EP1203101A1 - Ppar delta relie apc aux medicaments de chimio-prevention - Google Patents

Ppar delta relie apc aux medicaments de chimio-prevention

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Publication number
EP1203101A1
EP1203101A1 EP00957478A EP00957478A EP1203101A1 EP 1203101 A1 EP1203101 A1 EP 1203101A1 EP 00957478 A EP00957478 A EP 00957478A EP 00957478 A EP00957478 A EP 00957478A EP 1203101 A1 EP1203101 A1 EP 1203101A1
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EP
European Patent Office
Prior art keywords
pparδ
reporter gene
apc
tcf
binding
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EP00957478A
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German (de)
English (en)
Inventor
Tong-Chuan He
Kenneth W. Kinzler
Bert Vogelstein
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Johns Hopkins University
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Johns Hopkins University
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Priority claimed from PCT/US2000/022411 external-priority patent/WO2001012858A1/fr
Publication of EP1203101A1 publication Critical patent/EP1203101A1/fr
Withdrawn legal-status Critical Current

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Definitions

  • the invention relates to the area of cancer and gastroenterological therapeutic agents. More particularly, the invention relates to the area of screening assays for therapeutic agents.
  • colorectal cancer is the second leading cause of cancer deaths in the United States. Over half of the U.S. population will develop a colorectal tumor during their lifetime, and these tumors will progress to malignancy in approximately 10% of the cases. The high prevalence of this disease and aging nature of the population make effective prevention an important public health and economic concern.
  • APC has been shown to associate with at least a dozen proteins, its association with ⁇ -catenin seems to be of special importance for its tumor suppressor function.
  • the ⁇ -catenin protein was originally identified through its association with E-cadherin and role in cellular adhesion (reviewed in Kemler, 1993).
  • ⁇ -catenin has a separate cellular role, serving as a signal transducer in the Wg/WNT pathway. In the colon, ⁇ -catenin binds to the
  • Tcf-4 transcription factor providing a domain which activates genes containing Tcf-4 binding sites in their regulatory regions (Behrens et al, 1996; Molenaar et al, 1996).
  • the product of the wild type APC gene inhibits this ⁇ -catenin/Tcf-4 mediated transcription, while disease-associated APC mutants are deficient in this ability
  • One embodiment of the invention is an isolated subgenomic polynucleotide comprising a PPAR ⁇ binding element comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:l-21 and nucleotides 3-9 of SEQ ID NO:21
  • Figure 3B and an RXR binding element comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:22-42 and nucleotides 3-7 of SEQ ID NO:50 ( Figure 3A).
  • Another embodiment of the invention is an isolated subgenomic polynucleotide comprising at least 2 copies of a PPAR ⁇ binding element comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:l-21 and nucleotides 3-9 of SEQ ID NO:21 ( Figure 3B).
  • nucleic acid construct comprising at least one PPAR ⁇ binding element comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1-21 and nucleotides 3-9 of SEQ
  • the PPAR ⁇ binding element is upstream from the minimal promoter, and the minimal promoter is upstream from the reporter gene.
  • the minimal promoter regulates transcription of the reporter gene.
  • the invention provides a method of pre-screening agents for therapeutic use. Binding of a PPAR ⁇ protein to a DNA molecule comprising a PPAR ⁇ binding element is measured in the presence and in the absence of a test substance. The amount of binding of the PPAR ⁇ protein in the presence of the test substance is compared to amount of binding of the PPAR ⁇ protein in the absence of the test substance. A test substance which decreases the amount of binding is a candidate agent for use in cancer therapy. A test substance which increases the amount of binding is a candidate agent for ameliorating negative side effects of NSAIDs.
  • the invention provides another method of pre-screening agents for therapeutic use. A transfected cell is contacted with a test substance.
  • the transfected cell contains a PPAR ⁇ protein and a reporter construct comprising a reporter gene.
  • the reporter gene encodes an assayable product, a minimal promoter upstream from and regulating transcription of the reporter gene, and at least one copy of a PPAR ⁇ binding element upstream of the minimal promoter. Whether expression of the reporter gene is decreased or increased by the test substance is determined.
  • a test substance which decreases the amount of expression of the reporter gene is a candidate agent for use in cancer therapy.
  • a test substance which increases the amount of expression of the reporter gene is a candidate agent for ameliorating negative side effects of NSAIDs.
  • the invention provides yet another method of pre-screening agents for therapeutic use.
  • RNA polymerase, ribonucleotides, and PPAR ⁇ protein are added to a reporter construct.
  • the reporter construct comprises a reporter gene which encodes an assayable product and at least one copy of a PPAR ⁇ binding element upstream from a minimal promoter.
  • the minimal promoter is upstream from and controls transcription of the reporter gene.
  • the step of adding is effected in the presence and absence of a test substance. Whether transcription of the reporter gene is decreased or increased in the presence of the test substance is determined.
  • a test substance which decreases the amount of transcription of the reporter gene is a candidate agent for use in cancer therapy.
  • a test substance which increases the amount of transcription of the reporter gene is a candidate agent for ameliorating negative side-effects of NSAIDs.
  • Even another embodiment of the invention is a method of identifying candidate drugs for use in FAP patients, patients with APC or ⁇ -catenin mutations, or patients with increased risk of developing cancer.
  • a cell having no wild-type APC or a mutant ⁇ -catenin is contacted with a test compound. Transcription in the cell of a Tcf-responsive reporter gene is measured.
  • the Tcf-responsive reporter gene comprises a Tcf-4 binding element selected from the group consisting of CTTTGAT (TREl) and CTTTCAT (TRE2).
  • a test compound which decreases transcription of the reporter gene is a candidate drug for cancer therapy.
  • Still another embodiment of the invention is a method of identifying candidate drugs for use in for use in FAP patients, patients with APC or ⁇ -catenin mutations, or patients with decreased risk of developing cancer.
  • a Tcf-responsive reporter gene is contacted with a test compound under conditions in which the reporter gene is transcribed in the absence of the test compound.
  • the Tcf-responsive reporter gene comprises a Tcf-4 binding element selected from the group consisting of CTTTGAT (TRE1) and CTTTCAT (TRE2). Transcription of the Tcf-responsive reporter gene is measured.
  • a test compound which decreases transcription of the Tcf-responsive reporter gene is a candidate drug for cancer therapy.
  • the invention thus provides tools and methods for identifying potential therapeutic agents for cancer treatment and for ameliorating negative side effects of NSAIDs.
  • Figures lA-C Expression of PPAR ⁇ inhuman colorectal cancer cells.
  • FIG. 1A Decreased expression of PPAR ⁇ following induction of APC in human colorectal cancer cells.
  • Expression of APC (HT29-APC) or ⁇ -galactosidase (HT29-GAL) was induced with 110 ⁇ M ZnCl 2 for the indicated times in HT29 colorectal cancer cells containing the respective genes under the control of a modified metallothionein promotor.
  • Total RNA (10 ⁇ g) was isolated and analyzed by Northern blot analysis with probes specific for PPAR ⁇ and PPAR ⁇ .
  • Figure IB Increased expression of PPAR ⁇ in primary human colorectal cancers.
  • RNA (10 ⁇ g) was isolated and analyzed by Northern blot analysis with probes specific to PPAR ⁇ and PPAR ⁇ .
  • FIGS. 2A-E APC Regulates PPAR ⁇ expression through ⁇ -catenin/Tcf-4-mediated transcription.
  • Figure 2A PPAR ⁇ promotor. A restriction map of the 3.1 kb region upstream of the first exon of PPAR ⁇ is shown. Restriction fragments BE, NE, HE, DE, BN, NH, HD and NP were used to construct reporters for measuring APC and ⁇ -catenin responsiveness. Filled boxes represent potential Tcf-4 binding sites, and open sites represent the same sites engineered to contain mutations that abolish Tcf-4 binding. mNP represents fragment NP with both potential Tcf-4 binding sites mutated.
  • TRE1 and TRE2 contain four repeats of the two Tcf-4 binding sites, respectively. mTREl and mTRE2 are mutant forms of TRE1 and TRE2.
  • Figure 2A PPAR ⁇ promotor. A restriction map of the 3.1 kb region upstream of the first exon of PPAR ⁇ is shown. Restriction
  • the PPAR ⁇ promotor is repressed by APC and dominant negative Tcf-4.
  • SW480 colorectal cancer cells were transfected with the indicated PPAR ⁇ promotor luciferase reporters (0.4 ⁇ g), with a ⁇ -galactosidase expression vector (0.2 ⁇ g pCMV ⁇ ), and with 1.0 ⁇ g of either a vector control (Vector), an expression vector for APC (APC) or for a dominant negative form of Tcf-4 (dnTcf). Luciferase activity is reported relative to the vector control after normalizing for transfection efficiency through ⁇ -galactosidase activity. Bars represent the means of three independent replicates, with error bars being the unbiased standard deviations. Figure 2C.
  • APC and dnTcf responsiveness is mediated by two putative Tcf-4 binding sites.
  • PPAR ⁇ promotor fragments with intact and mutated Tcf-4 binding sites were tested for APC and dnTcf-4 responsiveness as described in Figure 2B. Bars represent the means of three independent replicates, with error bars representing the unbiased standard deviations.
  • Figure 2D ⁇ -catenin transactivation maps to the same promotor regions mediating APC and dnTcf responsiveness.
  • the 293 human cell line was transfected with the indicated PPAR ⁇ promotor luciferase reporters (0.4 ⁇ g), with a ⁇ -galactosidase expression vector (0.2 ⁇ g pCMV ⁇ ) and with 1.0 ⁇ g of either a no insert control (Vector) or an oncogenic ⁇ -catenin ( ⁇ -catenin) expression vector. Luciferase activity was reported as described for Figure 2B. Bars represent the means of three independent replicates, with error bars representing the unbiased standard deviations. Figure 2E. Putative Tcf-4 binding sites in the PPAR ⁇ promotor bind Tcf-4.
  • GEMS A was performed using 32 P-labeled probes containing either putative Tcf-4 binding sites TREl or TRE2. GEMS A was performed in the presence of a GST fusion protein containing the Tcf-4 DNA binding domain as indicated. Wild type (wt) or mutant (mut) competitors corresponding to the Tcf-4 binding sites were used as indicated.
  • Figures 3 A-G Development of a PPAR ⁇ -Specific Reporter.
  • Figure 3 A RXR consensus binding site. PCR products of a randomized ohgonucleotide template that bound a GST fusion protein containing the DNA binding domain of RXR were selected, cloned, and sequenced. The sequences of twenty-eight clones are shown, manually aligned to derive the consensus binding sequence indicated at the bottom.
  • FIG. 3B PPAR ⁇ consensus binding site. PCR products of a randomized ohgonucleotide template that bound a GST fusion protein containing the DNA binding domain of PPAR ⁇ were selected, cloned and sequenced. The sequences of twenty clones are shown, manually aligned to derive the consensus binding sequence indicated at the bottom.
  • Figure 3C Binding Specificity of PPAR ⁇ , PPAR ⁇ and PPAR ⁇ .
  • Oligonucleotides containing the indicated binding elements were 32 P-labeled and incubated with GST fusion proteins containing either the PPAR ⁇ , PPAR ⁇ , PPAR ⁇ , RXR, or no DNA binding domain (-). DNA binding was assessed by GEMS A, where "Probe” indicates the unbound probe and "Shifted” indicates bound probe.
  • Figure 3D DRE confers PPAR ⁇ responsiveness.
  • the 293 human cell line was transfected with the indicated (DRE or ACO) luciferase reporters (0.3 ⁇ g), with a ⁇ -galactosidase expression vector (0.2 ⁇ g pCMV ⁇ ), and with 1.0 ⁇ g of either a vector control (Vector), PPAR ⁇ , or PPAR ⁇ expression vectors. Luciferase activity was calculated as described in Figure 2B. Bars represent the means of three independent replicates with the error bars representing the unbiased standard deviations. Figure 3E .
  • Oligonucleotides containing the indicated binding elements were 32 P- labeled and incubated with in vitro translated PPAR ⁇ , PPAR ⁇ , and RXR ⁇ as indicated. The binding was supplemented with PPAR ⁇ ligand cPGI (10 ⁇ M) and PPAR ⁇ ligand BRL 49653 (10 ⁇ M) were added as indicated. DNA binding was assessed by GEMS A where "Probe” indicates the unbound probe and "Shifted” indicates bound probe. Figure 3F. DRE confers PPAR ⁇ but not PPAR ⁇ responsiveness.
  • the 293 human cell line was transfected with DRE luciferase reporter (0.3 ⁇ g), a ⁇ -galactosidase expression vector (0.2 ⁇ g pCMV ⁇ ), and with 1.0 ⁇ g of either empty vector (Control), PPAR ⁇ , or PPAR ⁇ expression vectors.
  • DRE luciferase reporter 0.3 ⁇ g
  • a ⁇ -galactosidase expression vector 0.2 ⁇ g pCMV ⁇
  • 1.0 ⁇ g of either empty vector (Control), PPAR ⁇ , or PPAR ⁇ expression vectors where indicated, cells were treated with the PPAR ⁇ ligand cPGI (20 ⁇ M) or the PPAR ⁇ ligand BRL 49653 (20 ⁇ M).
  • Luciferase activity was reported as relative luciferase activity after correction for transfection efficiency using ⁇ -galactosidase activity. Bars represent the means of three independent replicates, with the error bars representing the unbiased standard deviations.
  • ACO confers PPAR ⁇ but not PPAR ⁇ responsiveness.
  • the 293 human cell line was transfected with ACO luciferase reporter (0.3 ⁇ g), a ⁇ -galactosidase expression vector (0.2 ⁇ g pCMV ⁇ ), and with 1.0 ⁇ g of either empty vector (Control), PPAR ⁇ , or PPAR ⁇ expression vectors. Where indicated, cells were treated with the PPAR ⁇ ligand cPGI (20 ⁇ M) or the PPAR ⁇ ligand BRL 49653 (20 ⁇ M). Luciferase activity was reported as relative luciferase activity after correction for transfection efficiency using ⁇ -galactosidase activity. Bars represent the means of three independent replicates, with the error bars representing the unbiased standard deviations.
  • FIGs 4A-C PPAR ⁇ activity is regulated by APC, ⁇ -catenin and sulindac.
  • Figure 4 A APC and dnTcf specifically repress PPAR ⁇ activity.
  • PPAR ⁇ and PPAR ⁇ activity was assessed with the DRE and ACO luciferase reporters, respectively.
  • SW480 colorectal cancer cells were transfected with the indicated luciferase reporters (0.4 ⁇ g of DRE or ACO), with a ⁇ -galactosidase expression vector (0.2 ⁇ g pCMV ⁇ ), and with
  • Luciferase activity was calculated as described in Figure 2B. Bars represent the means of three independent replicates, with the error bars being the unbiased standard deviations. Figure 4B. ⁇ -catenin expression increases PPAR ⁇ activity.
  • the 293 human cell line was transfected with the indicated luciferase reporters
  • FIG. 4C Sulindac specifically represses PPAR ⁇ activity.
  • PPAR ⁇ and PPAR ⁇ activity was assessed as transcriptional activity of the DRE and ACO luciferase reporters, respectively.
  • HCT116 and SW480 colorectal cancer cells were transfected with the indicated luciferase reporters (1.0 ⁇ g of DRE or ACO) and with a ⁇ -galactosidase expression vector (0.2 ⁇ g pCMV ⁇ ). Cells were allowed to recover for 20 hours after transfection and were then treated for 10 hours with the indicated concentrations ( ⁇ M) of sulindac sulfide. Luciferase activity was reported relative to the control (0) after normalizing for transfection efficiency.
  • FIGS 5A-E Fluorescence microscopy of uninfected (Figure 5A), AdGFP (Figure 5B) or AdPPAR ⁇ (Figure 5C) infected HCT116 cells treated with 125 uM of sulindac sulfide, showing that PPAR ⁇ can partially protect colon cancer cells from sulindac-induced apoptosis.
  • HCT116 and SW480 cells were either mock infected (Uninfected) or infected with adenovirus expressing GFP (AdGFP) or PPAR ⁇ (AdPPAR ⁇ ). Twenty hours after infection, cells were treated for 42 hours with sulindac sulfide.
  • FIG. 5D Bars represent the fraction of apoptotic nuclei after treatment with the indicated adenoviruses and concentration of sulindac sulfide ( ⁇ M).
  • Figure 5E PPAR ⁇ rescues sulindac sulfide inhibition of clonal growth. Cells were infected with the indicated adenovirus, treated with the indicated concentrations of sulindac sulfide, and plated. Clonal growth was scored as colony formation after six days. Colonies were visualized by staining with Crystal Violet (upper panel) and enumerated (lower panel).
  • Figures 6A-E Mechanism of suppression of PPAR ⁇ by NSAIDs.
  • Figure 6A Mechanism of suppression of PPAR ⁇ by NSAIDs.
  • NSAIDs do not affect PPAR ⁇ expression.
  • HCT116 and SW480 cells were treated with the indicated concentration ( ⁇ M) of sulindac sulfide for 36 hours, and RNA was isolated. Northern blot analysis was performed on 10 ⁇ g of total RNA with a probe specific for PPAR ⁇ .
  • Figure 6B NSAIDs suppress PPAR ⁇ DNA binding.
  • the DRE binding element was 32 P-labeled and incubated with no lysate (Probe only), a non-programmed in vitro translation lysate (Blank Lysate) or in vitro translated PPAR ⁇ ( ⁇ ), RXR (RXR), or both ( ⁇ + RXR).
  • PPAR ⁇ + RXR was included in all lysates treated with the indicated NSAIDs. DNA binding was assessed by GEMSA, where "Probe” indicates the unbound probe and “Shifted” indicates bound probe. Figure 6C. NSAIDs do not suppress PPAR ⁇ DNA binding. DNA binding activity was assessed as in Figure
  • NSAIDs suppress PPAR ⁇ DNA binding.
  • the DRE binding element was 32 P-labeled and incubated with no lysate (Probe only) or in vitro translated PPAR ⁇ ( ⁇ ), RXR ⁇ (RXR ⁇ ), or both ( ⁇ + RXR ⁇ ).
  • PPAR ⁇ + RXR ⁇ + cPGI (10 ⁇ M) was included in all lysates treated with the indicated NSAIDs. DNA binding was assessed by GEMSA, where
  • RXR ⁇ RXR ⁇
  • PPAR ⁇ + RXR ⁇ + BRL 49653 10 ⁇ M was included in all lysates treated with the indicated NSAIDs. DNA binding was assessed by GEMSA, where "Probe” indicates the unbound probe and "Shifted” indicates bound probe.
  • Figure 7 Unified model for APC- and NSATD-mediated suppression of colorectal cancer. Elements indicated in blue have been shown to have a tumor suppressive effect, whereas elements in red have been shown to promote tumor formation. The effects of items in boxes have been demonstrated by genetic alterations.
  • LOX 5'-lipoxygenase
  • sPLA2 secretory phospholipase 2
  • COX cyclooxygenase.
  • PPAR ⁇ peroxisome proliferator-activated receptor delta
  • NUC1 peroxisome proliferator-activated receptor delta
  • FAAR peroxisome proliferator-activated receptor delta
  • PPAR ⁇ belongs to the nuclear receptor superfamily, which includes the steroid hormone, thyroid hormone, retinoid, and PPAR subfamilies as well as a growing number of orphan receptors (Kastner et al, 1995; Lemberger et al, 1996; Mangelsdorf et al, 1995).
  • the PPAR subfamily comprises at least three distinct subtypes found in vertebrate species: PPAR ⁇ (Dreyer et al, 1992), PPAR ⁇ (Amri et al, 1995; Jow and Mukherjee, 1995; Schmidt et al, 1992), and PPAR ⁇ (Tontonoz et al, 1994).
  • the nuclear receptor family members function as ligand-dependent sequence-specific activators of transcription (Lemberger et al, 1996; Mangelsdorf et al, 1995).
  • the PPARs were initially shown to be activated by peroxisome proliferators and hypolipidemic drugs of the fibrate class, and later by natural fatty acids and prostaglandins (Forman et al, 1997; Forman et al, 1995; Keller et al, 1993; Kliewer et al., 1995; Kliewer et al, 1997; Xu et al, 1999; Yu et al, 1995).
  • this information can be used to pre-screen agents for use in cancer therapy or the treatment of other conditions in which decreased cellular proliferation is desired, such as hyperplastic or dysplastic conditions.
  • agents that specifically target PPAR ⁇ can lead to more efficacious and less toxic means for colorectal cancer chemoprevention.
  • Subgenomic polynucleotides and nucleic acid constructs can be used to identify test substances which down-regulate the transcriptional activity of PPAR ⁇ .
  • Subgenomic polynucleotides of the invention contain less than a whole chromosome and can be single- or double-stranded genomic or cDNA.
  • the polynucleotides are isolated free of other cellular components, such as membrane components, proteins, and lipids. They can be made by a cell and isolated, or synthesized in the laboratory using an amplification method such as PCR or using an automatic synthesizer. Methods for purifying and isolating DNA are routine and are known in the art.
  • the isolated subgenomic polynucleotides contain a PPAR ⁇ binding element and an RXR binding element.
  • the nucleotide sequence of the PPAR ⁇ binding element can be selected, for example, from any of the nucleotide sequences shown in Figure 3B (SEQ ID NOS: 1-21), including the consensus nucleotide sequence CGCTCAC (nucleotides 3-9 of SEQ ID NO:21).
  • PPAR ⁇ binding elements with other nucleotide sequences which bind PPAR ⁇ protein can also be used in subgenomic polynucleotides of the invention.
  • binding elements can be identified, for example, by carrying out assays which can detect PPAR ⁇ protein-DNA binding, such as DNA footprinting, electrophoretic mobility shift assays, or immunoprecipitation of PPAR ⁇ -DNA complexes using antibodies specific for PPAR ⁇ . Such methods are well known in the art.
  • the nucleotide sequence of the RXR binding element can be selected from any of the nucleotide sequences shown in Figure 3A (SEQ ID NOS:22-50), including the consensus sequence GGTCA (nucleotides 3-7 of SEQ ID NO:50).
  • Other RXR binding elements which bind RXR can be identified as described for PPAR ⁇ binding elements, above.
  • the PPAR ⁇ and RXR binding elements can be located directly adjacent to each other in the subgenomic polynucleotide, as shown in SEQ ID NO:78, or can be separated by any number of nucleotides which still permits functional binding of a PPAR ⁇ /RXR heterodimer, such as 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides.
  • the isolated subgenomic polynucleotide can comprise 1, 2, 3, 4, or more copies of the PPAR ⁇ binding element. Multiple copies of the RXR binding element can also be included.
  • Isolated subgenomic polynucleotides comprising a PPAR ⁇ binding element can be attached to a solid support and used to selectively bind PPAR ⁇ and remove it from other cellular components.
  • Suitable solid supports include, but are not limited to, insoluble polymers, such as a column chromatography matrix, glass or plastic slides, tissue culture plates, microtiter wells, tubes, or particles such as beads, including but not limited to latex, polystyrene, or glass beads.
  • any method known in the art can be used to attach a subgenomic polynucleotide to the solid support, including use of covalent and non-covalent linkages, passive abso ⁇ tion, or pairs of binding moieties attached respectively to the subgenomic polynucleotide and the solid support.
  • PPAR ⁇ binding elements of the invention can be present in a nucleotide construct, which can be prepared using standard recombinant DNA techniques.
  • Nucleic acid constructs can be linear or circular molecules, with or without replication sequences.
  • Nucleic acid constructs of the invention contain at least 1, 2, 3, or 4 or more copies of the PPAR ⁇ binding element.
  • a nucleic acid construct can comprise a reporter gene which encodes an assayable product, such as ⁇ -galactosidase, luciferase, ⁇ -glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), or chloramphenicol acetyltransferase (CAT).
  • GFP green fluorescent protein
  • BFP blue fluorescent protein
  • GST glutathione-S-transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • Many such reporter genes are known in the art.
  • the reporter gene can be under the control of a minimal promoter, such that in the absence of PPAR ⁇ the reporter gene is not expressed or is expressed only at low levels.
  • reporter gene constructs can be used, for example, in methods for pre- screening agents for use in cancer therapy, which are described below.
  • the minimal promoter is upstream from the reporter gene, and at least one copy of the PPAR ⁇ binding element is upstream from the minimal promoter.
  • Suitable minimal promoters include, for example, the minimal CMV promoter (Boshart et al, 1985) and the promoters for TK (Nordeen, 1988), IL-2, and MMTV.
  • the reporter construct can include one or more RXR binding elements upstream of the minimal promoter.
  • a reporter gene is under the control of a Tcf-4 binding element.
  • the Tcf-4 binding element can be CTTTGAT (TRE1) or CTTTCAT
  • Tcf-4-responsive reporter constructs can comprise at least 1 , 2, 3, or 4 or more of either or both Tcf-4 binding elements, or can comprise nucleotides - 1543 to -759 of PPAR ⁇ .
  • the invention provides various methods of pre-screening agents for use in cancer therapy. These methods measure either PPAR ⁇ protein binding to its binding element or PPAR ⁇ -dependent transcription in response to a test substance. It is also possible to screen agents for use in cancer therapy by measuring transcription of PPAR ⁇ itself in response to a test substance.
  • Test substances which can be screened can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. Test substances can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, or can be produced recombinantly or synthesized by chemical methods known in the art.
  • binding of a PPAR ⁇ protein to a DNA molecule comprising a PPAR ⁇ binding element is measured in the presence and absence of a test substance. Binding can be measured either m a crude nuclear extract of a mammalian tissue, including human tissue, or a human or other mammalian cell line. Preferably the extract either lacks wild-type APC or contains a mutant ⁇ -catenin which permits transcription of PPAR ⁇ even in the presence of wild-type APC.
  • suitable extracts can be prepared from colorectal cancer tissue obtained from mammals, including humans, or from colorectal cancer cell lines, such as HT29, SW480, HCTl 16, and DLDl cells.
  • PPAR ⁇ protein can be purified from tissues or cell lines, chemically synthesized, or produced recombinantly, for example using the primer pairs shown in SEQ ID NOS:70 and 71 and in SEQ ID NOS:76 and 77 to amplify the human PPAR ⁇ coding sequence in an in vitro transcription-coupled translation system (see Example
  • Measurement of the binding of the PPAR ⁇ protein to the PPAR ⁇ binding element can be carried out using any method known in the art for detecting DNA- protein binding, such as gel electrophoretic mobility shift assays (GEMSA), DNA footprinting, or immunoprecipitation of bound and unbound PPAR ⁇ protein using PPAR ⁇ -specific antibodies.
  • PPAR ⁇ -specific probes for use in GEMSA or footprinting assays preferably comprise a detectable label. Either radiolabels or nonisotopic labels, such as chemiluminescent, fluorescent, or enzymatic labels, can be used.
  • binding can be measured in the presence of known agonists or antagonists of PPAR ⁇ regulated transcription.
  • Suitable antagonists include NSAIDs, such as sulindac, indomethacin, and other COX inhibitors (for a complete list, see Goodman & Gilman's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 9th ed, McGraw Hill, and Cada et al, FACTS AND COMPARISONS, J.B. Lippincott, 1999, including the July 1999 update).
  • NSAIDs such as sulindac, indomethacin, and other COX inhibitors
  • the amount of binding of the PPAR ⁇ protein to the PPAR ⁇ binding element in the presence of the test substance is compared to the amount of binding of the PPAR ⁇ protein to the PPAR ⁇ binding element in the absence of the test substance.
  • the comparison can be quantitative, for example by reference to a standard curve, or qualitative.
  • a test substance which decreases the amount of binding of PPAR ⁇ protein to the PPAR ⁇ binding element is a candidate drug for use in cancer therapy.
  • binding is decreased by at least 25, 50, 75, 85, 90, 95, 97, or 98 percent.
  • a transfected cell containing a Tcf-responsive reporter construct and a PPAR ⁇ protein is contacted with a test substance.
  • the cell can be either stably or transiently transfected.
  • Introduction of reporter constructs can be carried out in culture or in vivo.
  • the transfected cell either lacks wild-type APC or contains a mutant ⁇ -catenin.
  • Appropriate cells are, for example, colorectal cancer cells, present either in situ in a mammalian body or in vitro in a tissue culture preparation.
  • Colorectal cancer cells can be isolated from patients and placed in tissue culture or established colorectal cancer cell lines, such as HT29, SW480, HCTl 16, and DLDl, can be used.
  • Methods of transfecting nucleic acid constructs into cells are well known and include, but are not limited to, transfection with naked or encapsulated nucleic acids, cellular fusion, protoplast fusion, viral infection, and electroporation.
  • the PPAR ⁇ protein can be PPAR ⁇ protein which is either endogenous to the cell or which is added to the cell, for example by transfecting the cell with a nucleic acid construct encoding PPAR ⁇ protein, or both.
  • Expression of the reporter gene can be determined by any method suitable for detecting the assayable product of the particular reporter gene used, including biochemical, immunological, or visual detection methods. Expression of the reporter gene can also be determined by detecting its mRNA, for example using Northern or dot blots or in situ hybridization. A test substance which decreases the amount of expression of the reporter gene is a candidate drug for use in cancer therapy. The decrease in expression of the reporter gene can be determined qualitatively or quantitatively, for example by reference to a standard curve. Preferably, the test substance decreases expression of the reporter gene by at least 25, 50, 75, 85, 90, 95,
  • expression of the reporter gene can be measured in the presence of an agonist or antagonist of PPAR ⁇ regulated transcription.
  • agents are pre-screened for use in cancer therapy by measuring transcription of the reporter gene in the presence of RNA polymerase, ribonucleotides, and PPAR ⁇ protein.
  • the PPAR ⁇ protein can be purified, synthesized chemically, produced recombinantly, or synthesized by an in vitro translation reaction.
  • RNA polymerases and ribonucleotides are readily available commercially. The addition of the RNA polymerase, ribonucleotides, and PPAR ⁇ protein to the reporter construct is effected in the presence and absence of the test substance, and transcription of the reporter gene is determined.
  • transcription can be determined, for example, using Northern or dot blots, or by measuring the assayable product of the reporter gene.
  • a test substance which decreases the amount of transcription of the reporter gene preferably by at least 25, 50, 75, 85, 90, 95, 97, or 98 percent, is a candidate for use in cancer therapy.
  • transcription of the reporter gene can be measured in the presence of a known agonist or antagonist of PPAR ⁇ regulated transcription.
  • the invention also provides methods for identifying candidate drugs for use in
  • a cell having no wild-type APC or which has a mutant ⁇ -catenin is contacted with a test compound, and transcription in the cell of a Tcf-responsive reporter gene is measured.
  • Constructs comprising Tcf-response reporter genes can be introduced into the cells as described above, and the cell can be contacted with the test compound.
  • the Tcf-responsive reporter gene can be contacted with the test compound in a reconstituted in vitro system under conditions in which the reporter gene is transcribed in the absence of the test compound. Conditions which permit in vitro transcription are well known in the art (see Example 1).
  • a cell which has no wild-type APC either produces an APC protein defective in ⁇ -catenin binding or regulation or produces no detectable APC protein at all.
  • Cells which have no wild-type APC include primary colorectal cells isolated from FAP patients or other patients whose colorectal cells bear APC mutations, as well as cell lines such as HT29, SW480, or DLDl.
  • a cell which has mutant ⁇ -catenin produces a ⁇ -catenin protein which is super-active or which is defective in APC binding or which is resistant to APC regulation.
  • Cells which have mutant ⁇ -catenin include primary colorectal cells isolated from FAP patients or other patients whose colorectal cells produce mutant ⁇ -catenin.
  • Other cells which have no wild-type APC or which have mutant ⁇ -catenin can be identified by assaying candidate cells for production of wild- type APC or ⁇ -catenin protein or mRNA, by detecting mutations in APC or ⁇ -catenin coding sequences, or by assaying Tcf-4/ ⁇ -catenin-dependent transcription, using standard molecular biological or immunological techniques.
  • Transcription of the Tcf-responsive reporter gene is measured in the presence of the test compound and compared with transcription of the Tcf-responsive reporter gene in the absence of the test compound.
  • reporter gene mRNA or the encoded assayable product can be measured.
  • a test compound which decreases transcription of the reporter gene is a candidate drug for treating FAP patients, patients with APC or ⁇ -catenin mutations, or patients with increased risk of developing cancer.
  • reporter gene expression is decreased by at least 25, 50, 75, 85, 90, 95, 97, or 98 percent.
  • the invention also provides methods for identifying test compounds which can be used to encourage cell proliferation or to prevent apoptosis of cells which are dying prematurely in a disease state such as Alzheimer's Disease, AIDS, muscular dystrophy, amyotrophic lateral sclerosis, or other muscle wasting diseases, autoimmune diseases, heart attack, stroke, ischemic heart disease, kidney failure, septic shock, or a disease in which the cell is infected with a pathogen, such as a virus, bacterium, fungus, mycoplasm, or protozoan, to promote healing of the stomach or intestines, or to ameliorate negative side effects of NSAIDs, such as gastric and intestinal ulceration.
  • a pathogen such as a virus, bacterium, fungus, mycoplasm, or protozoan
  • PPAR ⁇ agonists can also be used to block harmful effects of NSAJDS.
  • PPAR ⁇ DNA binding activity and PPAR ⁇ -dependent transcription are measured as described above for the methods for screening test compounds as cancer therapeutics.
  • test compounds which increase transcription of PPAR ⁇ protein, PPAR ⁇ protein binding to a PPAR ⁇ binding element, or expression of a reporter gene which is under the control of a PPAR ⁇ binding element are identified as candidates for use in encouraging cell proliferation.
  • HCTl 16, SW480, and DLDl were maintained in McCoy's 5A medium (Life Technologies, MD) supplemented with 10% fetal bovine serum (HyClone, UT), 100 units / ml penicillin, and 100 ⁇ g/ml of streptomycin.
  • Human embryonic kidney cells 293 were maintained in DMEM (Life Technologies) supplemented with 10% fetal bovine serum, 100 units / ml penicillin, and 100 ⁇ g/ml of streptomycin.
  • Sulindac derivatives and indomethacin were purchased from BIOMOL.
  • BRL49653 and cPGI were purchased from American Radiolabeled Chemicals and Cayman Chemical Company, respectively. Unless otherwise indicated, all chemicals were purchased from
  • SAGE Serial Analysis of Gene Expression
  • PPAR ⁇ and residues 1-224 of RXR and cloning them into pGEX-2TK vector As controls, GST fusion proteins containing the DNA-binding domains of human PPAR ⁇ (amino acids 1-249) and PPAR ⁇ (amino acids 1-248) were also constructed. The fusion proteins were produced and purified according to the manufacturer's protocol. To identify the potential consensus DNA sequence motifs recognized by PPAR ⁇ and RXR, a previously described in vitro site selection procedure was utilized. Briefly, for binding to the PPAR ⁇ and RXR proteins, the following oligonucleotide was synthesized: 5 '-TAGTAAACACTCTATCAATTGG(N) 20 TCTAG-
  • AAAGCTTGTCGACGC-3' (SEQ ID NO:51), where "N” represents an equimolar mixture of each nucleotide.
  • N represents an equimolar mixture of each nucleotide.
  • a random duplex pool was generated by PCR amplification with primers that hybridized to the flanking sequences.
  • the fusion proteins were mixed with the random duplex pool and subjected to GEMSA (see below).
  • the ACO probe was formed by annealing S'-GCGGACCAGGACAAAGGTCACGTTC-S 1 (SEQ ID NO:80) and 5'-CGAACGTGACCT ⁇ GTCCTGGTCCG-3' (SEQ ID NO:81).
  • AGCGCTCACAGGTCAATTCGGTGAGCGCTCACAGGTCAATTCG-3' (SEQ ID N O : 5 4 ) a n d 5 ' - C T A GCGAATTGACCTGTGAGCGCTCACCGAATTGACCTGTGAGC-GCTCACG-3' (SEQ ID NO:55).
  • the following oligonucleotides containing a PPAR ⁇ and PPAR ⁇ responsive element from the acyl-CoA oxidase promotor were also synthesized: 5 '-CTAGCGGACCAGGACAAAGGTCACGTTCGGA- CCAGGACAAAGGTCACGTTCG-3' (SEQ ID NO:56) and
  • Reporter plasmid, effector plasmid and ⁇ -gal control plasmid were transfected into cells using LipofectAmine (Life Technologies). Twenty-four hours after transfection, cells were lysed and collected for assays of luciferase activity using Promega' s Luciferase Assay System.
  • 5'-TTTTTTTTAGTACAAGTCCTTGTAGATCTCC-3' (SEQ ID NO:73) for PPAR ⁇ ; a n d 5'-GGATCCTAATACGACTCACTATAGGGAGACCACCATGGACACCAAACAT- TTCCTGCCGC-3' (SEQ ID NO:74) and 5'-TTTTTTTTAAGTCA- TTTGGTGCGGCGCCTCC-3 ' (SEQ ID NO:75) for RXR ⁇ .
  • the full-length proteins were produced according to the manufacturer's protocol.
  • PPAR ⁇ expression was evident as early as 3 hours after APC induction whereas no change was detectable in HT29 ⁇ -Gal cells even 9 hours after induction.
  • expression of PPAR ⁇ was not affected by expression of APC, and the other known PPAR subfamily member, PPAR ⁇ , was not expressed at detectable levels in the presence or absence of wild type APC ( Figure 1A and data not shown).
  • APC inhibits Tcf-41 ⁇ -Catenin Mediated Transcription of the PPAR ⁇ Gene
  • a 3.1-kb genomic fragment containing the region upstream of the PPAR ⁇ transcription start site (GenBank Accession # ) and used it to analyze APC responsiveness ( Figure 2).
  • a luciferase reporter construct containing this fragment (BE) upstream of a minimal promotor was markedly repressed by APC expression ( Figures 2A and 2B).
  • Similar analysis of a series of nested deletions and promotor fragments identified two APC-responsive fragments (Fragment NH and HD, Figures 2A and 2B).
  • fragment NP fragment spanning these two sites with either intact Tcf-4 binding sites (fragment NP) or with alterations predicted to destroy the putative Tcf-4 binding sites (fragment mNP). Fragment NP demonstrated marked APC repression which was completely abrogated by disruption of the putative Tcf-4 binding sites (Figure 2C).
  • either of the putative Tcf-4 binding sites in isolation could confer APC responsiveness in a sequence specific manner (compare TRE1 vs. mTREl and TRE2 vs. mTRE2 in Figure 2C).
  • PPAR response element ACO from the acyl-CoA oxidase gene promotor contains two copies of the core binding sequence AGGTCA separated by one base pair (Juge-Aubry et al, 1997; Mangelsdorf, 1995; Lemberger et al, 1996; Tugwood et al, 1992). PPAR ⁇ and PPAR ⁇ bind this consensus efficiently whereas PPAR ⁇ does not (see below).
  • To define a PPAR ⁇ responsive element we performed in vitro binding site selection for both PPAR ⁇ and RXR. Analysis of 28 binding sites selected with a GST fusion protein containing the DNA binding domain of RXR identified (A/G)GGTCA as the core consensus for RXR ( Figure 3 A).
  • PPAR ⁇ Function is Specifically Regulated by the APC/ ⁇ -Catenin/Tcf-4 Pathway
  • NSAIDs Suppress PPAR ⁇ Activity
  • the effectiveness of NSAIDs at suppressing colorectal tumorigenesis has raised the suspicion that these compounds may somehow be linked to the genetic alterations that drive tumorigenesis in this organ.
  • the identification of PPAR ⁇ as a target of the APC tumor suppressive pathway suggested a specific link.
  • Both precursors and products involved in eicosanoid metabolism have recently been shown to be ligands for PPARs (Forman et al, 1997; Forman et al, 1995; Keller et al, 1993; Kliewer et al,
  • PPAR ⁇ function has been shown to effectively suppress intestinal tumorigenesis in both humans (Giardiello et al, 1993; Labayle et al, 1991; Nugent et al, 1993; Rigau et al, 1991; Thorson et al, 1994; Waddell et al, 1989; Winde et al, 1993; Winde et al, 1995) and mice (Beazer-Barclay et al, 1996; Chiu et al, 1997; Jacoby et al, 1996; Mahmoud et al, 1998), and this inhibition is associated with the induction of apoptosis (Mahmoud et al, 1998; Pasricha et al, 1995).
  • sulindac sulfide the active metabolite of sulindac
  • Sulindac sulfide treatment resulted in a dose-dependent repression of PPAR ⁇ activity in colorectal cancer cells, as assessed with the DRE reporter ( Figure 4C).
  • a similar dose dependent suppression of PPAR ⁇ was observed with indomethacin, another NSATD (data not shown).
  • AdPPAR ⁇ adenovirus
  • GFP green fluorescent protein
  • ⁇ -catenin/Tcf-4 activity leads to increased transcription of growth-promoting genes. Accordingly, restoration of APC function to colorectal cancer cells with defective APC function results in growth suppression and apoptosis (Morin et al, 1996).
  • the genes which have been postulated to mediate the growth-promoting effects of ⁇ -catenin/Tcf-4 activity include those encoded by the c-MYC oncogene (He et al, 1998) and the cyclin DI gene (Tetsu and McCormick, 1999), among others (WISP, c-jun and fra-1) (Mann et al, 1999; Pennica et al, 1998).
  • PPAR ⁇ represents a ⁇ -catenin/Tcf-4 target with particular importance for chemoprevention.
  • APC or ⁇ -catenin mutations can result in increased PPAR ⁇ activity
  • NSAIDs can compensate for this defect by suppressing PPAR ⁇ activity and promoting apoptosis. This suppression of PPAR ⁇ is mediated in part by the ability of some NSAIDs to directly inhibit the DNA binding activity of PPAR.
  • fatty acids and eicosanoids can act as ligands and modifiers of PPAR activity (Forman et al, 1997; Forman et al, 1995; Keller et al, 1993; Kliewer et al, 1995; Kliewer et al, 1997; Prescott and White, 1996; Xu et al, 1999; Yu et al, 1995; and unpublished observations of the inventors), PPAR ⁇ activity might be repressed by the NS AJD-mediated changes in eicosanoid metabolism. This model can help explain several features of NSATD mediated chemoprevention.
  • the sulindac derivative sulindac sulfone which is devoid of COX-inhibitory activity, has apoptotic activity in vitro and chemopreventive activity in vivo when used at high concentrations, and has been proposed as a chemopreventive agent that lacks the toxicity associated with traditional NSAIDs (Mahmoud et al, 1998; Piazza et al, 1997; Piazza et al, 1995).
  • Sulindac sulfone inhibited PPAR ⁇ activity, albeit at higher concentrations that required for sulindac sulfide, consistent with its reduced chemopreventive and apoptosis-promoting activity.
  • the ability of COX2 expression to modulate apoptosis (Tsujii and Dubois, 1995) and intestinal tumorigenesis (Oshima et al, 1996) may be partially related to its ability to alter the spectrum of Ugands for PPAR ⁇ and other PPARs.
  • the PPAR ⁇ ligand cPGI can partially rescue infertility resulting from COX-2 deficiency (Lim et al, 1999).

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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

PPARδ a été identifié comme une cible de la suppression d'APC par l'analyse des profils globaux d'expression génique dans les cellules humaines de cancer colorectal. L'expression de PPARδ est élevée dans les cancers colorectaux primaires, et notoirement réprimés par l'APC dans les cellules de cancer colorectal. Cette répression utilise une médiation de deux éléments réagissant au Tcf-4 dans le promoteur du PPARδ. Les rapporteurs contenant ces éléments réagissant au Tcf-4 sont supprimés par le sulindac, un anti-inflammatoire non stéroïdien (AINS) capable de réduire la taille et le nombre des tumeurs du colon chez l'homme et les autres animaux présentant des mutations APC. En outre, le sulindac est capable d'annihiler spécifiquement l'aptitude du PPARδ à se lier à ses séquences de reconnaissance de cognat. Ces découvertes suggèrent un modèle où les AINS bloquent la carcinogenèse grâce à une modification post-transcriptionnelle d'un gène normalement régulée par l'APC. Cette nouvelle cible moléculaire des AINS convient à la mise au point de nouveaux chimio-préventifs pour les tumeurs colorectales.
EP00957478A 1999-08-16 2000-08-16 Ppar delta relie apc aux medicaments de chimio-prevention Withdrawn EP1203101A1 (fr)

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