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Transketolase Regulates the Metabolic Switch to Control Breast Cancer Cell Metastasis Via the Alpha-Ketoglutarate Signaling Pathway

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Transketolase Regulates the Metabolic Switch to Control Breast Cancer Cell Metastasis Via the Alpha-Ketoglutarate Signaling Pathway Author Manuscript Published OnlineFirst on March 29, 2018; DOI: 10.1158/0008-5472.CAN-17-2906 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Transketolase Regulates the Metabolic Switch to Control Breast Cancer Cell Metastasis via the Alpha-ketoglutarate Signaling Pathway Chien-Wei Tseng1,2, Wen-Hong Kuo3, Shih-Hsuan Chan1,2,4, Hong-Lin Chan5, King-Jen Chang6, Lu-Hai Wang*1,2 1Graduate Institute of Integrated Medicine, China Medical University, Taichung, 404, Taiwan 2Institute of Molecular and Genomic Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli Country 350, Taiwan 3Department of Surgery, National Taiwan University Hospital, Taipei 100, Taiwan 4Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 300, Taiwan 5Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan 6 Department of Surgery, Taiwan Adventist Hospital, Taipei 105, Taiwan *Corresponding author Running title: TKT regulates breast cancer metastasis via α-KG signaling Statement of significance: Findings uncover the clinical significance of TKT in breast cancer progression and metastasis and demonstrate effective combined therapies against TKT and α-KG. Abbreviations list: transketolase, TKT; alpha-ketoglutarate, α-KG; succinate dehydrogenase, SDH; fumarate hydratase, FH; HIF prolyl hydroxylase 2, PHD2; triple negative breast cancer, TNBC; aldolase A, ALDOA; triose phosphate isomerase, TPIS; α-enolase, ENOA; pyruvate dehydrogenase E1, ODPB; pentose phosphate pathway, PPP; differential gel electrophoresis, DIGE; enhanced chemiluminescence, ECL; Institutional Animal Care and Use Committee, IACUC; Oxygen consumption rate, OCR; extracellular acidification rate, ECAR; carbonyl 1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 29, 2018; DOI: 10.1158/0008-5472.CAN-17-2906 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. cyanide-p-trifluoromethoxyphenylhydrazone, FCCP; 2-deoxy glucose, 2-DG; bioluminescence imaging, BLI; vascular endothelial growth factor, VEGF; G-protein-coupled receptor, GPCR; L-2HG dehydrogenase, L2HGDH; D-2HG dehydrogenase, D2HGDH; JmjC domain-containing histone demethylase, KDMs; glucose-6-phosphate, G6P; hepatocellular carcinoma, HCC; reactive oxygen species, ROS; pyruvate kinase M2, PKM2; 5-methylcytosine, 5mC; 5-carboxylcytosine, 5caC; docetaxel, Doc; doxorubicin, Dox; oxythiamine, OT Address Correspondence to: Lu-Hai Wang, China Medical University, No. 91, Hsueh-Shih Road, Taichung, 40402, Taiwan, Phone: 886-4-22057153, Fax: 886-4-22060248, E-mail: [email protected] or [email protected] . Conflict of interest statement The authors declare no potential conflicts of interest. 2 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 29, 2018; DOI: 10.1158/0008-5472.CAN-17-2906 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Although metabolic reprogramming is recognized as a hallmark of tumorigenesis and progression, little is known about metabolic enzymes and oncometabolites that regulate breast cancer metastasis, and very few metabolic molecules have been identified as potential therapeutic targets. In this study, the transketolase (TKT) expression correlated with tumor size in the 4T1/BALB/c syngeneic model. In addition, TKT expression was higher in lymph node metastases compared with primary tumor or normal tissues of patients, and high TKT levels were associated with poor survival. Depletion of TKT or addition of alpha-ketoglutarate (α-KG) enhanced the levels of tumor suppressors succinate dehydrogenase (SDH) and fumarate hydratase (FH), decreasing oncometabolites succinate and fumarate and further stabilizing HIF prolyl hydroxylase 2 (PHD2) and decreasing HIF-1α, ultimately suppressing breast cancer metastasis. Reduced TKT or addition of α-KG mediated a dynamic switch of glucose metabolism from glycolysis to oxidative phosphorylation. Various combinations of the TKT inhibitor oxythiamine, docetaxel, and doxorubicin enhanced cell death in triple-negative breast cancer (TNBC) cells. Furthermore, oxythiamine treatment led to increased levels of α-KG in TNBC cells. Together, our study has identified a novel TKT-mediated α-KG signaling pathway that regulates breast cancer oncogenesis and can be exploited as a modality for improving 3 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 29, 2018; DOI: 10.1158/0008-5472.CAN-17-2906 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. therapy. Keywords: TNBC, TKT, α-KG, metabolism, metastasis Introduction Breast cancer patients have five-year survival rate over 90%; however, for patients with distant metastasis, their survival rate decreases to only about 25% because of the lack of effective strategies against breast cancer metastasis and recurrence (1). Tumor cells with altered metabolic program have high requirements of glucose metabolism for rapid proliferation. Despite some studies aiming at elucidating the correlation between aberrant metabolic behavior and tumor progression, how metabolic processes regulate breast cancer cells growth and metastasis is not fully understood. A number of studies show that oncogenic signaling in cancers drives metabolic reprogramming to generate large amounts of biomass during rapid tumor growth (2). For example, HIF-1α elevates the expression of glycolytic enzymes including aldolase A, phosphoglycerate kinase 1, and pyruvate kinase (3). In addition, a number of studies revealed that genetic defects in TCA cycle enzymes, such as SDH and FH, were also associated with tumor progression (4,5). In this study, we used proteomic approach to identify certain differentially expressed metabolic enzymes involved in tumor progression such as aldolase A 4 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 29, 2018; DOI: 10.1158/0008-5472.CAN-17-2906 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. (ALDOA), triose phosphate isomerase (TPIS), α-enolase (ENOA), transketolase (TKT) and pyruvate dehydrogenase E1 (ODPB). Among them, TKT is a metabolic enzyme involved in the non-oxidative branch of the pentose phosphate pathway (PPP) and connects PPP with glycolysis. Previous studies revealed that TKT was associated 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 tumor metastasis in breast cancer. In this study, we reveal clinical significance and regulatory mechanism of TKT in progression and metastasis of breast cancer via α-KG signaling. TKT plays important roles in regulating dynamic switch of glucose metabolism. The combined therapy based on the new targets TKT or α-KG could be developed as an improved therapeutic approach for TNBC. Materials and Methods Cell culture and transfection The human breast cancer MDA-MB-231, Hs578T and MCF-7 cells and mouse breast cancer 4T1 cells were from ATCC (Manassas, VA). The 4T1 is a highly tumorigenic and invasive cell line capable of metastasizing from the primary mammary gland tumor to liver, lung, lymph nodes and brain. The highly metastatic 5 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 29, 2018; DOI: 10.1158/0008-5472.CAN-17-2906 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. cell line MDA-MB-231-IV2-3 was previously established and described (8). All cell lines were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine o serum (Biological Industries, Israel) at 37 C with 5% CO2. Cell lines were clear of mycoplasma as determined by the Venor GeM kit (MB Minerva biolabs) and were further authenticated in 2017 by Taiwan Bioresource Collection and Research Centre (BCRC) using a short tandem repeat method. For transfection assay, cells were transfected with 20 μM siTKT or 20 μM siRNA control or TKT/pCMV plasmid (1 μg/μL) using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, Waltham, MA). Protein extraction Cell samples were lysed in lysis buffer containing 7M urea, 2M thiourea, 4% w/v CHAPS, 10 mM Tris-HCl pH 8.3 and 1 mM EDTA. Protein lysates were extracted, sonicated and centrifuged and the protein concentration was determined using Coomassie Protein Assay Reagent (BioRad). 2-D DIGE, gel image analysis and protein identification by MALDI-TOF-MS The protein profiles of tumor tissues with 0.5, 1 and 2 cm in size were analyzed using 2-D differential gel electrophoresis (DIGE). Protein samples were labeled with cyanine dyes Cy2, Cy3 and Cy5 and all procedures have been described previously (9,10). The Cy-Dye-labeled 2-DE gels were visualized according to the previous 6 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author
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