Published OnlineFirst May 28, 2014; DOI: 10.1158/0008-5472.CAN-13-3694 Cancer Therapeutics, Targets, and Chemical Biology Research Selenium Suppresses Leukemia through the Action of Endogenous Eicosanoids Ujjawal H. Gandhi1, Naveen Kaushal1, Shailaja Hegde1, Emily R. Finch1, Avinash K. Kudva1, Mary J. Kennett1, Craig T. Jordan2, Robert F. Paulson1, and K. Sandeep Prabhu1 Abstract Eradicating cancer stem-like cells (CSC) may be essential to fully eradicate cancer. Metabolic changes in CSC could hold a key to their targeting. Here, we report that the dietary micronutrient selenium can trigger apoptosis of CSC derived from chronic or acute myelogenous leukemias when administered at supraphysiologic but non- toxic doses. In leukemia CSC, selenium treatment activated ATM-p53–dependent apoptosis accompanied by increased intracellular levels of reactive oxygen species. Importantly, the same treatment did not trigger apoptosis in hematopoietic stem cells. Serial transplantation studies with BCR–ABL-expressing CSC revealed that the selen- ium status in mice was a key determinant of CSC survival. Selenium action relied upon the endogenous production 12 of the cyclooxygenase-derived prostaglandins D -PGJ2 and 15d-PGJ2. Accordingly, nonsteroidal anti-inflammatory drugs and NADPH oxidase inhibitors abrogated the ability of selenium to trigger apoptosis in leukemia CSC. Our results reveal how selenium-dependent modulation of arachidonic acid metabolism can be directed to trigger apoptosis of primary human and murine CSC in leukemia. Cancer Res; 74(14); 1–12. Ó2014 AACR. 12 Introduction Cyclopentenone prostaglandins (CyPG), D -PGJ2 and 15d- Leukemia is a hierarchical disease in which leukemia stem PGJ2, are derived from the polyunsaturated fatty acid, arachi- cells (LSC) occupy the apex and give rise to bulk leukemia donic acid (ARA), via the sequential action of cyclooxygenases cells that are responsible for the pathology of the disease. (COX) and PGD2 synthases (PGDS) followed by nonenzymatic D12 However, unlike LSCs, bulk leukemia cells are unable to conversion of PGD2 to -PGJ2 and 15d-PGJ2 (4, 5). We have D12 initiate leukemia when transplanted into secondary recipi- recently demonstrated the ability of -PGJ2 and 15d-PGJ2 to ents. Because of this property, LSCs represent a challenge to selectively activate the p53-dependent pathway of apoptosis in current therapy in that they can actively resist chemother- LSCs, without affecting the (normal) HSCs in a murine model – apy regimens and cause relapse of the disease (1, 2). In of CML and in an unrelated model of Friend virus induced chronic myelogenous leukemia (CML), the BCR–ABL1 fusion erythroleukemia (6). The importance of the COX pathway in protein generated from the 9:22 translocation is capable of leukemia has been highlighted in a few epidemiologic studies generating LSCs when expressed in hematopoietic stem cells in which increasing incidences of leukemia were associated fl (HSC;ref.3).Althoughcurrent treatment using tyrosine with non-steroidal anti-in ammatory drugs (NSAID) con- kinase inhibitors (TKI) can block the production of bulk sumption (7, 8), which led us to study strategies to increase leukemia cells and lead to remission of CML, these agents do endogenous CyPGs for treatment of leukemia. Previous studies not affect LSCs (1). Therefore, new therapies are needed that have shown supplementation of mice or macrophages with target LSCs to prevent relapse following traditional TKI selenium, a micronutrient that functions through insertion therapy. into redox-active selenoproteins, led to enhanced production fl of CyPGs as opposed to proin ammatory PGE2, TXA2, or PGF2a through a process termed "eicosanoid class switching" (9, 10). Authors' Affiliations: 1Department of Veterinary and Biomedical Therefore, we hypothesized that supraphysiologic levels of Sciences, Center for Molecular Immunology and Infectious Disease and selenium may play a key role in treating leukemia through Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, Pennsylvania; and 2Division of Hema- the ability of endogenous CyPGs to target LSCs. In fact, low tology, Hematologic Malignancies, and Stem Cell Transplantation, Uni- serum selenium levels have been noted in leukemic patients versity of Colorado, Anshutz Medical Campus, Aurora, Colorado (11–16). On the other hand, use of selenium-cystine (as dis- Note: Supplementary data for this article are available at Cancer Research elenodialanine) in leukemic patients decreased total leukocyte Online (http://cancerres.aacrjournals.org/). count, immature leukocytes in patients with CML and acute U.H. Gandhi, N. Kaushal, and S. Hegde contributed equally to this work. myelogenous leukemia (AML; ref. 17). Antileukemic effect of Corresponding Authors: K. Sandeep Prabhu, Penn State University, 115 sodium selenite (Na2SeO3) was suggested to activate p53 to Henning Building, University Park, PA 16802. Phone: 814-863-8976; Fax: cause apoptosis (18–24). However, the mechanism by which 814-863-6140; E-mail: [email protected]; and R.F. Paulson, [email protected]. selenium activates p53 is not well understood. doi: 10.1158/0008-5472.CAN-13-3694 In addition to being the guardian of the genome, p53 also Ó2014 American Association for Cancer Research. regulates glycolysis and aerobic respiration through oxidative www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst May 28, 2014; DOI: 10.1158/0008-5472.CAN-13-3694 Gandhi et al. phosphorylation (OXPHOS) in cancer stem-like cells (CSC; Induction of Friend erythroleukemia in mice ref. 25). This process is accompanied by an increase in the After completion of the diet-feeding schedule, Balb/c mice expression of Tigar (TPp53-inducible glycolysis and apoptosis were injected with FVP via the retroorbital sinus. On day 15 regulator), Rm2b (Ribonucleotide-diphosphate reductase sub- after infection, splenomegaly and changes in the hematologic unit RM2B), and Sco2 (synthesis of cytochrome oxidase-2) that parameters were assessed as described earlier (32). induce cell-cycle arrest followed by reactive oxygen species (ROS)–dependent apoptosis of irreparable cells (26–28). How- Expression of BCR–ABL fusion protein in HSC and ever, p53 also upregulated an antioxidant response through a induction of CML in mice sestrin-dependent Nrf-2 activation pathway (29). Thus, by Retroviral stocks were generated by transfecting HEK293T establishing complex regulatory networks involving temporal- cells with MIGR–BCR–ABL–IRESGFP or MIGR–IRESGFP ly segregated responses, p53 can cooperate with redox changes murine stem cell virus retroviral expression system (MSCV) to affect cellular metabolism and survival involving intricate empty plasmid (kind gift from Dr. Warren Pear, University of control of intracellular ROS levels, resulting in diverse out- Pennsylvania, Philadelphia, PA) using Fugene 6 transfection comes. In fact, the sensitivity of LSCs to agents that target reagent (Roche). Isolation and transduction of HSCs with these aberrant antioxidant (glutathione) metabolism (30) further viruses were performed as described earlier (3, 6). LSCs þ þ þ À highlight the importance of intracellular ROS as a means of (GFP Kit Sca1 Lin ) were isolated from spleen and bone inducing apoptosis in LSCs. marrow using FACS (BD Influx Cell Sorter) as described Here, we show that selenium-dependent modulation of the earlier (3, 6). ARA pathway is central to the ablation of LSCs in vivo. Treatment of selenium-supplemented mice with indometha- Serial transplantation assays þ cin, an NSAID, blunted the protective effects of selenium BCR–ABL LSCs isolated from the bone marrow of a C57BL/ implicating endogenous COX metabolites in the activation of 6 CD45.1 donor (as described above) were transplanted into Se- þ p53 and apoptosis of LSCs. In addition, treatment of blood A mice on a CD45.2 background. The LSCs (CD45.1 ) isolated samples from human patients with AML and blast-crisis CML from the bone marrow of primary transplants were used for þ with lipid extracts (LE) from selenium-supplemented macro- secondary transplants in CD45.2 mice maintained on Se-A or þ À þ phages induced apoptosis in CD34 CD38 CD123 LSCs, Se-S diets. The LSCs sorted from the bone marrow (using flow which was blocked by NSAIDs. The proapoptotic effects of cytometry) from Se-A or total bone marrow (unsorted) in the selenium were, in part, related to exacerbated oxidative stress case of Se-S mice were used in a tertiary transplant into þ in LSCs that involved NADPH oxidases, particularly Nox1. In CD45.2 Se-A mice. Leukocytosis and the presence of LSCs contrast, selenium treatment did not affect normal HSCs, in the bone marrow and spleen were examined using flow suggesting that LSCs are uniquely sensitive to changes in cytometry. intracellular ROS. These studies suggest a new mechanism that underlies the selenium-dependent effects on the viability Histology and TUNEL staining of LSCs, which may open new opportunities for leukemia Formalin-fixed spleens were paraffin-embedded, sectioned, therapy. and used for hematoxylin and eosin or terminal deoxynucleo- tidyl transferase–mediated dUTP nick end labeling (TUNEL) staining to examine gross anatomical changes or apoptosis, Materials and Methods respectively, following induction of leukemia as described in Differential selenium status in mice the Supplementary Methods. Three-week-old male BALB/c mice (n ¼ 7/group) for poly-