Nov 17, 2008 - cancer therapy as well as a paracrine-mediated cell-extrinsic death ... and Genetics; Department of Pathology; Karmanos Cancer Institute; ...
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[Cancer Biology & Therapy 7:12, 2034-2038; December 2008]; ©2008 Landes Bioscience
Research Paper
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TNFSF10 (TRAIL), a p53 target gene that mediates p53-dependent cell death
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Kageaki Kuribayashi,1 Gabriel Krigsfeld,1 Wenge Wang,1 Jing Xu,2 Patrick A. Mayes,1 David T. Dicker,1 Gen Sheng Wu2 and Wafik S. El-Deiry1,* 1Laboratory
of Molecular Oncology and Cell Cycle Regulation; Departments of Medicine (Hematology/Oncology), Genetics and Pharmacology; Institute for Translational Medicine and Therapeutics and the Abramson Comprehensive Cancer Center; University of Pennsylvania School of Medicine; Philadelphia, Pennsylvania USA; 2Program in Molecular Biology and Genetics; Department of Pathology; Karmanos Cancer Institute; Wayne State University School of Medicine; Detroit, Michigan USA
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activation of oncogenic signaling, hypoxia or nucleotide depletion, p53 accumulates in the nucleus in a tetrameric form.2 Upon activation, p53 mediates a growth-suppressive effect on cells by blocking the cell cycle or it can lead the cells to undergo programmed cell death primarily by binding to particular DNA sequences and activating transcription of specific genes.3 In the case of cell cycle arrest, p21 appears sufficient to block cell cycle progression out of G1 until repair has occurred or the cellular stress has been resolved.4 However, the p53-dependent apoptotic response is more complex and involves transcriptional activation of multiple pro-apoptotic target genes, tissue and signal specificity as well as additional events that are less well understood. In death receptor-mediated apoptosis, there are two cell type specific signaling pathways to activate effector caspases leading to cell death, so-called type I and type II pathways.5 After the ligand binds to their cognate receptor, death inducing signaling complex (DISC) forms, which consists of receptor, FADD and caspase-8. In type I (extrinsic) pathway, caspase-8 activation is sufficient to kill cells with subsequent activation of effector caspase-3, 6 and 7. This pathway is independent of mitochondria and is not blocked by overexpression of Bcl-2 or caspase-9 inhibitor. In type II (intrinsic) pathway, activation of caspase-8 is not enough to activate effector caspases and amplifies death signaling via mitochondria. Bid bridges the death signal from DISC to mitochondoria, as it is cleaved by caspase-8, myristoylated and translocates to mitochondoria. Bax and Bak induce mitochondorial outer membrane permeabilization (MOMP), release cytochrome c to the cytosol resulting in caspase-9 and subsequent effector caspase activation. The type II pathway can be blocked by overexpression of Bcl-2, BclXL or caspase-9 inhibitor. There have been many attempts to unravel the essential genes that mediate p53-dependent cell death. It is clear that p53 activates multiple components of the death signaling pathways. We have been interested in whether p53 can activate a death signal that may mediate cell death in a paracrine fashion. In this study, we have identified TNF related apoptosis inducing ligand (TRAIL) as a p53-target gene that may play a central role in mediating p53-dependent cell death. TRAIL is part of the host immune system that suppresses cancer and its metastases through binding of pro-apoptotic TRAIL death receptors DR5 and DR4. TRAIL receptors such as DR5 are directly
Introduction
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We have identified TNFSF10 (TRAIL) as a p53-transcriptional target gene. There are two p53 DNA-binding sites in the human TNFSF10 promoter region, at 346 and 625 bp upstream of the transcription start site. A human p53-expressing adenovirus (Ad-p53) induced TRAIL mRNA and protein expression in HCT116 p53-/- human colon cancer cells. A human TRAILpromoter reporter assay showed increased luciferase activity with the promoter vector that contains two p53 DNA-binding motifs, following Ad-p53 infection, compared to the control adenovirus infection. Using HCT116 cells, gene silencing of TNFSF10 by siRNA suppressed caspase 3 and 7 activity, even after treatment with the DNA-damaging chemotherapeutic agent adriamycin. TRAIL protein expression was elevated in adriamycin-treated breast cancer cells. In vivo, TRAIL expression was induced in mouse natural killer cells at 24 hours after systemic treatment with 5-Fluorouracil. p53-dependent TRAIL induction in natural killer cells after chemotherapy exposure provides a link between the tumor suppressor p53 and the host immune response during cancer therapy as well as a paracrine-mediated cell-extrinsic death response. Our findings provide new mechanistic insights into the signaling of p53-dependent cell death and tumor suppression, including the involvement of the host immune system and natural killer cells in vivo in the anti-tumor efficacy of chemotherapy.
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Key words: p53, TRAIL, apoptosis, p53-dependent cell death
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The p53 pathway plays a critical role in silencing carcinogenesis, as half of human tumors bear inactive p53 caused directly by its mutations and the other half have insufficient p53 functions as a consequence of the protein binding to viral or cellular proteins or as a result of alteration in its regulators.1 p53 function is usually switched off, although when the cells get exposed to stress such as DNA damage induced by ionizing radiation or ultraviolet rays,
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*Correspondence to: Wafik S. El-Deiry; 415 Curie Blvd. CRB 437A; Philadelphia, Pennsylvania 19104 USA; Tel.: 215.898.9015; Fax: 215.573.9139; Email: wafik@ mail.med.upenn.edu Submitted: 11/17/08; Accepted: 11/19/08
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Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/article/7460
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TRAIL promoter regulation by p53 mediates cell death
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Figure 1. TRAIL is a p53 dependent gene. (A) HCT116 and HCT116 p53-/- cells were treated with either ADR or 5FU and subjected for Western blot analysis. TRAIL was induced in a p53-dependent manner. (B) HCT116 p53-/- cells were infected with Ad-GFP or Ad-p53 and subjected for RT-PCR analysis. Asterisk shows the specific band for TRAIL. (C) HCT116 p53-/- cells were infected with Ad-GFP or Ad-p53 and subjected for Western blot analysis. (D) p53-inducible H1299 cells were treated with tetracycline to induce p53 and TRAIL expression was analyzed by RT-PCR (Top). (E) HCT116 cells were treated with ADR or 5FU and TRAIL expression was analyzed by RT-PCR (bottom).
regulated by p53 and so the induction of TRAIL in a p53-dependent manner suggests that p53 not only controls pro-apoptotic receptor expression but also expression of the TRAIL ligand.
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TRAIL is a p53 target gene. We hypothesized that TRAIL may be involved in the apoptotic response triggered by DNA damage. We treated HCT116 cells with 5FU and adriamycin and subjected them to Western blot. As shown in Figure 1A, 5FU and adriamycin treatment induced TRAIL protein expression in a p53-dependent manner. From this result, we further investigated the possibility that TRAIL may be a p53 target gene. There were two potential p53 binding sites in TNFSF10 promoter region, 346 and 625 bp upstream of UTR start site (Fig. 2A). To confirm that TRAIL is a p53 target gene, we transduced HCT116 p53-/- cells with Ad-GFP or Ad-p53. As shown in Figure 1B, TRAIL mRNA was upregulated by p53-induction as compared to the GFP expressing Ad. Western blot also showed that upregulation of p53 by Ad-53 (Fig. 1C). Upregulation of TRAIL mRNA was also seen in H1299 cells after induced p53 expression, or ADR and 5FU treatment of HCT116 cells (Fig. 1D and E). p53 regulates TRAIL gene transcription. We tested TRAIL promoter-reporter constructs for their potential to be regulated by p53. Reporter assay showed that Ad-p53 induced higher luciferase activity than Ad-GFP in pGL3-1102 that contains two p53 binding sites (Fig. 2). To test whether DNA damage induces TRAIL in cells with wild-type p53, we treated MCF-7 with adriamycin and
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Results
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Figure 2. TRAIL is a p53-target gene. (A) Schematic presentation of p53 binding sites of TRAIL promoter region. (B) Schematic presentation of the vetors for TRAIL reporter assay. (C) HCT116 p53-/- cells infected with either Ad-GFP or Ad-p53 and subjected for reporter assay. Actual image is shown on the left. The signals were quantified and shown as bar graph (right). *, statistically significant (p < 0.05); **, statistically not significant (p > 0.05). www.landesbioscience.com
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TRAIL promoter regulation by p53 mediates cell death
Figure 3. Induction of TRAIL by chemotherapeutic agents. (A) Induction of TRAIL and increased p53 phosphorylation at Ser 15 by adriamycin in MCF-7 cells. (B) MCF-10 cells were treated with adraimycin, MS275 or their combination. Induction of TRAIL was determined by Western blot.
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showed that TRAIL was induced by adriamycin, which correlated with p53 phosphorylation (Fig. 3A). Further, we showed that in MCF-10 cells, TRAIL was also induced by adriamycin, the histone deacetylase inhibitor MS275 or their combination (Fig. 3B). To further evaluate the effects of DNA damage on TRAIL reporter gene induction by endogenous p53 we designed assays relying on reporter induction following endogenous p53 stimulation by the DNA damaging agent adriamycin (Fig. 4). We tested the effect of adriamycin at different concentrations and time points on reporter activity in wild-type p53-expressing HCT116 cells, while at the same time evaluating the effect of the drug on cell viability. The results in Figure 4 show a consistent increase in TRAIL promoter
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Figure 4. Endogenous p53 regulates TRAIL promoter activity and this correlates with reduced cell viability in HCT116 cells. HCT 116 Bax-/- cells were transfected with the TRAIL/Luciferase promoter-reporter (PGL3-1102-luc). Cells were exposed to different concentrations of Adriamycin. D-luciferin was used as a substrate for luciferase to measure bioluminescent intensity, which correlates with the TRAIL promoter activity, while the MTT assay determined cell viability. (A) Images of the luminescence from the D-Luciferin and MTT assays are shown at the 12 (top), 24 (middle) and 48 hours (lower) time points. The ratio of luminescence intensity in treated vs. untreated wells were calculated and represented as the mean +/- standard deviation at 12 (B), 24 (C) and 48 hours (D). 2036
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TRAIL promoter regulation by p53 mediates cell death
Discussion
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Figure 5. TRAIL is involved in p53-dependent cell death. (A) HCT116 cells were transfected with siRNA against control or TRAIL siRNA and caspase activity was measured. (B) Balb c nu/nu mice were treated with either vehicle or 10 mg/kg 5FU and the natural for 24 hours and sacrificed. The spleen was dissociated and analyzed by flow cytometry using the antibodies against TRAIL and NK cells.
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In this study, we have identified TRAIL as a p53 target gene that mediates cell death in the p53-dependent DNA damage response. There have been many efforts to deduce the essential genes transducing p53-dependent apoptotic response. We have shown that TRAIL receptor DR5 plays an important role in mediating cell death in vivo in the ionizing radiation response.7 Our study shows that TRAIL mediates cell death in the p53 damage response and the extent of suppression of caspase 3/7 activity was quite prominent after TRAIL gene silencing. p53-null mice are tumor prone. However, among various p53 target genes, none of the single-gene targeted mice have been shown to be tumor prone in a manner similar to p53. We recently observed that TRAIL receptor-/- mice are tumor prone in several situations including exposure to ionizing radiation, in the E-myc background or following exposure to a hepatocarcinogen.8 This pathway is certainly relevant and important with regard to tumor suppression in vivo. Our study reveals a novel mechanism regarding how chemotherapy kills cancer cells. TRAIL was upregulated in HCT 116, MCF-7 and MCF-10 cells by chemotherapeutic drugs and gene silencing of TRAIL conferred resistance to adriamycin in HCT116 cells. These results show that chemotherapeutic drugs induce cell death in cancer cells by inducing TRAIL. There was still some activation of caspase 3/7 activity in TRAIL silenced cells. This activity might result from the activation of other components of the apoptotic pathway by p53. There is a phenomenon named the bystander effect following radiotherapy such that a non-irradiated tumor regresses after radiating of a neighboring tumor.9 We show in our manuscript that TRAIL is a p53 target gene induced by DNA damaging agents 5-FU and ADR. Solubilized TRAIL from an irradiated tumor may mediate a more potent bystander effect in tumors with wild-type versus those that harbor mutant p53 genes. We show that TRAIL was also expressed in NK cells treated with 5FU, in vivo. These natural killer cells might be involved in a more potent role in cancer therapy due to the p53-dependent upregulation of the TRAIL gene. We have reported that myc sensitizes cancer cells to TRAIL treatment by downregulating anti-apoptotic genes.10 It is also reported that amplification of c-myc sensitizes colon cancer to 5FU in the presence of wild-type p53.11 Our study connects these two studies by showing 5FU upregulates TRAIL expression since c-myc can activate p53 and we now show that p53 can upregulate TRAIL expression. The p53-binding motif within the TRAIL promoter region was atypical from that of consensus region, as one of the two motifs showed only 70% similarity. A lower affinity of the site to p53 may allow cells to avoid easy suicide by lack of sufficient TRAIL expression in the absence of a strong DNA damage response. In conclusion, we identified TRAIL as a critical mediator of the p53 response in the apoptotic pathway. This study provides new insights into p53-dependent cell death as well as the mechanism how DNA damaging agents kill cancer cells.
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reporter activity with increasing adriamycin dose and at increasing time points up to 48 hours while at the same time we observed reduction in cell viability. These results further confirmed that the human TRAIL promoter is regulated by p53 and that induction of the TRAIL promoter is associated with loss of cell viability. TRAIL induction contributes to cell death in the p53-dependent DNA damage response. We next investigated whether TRAIL induction may be involved in cell death during the p53-dependent DNA damage response. TRAIL or control siRNA were transduced to HCT 116 cells and they were treated with adriamycin. As shown in Figure 5, control siRNA did not have effect on adriamycin-induced caspase 3/7 activity. However, TRAIL-silenced cells had an increased number of the cells at 24 hours after transfection, showed decreased levels of caspase 3/7 activity and the adriamycin treatment had much less caspase 3/7 activation than the control siRNA. Thus TRAIL siRNA showed a prominent effect in suppressing caspase 3/7 activity following exposure to the potent DNA damaging anti-tumor agent adriamycin. These results show that TRAIL may play a central role in mediating cell death during the p53-dependent DNA damage response. TRAIL is induced in mice treated with 5FU. As TRAIL was induced during the DNA damage response in a p53 dependent manner, we hypothesized that TRAIL may also be induced in natural killer cells after chemotherapy. We treated mice with 5FU and checked TRAIL expression level in NK cells by FACS analysis. As shown in Figure 5B, after 24 hours of 5FU treatment, TRAIL expression in NK cells increased from 0.5% to 3.0%. As 5FU did not alter the number of total NK cells, the result shows that 5FU induced TRAIL expression in NK cells. www.landesbioscience.com
Materials and Methods Cells and cell culture. Human colonic cancer cell line HCT116 was purchased from ATCC. p53-/- HCT116 and Bax-/- HCT116 cells
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with the Xenogen IVIS camera system. Cells were then grown on black 96-well plates with a clear bottom (Corning Incorporated, Corning, NY) at 10,000 cells per well. Images were taken again with the Xenogen IVIS camera system and luminescence intensity was quantified using the Living Image software from Xenogen to select a population with a basal level of luciferase expression. The selected population was then plated on four 96-well plates. Adriamycin, a DNA damaging agent, was added to all plates in concentrations ranging from 0–1 μg/ml. One plate was used for the luciferase assay where D-luciferin was added to each well at 12, 24 and 48 hours time points followed by luminescent intensity measurements. The other three plates were used for the MTT assay (Promega) following the vendor’s instructions. Gene silencing using siRNA and caspase3/7 Glo assay. For transient silencing of the TNFSF10, siRNA were purchased from Santa Cruz Biotechnology. RNA TransPass RI Transfection Reagent (New England BioLabs) was used for transfection of the siRNA, according to the manufacturer’s instruction. On day 1, 7,500/well HCT116 cells were plated in each wells of 96 well plate. On day two, siRNA were transfected and on day three, the cells were treated with 1 mg/ml adriamycin. On day four, caspase 3/7 activity was measured using Caspase-Glo 3/7 Assay (Promega, Madison, WI) according to the manufacturer’s instructions. The luminescent signal was detected, visualized and measured using Xenogen in vivo Imaging System (Xenogen) and shown as bar graphs. Detection of TRAIL expression in natural killer (NK) cells. Balb c nu/nu mice were treated with or without 10 mg/kg 5FU, ip, for 24 hours and sacrificed. The spleens were cut and gently disrupted with a 10-ml syringe plunger in supplemented RPMI 1640 and the cell suspensions were filtered through a 100-μm mesh. They were incubated with FITC anti-mouse pan NK cell antibody (CD49b, a2 integrin, eBioscience, San Diego, CA) and PE anti-mouse TRAIL antibody (CD253, eBioscience). Stained cells were then analyzed using an Epic Elite flow cytometer (Beckman-Coulter). References 1. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000; 408:307-10. 2. Bode AM, Dong Z. Post-translational modification of p53 in tumorigenesis. Nat Rev Cancer 2004; 4:793-805. 3. El-Deiry WS. The role of p53 in chemosensitivity and radiosensitivity. Oncogene 2003; 22:7486-95. 4. El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell 1993; 75:817-25. 5. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 1998; 17:1675-87. 6. Wang W, Takimoto R, Rastinejad F, El-Deiry WS. Stabilization of p53 by CP-31398 inhibits ubiquitination without altering phosphorylation at serine 15 or 20 or MDM2 binding. Mol Cell Biol 2003; 23:2171-81. 7. Finnberg N, Gruber JJ, Fei P, Rudolph D, Bric A, Kim SH, Burns TF, Ajuha H, Page R, Wu GS, Chen Y, McKenna WG, Bernhard E, Lowe S, Mak T, El-Deiry WS. DR5 knockout mice are compromised in radiation-induced apoptosis. Mol Cell Biol 2005; 25:2000-13. 8. Finnberg N, Klein-Szanto AJ, El-Deiry WS. TRAIL-R deficiency in mice promotes susceptibility to chronic inflammation and tumorigenesis. J Clin Invest 2008; 118:111-23. 9. Prise KM, Schettino G, Folkard M, Held KD. New insights on cell death from radiation exposure. Lancet Oncol 2005; 6:520-8. 10. Ricci MS, Kim SH, Ogi K, Plastaras JP, Ling J, Wang W, Jin Z, Liu YY, Dicker DT, Chiao PJ, Flaherty KT, Smith CD, El-Deiry WS. Reduction of TRAIL-induced Mcl-1 and cIAP2 by c-Myc or sorafenib sensitizes resistant human cancer cells to TRAIL-induced death. Cancer Cell 2007; 12:66-80. 11. Arango D, Corner GA, Wadler S, Catalano PJ, Augenlicht LH. c-myc/p53 interaction determines sensitivity of human colon carcinoma cells to 5-fluorouracil in vitro and in vivo. Cancer Res 2001; 61:4910-5.
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were kindly provided by B. Vogelstein (Johns Hopkins University, Baltimore, MD). HCT116, p53-/- HCT116 and Bax-/- HCT116 cells were cultured in McCoy’s5A medium supplemented with 10% FCS and antibiotics in humidified 5% CO2 at 37°C, respectively. MCF-7 and MCF-10 were obtained from Karmanos Cancer Institute and maintained in DMEM-F12 medium supplemented with 0.1 μg/ ml cholera toxin, 10 μg/ml insulin, 0.5 μg/ml hydrocortisone, 0.02 μg/ml epidermal growth factor and 10% horse serum. Construction of adenovirus (Ad) p53. We have created Ad vectors that overexpress p53.6 Briefly, for Ad/GFP-p53 construction, green fluorescent protein (GFP) was fused to the N terminus of p53 and the open reading frame of the fused protein was inserted into pAdTrack-CMV vector. The pAdTrack-CMV constructs were recombined with pAdEasy-1 (Stratagene, La Jolla, CA) in Escherichia coli BJ5183 cells to get recombined Ad plasmid. The pAdTrack-CMV that only expresses GFP without p53 expression was used as a control vector. The plasmids were linearized by Pac I digestion and transfected to 293 cells to obtain Ads. HCT116 cells were infected with lowest amount of MOI that induces nearly 100% of induction of GFP, confirmed by fluorescent microscope. Western blot analysis. Western blot analysis was carried out by standard methods. Anti-Ran antibodies were purchased from Becton Dickinson (Franklin Lakes, NJ). Anti-p53 (DO1) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-TRAIL antibody was purchased from R and D systems (Minneapolis, MN). RT-PCR analysis. HCT116 cells were treated with 5FU, adriamycin, Ad-GFP or Ad-p53 for 16 h. The cells were washed, collected and total RNA was prepared by Trizol LS Reagent (Invitrogen) according to the manufacturer’s instruction. 5'-ATG GCT ATG ATG GAG GTC CA was used as a forward and 5'-TTA GCC AAC TAA AAA GGC CC was used as a reverse primer. PCR condition consists of 95°C 30 sec, 55°C 1 min, 72°C 1 min, for 34 cycles. The samples were separated by 2% agarose gel containing ethidium bromide and the bands were detected by Molecular Imager Gel Documentation System (Bio-Rad Laboratories). TRAIL reporter assay. Promoter regions of TRAIL (-1 to -504, -1 to -1102, -1 to -2100) were cloned into XhoI-HindIII site of pGL3 vectors to make PGL3-504, pGL3-1102, pGL3-2100, respectively. On day 1, 1.5 x 105/well HCT116 p53-/- cells were plated in each wells of six well plate. On day two, the vectors were transfected to the cells using Lipofectamine 2000 according to the manufacturer’s instructions. On day three, the cells were trypsinized and re-seeded with 20 mg/ml of D-luciferin (Gold Biotechnology, St. Louis, MO) in each wells of 96 well plate. The cells were infected with either Ad-GFP or Ad-p53 for 16 hours. After the infection, the luminescent signal was detected, visualized and measured using Xenogen in vivo Imaging System (Xenogen) and the values are shown as bar graphs. TRAIL activity screen. A vector containing the promoter region of TRAIL (-1 to -1102) was cloned with Renilla luciferase (PGL31102-luc vector) to detect TRAIL activity. For experiments in Figure 4, HCT116 Bax-/- cells were plated on tissue culture treated dishes. 24 hours later, cells were transfected with the PGL3-1102-luc vector using Lipofectamine-2000 (Invitrogen) following the vendor’s instructions. Puromycin resistant cells were selected. D-luciferin (1:500 dilution) was added and luminescence images were captured
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