Published OnlineFirst September 5, 2013; DOI: 10.1158/1541-7786.MCR-13-0172 Molecular Cancer Cell Death and Survival Research Inhibition of mTOR Pathway Sensitizes Acute Myeloid Leukemia Cells to Aurora Inhibitors by Suppression of Glycolytic Metabolism Ling-Ling Liu1,2, Zi-Jie Long1,3, Le-Xun Wang1,3, Fei-Meng Zheng2, Zhi-Gang Fang1, Min Yan2, Dong-Fan Xu1,3, Jia-Jie Chen1,3, Shao-Wu Wang4,5, Dong-Jun Lin1,3, and Quentin Liu1,2,3,4 Abstract Aurora kinases are overexpressed in large numbers of tumors and considered as potential therapeutic targets. In this study, we found that the Aurora kinases inhibitors MK-0457 (MK) and ZM447439 (ZM) induced polyploidization in acute myeloid leukemia (AML) cell lines. The level of glycolytic metabolism was significantly increased in the polyploidy cells, which were sensitive to glycolysis inhibitor 2-deoxy-D-glucose (2DG), suggesting that polyploidy cells might be eliminated by metabolism deprivation. Indeed, inhibition of mTOR pathway by mTOR inhibitors (rapamycin and PP242) or 2DG promoted not only apoptosis but also autophagy in the polyploidy cells induced by Aurora inhibitors. Mechanically, PP242 or2DGdecreased the level of glucose uptake and lactate production in polyploidy cells as well as the expression of p62/SQSTM1. Moreover, knockdown of p62/ SQSTM1 sensitized cells to the Aurora inhibitor whereas overexpression of p62/SQSTM1 reduced drug efficacy. Thus, our results revealed that inhibition of mTOR pathway decreased the glycolytic metabolism of the polyploidy cells, and increased the efficacy of Aurora kinases inhibitors, providing a novel approach of combination treatment in AML. Mol Cancer Res; 11(11); 1326–36. Ó2013 AACR. Introduction apoptosis of acute myeloid leukemia (AML) cells (6). Our previous study showed that VX-680 (MK-0457), a rela- Anticancer drug that targets cell-cycle regulators has fi been widely applied in the treatment of the hematologic tively speci c Aurora A inhibitor, induced apoptotic cell malignancies (1–3). Aurora kinases A–C, which play death in AML cells, especially in the primary leukemic blasts with high Aurora A expression (4, 7). Although important roles in the process of mitosis, have been fi implicated in the tumorigenesis (1–3). Aurora A and B Aurora kinases inhibitors have signi cant therapeutic potential in AML, single-agent activity seems to be uni- proteins were overexpressed in a number of tumors, fi including leukemia cells, which were correlated with aneu- formly modest and the ef cacy needs improving (6). ploidy in tumor tissues and poor prognosis (4, 5). Previous Recently, we also showed that Aurora A inhibition trig- report showed that Aurora kinases inhibitors could sup- gered drug resistance could be reversed by autophagy press the growth of leukemia cells (1). ZM447439 (ZM), inhibitors in breast cancer cells (8). In addition, combi- the dual Aurora A and B inhibitor, inhibited the cell nation of Aurora kinases inhibitors with vincristine or proliferation, induced polyploidization, and promoted dasatinib presented synergistic effect to inhibit cell viability in human pre-B acute lymphoblastic leukemia (9). Thus, new strategy for drug combination is urgently required to Authors' Affiliations: 1Department of Hematology, the Third Affiliated overcome drug resistance induced by Aurora A inhibition. Hospital, Sun Yat-sen University, Guangzhou, China; 2State Key Labora- tory of Oncology in South China, Cancer Center, Sun Yat-sen University, Polyploidy is a prominent characteristic of human cancers Guangzhou, China; 3Sun Yat-sen Institute of Hematology, Guangzhou, and proposed as a driver of tumorigenesis (10). Study China; 4Institute of Cancer Stem Cell, Dalian Medical University, Dalian, showed that somatic polyploidy was associated with China; and 5Department of Radiology, the First Affiliated Hospital, Dalian Medical University, Dalian, China readjustment of main metabolic pathways. For example, mitochondrial process was depressed, and carbohydrate Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). degradation and lipid biosynthesis were increased in the polyploidy cells (11). Warburg reported that tumors pre- L.-L. Liu and Z.-J. Long have contributed equally to this article. ferred aerobic glycolysis to mitochondrial oxidative phos- Corresponding Authors: Quentin Liu, Department of Hematology, the phorylation (12). Based on the reports, suppression of the Third Affiliated Hospital, Sun Yat-sen University, 600 Tianhe Road, Guangzhou 510630, China. Phone: 86-20-8525-3601; Fax: 86-20- aerobic glycolytic metabolism might help for restraining the 87343171; E-mail: [email protected]; and Dong-Jun Lin, proliferation of polyploidy cancer cells. Aurora kinases are [email protected] crucial for cytokinesis in a number of reports. Aurora kinases doi: 10.1158/1541-7786.MCR-13-0172 defection induced mitosis arrest and promoted polyploidy Ó2013 American Association for Cancer Research. formation (13). Therefore, readjustment of the metabolic 1326 Mol Cancer Res; 11(11) November 2013 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst September 5, 2013; DOI: 10.1158/1541-7786.MCR-13-0172 mTOR Inhibition Enhances the Efficacy of Aurora Inhibitors status in polyploidy cells induced by Aurora kinases inhibi- Peripheral blood mononuclear cells (PBMC) were enriched tion could also be a promise approach to cancer therapy. by Ficoll–Hypaque density gradient centrifugation. The mTOR, a well-conserved serine/threonine kinase with 2 complexes, mTOR complex 1 (mTORC1) and Cell-proliferation assay mTOR complex 2 (mTORC2), is essential for cell growth, Cell viability was assessed by Cell counting kit-8 protein translation, autophagy, and metabolism (14). To (CCK8; Dojindo Biotechnology) according to the man- adapt the condition of hypermetabolic demand and thrive ufacturer's instructions. The optical density at 450 nm was rapidly, oncogenes in growth and nutrient-sensing signaling measured using a multiwell plate reader (Micro-plate pathways was activated (15). In numerous cancer types, Reader; Bio-Rad). mTOR signaling pathway was activated by deregulation of its upstream factors, such as Akt activation or deficiency of Cell-cycle analysis tuberous sclerosis complex 2 (TSC2; refs. 16 and 17). Cell cycle was analyzed by flow cytometer. Cells were Activation of mTORC1, a metabolic hallmark of cancer, treated with drugs for 24 hours. Then, cells were harvested increased glycolysis, and lipid biosynthesis (15). Although and stained with mixture of 50 mg/mL propidium iodide mTORC2 was not thoroughly studied in the metabolism of (PI), 0.2% Triton X-100, and 100 mg/mL RNAase inhib- tumor, RNAi against one of mTORC2 components also itor. Cell-cycle analysis was conducted using a FACS flow impaired the glucose and lipid metabolism in muscle and cytometer equipped with Modfit LT for Mac V2.0 software fat cells (18, 19). Furthermore, mTOR was associated with (BD Biosciences). the development of polyploidization. Ma and colleagues showed that S6K1, a downstream of mTOR, was involved Microscopic analysis of cells and nuclear morphology in polyploidization through its phosphorylation at Thr421/ Cells were treated with Aurora kinases inhibitors for 24 Ser424 (20). In addition, activation of mTOR pathway was hours, followed by PP242 or 2DG for another 24 hours. þ required for TSC2 /EK [EKer rat model carrying a germline After 48 hours, the cells were photographed under an insertional deletion in one copy of the Tsc2 (tuberous inverted microscopy (Olympus). Nuclear fragmentation was sclerosis 2) gene] smooth muscle cells of pulmonary lym- stained by Hoechst 33342 (Sigma) with 10 mg/mL for 15 phangioleiomyomatosis to undergo tetraploidization (21). minutes at 37C. Slides were viewed using a fluorescence Here, we found that mTOR signaling pathway was critical microscope. for maintaining the glycolytic metabolism state of polyploidy cancer cells induced by inhibition of Aurora kinases. More- Monodansylcadaverine staining over, we provided evidences that combination of mTOR Cells were incubated with the monodansylcadaverine inhibitors and Aurora kinases inhibitors effectively elimi- (MDC; Sigma) with a final concentration of 0.05 mmol/L nated leukemia cells by inducing apoptosis and autophagy. for 10 minutes at 37C. Then, cells were viewed on slides These results suggested a new approach of targeted thera- under a laser scanning confocal microscope (ZEISS, peutic drugs combination for AML treatment. Germany), and tested by flow cytermetry. MDC dye was applied to stain the late autophagic vacuoles. Materials and Methods Western blotting analysis Reagents and cell culture Total cellular proteins were isolated with RIPA buffer. Reagents included MK-0457 (Selleck Chemicals, South Lysates were subjected to SDS-PAGE gel electrophoresis, Loop West, Houston), ZM447439 (AstraZeneca, Canada), and were transferred to nitrocellulose membranes. The PP242, 2-deoxy-D-glucose (2DG), and rapamycin (Sigma). membranes were blocked with 5% bovine serum albumin U937 cell line was obtained from the American Type and then incubated overnight with LC3 (Novus), p62 (Santa Culture Collection (ATCC). 293FT cell line was obtained Cruz), Aurora A (Upstate), p-mTOR (Ser2448, Ser2481), from Invitrogen. NB4 and HL-60 cell lines were provided by mTOR, p-Aurora A (Thr288), cleaved caspase 3 (Asp175), Shanghai Institute of Hematology, Ruijin Hospital. U937, cleaved