TRAIL Promoter Research Articles

C-Myc Modulation and Acetylation Is a Key HDAC Inhibitor Target in Cancer.

Purpose: Histone deacetylase inhibitors (HDACi) are promising anticancer drugs. Although some HDACi have entered the clinic, the mechanism(s) underlying their tumor selectivity are poorly understood.Experimental Design and Results: Using gene expression analysis, we define a core set of six genes commonly regulated in acute myeloid leukemia (AML) blasts and cell lines. MYC, the most prominently modulated, is preferentially altered in leukemia. Upon HDACi treatment, c-Myc is acetylated at lysine 323 and its expression decreases, leading to TRAIL activation and apoptosis. c-Myc binds to the TRAIL promoter on the proximal GC box through SP1 or MIZ1, impairing TRAIL activation. HDACi exposure triggers TRAIL expression, altering c-Myc-TRAIL binding. These events do not occur in normal cells. Excitingly, this inverse correlation between TRAIL and c-Myc is supported by HDACi treatment ex vivo of AML blasts and primary human breast cancer cells. The predictive value of c-Myc to HDACi responsiveness is confirmed in vivo in AML patients undergoing HDACi-based clinical trials.Conclusions: Collectively, our findings identify a key role for c-Myc in TRAIL deregulation and as a biomarker of the anticancer action of HDACi in AML. The potential improved patient stratification could pave the way toward personalized therapies. Clin Cancer Res; 23(10); 2542-55. ©2016 AACR.

Role of PU.1 Down-Regulation for the Development of Primary Effusion Lymphoma

Introduction: Primary effusion lymphoma (PEL) is a subtype of aggressive non-Hodgkin lymphoma that shows serous lymphomatous effusion without tumor masses in body cavities especially in advanced acquired immunodeficiency syndrome (AIDS). PEL is universally associated with Kaposi sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) infection. The prognosis of PEL is extremely poor due to its aggressiveness and resistance to conventional chemotherapy. Therefore, the understanding of the molecular pathogenesis of PEL is vital to develop novel therapeutic strategies. PU.1, one of the Ets family transcription factors, has been reported to be down-regulated in some B-cell malignancies and acute myeloid leukemia. In this study, we examined the methylation of the PU.1 promoter and enhancer regions in PEL cell lines and performed the restoration of PU.1 in PEL cells in vitro and in vivo.Methods:PU.1 mRNA expressions of eight PEL cell lines were examined by polymerase chain reaction (PCR). PU.1 was conditionally expressed using tet-on system in three PEL cell lines. The bisulfite sequencing was performed to analyze the methylation of the PU.1 promoter and enhancer regions in PEL cells. The DNA methyltransferase inhibitor, 5-Azacytidine, was used as an experimental tool for demethylation. The anti-proliferative activities of restoring PU.1 or 5-Azacytidine in PEL cell lines were measured by the methylthiotetrazole method. Apoptosis was quantified using Annexin V antibody. To evaluate the association of PU.1 with TRAIL promoter, we analyzed its recruitment to the promoter by luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. BCBL-1 cells that expressed tet-inducible PU.1 were injected intraperitoneally into NOD/Rag-2/Jak3double-deficient mice to assess the in vivo efficacy of restoring PU.1. Organ invasion by BCBL-1 cells was evaluated by immunohistochemistry.Results: The expression of PU.1 mRNA in PEL cell lines was weaker than in the human monoblast leukemia cell line, U937, which expresses PU.1 mRNA. The promoter and enhancer regions of the PU.1 gene were highly methylated in PEL cells. The treatment with 5-Azacytidine up-regulated the expression of PU.1 mRNA. Restoration of PU.1 or treatment with 5-Azacytidine inhibited cell proliferation. PU.1 up-regulated TRAIL gene expression among apoptosis-related genes and caused apoptosis. PU.1 induced the transactivation of TRAIL promoter in the reporter assay. Disruption of the PU.1 binding site repressed its transactivation. PU.1 was recruited to TRAIL promoter in the ChIP assay. Although PU.1 induced the master regulator of viral replication, ORF50 expression, the expression of ORF57, which is essential for virus production as a posttranscriptional activator, decreased. PU.1 suppressed ORF 57 by the occupancy of IRF7 in the ORF57 promoter region. Restoration of PU.1 inhibited the ascites formation and organ invasion significantly in vivo compared with control mice.Conclusions: The up-regulation of PU.1 induces growth inhibition and apoptosis via the direct transactivation of TRAIL and modulates viral transactivation via the up-regulation of IRF7. PU.1 is a tumor suppressor in PEL and its down-regulation has a critical role in PEL development. The targeting of PU.1 could be a therapeutic approach in patients with PEL. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2754: Induction of Sp1 factor by CYP1B1 inhibits TRAIL-mediated apoptotic pathway

Abstract Human cytochrome P450 1B1 (CYP1B1) belongs to the CYP1 family and is known as a major enzyme for estradiol 4-hydroxylation. It has been reported that CYP1B1 expression is higher in the tumor tissues than the normal ones, especially in hormone-related cancers such as breast, ovarian and prostate. Previously, we identified that CYP1B1 induces cell proliferation by activation of Wnt/β-catenin signal pathway. To explore the role of CYP1B1 on cancer cell proliferation further, we investigated whether CYP1B1 blocks apoptotic pathways. Using Western blot and RT-PCR, the expression of TRAIL was examined in MCF-7 cells, and we found that CYP1B1 overexpression or treatment with a CYP1B1 inducer, 7, 12-dimethylbenz[α]anthracene (DMBA) caused suppression of TRAIL and its receptors (DR4 and DR5) expression. However, when cells were treated with CYP1B1 siRNA or a selective CYP1B1 inhibitor, tetramethoxystilbene (TMS), expression of TRAIL and its receptors were significantly increased. Sp1, a zinc finger-containing transcription factor, has been reported to suppress TRAIL gene expression by binding TRAIL promoter. Interestingly, CYP1B1 overexpression and DMBA induced Sp1 expression and TMS was able to prevent DMBA-induced Sp1 expression. Sp1 knockdown with siRNA suppressed c-FLIP, an inhibitor protein of TRAIL-mediated apoptosis while it enhanced TRAIL and DR4 expression. These results were reversed by Sp1 overexpession. Moreover, attenuation of Sp1 DNA binding activity using mithramycin A, a Sp1 binding inhibitor, resulted in recovery of TRAIL expression suppressed by DMBA. Taken together, these data suggest that CYP1B1 prevents apoptosis via TRAIL suppression and this is mediated by Sp1 induction. Citation Format: Yeo-Jung Kwon, Nahee Park, Sangyun Shin, Dong-Jin Ye, Mihye Hong, Young-Jin Chun. Induction of Sp1 factor by CYP1B1 inhibits TRAIL-mediated apoptotic pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2754. doi:10.1158/1538-7445.AM2014-2754

Survival factor NFIL3 restricts FOXO-induced gene expression in cancer

Depending on the circumstance, FOXO (Forkhead O) (FOXO1, FOXO3, and FOXO4) transcription factors activate the expression of markedly different sets of genes to produce different phenotypic effects. For example, distinct FOXO-regulated transcriptional programs stimulate cell death or enhance organism life span. To gain insight into how FOXOs select specific genes for regulation, we performed a screen for genes that modify FOXO activation of TRAIL, a death receptor ligand capable of inducing extrinsic apoptosis. We discovered that the bZIP transcriptional repressor NFIL3 (nuclear factor interleukin 3-regulated) hindered FOXO transcription factor access to chromatin at the TRAIL promoter by binding to nearby DNA and recruiting histone deacetylase-2 (HDAC2) to reduce histone acetylation. In the same manner, NFIL3 repressed expression of certain FOXO targets--e.g., FAS, GADD45α (growth arrest and DNA damage-inducible, α), and GADD45β--but not others. NFIL3, which we found to be overexpressed in different cancers, supported tumor cell survival largely through repression of TRAIL and antagonized hydrogen peroxide-induced cell death. Moreover, its expression in cancer was associated with lower patient survival. Therefore, NFIL3 alters cancer cell behavior and FOXO function by acting on chromatin to restrict the menu of FOXO target genes. Targeting of NFIL3 could be of therapeutic benefit for cancer patients.

AML1/ETO sensitizes via TRAIL acute myeloid leukemia cells to the pro-apoptotic effects of hypoxia

We determined the effects of severe hypoxia (∼0.1% O2) on acute myeloid leukemia cells expressing the AML1/ETO oncogene. Incubation of Kasumi-1 cells in hypoxia induced growth arrest, apoptosis and reduction of AML1/ETO protein expression. The conditional expression of AML1/ETO in U937-A/E cells showed that hypoxia induces marked apoptosis in AML1/ETO-expressing cells only, pointing to AML1/ETO as a factor predisposing cells to hypoxia-induced apoptosis. In AML1/ETO-expressing cells, hypoxia enhanced TRAIL expression and its proapoptotic effects. AML1/ETO was found to bind TRAIL promoter and induce TRAIL transcription, although TRAIL expression was restrained by a concomitant relative transcription block. In hypoxia, such a TRAIL repression was removed and an increase of TRAIL expression was induced. Finally, blocking anti-TRAIL antibodies markedly reduced (Kasumi-1 cells) or completely inhibited (U937-A/E cells) hypoxia-induced apoptosis. Taken together, these results indicated that hypoxia induces apoptosis in AML1/ETO-expressing cells via a TRAIL/caspase 8-dependent autocrine loop and that TRAIL is a key regulator of hypoxia-induced apoptosis in these cells.

Abstract 1935: Potent anti-tumor effects of TIC10 require Foxo3a and TRAIL gene upregulation

Abstract TNF-related apoptosis-inducing ligand (TRAIL; Apo2L) is an endogenous protein that induces tumor-selective apoptosis and is currently being tested in clinical trials as an anti-tumor agent. Despite promising preclinical activity, recombinant human TRAIL suffers from efficacy-limiting properties such as short serum half-life, stability, cost, and limitations in delivery, e.g. to the central nervous system. We hypothesized that induction of the human TRAIL gene by small molecules may overcome these limitations of TRAIL-based protein therapy. A cell-based luciferase reporter screen using the human TRAIL promoter identified TIC10 as a p53-independent small molecule inducer of the TRAIL gene. TIC10 induces TRAIL on the surface of normal and tumor cells, in sera of non-tumor-bearing mice, and throughout the tumor microenvironment. Co-culture experiments suggest that TRAIL upregulation by normal cells can contribute to the anti-tumor response of TIC10 through a TRAIL-mediated bystander effect. TIC10-induced TRAIL causes tumor-specific and TRAIL-mediated cell death as indicated by inhibition of cytotoxic effects following TRAIL gene knockdown in vitro and in vivo. Expression profiling of TIC10-induced transcriptional changes revealed altered expression levels of Foxo3a target genes, including DR5. We found that TIC10 causes the nuclear translocation of Foxo3a, but not Foxo1, to upregulate TRAIL gene transcription as determined by chromatin-immunoprecipitation assays. Western blot analysis indicated that TIC10 potently inhibits the phosphorylation of Foxo3a at Ser253 and Ser294 that normally docks the transcription factor to 14-3-3 proteins in the cytosol. Dual inhibition of Akt and ERK by TIC10, by other individual small molecule kinase inhibitors in combination or targeted siRNA inhibited the phosphorylation of these critical serine residues and synergistically induced Foxo3a-dependent TRAIL and TRAIL-mediated cell death. TIC10 is a promising first-in-class anti-tumor agent with a broad range of anti-tumor effects in preclinical animal models including othrotopic GBM, immunocompetent lymphoma, xenografted colon, lung, or breast cancers, and exhibits synergy with taxanes as well as bevacizumab among other agents. TIC10 appears to have no measurable toxic effects in mice including evaluation of serum chemistries, liver function studies, LDH, tissue histology, weight of the animals or behavioral changes. We are pursuing the clinical translation of these important findings that indicate a favorable and wide therapeutic index for a novel anti-tumor agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1935. doi:1538-7445.AM2012-1935

Transformation-Dependent Silencing of Tumor-Selective Apoptosis-Inducing TRAIL by DNA Hypermethylation Is Antagonized by Decitabine

TNF-related apoptosis-inducing ligand (TRAIL) kills tumor cells selectively. We asked how emerging tumor cells escape elimination by TRAIL and how tumor-specific killing by TRAIL could then be restored. We found that TRAIL expression is consistently downregulated in HRAS(G12V)-transformed cells in stepwise tumorigenesis models derived from four different tissues due to DNA hypermethylation of CpG clusters within the TRAIL promoter. Decitabine de-silenced TRAIL, which remained inducible by interferon, while induction of TRAIL by blocking the HRAS(G12V)-activated mitogen-activated protein kinase pathway was subordinated to epigenetic silencing. Decitabine induced apoptosis through upregulation of endogenous TRAIL in cooperation with favorable regulation of key players acting in TRAIL-mediated apoptosis. Apoptosis induction by exogenously added TRAIL was largely increased by decitabine. In vivo treatment of xenografted human HRAS(G12V)-transformed human epithelial kidney or syngenic mice tumors by decitabine blocked tumor growth induced TRAIL expression and apoptosis. Our results emphasize the potential of decitabine to enhance TRAIL-induced apoptosis in tumors and thus provide a rationale for combination therapies with decitabine to increase tumor-selective apoptosis.

Type I IFNs signaling and apoptosis resistance in glioblastoma cells

Deletion of type I IFN genes and resistance to apoptosis induced by type I IFNs are common in glioblastoma. Here we have investigated the importance of the constitutive weak IFN-signaling in the apoptotic response to IFN-α in glioblastoma cells. U87MG cells hold a deletion of type I IFN genes, whereas in T98G cells the spontaneous IFN signaling is intact. In response to IFN-α U87MG cells produce much less TRAIL, while other IFN-inducible genes were efficiently up-regulated. Alterations in TRAIL promoter sequence and activity were not observed. DNA methylation can influence TRAIL transcription but without overt differences between the two cell lines. We also discovered that TRAIL mRNA stability is influenced by IFN-α, but again no differences can be appreciated between the two cell lines. By silencing IFNAR1 we provide evidences that the spontaneous IFN signaling loop is required to sustain elevated levels of TRAIL expression, possibly through the regulation of IRF-1. Despite the presence/absence of the constitutive IFN signaling, both cell lines were resistant to IFN-α induced apoptosis. Targeting the deisgylase USP18 can overcome resistance to IFN-induced apoptosis only in T98G cells. Alterations in elements of the extrinsic apoptotic pathway, such as Bid and c-FLIP contribute to apoptotic resistance of U87MG cells. Down-regulation of USP18 expression together with the induction of ER-stress efficiently restored apoptosis in U87MG cells. Finally, we demonstrated that the BH3-only protein Noxa provides an important contribution in the apoptotic response to ER-stress in USP18 silenced cells.

NFATc1 Regulation of TRAIL Expression in Human Intestinal Cells

TNF-related apoptosis-inducing ligand (TRAIL; Apo2) has been shown to promote intestinal cell differentiation. Nuclear factor of activated T cells (NFAT) participates in the regulation of a variety of cellular processes, including differentiation. Here, we examined the role of NFAT in the regulation of TRAIL in human intestinal cells. Treatment with a combination of phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187 (Io) increased NFAT activation and TRAIL expression; pretreatment with the calcineurin inhibitor cyclosporine A (CsA), an antagonist of NFAT signaling, diminished NFAT activation and TRAIL induction. In addition, knockdown of NFATc1, NFATc2, NFATc3, and NFATc4 blocked PMA/Io increased TRAIL protein expression. Expression of NFATc1 activated TRAIL promoter activity and increased TRAIL mRNA and protein expression. Deletion of NFAT binding sites from the TRAIL promoter did not significantly abrogate NFATc1-increased TRAIL promoter activity, suggesting an indirect regulation of TRAIL expression by NFAT activation. Knockdown of NFATc1 increased Sp1 transcription factor binding to the TRAIL promoter and, importantly, inhibition of Sp1, by chemical inhibition or RNA interference, increased TRAIL expression. These studies identify a novel mechanism for TRAIL regulation by which activation of NFATc1 increases TRAIL expression through negative regulation of Sp1 binding to the TRAIL promoter.

Abstract 267: E2F1 regulates human TRAIL promoter and mediates Interferon alpha 2a response

Abstract The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L) is a member of the TNF gene superfamily that induces apoptosis upon engagement of cognate death receptors. While TRAIL is relatively non-toxic to normal cells, it selectively induces apoptosis in many transformed cells and has been explored as an anti-cancer agent. Earlier studies have shown the upstream region of human TRAIL gene, contain NFAT, AP-1, and Sp1 binding sites which have been shown to regulate expression of multiple cytokines, but the transcriptional regulation of TRAIL has not yet been elucidated in detail. An examination of the human TRAIL promoter showed the presence of five binding sites for the E2F transcription factors. E2F1 is a transcription factor that plays a well-documented role in cell cycle regulation, differentiation and senescence. In addition to its proliferative effects, E2F1 has been shown to have potent apoptotic properties as well, under appropriate conditions. E2F1 can induce apoptosis via several p53-dependent or -independent mechanisms, either through direct transactivation of proapoptotic genes such as p73, apaf-1 (apoptosis protease-activating factor 1) and caspases or transcriptional repression. Studies presented here show that the human TRAIL promoter is E2F responsive in multiple lung cancer cell lines. Chromatin immunoprecipitation assays showed the recruitment of E2F1 as well as Rb to the TRAIL promoter and transient transfection of E2F1 could induce the expression of a TRAIL-luciferase reporter construct. TRAIL could be repressed by Rb, but this repression could be overcome by the transient transfection of ASK-1, Raf-1 or p38 kinases as well as cycling D or E, suggesting that this promoter is responsive to extracellular signals that can regulate Rb function. It has been shown that stimulation of natural killer cells by Interferon alpha-2a leads to upregulated TRAIL expression. We find that Interferon alpha 2a (IFN α 2a) stimulated lung cancer cell lines exhibited high TRAIL luciferase activity and depletion of E2F1 by siRNA techniques prevented the induction of TRAIL. These results suggest that E2F1 as a critical regulator of TRAIL expression and might facilitate death-receptor-mediated apoptosis. These observations raise the possibility that E2F-mediated regulation of TRAIL might have effects on the immune response of lung cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 267. doi:10.1158/1538-7445.AM2011-267

Functional implication of TRAIL −716 C/T promoter polymorphism on its in vitro and in vivo expression and the susceptibility to sporadic breast tumor

Recently, TRAIL function has been elucidated beyond its known classical role of mediating cellular homeostasis and immune surveillance against transformed cells. Here, we show how CC genotype of -716 TRAIL promoter SNP rendered risk for sporadic breast cancer as compared to the CT and TT genotypes (P (recessive model) = 0.018, OR = 1.4, 95% CI = 1.1-1.9; P (allele model) = 0.010, OR = 1.3, 95% CI = 1.1-1.7). The in silico prediction of the introduction of core Sp1/Sp3-binding motif suggested the functional significance of the SNP variation. This functional implication was validated by luciferase assay in HeLa (P = 0.026), MCF-7 (P = 0.022), HepG2 (P = 0.024), and HT1080 (P = 0.030) cells and also by real-time expression studies on tumor tissues (P = 0.01), revealing the transcriptionally repressed status of -716 T when compared to -716 C allele. The SNP-SNP interactions reflected an enhanced protective effect of CT and TT genotypes with the protective genetic backgrounds of TP53-BRCA2 (P = 0.002, OR = 0.2, 95% CI = 0.1-0.6), IFNG (P = 0.0000002, OR = 0.3, 95% CI = 0.2-0.4), and common variant Casp8 (P = 0.0003, OR = 0.5, 95% CI = 0.3-0.7). Interestingly, a comparison with clinical parameters showed overrepresented CT and TT genotypes in progressing (P = 0.041) and ER/PR negative tumors (P = 0.024/0.006). This was explained by increased apoptotic index, calculated as a ratio of selected pro-apoptotic and anti-apoptotic gene expression profiles, in CC genotyped tumors, favoring either intrinsic (P = 0.008,0.018) or extrinsic (P = 0.025,0.217) pathway depending upon the ER/PR status. Our study reveals for the first time that a promoter SNP of TRAIL functionally modulates the gene and consequently its role in breast cancer pathogenesis, cautioning to consider the -716 TRAIL SNP status in patients undergoing TRAIL therapy.

Abstract 5467: Breflate upregulates TRAIL gene transcription in Bax-null cells and induces TRAIL-mediated apoptosis independently of p53 in TRAIL-sensitive tumor lines

Abstract TNF-related apoptosis-inducing ligand (TRAIL) has been hailed as a promising cancer drug target due to its apoptosis-inducing capabilities which are aimed selectively at cancer cells. Methods of inducing endogenous TRAIL as well as introducing exogenous recombinant human TRAIL (rhTRAIL) are being explored in preclinical studies and in the case of the latter, clinical trials. While the gene encoding for DR5, a TRAIL death receptor, has been known to be regulated by p53, we have recently shown that TRAIL is regulated by p53 at base pairs 504-1102 of its promoter (Kuribayashi et al., Cancer Biology and Therapy. 7:2034-2038, 2008). p53 is mutated in over half of all cancers; therefore, a method of upregulating endogenous TRAIL via a mechanism independent of p53 holds great potential therapeutic value for cancer. We searched for small molecule p53-independent upregulators of TRAIL using a luciferase reporter assay which utilized a plasmid with the gene encoding for firefly luciferase under the transcriptional control of the first 504 base pairs of the human TRAIL promoter. Screening with the NCI Diversity Set II library yielded several hits with >2-fold TRAIL induction which were repeated and further analyzed for use as a drug and to gain mechanistic insight. Interestingly, one hit from this screen was breflate, the prodrug of Brefeldin A (BFA). BFA inhibits retrograde protein transport from the Golgi apparatus to the endoplasmic reticulum and is currently being investigated as a cancer therapeutic. Shao et al. (Exp. Cell Res. 227:190-196, 1996) have previously reported that BFA is a potent inducer of apoptosis in some cancer cell lines independently of p53, though the mechanistic relationship was not clear. Breflate clearly increased intracellular levels of TRAIL in HCT116 Bax−/− cells as determined by Western blot analysis, and in addition to the original reporter assay this is being corroborated by reverse transcriptase polymerase chain reaction (RT-PCR). Moreover, sub-G1 analysis found that breflate induced levels of apoptosis >2-fold relative to control levels at 10μM after 24 hours in HCT116 p53−/− cells. Further mechanistic and ongoing in vivo studies are being conducted with breflate alone and in combination with chemotherapy or targeted cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5467.

Conditionally Expressed PU.1 Transactivates TRAIL Gene and Induces Apoptosis in Myeloma Cell Lines.

PU.1 is an Ets family transcription factor, which is important for differentiation of both myeloid and lymphoid lineages. In the Friend leukemia model, the failure of PU.1 down-regulation in erythroblasts reportedly results in differentiation arrest, leading to erythroleukemia. In mice conditionally knocked-out of the 3.5 kb length of enhancer region located in14 kb 5′ upstream of the PU.1 gene, PU.1 is down-regulated in myeloid cells and B cells to 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like diseases. Since the 3.5 kb enhancer region contains a suppressor region for PU.1 expression in T cells, such mice ectopically express PU.1 in T cells and develop T cell lymphoma. Thus, the failure of proper expression of PU.1 in certain differentiation stages for certain cell lineages appears to result in hematological malignancies.We recently reported that human PU.1 is down-regulated in a majority of myeloma cell lines through the methylation of the promoter and enhancer region located in17 kb 5′ upstream of the PU.1 gene which is homologous to that in14 kb 5′ upstream of murine PU.1 gene. Conditionally expressed PU.1 induced cell growth arrest and apoptosis of two PU.1 low-negative myeloma cell lines, U266 and KMS12PE, suggesting that the down-regulation of PU.1 is essential for myeloma cell growth. We have also reported that PU.1 is expressed in normal plasma cells and PU.1 is down-regulated in myeloma cells of certain myeloma patients, who appear to have poor prognosis. In the present study, to elucidate the mechanisms of the cell growth arrest and apoptosis in PU.1-conditionally expressing myeloma cells, we performed DNA microarray analysis to compare gene expression levels before and after PU.1 expression, utilizing Illumina Sentrix® Human-6 Expression BeadChip. Of 47,296 genes, 479 genes were up-regulated (>2fold) and 1,697 genes down-regulated (<0.5 fold), either 1 or 3 days after PU.1 expression in U266 cells. Among cell-cycle related genes, p21WAF1/CIP1 was found up-regulated in U266 cells, which was confirmed at protein levels. Among apoptosis related genes, TRAIL was highly up-regulated in both U266 and KMS12PE cell lines. Stably expressed siRNA for TRAIL inhibited apoptosis of PU.1-expressing U266 cells, suggesting that TRAIL may have a crucial role in the PU.1- induced apoptosis. We subsequently examined how TRAIL was up-regulated in such PU.1-expressing myeloma cells. We performed chromatin immunoprecipitation assay and found that PU.1 directly binds to the promoter region of the TRAIL gene in U266 cells. We also determined the PU.1 binding site using electrophoretic mobility shift assays. An introduction of mutations in the PU.1 binding site abolished the binding. We also subcloned the TRAIL promoter and exon1 5′ non-coding sequence into pGL4.26 vector and performed reporter assays. Induction of PU.1 by removal of tetracycline in U266 cells induced 4-fold up-regulation of luciferase activity compared to that without PU.1 expression (28.7-fold compared to that with pGL4.26 vector alone) and mutations in the PU.1 binding site completely abolished the up-regulation. Taken together, we hereby conclude that PU.1 can directly transactivate the TRAIL gene in myeloma cells, leading to apoptosis.

Radiosensitization of melanoma cells through combined inhibition of protein regulators of cell survival

The incidence of melanoma continues to dramatically increase in most Western countries with predominantly Caucasian populations. However, only limited therapies for the metastatic stage of the disease are currently available. The main purpose of this study is to determine approaches that can substantially increase radiosensitivity of melanoma cells. The PI3K-AKT, NF-kappaB and COX-2 pathways, which are involved in the radioprotective response, are highly active in melanoma cells. Pharmacological suppression of COX-2 and PI3K-AKT, or RNAi-mediated knockdown of COX-2, substantially increased levels of G2/M arrest of the cell cycle and decreased clonogenic survival of gamma-irradiated melanomas, predominantly via a necrotic mechanism. On the other hand, resveratrol, a polyphenolic phytoalexin, selectively targets numerous cell signaling pathways, decreasing clonogenic survival primarily via an apoptotic mechanism. In melanoma cells, resveratrol inhibits STAT3 and NF-kappaB-dependent transcription, culminating in suppression of cFLIP and Bcl-xL expression, while activating the MAPK- and the ATM-Chk2-p53 pathways. Resveratrol also upregulates TRAIL promoter activity and induces TRAIL surface expression in some melanoma cell lines, resulting in a rapid development of apoptosis. Sequential treatment of melanoma cells, first with gamma-irradiation to upregulate TRAIL-R surface expression, and then with resveratrol to suppress antiapoptotic proteins cFLIP and Bcl-xL and induce TRAIL surface expression, had dramatic effects on upregulation of apoptosis in some melanoma lines, including SW1 and WM35. However, for melanoma lines exhibiting suppressed translocation of TRAIL to the cell surface, a necrotic mechanism of cell death was primarily involved in radiation response. Hence, surface expression of TRAIL induced by resveratrol appears to be a decisive event, one which determines an apoptotic versus a necrotic response of melanoma cells to sequential treatment.

Activated Protein C Decreases Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand by an EPCR- Independent Mechanism Involving Egr-1/Erk-1/2 Activation

Background- APC is an antithrombotic and antiinflammatory serine protease that plays an important role in vascular function. We report that APC can suppress the proapoptotic mediator TRAIL in human umbilical vein endothelial cells, and we have investigated the signaling mechanism. APC inhibited endothelial TRAIL expression and secretion and its induction by cell activation. To explore the mechanism, we examined factors associated with TRAIL regulation and demonstrated that APC increased the level of EGR-1, a transcriptional factor known to suppress the TRAIL promoter. APC also induced a significant increase in phosphorylation of ERK-1/2, required to activate EGR-1 expression. Activation of ERK-1/2 was dependent on the protease activated receptor-1 (PAR-1), but independent of the endothelial protein C receptor (EPCR). Using siRNA, we found that the effect of APC on the EGR-1/ERK signaling required for TRAIL inhibition was dependent on the S1P1 receptor and S1P1 kinase. Our data suggest that APC may provide cytoprotective activity by activating the ERK pathway, which upregulates EGR-1 thereby suppressing the expression of TRAIL. Moreover, we provide evidence that APC can induce a cell signaling response through a PAR-1/S1P1-dependent but EPCR-independent mechanism.

Inhibition ofTrailGene Expression by Cyclopentenonic Prostaglandin 15-Deoxy-Δ12,14-Prostaglandin J2in T Lymphocytes

15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a cyclopentenonic prostaglandin endowed with powerful anti-inflammatory activities, as shown in animal models of inflammatory/autoimmune diseases, where pharmacological administration of this prostanoid can ameliorate inflammation and local tissue damage via activation of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) and/or covalent modifications of cellular proteins. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily expressed in most of the cells, including those of immune system such as T lymphocytes, in which it is up-regulated upon antigen-specific stimulation. This cytokine plays an important role in regulating various physiological and immunopathological processes, such as immunosurveillance of tumors and tissue destruction associated with different inflammatory and autoimmune diseases. Here, we demonstrate that 15d-PGJ(2) inhibits trail mRNA and protein expression by down-regulating the activity of its promoter in human T lymphocytes. Our data indicate that both the chemically reactive cyclopentenone moiety of 15d-PGJ(2) and the activation of PPARgamma may be involved in this repressive mechanism. We identified nuclear factor kappaB (NF-kappaB) as a direct target of the prostanoid. 15d-PGJ(2) significantly decreases the expression and/or DNA binding of c-rel, RelA, and p50 transcription factors to the NF-kappaB1 site of trail promoter. Moreover, 15d-PGJ(2)-mediated activation of the transcription factor heat shock factor-1 may contribute to inhibit trail promoter activity in transfected Jurkat T cells. These results suggest that modulation of TRAIL gene expression by 15d-PGJ(2) in T cells may provide a novel pharmacological tool to modify the onset and the progression of specific autoimmune and inflammatory disorders.

Interferon-α Induces TRAIL Expression and Cell Death Via an IRF-1-Dependent Mechanism in Human Bladder Cancer Cells

Apoptosis induced by interferon-alpha (IFNα) is associated with induction of TRAIL in a number of different cell types. Here we examined whether or not TRAIL was required for apoptosis in a model human bladder cancer cell line (UM-UC-12) and defined the molecular mechanisms involved in IFNα-induced TRAIL expression. Exposure to IFNα resulted in concentration-dependent induction of TRAIL and apoptosis. Inhibition of TRAIL or downstream components of the TRAIL cell death pathway (FADD, caspase-8) via siRNA-mediated knockdown attenuated IFNα-induced cell death, thereby implicating TRAIL in the response. IFNα induced rapid STAT-1 phosphorylation and DNA binding activity and subsequent accumulation of IRF-1. Transfection with siRNAs directed against STAT-1 or IRF-1 inhibited IFNα-induced TRAIL production and cell death, and chromatin immunprecipitation (ChIP) analyses demonstrated that IFNα induced direct, time-dependent binding of both transcription factors to the TRAIL promoter. Together, our results demonstrate that IFNα induces TRAIL expression via a STAT-1/IRF-1-dependent mechanism in human bladder cancer cells, and this induction of TRAIL plays an important role in IFNα-induced cell killing.

Identification of TRAIL as an Interferon Regulatory Factor 3 Transcriptional Target

Interferon production and apoptosis in virus-infected cells are necessary to prevent progeny virus production and to eliminate infected cells. Paramyxovirus infection induces apoptosis through interferon regulatory factor 3 (IRF-3), but the exact mechanism of how IRF-3 functions is unknown. We show that IRF-3 is involved in the transcriptional induction of TRAIL, a key player in the apoptosis pathway. IRF-3 upregulates TRAIL transcription following viral infection and binds an interferon-stimulated response element in the TRAIL promoter. The mRNA for TRAIL and its receptor, DR5, are induced following viral infection. These studies identify TRAIL as a novel IRF-3 transcriptional target.

Tumor suppressor IRF-1 mediates retinoid and interferon anticancer signaling to death ligand TRAIL.

Retinoids and interferons are signaling molecules with pronounced anticancer activity. We show that in both acute promyelocytic leukemia and breast cancer cells the retinoic acid (RA) and interferon signaling pathways converge on the promoter of the tumoricidal death ligand TRAIL. Promoter mapping, chromatin immunoprecipitation and RNA interference reveal that retinoid-induced interferon regulatory factor-1 (IRF-1), a tumor suppressor, is critically required for TRAIL induction by both RA and IFNgamma. Exposure of breast cancer cells to both antitumor agents results in enhanced TRAIL promoter occupancy by IRF-1 and coactivator recruitment, leading to strong histone acetylation and synergistic induction of TRAIL expression. In coculture experiments, pre-exposure of breast cancer cells to RA and IFNgamma induced a dramatic TRAIL-dependent apoptosis in heterologous cancer cells in a paracrine mode of action, while normal cells were not affected. Our results identify a novel TRAIL-mediated tumor suppressor activity of IRF-1 and suggest a mechanistic basis for the synergistic antitumor activities of certain retinoids and interferons. These data argue for combination therapies that activate the TRAIL pathway to eradicate tumor cells.

FOXO Proteins Regulate Tumor Necrosis Factor-related Apoptosis Inducing Ligand Expression: IMPLICATIONS FOR PTEN MUTATION IN PROSTATE CANCER

Mutations in PTEN occur in 60-80% of prostate cancers and lead to a constitutive activation of the phosphatidylinositol 3-kinase pathway and a resultant loss of activity of the FOXO family of forkhead transcription factors FKHRL1 and FKHR. To provide insight into the role of PTEN mutations in prostate cancer, we used microarrays to identify genes regulated by FKHRL1 and FKHR in LAPC4 prostate carcinoma cells. These studies revealed that adenoviral overexpression of FKHRL1 and FKHR in the LAPC4 prostate cancer cell line resulted in apoptosis and induced the expression of many genes that affect cellular proliferation or survival. The expression of one of these FOXO-regulated genes, TRAIL, a pro-apoptotic member of the tumor necrosis factor family, was decreased in human metastatic prostate tumors. The altered expression of TRAIL in these tumors correlated directly with decreased PTEN expression and the resultant loss of FKHRL1 and FKHR activity. Analysis of the effects of FOXO proteins on the TRAIL promoter localized the FKHRL1 responsive element of the TRAIL promoter to nucleotides -138 to -121 and demonstrated that TRAIL is a direct target of FKHRL1. These findings suggest that the decreased activity of FKHRL1 and FKHR in prostate cancers resulting from loss of PTEN leads to a decrease in TRAIL expression that may contribute to increased survival of the tumor cells.