Title: Peroxisome Proliferator-activated Receptor γ Agonists Promote TRAIL-induced Apoptosis by Reducing Survivin Levels via Cyclin D3 Repression and Cell Cycle Arrest
Abstract: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapy that preferentially induces apoptosis in cancer cells. However, many neoplasms are resistant to TRAIL by mechanisms that are poorly understood. Here we demonstrate that human breast cancer cells, but not normal mammary epithelial cells, are dramatically sensitized to TRAIL-induced apoptosis and caspase activation by peroxisome proliferator-activated receptor γ (PPARγ) agonists of the thiazolidinedione (TZD) class. Although TZDs do not significantly alter the expression of components of the TRAIL signaling pathway, they profoundly reduce protein levels of cyclin D3, but not other D-type cyclins, by decreasing cyclin D3 mRNA levels and by inducing its proteasomal degradation. Importantly, both TRAIL sensitization and reduction in cyclin D3 protein levels induced by TZDs are likely PPARγ-independent because a dominant negative mutant of PPARγ did not antagonize these effects of TZDs, nor were they affected by the expression levels of PPARγ. TZDs also inhibit G1 to S cell cycle progression. Furthermore, silencing cyclin D3 by RNA interference inhibits S phase entry and sensitizes breast cancer cells to TRAIL, indicating a key role for cyclin D3 repression in these events. G1 cell cycle arrest sensitizes breast cancer cells to TRAIL at least in part by reducing levels of the anti-apoptotic protein survivin: ectopic expression of survivin partially suppresses apoptosis induced by TRAIL and TZDs. We also demonstrate for the first time that TZDs promote TRAIL-induced apoptosis of breast cancer in vivo, suggesting that this combination may be an effective therapy for cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapy that preferentially induces apoptosis in cancer cells. However, many neoplasms are resistant to TRAIL by mechanisms that are poorly understood. Here we demonstrate that human breast cancer cells, but not normal mammary epithelial cells, are dramatically sensitized to TRAIL-induced apoptosis and caspase activation by peroxisome proliferator-activated receptor γ (PPARγ) agonists of the thiazolidinedione (TZD) class. Although TZDs do not significantly alter the expression of components of the TRAIL signaling pathway, they profoundly reduce protein levels of cyclin D3, but not other D-type cyclins, by decreasing cyclin D3 mRNA levels and by inducing its proteasomal degradation. Importantly, both TRAIL sensitization and reduction in cyclin D3 protein levels induced by TZDs are likely PPARγ-independent because a dominant negative mutant of PPARγ did not antagonize these effects of TZDs, nor were they affected by the expression levels of PPARγ. TZDs also inhibit G1 to S cell cycle progression. Furthermore, silencing cyclin D3 by RNA interference inhibits S phase entry and sensitizes breast cancer cells to TRAIL, indicating a key role for cyclin D3 repression in these events. G1 cell cycle arrest sensitizes breast cancer cells to TRAIL at least in part by reducing levels of the anti-apoptotic protein survivin: ectopic expression of survivin partially suppresses apoptosis induced by TRAIL and TZDs. We also demonstrate for the first time that TZDs promote TRAIL-induced apoptosis of breast cancer in vivo, suggesting that this combination may be an effective therapy for cancer. Several members of the tumor necrosis factor (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; PPARγ, peroxisome proliferator-activated receptor γ; TZDs, thiazolidinediones; TGZ, troglitazone; RSG, rosiglitazone; HMECs, human mammary epithelial cells; PARP, poly(ADP-ribose)polymerase; NCD, nocodazole; WT, wild type; DN, dominant negative; IAP, inhibitor of apoptosis; DISC, death-inducing signaling complex; DR, death receptor. 1The abbreviations used are: TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; PPARγ, peroxisome proliferator-activated receptor γ; TZDs, thiazolidinediones; TGZ, troglitazone; RSG, rosiglitazone; HMECs, human mammary epithelial cells; PARP, poly(ADP-ribose)polymerase; NCD, nocodazole; WT, wild type; DN, dominant negative; IAP, inhibitor of apoptosis; DISC, death-inducing signaling complex; DR, death receptor.-α family of cytokines, including TNF-α, FasL/CD95L, and TNF-related apoptosis-inducing ligand (TRAIL/Apo2L), induce apoptosis in target cells by binding to cell surface receptors and activating caspases (1Ashkenazi A. 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Cell Death Differ. 2003; 10: 66-75Crossref PubMed Scopus (749) Google Scholar).TRAIL is expressed in hepatic natural killer cells and is essential for natural killer cell-mediated tumor surveillance: inhibition of TRAIL with blocking antibodies or germ-line deletion of TRAIL sensitizes mice to carcinogen-induced fibrosarcomas and promotes primary tumor growth and metastasis in experimental models (11Takeda K. Hayakawa Y. Smyth M.J. Kayagaki N. Yamaguchi N. Kakuta S. Iwakura Y. Yagita H. Okumura K. Nat. Med. 2001; 7: 94-100Crossref PubMed Scopus (587) Google Scholar, 12Cretney E. Takeda K. Yagita H. Glaccum M. Peschon J.J. Smyth M. J. Immunol. 2002; 168: 1356-1361Crossref PubMed Scopus (517) Google Scholar). Consistent with its normal physiologic function, recombinant soluble TRAIL potently induces apoptosis in a variety of human cancer cells in vitro and in xenograft carcinomas in vivo (13Ashkenazi A. Pai R.C. Fong S. Leung S. Lawrence D.A. Marsters S.A. Blackie C. Chang L. 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In addition, we show for the first time that TZDs sensitize human breast carcinomas to TRAIL-induced apoptosis in vivo, suggesting that the combination of TZDs and TRAIL may be an effective therapy for cancer.EXPERIMENTAL PROCEDURESCell Culture and Reagents—Human MDA-MB-435, MDA-MB-468, and T47D breast carcinoma cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium (Mediatech, Herndon, VA) supplemented with 4.5 g/liter glucose and 4 mm l-glutamine, 100 units/ml penicillin/streptomycin, and 10% fetal calf serum (Invitrogen, Carlsbad, CA). Human mammary epithelial cells (Cambrex, East Rutherford, NJ) were cultured according to the manufacturer's instructions. Cell cultures were grown in 5% CO2 atmosphere at 37 °C. The PPARγ agonists used in these studies were troglitazone (TGZ) (kindly provided by Dr. Andrea Dunaif, Northwestern University) and rosiglitazone (RSG) (Avandia, GlaxoSmithKline, Research Triangle Park, NC).Expression and Large Scale Purification of Recombinant TRAIL— BL-21 competent cells (Novagen, Madison, WI) were transformed with a pET15b plasmid (Novagen) containing a partial TRAIL cDNA encoding amino acids 95–281 (41Pan G. O'Rourke K. Chinnaiyan A.M. Gentz R. Ebner R. Ni J. Dixit V.M. Science. 1997; 276: 111-113Crossref PubMed Scopus (1545) Google Scholar). Protein expression was induced by adding 2 mm isopropyl-1-thio-β-d-galactopyranoside to exponentially growing bacterial cultures (OD600 0.5–0.7), and His-tagged TRAIL was purified under native conditions using the QIAexpressionist system (Qiagen, Valencia, CA) according to the manufacturer's instructions. Fractions were analyzed by SDS-PAGE and protein assay (Bio-Rad) to determine the amount of purified TRAIL; those fractions with high concentrations of TRAIL were pooled. Aliquots were stored in 10% glycerol at –80 °C.Apoptosis Experiments—Cancer cells were treated with TGZ (0–50 μm) or RSG (0–50 μm) for 48 h and then treated with TRAIL (0–2.5 μg/ml) for 16 additional h. To score apoptosis, nuclei were visualized by staining with Hoescht 33258 (Sigma-Aldrich) as described previously (42Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 43Byun Y. Chen F. Chang R. Trivedi M. Green K.J. Cryns V.L. Cell Death Differ. 2001; 8: 443-450Crossref PubMed Scopus (283) Google Scholar), and the percentage of nuclei with apoptotic condensation or fragmentation was determined. At least 200 nuclei were scored per experiment and all experiments were performed three times; the data are presented as the mean ± S.E. Statistical significance of differences was examined by two-tailed, paired Student's t test. Cell death was also measured by flow cytometry using the Annexin-PE apoptosis detection kit I (BD Biosciences) according to the manufacturer's protocol. Annexin V-positive cells were scored as apoptotic.PPARγ Adenovirus Experiments—Adenoviral constructs expressing wild-type murine PPARγ (Ad-WT PPARγ), dominant-negative murine L466A PPARγ (Ad-DN PPARγ), or control β-galactosidase (Ad-βGal) were described previously (44Park Y. Freedman B.D. Lee E.J. Jameson J.L. Diabetologia. 2003; 46: 365-377Crossref PubMed Scopus (252) Google Scholar). MDA-MB-468 breast cancer cells were infected with Ad-βGal, Ad-WT PPARγ, or Ad-DN PPARγ at 25 plaque-forming units per cell as detailed (44Park Y. Freedman B.D. Lee E.J. Jameson J.L. Diabetologia. 2003; 46: 365-377Crossref PubMed Scopus (252) Google Scholar). Seven hours later, adenovirus-infected cells were treated with vehicle alone, TGZ alone, TRAIL alone, or the combination of TGZ and TRAIL, and apoptosis was scored as described above.Immunoblotting—Cell lysates were prepared in modified RIPA buffer (50 mm Tris, 0.1% SDS, 150 mm NaCl, 0.5% deoxycholate, and 1% Nonidet P-40) and analyzed by immunoblotting as described (45Cryns V.L. Bergeron L. Zhu H. Li H. Yuan J. J. Biol. Chem. 1996; 271: 31277-31282Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The following antibodies were used for immunoblotting: DR4, DR5, DcR2 (Stressgen, Victoria, Canada), tubulin, FLAG, phospho-RB (Sigma-Aldrich), caspase-3, p27, RIP, Bcl-XL, cyclin D3, cyclin A, CDK2, CDK4, FADD, TRADD, DcR1, FLIP, caspase-8, Bak, PARP, p21 (BD Biosciences, San Jose, CA), XIAP and survivin (R&D Systems, Minneapolis, MN), Bcl-2, PPARγ, cyclin D1, cyclin E, and Bax (Santa Cruz Biotechnology, Santa Cruz, CA), and BID (kindly provided by Dr. Honglin Li, Northwestern University). To determine whether inhibition of cyclin D3 expression by TGZ was mediated by the ubiquitin-proteasome pathway, cells were preincubated with vehicle or 50 μm TGZ for 48 h, and then treated with the proteasomal inhibitor epoxomicin (200 nm, EMD Biosciences, San Diego, CA) for an additional 16 h.Real-time RT-PCR—MDA-MB-435 breast cancer cells were treated with vehicle or 50 μm TGZ for 64 h. Total RNA was extracted from these cells with the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. cDNA was prepared using Superscript First-Strand Synthesis System for RT-PCR (Invitrogen). CyclinD3 mRNA and human 18 S rRNA (for normalization) were amplified by real-time PCR using iQ Supermix and an iCycler iQ real-time PCR detection system (Bio-Rad) according to the manufacturer's protocol. Primers and probes for Cyclin D3 and 18 S rRNA were designed with Primer Express TM1.5 software (Applied Biosystems) set at default parameters to select optimized primer and probe sets. The cyclin D3 primers were 5′-TGGTCCTAGGGAAGCTCAAGTG-3′ (forward) and 5′-CTGTAGCACAGAGGGCCAAAA-3′ (reverse). The probe for cyclin D3 was 5′-[FAM]-CTTCATTCTGCACCGGCTCTCTCTGC-[BHQ]-3′. The primers for 18 S rRNA were 5′-CGGCTACCACATCCAAGGAA-3′ (forward) and 5′-GCTGGAATTACCGCGGCT-3′ (reverse). The 18 S rRNA probe was 5′-[FAM]-TGCTGGCACCAGACTTGCCCTC-[Qsy7]-3′. 100 ng of total cDNA, 100 nm probe, and 200 nm primers were used in PCR reactions (40 cycles of 95 °C for 15 s and 60 °C for 1 min).Construction of pSUPER-Cyclin D3 and RNAi Experiments— pSUPER was made as described (46Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3941) Google Scholar) and verified by DNA sequencing. To create pSUPER-cyclin D3 the following oligonucleotides were annealed: 5′-gatccccGACCAGCACTCCTACAGATttcaagagaATCTGTAGGAGTGCTGGTCtttttggaaa-3′ and 5′-agcttttccaaaaaGACCAGCACTCCTACAGATtctcttgaaATCTGTAGGAGTGCTGGTCggg-3′ (the capitalized letters are the cyclin D3 target sequence corresponding to nucleotides 840–858). After annealing, the oligonucleotides were ligated into the corresponding BglII and HindIII sites in pSUPER, and the sequence of the silencing construct was confirmed by automated DNA sequencing. MDA-MB-435 breast carcinoma cells were co-transfected with 0.2 μg of pEGFP-N1 (BD Bioscience) and 0.8 μg of pSUPER-cyclin D3 or pSUPER vector using Lipofectamine Plus reagent (Invitrogen). Forty-eight hours later, GFP-positive cells were sorted by FACS analysis and analyzed for cyclin D1, cyclin D3 and survivin expression by immunoblotting. Alternatively, for apoptosis experiments, MDA-MB 435 cells were co-transfected with pEGFP-N1 and pSUPER-cyclin D3 (or pSUPER vector), and 48 h later, treated with vehicle (phosphate-buffered saline) or 2.5 μg/ml TRAIL for an additional 24 h. GFP-positive cells were identified by immunofluorescence as detailed previously (47Chen F. Kamradt M. Mulcahy M. Byun Y. Xu H. McKay M.J. Cryns V.L. J. Biol. Chem. 2002; 277: 16775-16781Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and nuclei were scored for apoptosis as described under "Apoptosis Experiments."Cell Cycle Blockade—MDA-MB-435 cells were treated with vehicle, double thymidine block (overnight 2 mm thymidine treatment on two consecutive nights with normal growth media between thymidine treatments) or nocodazole (0.1 μg/ml) for 24 h, and cells were then treated with 2.5 μg/ml TRAIL for an additional 8 h. Cell cycle distribution was analyzed by propidium iodide staining for DNA content using flow cytometry, and cell death was measured using the Annexin-PE Apoptosis Detection Kit I (BD Bioscience).Survivin Transfection Experiments—MDA-MB-435 cells were co-transfected with 0.2 μg of pEGFP-N1 (BD Bioscience) and 0.8 μg of pcDNA3 vector or pcDNA3-survivin using Lipofectamine Plus reagent (Invitrogen). 24 h later, transfected cells were treated with TGZ (0–50 μm) for 48 h and then treated with TRAIL (0–2.5 μg/ml) for an additional 16 h. Apoptosis was measured in transfected (GFP-positive) cells by flow cytometry using the Annexin-PE Apoptosis Detection Kit I (BD Bioscience).Xenograft Tumor Experiments—2.5 × 106 MDA-MB-435 breast carcinoma cells were implanted subcutaneously (s.c.) into the mammary fat pads of a single 4–5-week old female athymic nude mouse (Harlan Sprague-Dawley, Madison, WI) and xenograft tumors established. 1-mm3 pieces of the xenograft tumor were then transplanted s.c. into both mammary fat pads of 4–5-week old female athymic nude mice. Two weeks after tumor implantation, mice were randomized into 6 treatment groups (10 mice per group) for 5 weeks: 0.75% methylcellulose vehicle (Sigma-Aldrich), RSG 20 mg/kg/day by oral gavage, RSG 50 mg/kg/day by oral gavage, TRAIL 5 mg/kg/day intraperitoneally, RSG 20 mg/kg/day orally + TRAIL 5 mg/kg/day intraperitoneal, or RSG 50 mg/kg/day orally + TRAIL 5 mg/kg/day intraperitoneal. Tumors were measured weekly with Vernier calipers, and tumor volume was calculated using the equation: tumor volume (mm3) = (length × width2) × π /6. To determine the effect of these treatments on the induction of apoptosis, female athymic nude mice with established MDA-MB-435 xenograft tumors were treated for 2 weeks with vehicle, RSG 50 mg/kg/day by oral gavage, TRAIL 10 mg/kg/day intraperitoneal, or RSG and TRAIL. Xenograft tumors were fixed in formalin, embedded in paraffin, and sectioned by standard methods. Apoptotic nuclei were identified by TUNEL assay (in Situ Cell Death Detection kit, TMR Red, Roche Applied Science) according to the manufacturer's instructions. Tumors were also analyzed for cyclin D3 and survivin levels as described in "Immunoblotting." All procedures involving animals were approved by the Animal Care and Use Committee of Northwestern University.RESULTSPPARγ Agonists Sensitize Human Breast Cancer Cells, but Not Normal Human Mammary Epithelial Cells, to TRAIL-induced Apoptosis in Vitro—To examine whether PPARγ agonists might restore the sensitivity of TRAIL-resistant human cancer cells to TRAIL-induced apoptosis, we incubated estrogen receptor (ER)-negative human MDA-MB-435 breast carcinoma cells with the synthetic PPARγ agonist troglitazone (TGZ, 0–50 μm) for 48 h and then added TRAIL (0–2.5 μg/ml) for an additional 16 h. MDA-MB-435 cancer cells were highly resistant to TRAIL alone: <10% of these cells were apoptotic after treatment with 2.5 μg/ml TRAIL (Fig. 1A). Similarly, TGZ alone (at concentrations ≤50 μm) did not induce apoptosis in these cancer cells. However, TGZ (50 μm but not 10 μm) dramatically sensitized MDA-MB-435 cells to TRAIL-induced apoptosis, with >60% apoptosis induction in cells treated with 50 μm TGZ and 2.5 μg/ml TRAIL. Similar results were observed when apoptosis was measured by annexin V labeling (Fig. 1B). TGZ also sensitized T47D and MDA-MB-468 breast cancer cells to TRAIL (data not shown). A second synthetic PPARγ agonist, rosiglitazone (RSG), also acted synergistically with TRAIL to promote apoptosis in human T47D breast cancer cells, an ER-positive, TRAIL-resistant breast cancer cell line (Fig. 1C). Like TGZ, RSG did not induce apoptosis when used as a single agent, but sensitized T47D breast cancer cells to TRAIL-induced apoptosis (some synergy was seen with 10 μm RSG). Taken together, these findings indicate that TZDs promote TRAIL-induced apoptosis in multiple TRAIL-resistant breast cancer cell lines.Because much of the enthusiasm for TRAIL stems from its ability to preferentially indu