Oncogene (2014) 33, 3538–3549 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

ORIGINAL ARTICLE Inhibition of the mitochondrial dihydroorotate dehydrogenase by doxorubicin and brequinar sensitizes cancer cells to TRAIL-induced apoptosis

THe1, S Haapa-Paananen1, VO Kaminskyy2, P Kohonen1,3,VFey1, B Zhivotovsky2, O Kallioniemi1,4 and M Pera¨la¨1

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent in selectively killing tumor cells. However, TRAIL monotherapy has not been successful as many cancer cells are resistant to TRAIL. Chemotherapeutic agents, such as doxorubicin have been shown to act synergistically with TRAIL, but the exact mechanisms of actions are poorly understood. In this study, we performed high-throughput small interfering RNA screening and genome-wide profiling on doxorubicin- treated U1690 cells to explore novel mechanisms underlying doxorubicin-TRAIL synergy. The screening and expression profiling results were integrated and dihydroorotate dehydrogenase (DHODH) was identified as a potential candidate. DHODH is the rate-limiting enzyme in the pyrimidine synthesis pathway, and its expression was downregulated by doxorubicin. We demonstrated that silencing of DHODH or inhibition of DHODH activity by brequinar dramatically increased the sensitivity of U1690 cells to TRAIL-induced apoptosis both in 2D and 3D cultures, and was accompanied by downregulation of c-FLIPL as well as by mitochondrial depolarization. In addition, uridine, an end product of the pyrimidine synthesis pathway was able to rescue the sensitization effects initiated by both brequinar and doxorubicin. Furthermore, several other cancer cell lines, LNCaP, MCF-7 and HT-29 were also shown to be sensitized to TRAIL by brequinar. Taken together, our findings have identified a novel target and its inhibitor, brequinar, as a potential agent in TRAIL-based combinatorial cancer therapy and highlighted for the first time the importance of mitochondrial DHODH enzyme and pyrimidine pathway in mediating TRAIL sensitization in cancer cells.

Oncogene (2014) 33, 3538–3549; doi:10.1038/onc.2013.313; published online 9 September 2013 Keywords: doxorubicin; pyrimidine pathway; mitochondria; brequinar; DHODH; TRAIL

INTRODUCTION in the synergistic effects of the combinatorial treatment are not Tumor necrosis factor (TNF)-related apoptosis-inducing ligand fully understood, although it has been demonstrated that some (TRAIL) is a member of the TNF superfamily, and apoptosis is of these compounds are able to restore tumor cell sensitivity elicited upon binding to its death domain-containing transmem- to TRAIL at the level of DRs/death-inducing signaling complex brane death receptors (DRs). Only the binding of TRAIL to DR4 or complex, mitochondria as well as several survival-signaling 15 DR5 leads to the formation of death-inducing signaling complex pathways. consisting of the activated receptor, Fas-associated protein with In the current study, we aimed to explore novel mechanisms of death domain, and caspase-8/10. Activated caspase-8/10 proteo- action of doxorubicin in sensitizing resistant small cell lung cancer lytically cleaves caspase-3 or the BCL-2 homology domain (SCLC) U1690 cells to TRAIL by using bioinformatic analysis that 3-interacting domain death agonist, resulting in the hallmarks of combines functional high-throughput small interfering RNA apoptosis.1–3 TRAIL has over the years gained extensive interests (siRNA) screening results with gene expression profiling. Our as a therapeutic agent, because it kills preferentially tumor cells results showed that doxorubicin was able to downregulate the while sparing most of the normal cells intact,4,5 and can elicit the expression of dihydroorotate dehydrogenase (DHODH) and apoptotic program even in cells deficient in p53, which is required thereby sensitizing U1690 cells to TRAIL-mediated apoptosis. for successful conventional chemotherapy.6 However, further DHODH is the fourth and rate-limiting enzyme in the de novo studies have demonstrated that TRAIL monotherapy is not biosynthesis of and is localized at the inner successful because a wide range of tumor cells are resistant to mitochondrial membrane. It functions to catalyze oxidation of TRAIL treatment.7–12 Despite the discouraging findings, TRAIL dihydroorotate to orotate, that subsequently induces formation of remains to be an attractive therapeutic agent, because it is well uridine and cytidine nucleosides.16 Pyrimidine is needed for RNA tolerated and in conjunction with other anticancer modalities can and DNA synthesis and thus is essential for cell proliferation and improve the efficiency of therapy. To date, a variety of cytotoxic metabolism, making DHODH an attracting target for drug agents have shown synergy with TRAIL including DNA-damaging development in cancer, parasitic and immunological diseases.17 agents, HDAC inhibitors, proteasome inhibitors, and NF-kB and Several DHODH inhibitors have been developed including PI-3K pathway inhibitors.13,14 The molecular mechanisms resulting brequinar, leflunomide and its active metabolite A77 1726 and

1Medical Biotechnology, VTT Technical Research Centre of Finland, Turku, Finland; 2Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden; 3Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden and 4FIMM-Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland. Correspondence: Dr T He, Medical Biotechnology, VTT Technical Research Centre of Finland, Ita¨inen Pitka¨katu 4C, PO Box 106, FI-20521 Turku, Finland. E-mail: tao.he@vtt.fi Received 20 December 2012; revised 13 June 2013; accepted 13 June 2013; published online 9 September 2013 Dihydroorotate dehydrogenase inhibition sensitizes cells to TRAIL THeet al 3539 teriflunomide.18 Brequinar is an immunosuppressive and anti- RESULTS proliferative drug, which effectively functions to protect allograft Gene expression profiling and high-throughput siRNA screening and xenograft from rejections following transplantation,19 and has (HTS) of TRAIL-resistant U1690 cells reveal genes that contribute to been shown to inhibit the proliferation of T-cells, antibody doxorubicin-induced TRAIL sensitization production as well as tumor growth.20 In our study, we found In order to investigate the mechanistic underpinnings of cancer that the downregulation of DHODH expression either by siRNAs cell TRAIL resistance, U1690 SCLC cell line was selected as our cell or doxorubicin or using its inhibitor brequinar significantly model because it expresses death-inducing signaling complex sensitized U1690 cells to TRAIL-mediated apoptosis. The induced components, including pro-caspase-8, Fas-associated protein with apoptosis was accompanied by downregulation of c-FLIPL and death domain, low-level surface DR5 but no detectable surface significant effect on mitochondria. Addition of uridine abolished expression of DR49 and is resistant to TRAIL but can be sensitized such sensitization effect signifying the importance of DHODH by doxorubicin (Supplementary Figure 1). inhibition in TRAIL sensitization. Combinatorial treatment of TRAIL To evaluate how doxorubicin affects the overall gene expres- and brequinar also induced apoptosis in three dimensional (3D) sion, U1690 cells were treated with doxorubicin (1 mM) for 12 and organotypical cultures of U1690 cells. Furthermore, the ability 24 h and the genome-wide expression changes were analyzed. of brequinar to enhance TRAIL-induced apoptosis was confirmed Genes with consistently up- or downregulated expression for in several other cancer cell lines, but had no effect on human more than 1.5-fold by doxorubicin are illustrated in Figure 1a non-malignant mammary epithelial cells (HMECs). Taken together, (Supplementary Material and Methods). Approximately 80% of the we have provided evidence showing that inhibition of genes with altered expression patterns from the 12 h time point mitochondrial enzyme DHODH links metabolism of pyrimidines were common to the 24 h time point (Figure 1b). To facilitate and TRAIL apoptotic signaling, suggesting utilization of DHODH further analysis, we averaged the expression values from both of inhibitors in TRAIL combinatorial therapy. These data revealed the time points and a total of 1887 genes showed at least 1.5-fold a new role of mitochondria in death-receptor-mediated cell changes in response to doxorubicin. The ingenuity pathway death. analysis (IPA) of these genes revealed several significantly altered

Figure 1. Gene expression profiling and HTS siRNA screen of U1690 cells. (a) Treatment of U1690 cells with doxorubicin (1 mM) for 12 and 24 h and a hieratical clustering of genes with at least 1.5-fold changes (doxorubicin versus control) in any of the time point is shown. Red color denotes for upregulation and blue for downregulation. (b) The overlap of gene expression changes between doxorubicin 12 and 24 h treatment. (c) An overview of the normalized U1690 TRAIL sensitization screen results (T-C_min, Loess score). Positive TRAIL sensitization controls (BIRC2, c-FLIP) were pointed out, scrambled controls and buffer only samples were colored in green, target genes in gray and the 288 sensitization hits in red. Several gray spots under the median-3SD were AllStars Cell Death control siRNAs. (d) Integration of the gene expression Illumina data (x axis, doxorubicin/control) with the siRNA screening results (y axis, relative cell viability) in U1690 cells. Twenty two hits that were both TRAIL sensitizers and were downregulated by doxorubicin by at least 1.5-fold were colored in red.

& 2014 Macmillan Publishers Limited Oncogene (2014) 3538 – 3549 Dihydroorotate dehydrogenase inhibition sensitizes cells to TRAIL THeet al 3540 important cell functions and pathways and are presented in representation of results from the entire druggable genome Supplementary File 1. screen with raw and normalized values as well as integration of To verify the importance of the observed gene expression the gene expression data with fold changes can be found in changes in doxorubicin–TRAIL synergy, we performed a high- Supplementary File 4, from which TRAIL sensitization scores, throughput siRNA screen in U1690 cells in the presence and doxorubicin-induced expression fold changes as well as the hit absence of TRAIL. The siRNA library purchased from Qiagen lists can be filtered out. Based on the literature, only one gene, (Hilden, Germany) targets the druggable genome, with 6951 importin b1(KPNB1), out of the 22 identified has been shown to genes that are known or putative to be responsive to small- be involved in TRAIL-mediated signaling,21 proving that our molecule drugs. Transfection efficiency of U1690 cells on 384-well approach is able to generate true and novel hits. To further narrow plate format was confirmed showing that silencing of the key down genes to be validated in the following studies, we analyzed mitotic regulator PLK1 and the AllStars Cell Death siRNA control these 22 genes using the IPA program to explore whether there reduced the cell viability in comparison with the negative control were known inhibitors available, because existing small molecule by 43% and 93%, respectively (Supplementary Figure 2). Four compounds exhibit advantageous properties in therapeutics over siRNAs were available for each gene and were pooled together siRNAs, which still have limited in vivo applications.22 The IPA into a single well to yield a final concentration of 50 nM. Cell revealed that only 2 out of 22 genes had known inhibitors viability was analyzed using CellTiter-Glo cell proliferation assay available, with one of them being DHODH (Table 1). Due to the after the treatments. Statistical tools were applied for hit importance of DHODH and its inhibitors in the clinics, we identifications, and are described in the Supplementary Material continued our validation studies on this particular gene. and Methods. An overview of normalized sensitization results is illustrated in Figure 1c. The screen was performed in two Silencing of DHODH sensitizes U1690 cells to TRAIL replicates and siRNAs targeting the known apoptosis pathway factors such as c-FLIP (CFLAR) and BIRC2 were found among the To verify the observed sensitization effects of DHODH from the sensitization hits confirming the screens to be successful siRNA screening, we selected two individual siRNAs targeting (Figure 1c). Altogether 288 reproducible sensitization hits were DHODH and monitored their effects on both cell viability and identified and marked in red (Figure 1c). The detailed list of the apoptosis in the presence and absence of TRAIL. U1690 cells were 288 genes is presented in the Supplementary File 2. The IPA transfected with the siRNAs for 48 h followed by TRAIL addition analysis results for major cell functions, pathways and networks of (100 ng/ml, 24 h). In the presence of TRAIL, silencing of DHODH by the 288 TRAIL sensitization hits are presented in Supplementary both siRNAs reduced the cell proliferation (Figure 2a) and File 3. increased caspase-3/7-like activities (Figure 2b). The silencing Next, we integrated the siRNA-screening data with the ability of the siRNAs was verified by qRT–PCR (Figure 2c). doxorubicin-treated gene expression profiles to gain an overview of genes that have a potential role in doxorubicin sensitization in DHODH inhibitor brequinar enhances TRAIL-induced apoptosis in U1690 cells. A hit was defined if it was both a TRAIL sensitization U1690 cells hit and its expression was downregulated by at least 1.5-fold by Next, we wanted to examine whether DHODH inhibitors could be doxorubicin treatment. The integration resulted in 22 genes utilized in synergizing TRAIL activity. U1690 cells were treated with ranging from factors to nuclear transporters to 100 mM of leflunomide, its metabolite A77 1726 and brequinar for various (Figure 1d, red spots and Table 1). A detailed 2 h before treatment with different concentrations of TRAIL, and

Table 1. Genes that contribute to doxorubicin-induced TRAIL sensitization

Symbol Entrez gene name Location Type(s) Drug(s)

ADSL Adenylosuccinate lyase Cytoplasm Enzyme AURKB Aurora kinase B Nucleus Kinase AZD-1152, tozasertib, GSK1070916 BUB3 Budding uninhibited by benzimidazoles Nucleus Other 3 homolog (yeast) C10orf57 Chromosome 10 open reading frame 57 Cytoplasm Other CCT2 Chaperonin containing TCP1, subunit 2 (b) Cytoplasm Kinase CSE1L CSE1 chromosome segregation 1-like (yeast) Nucleus Transporter DHODH Dihydroorotate dehydrogenase (quinone) Cytoplasm Enzyme Brequinar, leflunomide GORASP2 Golgi reassembly stacking protein 2, 55 kDa Cytoplasm Other GRK5 G protein-coupled receptor kinase 5 Plasma membrane Kinase JAZF1 JAZF finger 1 Nucleus Transcription regulator KIF11 Kinesin family member 11 Nucleus Other KPNB1 Karyopherin (importin) b 1 Nucleus Transporter NUP153 Nucleoporin 153 kDa Nucleus Transporter PPFIA1 Protein phosphatase, receptor type, f Plasma membrane Phosphatase polypeptide (PTPRF), interacting protein (liprin), alpha 1 PSMA1 Proteasome (prosome, macropain) subunit, alpha type, 1 Cytoplasm Peptidase RACGAP1 Rac GTPase activating protein 1 Cytoplasm Transporter SCAP SREBF chaperone Cytoplasm Other TFDP1 Transcription factor Dp-1 Nucleus Transcription regulator THOP1 Thimet oligopeptidase 1 Cytoplasm Peptidase TRAM1 Translocation-associated membrane protein 1 Cytoplasm Other UBTD2 domain containing 2 Unknown Other VEGFB Vascular endothelial growth factor B Extracellular Other Abbreviation: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

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Figure 2. Silencing of DHODH sensitizes U1690 cells to TRAIL. (a) U1690 cells were transiently transfected with two different siRNAs against DHODH, and 48 h post transfection, TRAIL (100 ng/ml) was added to the cells. The cells were further incubated for 24 h and cell viability was measured using CellTiter-Glo Luminescence Assay. (b) Same as in (a), and apoptosis was measured using Caspase-Glo 3/7 Assay. (c) Taqman quantitative real-time PCR after DHODH silencing. U1690 cells were transfected with siDHODH (13 nM) or negative control siRNA for 48 h. Total RNA was isolated and analyzed using (ACTB) as a reference. The results (a–c) were averages of three replicates and were representative of three biological repeats (Student’s t-test; *Po0.05, **Po0.01, ***Po0.001). RLU: relative luminescence units. cell viability was measured 24 h afterwards (Figure 3a). Both (Figure 4b). As DHODH is a mitochondrial enzyme, we decided to leflunomide and A77 1726 were able to induce more pronounced explore the effect of brequinar on mitochondrial membrane cell death in the absence of TRAIL comparing with brequinar, but potential using TMRE staining. Although treatment with brequinar had no effect to potentiate TRAIL-induced cell death. However, by itself did not cause any significant effect on mitochondria, brequinar, as a single agent, did not affect significantly on cell the combining treatment with brequinar and TRAIL dramati- viability but when treated sequentially with TRAIL, cell death was cally increased the number of cells with drop of mitochondrial dramatically increased. Therefore, brequinar was selected in the membrane potential, suggesting that inhibition of DHODH also following studies. facilitates TRAIL-mediated apoptosis at the level of mitochondria The concentration dependence of brequinar on TRAIL-induced (Figure 4c). Furthermore, based on the previous report that apoptosis was assessed by treating U1690 cells with three doxorubicin increases the expression of DR5,9 we evaluated the brequinar concentrations in combination with increasing doses expression of DR5 upon brequinar treatment. U1690 cells were of TRAIL for 24 h and apoptosis was measured by caspase-3/7 either untreated or treated with brequinar (100 mM,24h)or activation. Already at 10 mM, brequinar was able to enhance TRAIL- doxorubicin (1 mM, 24 h) and the total level of DR5 expression induced caspase activation, while 100 mM exhibited the highest (Figure 5a), and surface expression of DR4 and 5 (Figures 5b and c) synergistic effect with 50 ng/ml TRAIL. Higher concentrations of were examined by immunoblots and flow cytometry. As brequinar TRAIL did not result in additional synergy (Figure 3b). Furthermore, had its own green fluorescence, all the histograms of brequinar- PARP cleavage was also observed in response to brequinar treated samples were shifted to the right. The MFI values were and TRAIL treatment (Figure 3c), indicating the occurrence of normalized according with level of brequinar’s autofluorescence in apoptosis. green channel. Relative normalized surface DR expression is To examine the molecular mechanisms involving in the TRAIL presented in Figure 5c. These data indicated that in contrast to signaling pathway, U1690 cells were pretreated with brequinar doxorubicin, inhibition of DHODH with brequinar had no impact (100 mM, 2 h) with the following TRAIL treatment (100 ng/ml, 24 h) on the expression of DR4 and DR5 receptors. Taken together, and immunoblots were performed. Treatment with brequinar inhibition of DHODH by brequinar sensitized cells to TRAIL- caused a significant downregulation in protein levels of c-FLIPL, mediated apoptosis by reducing the level of c-FLIPL, and such a which was further decreased after addition of TRAIL (Figure 4a). response was also accompanied by the effect on mitochondria. Similar c-FLIPL downregulation was observed when U1690 cells were transfected with siRNAs against DHODH and treated with TRAIL (Figure 4a). However, the level of c-FLIPS was not changed in Uridine rescues brequinar-TRAIL-induced apoptosis response to brequinar. Furthermore, such reduction of c-FLIPL was As DHODH is a key enzyme in the de novo synthesis of uridine accompanied by processing of procaspase-8 (Figure 4a). Activa- monophosphate, which is pivotal for many cellular functions, we tion of caspase-8 was also confirmed by caspase-8 activity assay examined whether uridine could rescue the TRAIL sensitization

& 2014 Macmillan Publishers Limited Oncogene (2014) 3538 – 3549 Dihydroorotate dehydrogenase inhibition sensitizes cells to TRAIL THeet al 3542

Figure 3. Brequinar sensitizes U1690 cells to TRAIL-induced apoptosis. (a) U1690 cells were pretreated for 2 h with 100 mM leflunomide, A77 1726 or brequinar. The cells were subsequently incubated with 50 or 100 ng/ml of TRAIL for 24 h and cell proliferation was measured by CellTiter-Glo Luminescence Assay. (b) U1690 cells were pretreated with three brequinar concentrations, 10, 50 or 100 mM and the cells were subsequently treated with increasing concentrations of TRAIL for additional 24 h. Caspase-3/7 activity was measured using Caspase-Glo 3/7 Assay. (c) U1690 cells were pretreated with 10 or 100 mM of brequinar and the cells were subsequently treated with of TRAIL (50 ng/ml, 24 h). After the incubation period, cells were lysed and subjected to SDS–PAGE, transferred onto membranes and blotted with antibodies against PARP, DHODH or actin. (a, b) The results were averages of three replicates and were representative of three biological repeats. Student’s t-test; *Po0.05, **Po0.01, ***Po0.001.

effect induced by brequinar in U1690 cells. We incubated U1690 absence of TRAIL and/or uridine. As shown in Figure 7b, apoptosis cells with increasing concentrations of brequinar with or without induced by 0.1 mM doxorubicin and 50 ng/ml TRAIL was diminished uridine (100 mM, 2 h) followed by 24 h incubation with or without by uridine by 25%, whereas uridine was not able to reverse the TRAIL (50 ng/ml). Apoptosis was detected by caspase-3/7 activa- sensitization effect induced by 1 mM of doxorubicin. These data tion as shown in Figure 6a. Uridine alone did not affect the cell indicated that inhibition of DHODH has, at least in part, an viability, whereas it completely reversed apoptosis induced by important role in the sensitization of doxorubicin to TRAIL. Thus, combined treatment of brequinar (10 and 50 mM) and TRAIL. At we revealed that downregulation of DHODH induced by doxor- 100 mM of brequinar, the reverse effect by uridine was attenuated ubicin is among the mechanisms that contribute to the sensitiza- to 25%. Immunoblots in Figure 6b also showed that both PARP tion of cancer cells to TRAIL-mediated apoptosis. and caspase-3 cleavages enhanced by brequinar and TRAIL were rescued by uridine with significant less cleavages of PARP and caspase-3 at 10 mM of brequinar. The rescue effect was, however, Brequinar sensitizes multiple cancer cell lines to TRAIL and induces less obvious at 100 mM of brequinar. These results suggested that apoptosis of U1690 cells in 3D cell culture upon TRAIL treatment uridine production was important for U1690 cells to maintain its The results that have been shown were performed on SCLC U1690 resistance to TRAIL and reduction of uridine levels by inhibition of cells. To examine whether the effects observed using brequinar DHODH by brequinar at lower concentrations sensitized cells to was also true in other cancer cell models, we employed three TRAIL. different cancer cell lines, LNCaP (prostate cancer), MCF-7 (breast cancer) and HT-29 (colon cancer) cells. These cells were pretreated with brequinar (50 mM, 2 h) before incubation with TRAIL (50 ng/ml, Doxorubicin reduces DHODH expression and uridine reverses 24 h). Apoptosis induction was measured by caspase-3/7 activity doxorubicin sensitization effect at lower concentrations assay. As shown in Figure 8a, brequinar was able to elevate the The gene expression profiling data showed that expression of a apoptosis induction in LNCaP, MCF-7 and HT-29 cells to 1.9-, 1.2- variety of genes, including DHODH, was modulated by doxorubicin and 1.8-folds, respectively. These results implied that brequinar treatment. Therefore, we hypothesized that doxorubicin might may be used as a drug in TRAIL combinatorial treatment on pursue the sensitization effect via the DHODH pathway. The gene various cancer types. To examine whether the brequinar and expression profiling data were verified as both DHODH mRNA and TRAIL treatment can be tolerated by non-cancerous tissues, protein expression decreased in response to increasing doses of HMECs were employed. In contrary to cancer cell lines, HMECs doxorubicin (Figure 7a). To further validate the importance of the did not show any sensitization to TRAIL when pretreated with DHODH pathway in the doxorubicin-TRAIL synergy, we treated brequinar (50 mM, 2 h) indicating possible cancer-specific property U1690 cells with 0.1 and 1 mM of doxorubicin in the presence and of the combinatorial treatment (Figure 8a).

Oncogene (2014) 3538 – 3549 & 2014 Macmillan Publishers Limited Dihydroorotate dehydrogenase inhibition sensitizes cells to TRAIL THeet al 3543

Figure 4. Brequinar decreases expression of c-FLIPL, and induces activation of caspase-8 and mitochondrial depolarization in response to TRAIL treatment. (a) U1690 cells were pretreated with brequinar (100 mM, 2 h) and subsequently treated with TRAIL (100 ng/ml, 24 h). The expression of c-FLIPs and processing of caspase-8 was analyzed by immunoblot. GAPDH was used as a loading control. U1690 cells were transfected with siDHODH and treated with 100 ng/ml of TRAIL as described in Figure 2. After the incubation period, cells were lysed and subjected to SDS–PAGE, transferred onto membranes and blotted with antibodies against c-FLIP or actin. (b) U1690 cells were pretreated with brequinar (10, 50 or 100 mM, 2 h) and the cells were subsequently treated with 50 ng/ml of TRAIL for additional 24 h. Caspase-8 activity was measured using Caspase-Glo 8 Assay. The results were averages of three replicates and were representative of three biological repeats (Student’s t-test; **Po0.01). (c) U1690 cells were untreated, treated with TRAIL (100 ng/ml, 24 h) alone or pretreated with brequinar (100 mM, 2 h) and subsequently treated with TRAIL (100 ng/ml, 24 h). Mitochondrial membrane potential (MMP) was assessed using TMRE staining.

Figure 5. Brequinar does not alter the total or surface expression of TRAIL receptors. (a) U1690 cells were untreated, treated for 24 h with brequinar (100 mM) or doxorubicin (1 mM). The expression of DR5 was analyzed by immunoblot. GAPDH was used as a loading control. (b) Expression of surface DR4 were assessed by flow cytometry as described in Materials and methods. Filled histograms represent antibody control, green histograms represent staining with DR4 and red histograms represent staining with DR5 antibody. Relative surface DR expression is presented in (c).

& 2014 Macmillan Publishers Limited Oncogene (2014) 3538 – 3549 Dihydroorotate dehydrogenase inhibition sensitizes cells to TRAIL THeet al 3544

Figure 6. Uridine rescues apoptosis induced by brequinar and TRAIL combinatorial treatment. (a) U1690 cells were pretreated with brequinar (10, 50 or 100 mM) either alone or together with 100 mM uridine for 2 h. Subsequently, the cells were treated with 50 ng/ml of TRAIL for 24 h. Caspase-3/7 activity was measured using Caspase-Glo 3/7 Assay. The results were averages of three replicates and were representative of three biological repeats (Student’s t-test; **Po0.01, ***Po0.001). (b) U1690 cells were pretreated with 10 or 100 mM of brequinar with or without 100 mM uridine and the cells were subsequently treated with TRAIL (50 ng/ml, 24 h). After the incubation period, cells were lysed and subjected with SDS–PAGE, transferred to membranes, and blotted with antibodies against PARP, cleaved caspase-3 or actin.

To further characterize the response of cancer cells to brequinar mechanism underlying the synergistic effect is not well defined. and TRAIL towards an in vivo setting, we performed miniaturized Our study explored pertinent contributory mechanism of doxo- 3D cell culture of U1690 cells. Five days post culturing on rubicin in sensitizing resistance cells to TRAIL. Understanding of matrigel, U1690 cells generated large and irregular spheroids these mechanisms could provide the possibility to identify novel (Figure 8b), which were later subjected to treatments with drugs for combining with TRAIL for cancer treatment. brequinar and TRAIL. Cell morphology and apoptosis induction In this regard, a genome-wide gene expression profiling of were measured by microscopy and caspase activity assay. As doxorubicin-treated SCLC U1690 cells was performed and the single agents, brequinar and TRAIL had minor effects on the importance of the genes with altered expression was accessed by a morphology of U1690 spheres, whereas the combination treat- synthetic lethal siRNA high-throughput screen. HTS has been ment induced spheroidal changes such as membrane blebbing utilized in searching for novel factors in mediating TRAIL and structure rupture (Figure 8b). Quantification of the overall resistance23–25 and HTS format of compound screening has also apoptosis induction using caspase-3/7 activity assay confirmed been widely employed to identify synergistic TRAIL sensitizers.26–28 that the combined treatment induced an increase of caspase-3/7 In our study, we integrated the genome-wide gene expression activity in U1690 spheroids (Figure 8c). These results suggested profiles with the siRNA screening data to explore mechanisms of that combination therapy of TRAIL and brequinar can also be actions of doxorubicin in sensitizing resistant cells to TRAIL. The effective on 3D organotypic cultures of U1690 cells and may be integration identified 22 genes to be both downregulated by utilized as an initial evidence of using brequinar and TRAIL doxorubicin treatment and to induce U1690 cells TRAIL sensitivity towards an in vivo validation. upon expression silencing. With the exception of KPNB1, all other 21 genes have not been linked to TRAIL signaling previously. Thus, our findings provided novel insights to inspire new mechanistic DISCUSSION studies of TRAIL therapy in SCLC. Previous studies have shown that the chemotherapeutic drug DHODH is an important mediator in the pyrimidine pathway doxorubicin overcomes the TRAIL resistance and acts synergistically and as DHODH inhibitors are readily available, we continued the with TRAIL to induce more dramatic cancer cell death.15 Although validation procedure focusing on DHODH. Here we showed that modulation of surface DR levels and localization as well as DHODH has an important role in sensitizing SCLC U1690 cells to downregulation of anti-apoptotic genes, such as c-FLIPS/L and TRAIL-induced apoptosis, and highlighted an essential role of X-IAP, have been implicated to contribute to the sensitization of mitochondria. Silencing of DHODH using siRNAs in conjunction doxorubicin to TRAIL in cancer cells,15 the exact molecular with TRAIL treatment dramatically reduced cell proliferation and

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Figure 7. Doxorubicin regulates the expression of DHODH and uridine rescues apoptosis induced by doxorubicin and TRAIL combinatorial treatment. (a) U1690 cells were treated with varying concentrations of doxorubicin for 24 h and the relative DHODH mRNA as well as protein expression was analyzed by Taqman quantitative real-time PCR and immunoblot, respectively. (b) U1690 cells were pretreated with doxorubicin (0.1 or 1 mM) either alone or together with 100 mM uridine for 2 h. Subsequently, the cells were treated with 50 ng/ml of TRAIL for 24 h. Caspase-3/7 activity was measured using Caspase-Glo 3/7 Assay. The results were averages of three replicates and were representative of three biological repeats (Student’s t-test; **Po0.01). induced apoptosis. Doxorubicin significantly reduced both deacetylase32 in addition to doxorubicin. In this study, we showed protein and mRNA expression of DHODH in U1690 cells in a that DHODH inhibitor brequinar could also be utilized as a TRAIL concentration-dependent manner. Interestingly, uridine, which is sensitizer. The other two inhibitors, leflunomide and its active the most abundantly available salvageable pyrimidine nucleoside metabolite A771726 did not sensitize cells to TRAIL, and in fact in vivo, was able to reverse the sensitization effect mediated they were cytotoxic to cells as single agents. These data coincided by doxorubicin at a lower concentration. These results pinpointed with previous findings showing that leflunomide and A77 1726 to a potential role of DHODH in mediating TRAIL resistance in could be used in inducing apoptosis in melanoma and multiple a subset of cancer cell lines. The importance of pyrimidine myeloma cells.33–35 In one previous report, A77 1726 was shown pathway has become evident as serious diseases such as to enhance TRAIL-induced apoptosis in human hepatic stellate hereditary orotic aciduria, anemia and neurological disorders cells and thereby function in anti-fibrotic therapy.36 These data have been linked to defects in the pyrimidine metabolism.17 suggested that the differences in structures, cell line origin as well However, knowledge on whether pyrimidine pathway and meta- as molecular mechanism of actions of these DHODH inhibitors18 bolism could be involved in TRAIL signaling is relatively scarce might contribute to these distinct effects. with one report showing that TRAIL-induced apoptosis in HT-20 Brequinar was clinically developed due to its anti-tumor activity, cells was inhibited by via the pyrimidine pathway.29 and its effect on various cancer cell lines has previously been Our finding suggested that DHODH involved pyrimidine pathway shown, being more potent in half of the cell lines tested and less may contribute to TRAIL signaling and doxorubicin exerts its potent in the other half.37 However, the use of brequinar in clinical TRAIL sensitization effect via the DHODH pyrimidine pathway. We trials for cancer treatment has been limited. When advanced lung cannot, however, rule out the fact that other mechanisms, such cancer patients (including both NSCLC and SCLC patients) were as upregulation of DR59 as well as the B1900 gene expression treated using brequinar, insufficient killing of tumor cells was changes induced by doxorubicin, contribute to the effect of observed.38 We also observed that brequinar possessed significant sensitization, and we suggest that the DHODH pyrimidine lower toxicity towards U1690 cells and in fact this characteristic pathway is among the multiple mechanisms that cells select to would be optimal for a combinatorial therapy. We demonstrated respond to doxorubicin. that pretreatment with brequinar dramatically enhanced the To date, a number of DHODH inhibitors have been classified, susceptibility of U1690 cells to TRAIL-induced apoptosis, and the and they have proven efficacy for treatment of cancer30 and observed TRAIL resistance could already be overcome by a immunological disorders, such as rheumatoid arthritis and combination of brequinar and TRAIL. These observations were multiple sclerosis.31 Here, we investigated the role of DHODH further confirmed in 3D cell culture of U1690 cells. The induced inhibitors in sensitizing U1690 cells to TRAIL. Synergy between apoptosis was accompanied with activation of caspase-8 and TRAIL and other chemotherapeutic drugs in SCLC cells has only mitochondrial depolarization. Inhibition of DHODH by either 9 been shown with etoposide, DNA and brequinar or siRNAs reduced expression of c-FLIPL but had no

& 2014 Macmillan Publishers Limited Oncogene (2014) 3538 – 3549 Dihydroorotate dehydrogenase inhibition sensitizes cells to TRAIL THeet al 3546

Figure 8. Brequinar sensitizes multiple cancer cell lines to TRAIL and induces apoptosis of U1690 cells in 3D culture upon TRAIL treatment. (a) LNCaP, MCF-7, HT-29 and HMECs were pretreated with 50 mM brequinar for 2 h and the cells were subsequently treated with 50 ng/ml of TRAIL for an additional 24 h. Caspase-3/7 activity was measured using Caspase-Glo 3/7 Assay. The results were averages of three replicates and were representative of three biological repeats (Student’s t-test; **Po0.01, ***Po0.001). (b) Live cell imaging of U1690 spheres which were pretreated with 50 or 100 mM brequinar for 2 h followed by 50 or 100 ng/ml of TRAIL for an additional 48 h. (c) Same as in (b), and caspase-3/7 activity of U1690 spheroids treated was measured using Caspase-Glo 3/7 Assay (Student’s t-test; *Po0.05, **Po0.01).

effects on c-FLIPS. Short and long FLIPs have differential regulatory pathways, but our data demonstrated that reduction of FLIP level roles in apoptosis and small molecule therapy selectively targeting is important in sensitizing U1690 cells to TRAIL by brequinar. FLIP variants have been used in sensitizing cancer cell lines to Moreover, it has been shown that expression of surface DR5, TRAIL.39 We have not studied whether the observed down- but not DR4, was significantly enhanced due to doxorubicin 9 regulation of c-FLIPL undergoes via transcriptional or translational treatment in SCLC cells. However, in contrast to doxorubicin,

Oncogene (2014) 3538 – 3549 & 2014 Macmillan Publishers Limited Dihydroorotate dehydrogenase inhibition sensitizes cells to TRAIL THeet al 3547 which also by itself downregulated DHODH expression, brequinar manufacturer’s protocol. Integrity of the RNA before hybridization was had no effect on the total or surface expression of DR5. Another monitored using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) DHODH inhibitor, leflunomide, was also observed not to have according to manufacturer’s instructions. Purified total RNA (500 ng) was any impact on DR4 and DR5 expression in human hepatic stellate amplified with the TotalPrep Kit (Ambion, Austin, TX, USA) and the biotin cells,36 indicating that brequinar and leflunomide could labeled cRNA was hybridized to Sentrix HumanRef-8 Expression BeadChips (Illumina, San Diego, CA, USA). The assay was performed at the Finnish preferentially depend on other mechanisms, such as down- DNA Microarray Centre, Turku Centre for Biotechnology. The arrays were regulating FLIPs and inhibiting the pyrimidine pathway to scanned with the BeadArray Reader (Illumina) and the raw data were accelerate the TRAIL-induced apoptosis. The sensitization effect obtained by GenomeStudio (Illumina). The microarray data are accessible was reversed at lower concentrations of brequinar by the addition at the NCBI’s Gene Expression Omnibus through a GEO Series accession of uridine. These results further confirmed that DHODH expression number (GSE42531). and activity indeed contributed to the TRAIL resistance in U1690 cells. However, the rescue effect of uridine was less prominent Transient transfections with higher concentrations of brequinar, indicating additional U1690 cells were reverse-transfected using SilentFect (Bio-Rad Labora- mechanisms are likely activated. Furthermore, the brequinar TRAIL tories) with siRNAs against DHODH 13 nM, (Qiagen, SI00363391, synergy was also found in other cancer cell lines, LNCaP, MCF-7 SI00363398) on 384-well plates for 48 h and treated with 100 ng/ml TRAIL and HT-29 cells. Numerous compounds have been reported to for 24 h post-transfection. synergistically act with TRAIL to potentiate apoptosis in these cells, 40–45 including chemotherapeutic drugs and natural compounds. Cell viability and caspase activity assays Our results added the usage of brequinar into this continuously Viability or caspase-3/7/8 activities was measured using CellTiter-Glo growing list of agents that can be utilized in TRAIL combinatorial Luminescence Assay (Promega, Madison, WI, USA) or Caspase-Glo 3/7 therapy. Interestingly, the combined treatment of brequinar Assay (Promega) or Caspase-Glo 8 Assay (Promega) with Envision Plate- and TRAIL did not harm the mammary epithelial cells HMEC reader according to manufacturer’s instructions. suggesting normal cells possess own mechanisms to overcome the sensitization effect. Surface TRAIL receptor expression. In conclusion, we analyzed novel mechanisms of action of The level of surface expressed TRAIL receptors was detected by flow doxorubicin in TRAIL sensitization and identified a key role for the cytometry. After washing with phosphate buffer solution, cells were mitochondrial enzyme DHODH and the pyrimidine synthesis incubated (30 min, 4 1C) with primary anti-DR4 (Diaclone, Besancon, France, pathway. We also characterized DHODH inhibitor brequinar as a clone B-N36) or anti-DR5 (Diaclone, clone B-K29) antibodies. After washing, potential TRAIL sensitizer. Although brequinar as a single agent cells were incubated (30 min, 4 1C) with secondary antibody (Alexa- has limited therapeutic windows, it may serve as a senstitizer for Fluor488-conjugated donkey anti-rabbit IgG, Molecular Probes, Eugene, TRAIL in combinatorial cancer therapy. Similar approach could also OR, USA), and stained with 7-AAD (1 mg/ml, Molecular Probes). Analysis was be utilized in evaluating mechanisms of other existing or potential performed using flow cytometry (FACScan, Becton Dickinson, San Jose, CA, anti-cancer agents and thereby provide novel directions for USA). 7-AAD-negative cells were subjected to receptor analysis (Cell Quest development of combinatorial cancer therapy. software, San Jose, CA, USA). The results are expressed as histograms and related to appropriate controls lacking the specific primary antibody.

MATERIALS AND METHODS Assessment of mitochondrial membrane potential Cell culture and reagents U1690 cells were washed in phosphate buffer solution, incubated for 20 min at 37 1C with 25 nM of tetramethylrhodamine ethyl perchlorate U1690, LNCaP, MCF-7 and HT29 cells (ATCC, Manassas, VA, USA) were (TMRE, Molecular Probes, T-669) in phosphate buffer solution. Cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supple- analyzed by flow cytometry (FACScan, Becton Dickinson), and data were mented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM evaluated using Cell Quest software. L-glutamine and 1% penicillin/streptomycin. HMECs (ATCC) were cultured 1:1 in DMEM (4500 mg/l glucose, Sigma-Aldrich, St Louis, MO, USA)/Ham’s F12 (Gibco/Invitrogen) supplemented with 1% FBS, 2 mML-glutamine, Immunoblotting 10 mg/ml , 0.5 mg/ml hydrocortisone, 0.05 mg/ml choleratoxin and Cells were lysed and separated on SDS–polyacrylamide gels and western 0.01 mg/ml EGF (all from Sigma-Aldrich). Doxorubicin, brequinar, lefluno- blotting was performed using antibodies against cleaved caspase-3 mide and uridine were from Sigma-Aldrich, and A77 1726 was from Enzo (Asp175) (9661, Technology, Danvers, MA, USA), PARP Life Sciences (Farmingdale, NY, USA). Recombinant human TRAIL/TNFSF10 (P248, Sigma-Aldrich), DHODH (WH0001723M1, Sigma-Aldrich) DR5 was obtained from R&D Systems (Minneapolis, MN, USA). DHODH siRNAs (D3938, Sigma-Aldrich), GAPDH (2275-PC-100, Trevigen, Gaithersburg, and AllStars Negative and Cell Death Control siRNAs were purchased from MD, USA), actin (A1978 Sigma-Aldrich), caspase-8 and c-FLIP (kindly Qiagen. provided by Drs I Lavrik and P Krammer) antibodies. Secondary antibodies coupled to horseradish peroxidase were from Amersham Biosciences (Freiburg, Germany), and visualization was performed using enhanced High-throughput siRNA screening chemiluminescence procedure (Amersham Biosciences). The siRNA library targeting human druggable genome (Qiagen GmbH) consists of four siRNAs per gene. These four siRNAs were pooled into the same well (final assay concentration 50 nM) on 384-well white, clear- Real-time quantitative PCR analysis bottom assay plates (Greiner Bio-One GmbH, Frickenhausen, Germany), U1690 cells were reverse transfected with two different siRNAs against followed by addition of siLentFect (Bio-Rad Laboratories, Hercules, CA, DHODH (Qiagen, SI00363391, SI00363398) for 48 h or treated with USA) using a Multidrop 384 Microplate Dispenser (Thermo Fisher Scientific doxorubicin (0.2, 0.5 and 1 mM) for 24 h on 48-well plates. RNA samples Inc, Waltham, MA, USA) for 1 h at room temperature. Cell suspension was were extracted using RNeasy Mini Kit (Qiagen) and reverse transcribed to thereafter overlaid and the plates were incubated for 48 h at þ 37 1C. cDNA (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Recombinant human TRAIL with a final concentration of 100 ng/ml was Foster City, CA, USA). Taqman quantitative real-time PCR was performed subsequently added to plates designated for TRAIL treatment and they using ABI Prism 7900 (Applied Biosystems) with specific primers for were further incubated for 24 h at þ 37 1C. DHODH (For 50-CGGAGCCTTCAGGGAAAG-30, Rev 50-CACTCTCCGCAAGCC ATCC-30) and b-actin (ACTB) from the Universal Probe Library (Roche Applied Biosciences, Basel, Switzerland). The results were analyzed using Illumina gene expression SDS 2.3 and RQ manager software (Applied Biosystems). ACTB was used as Gene expression profiles of U1690 cells treated with or without 1 mM an endogenous control to determine the expression of DHODH by relative doxorubicin for 12 and 24 h were analyzed with two replicates. Total RNA quantitation method. Averaged data were obtained from three biological was extracted using RNeasy (Qiagen GmbH) according to the experiments with quadruplicates.

& 2014 Macmillan Publishers Limited Oncogene (2014) 3538 – 3549 Dihydroorotate dehydrogenase inhibition sensitizes cells to TRAIL THeet al 3548 Miniaturized 3D cell cultures and morphological image and 19 Cramer DV. Brequinar sodium. Pediatr Nephrol 1995; 9(Suppl): S52–S55. apoptosis analysis 20 Peters GJ, Nadal JC, Laurensse EJ, de Kant E, Pinedo HM. Retention of U1690 cells were plated on miniaturized 3D cultures as described earlier.46 in vivo antipyrimidine effects of Brequinar sodium (DUP-785; NSC 368390) in Cells were seeded with defined numbers (1500 cells/well). After 5 days, murine , bone marrow and colon cancer. Biochem Pharmacol 1990; 39: spheroids formed were pretreated with brequinar for 2 h and subsequent 135–144. TRAIL treatment for 48 h. The time lapse phase-contrast multicellular 21 Kojima Y, Nakayama M, Nishina T, Nakano H, Koyanagi M, Takeda K et al. structures images were acquired by Incucyte (Essen Instruments, Ann Importin beta1 protein-mediated nuclear localization of death receptor 5 Arbor, MI, USA) and caspase-3/7 activity was measured using Caspase-Glo (DR5) limits DR5/tumor necrosis factor (TNF)-related apoptosis-inducing ligand 3/7 Assay (Promega). (TRAIL)-induced cell death of human tumor cells. J Biol Chem 2011; 286: 43383–43393. 22 Kawasumi M, Nghiem P. Chemical genetics: elucidating biological systems with CONFLICT OF INTEREST small-molecule compounds. J Invest Dermatol 2007; 127: 1577–1584. The authors declare no conflict of interest. 23 Coussens MJ, Corman C, Fischer AL, Sago J, Swarthout J. MISSION LentiPlex pooled shRNA library screening in mammalian cells. J Vis Exp 2011; 3305. 24 Aza-Blanc P, Cooper CL, Wagner K, Batalov S, Deveraux QL, Cooke MP. Identifi- ACKNOWLEDGEMENTS cation of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening. Mol Cell 2003; 12: 627–637. We acknowledge Jouni Latoniitty and Rami Ma¨kela¨ for robotic technical assistance, 25 Ovcharenko D, Kelnar K, Johnson C, Leng N, Brown D. Genome-scale microRNA Ville Ha¨rma¨ and Johannes Virtanen for advices and help in 3D experiments, Pirjo and small interfering RNA screens identify small RNA modulators of TRAIL- Ka¨pyla¨ for excellent technical support and Drs I Lavrik and P Krammer for antibodies. induced apoptosis pathway. Cancer Res 2007; 67: 10782–10788. Finnish DNA Microarray Centre is acknowledged for performing TaqMan qRT–PCR 26 Booth NL, Sayers TJ, Brooks AD, Thomas CL, Jacobsen K, Goncharova EI et al. and Illumina gene expression analysis. 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

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