Published OnlineFirst March 29, 2018; DOI: 10.1158/0008-5472.CAN-17-2906 Cancer Metabolism and Chemical Biology Research Transketolase Regulates the Metabolic Switch to Control Breast Cancer Cell Metastasis via the a-Ketoglutarate Signaling Pathway Chien-Wei Tseng1,2, Wen-Hung Kuo3, Shih-Hsuan Chan1,2,4, Hong-Lin Chan5, King-Jen Chang6, and Lu-Hai Wang1,2 Abstract Although metabolic reprogramming is recognized as a hall- mately suppressing breast cancer metastasis. Reduced TKT or mark of tumorigenesis and progression, little is known about addition of aKG mediated a dynamic switch of glucose metab- metabolic enzymes and oncometabolites that regulate breast olism from glycolysis to oxidative phosphorylation. Various cancer metastasis, and very few metabolic molecules have been combinations of the TKT inhibitor oxythiamine, docetaxel, and identified as potential therapeutic targets. In this study, the doxorubicin enhanced cell death in triple-negative breast cancer transketolase (TKT) expression correlated with tumor size in the (TNBC) cells. Furthermore, oxythiamine treatment led to 4T1/BALB/c syngeneic model. In addition, TKT expression was increased levels of aKG in TNBC cells. Together, our study has higher in lymph node metastases compared with primary tumor identified a novel TKT-mediated aKG signaling pathway or normal tissues of patients, and high TKT levels were associated that regulates breast cancer oncogenesis and can be exploited as with poor survival. Depletion of TKT or addition of alpha-keto- a modality for improving therapy. glutarate (aKG) enhanced the levels of tumor suppressors succi- Significance: These findings uncover the clinical significance nate dehydrogenase and fumarate hydratase (FH), decreasing of TKT in breast cancer progression and metastasis and demon- oncometabolites succinate and fumarate, and further stabilizing strate effective therapy by inhibiting TKT or by adding aKG. HIF prolyl hydroxylase 2 (PHD2) and decreasing HIF1a, ulti- Cancer Res; 78(11); 2799–812. Ó2018 AACR. Introduction elevates the expression of glycolytic enzymes, including aldolase A, phosphoglycerate kinase 1, and pyruvate kinase (3). In addi- Patients with breast cancer have a 5-year survival rate over 90%; tion, a number of studies revealed that genetic defects in TCA however, for patients with distant metastasis, their survival rate cycle enzymes, such as succinate dehydrogenase (SDH) and decreases to only about 25% because of the lack of effective fumarate hydratase (FH), were also associated with tumor pro- strategies against breast cancer metastasis and recurrence (1). gression (4, 5). Tumor cells with altered metabolic program have high require- In this study, we used a proteomic approach to identify ments of glucose metabolism for rapid proliferation. Despite certain differentially expressed metabolic enzymes involved in some studies aiming at elucidating the correlation between tumor progression such as aldolase A (ALDOA), triose phos- aberrant metabolic behavior and tumor progression, how meta- phate isomerase (TPIS), a-enolase (ENOA), transketolase bolic processes regulate breast cancer cells growth and metastasis (TKT), and pyruvate dehydrogenase E1 (ODPB). Among them, is not fully understood. TKT is a metabolic enzyme involved in the nonoxidative branch A number of studies show that oncogenic signaling in cancers of the pentose phosphate pathway (PPP) and connects PPP drives metabolic reprogramming to generate large amounts of with glycolysis. Previous studies revealed that TKT was associ- biomass during rapid tumor growth (2). For example, HIF1a ated with metastasis of ovarian (6) and esophageal (7) cancers, as well as poor patient survival (6, 7). To date, no study has reported the effect of TKT-regulated metabolic signaling on 1Graduate Institute of Integrated Medicine, China Medical University, Taichung, tumor metastasis in breast cancer. Taiwan. 2Institute of Molecular and Genomic Medicine, National Health Research In this study, we reveal clinical significance and regulatory Institutes, Zhunan, Miaoli County, Taiwan. 3Department of Surgery, National mechanism of TKT in progression and metastasis of breast cancer 4 Taiwan University Hospital, Taipei, Taiwan. Institute of Molecular Medicine, via alpha-ketoglutarate (aKG) signaling. TKT plays important National Tsing Hua University, Hsinchu, Taiwan. 5Institute of Bioinformatics and 6 roles in regulating dynamic switch of glucose metabolism. The Structural Biology, National Tsing Hua University, Hsinchu, Taiwan. Department a of Surgery, Taiwan Adventist Hospital, Taipei, Taiwan. combined therapy based on the new targets TKT or KG could be developed as an improved therapeutic approach for triple- Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). negative breast cancer (TNBC). Corresponding Author: Lu-Hai Wang, China Medical University, No. 91, Hsueh- Shih Road, Taichung 40402, Taiwan. Phone: 8864-2205-7153; Fax: 8864-2206- Materials and Methods 0248; E-mail: [email protected] Cell culture and transfection doi: 10.1158/0008-5472.CAN-17-2906 The human breast cancer MDA-MB-231, Hs578T and MCF-7 Ó2018 American Association for Cancer Research. cells, and mouse breast cancer 4T1 cells were obtained from www.aacrjournals.org 2799 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst March 29, 2018; DOI: 10.1158/0008-5472.CAN-17-2906 Tseng et al. ATCC. The 4T1 is a highly tumorigenic and invasive cell line University Hospital (Taipei, Taiwan). Other samples were from capable of metastasizing from the primary mammary gland commercial tissue arrays (US Biomax; SuperBioChips), including tumor to liver, lung, lymph nodes, and brain. The highly meta- 19 normal, 90 tumors, and 50 lymph node metastatic tissues. The static cell line MDA-MB-231-IV2-3 was previously established and slides were stained with mouse monoclonal anti-TKT antibody described (8). All cell lines were cultured in DMEM (Invitrogen) (clone 7H1AA1, ab112997; Abcam) using an automatic slide supplemented with 10% FBS (Biological Industries) at 37C with stainer BenchMark XT (Ventana Medical Systems). The staining 5% CO2. Cell lines were clear of Mycoplasma as determined by the intensities were evaluated and quantified by one pathologist Venor GeM kit (Minerva Biolabs) and were further authenticated (Pathology Core Lab, National Health Research Institutes) and in 2017 by Taiwan Bioresource Collection and Research Centre two independent investigators. The IHC scores of TKT for each (BCRC) using a short tandem repeat method. For transfection specimen were graded as follows: no expression, weak (þ); assay, cells were transfected with 20 mmol/L siTKT or 20 mmol/L moderate (þþ); and strong (þþþ).The expression levels of TKT siRNA control or TKT/pCMV plasmid (1 mg/mL) using Lipofecta- in tumor cells were quantified as a percentage. Paraffin-embedded mine RNAiMAX transfection reagent (Thermo Fisher Scientific). sections of tumor cells with TKTL1 overexpression (Origene, RG205218) were stained with mouse monoclonal anti-TKT anti- Protein extraction body (1 mg/mL, 1:75 dilution; clone 7H1AA1, ab112997; Cell samples were lysed in lysis buffer containing 7 mol/L urea, Abcam) or rabbit polyclonal anti-TKTL1 antibody (1 mg/mL, 2 mol/L thiourea, 4% w/v CHAPS, 10 mmol/L Tris-HCl pH 8.3, 1:75 dilution; clone N1C1, GTX109459; Genetex). We first used and 1 mmol/L EDTA. Protein lysates were extracted, sonicated, the D'Agostino and Pearson omnibus normality test to reveal that and centrifuged and the protein concentration was determined the quantitative results of IHC TKT expression were not Gaussian using Coomassie Protein Assay Reagent (Bio-Rad). distribution (P ¼ 0.0015). Thus, we used nonparametric Mann– Whitney test to analyze the quantitative results. 2-D DIGE gel image analysis and protein identification by MALDI-TOF-MS Proliferation assay The protein profiles of tumor tissues with 0.5, 1, and 2 cm in Cell proliferation was detected using CellTiter 96 Aqueous One size were analyzed using 2D differential gel electrophoresis Solution Cell Proliferation Assay (Promega). Assay was per- (DIGE). Protein samples were labeled with cyanine dyes Cy2, formed according to manufacturer's protocol. A total of 1.4 Â Cy3, and Cy5, and all procedures have been described previously 104 cells were cultured in a 24-well plate and incubated for (9, 10). The Cy-Dye–labeled 2-DE gels were visualized according different times. CellTiter 96 Aqueous One Solution reagent was to the previous report (10). For protein identification, the peptide added and incubated for 1 hour at 37C. The quantity of formazan mixture was loaded onto a MALDI plate and samples were product, proportional to living cell numbers, was measured at 490 analyzed using an Autoflex III mass spectrometer (Bruker Dal- nm using 96-well plate reader. Each experiment was performed in tonics) and parameters were described according to the previous triplicate and the shown data were mean Æ SD. report (10). Cell invasion and migration assays Western blotting MDA-MB-231 and Hs578T cells were treated with 20 mmol/L Cells were lysed in the lysis buffer containing 7 mol/L urea, siTKT or 1 mmol/L aKG, or TKT/pCMV plasmid (1 mg/mL). After 2 mol/L thiourea, 4% w/v CHAPS, 10 mmol/L Tris-HCl (pH 8.3), 48 hours, these cells (1 Â 105 cells) were seeded on Boyden 1 mmol/L EDTA, and phosphatase and protease inhibitors chamber, incubated for 8 hours, and