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 pyrimidine biosynthesis enzyme 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 gene expression 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 protein 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 pyrimidines 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)