing this line of research, our current focus is to identify biochemical target(s) that differentially bind to WT or K422Q PPAR
.
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FOOTNOTES |
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¶¶ The Mina C. Wallace Professor of Cancer Prevention. To whom correspondence should be addressed: Dept. of Medicine/GI, MCN C-2104, Vanderbilt University Medical Center, 1161 21st Ave. S., Nashville, TN 37232-2279. Tel.: 615-322-5200; Fax: 615-343-6229; E-mail: raymond.dubois{at}mcmail.vanderbilt.edu.
1 The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; RXT, retinoid X receptor; WT, wild-type; FBS, fetal bovine serum; EMSA, electrophoretic mobility shift assay; HA, hemagglutinin; luc, luciferase; CEA, carcino-embryonic antigen; PGJ2, prostaglandin J2.
2 R. A. Gupta and R. N. DuBois, unpublished results.
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REFERENCES |
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