Published OnlineFirst April 6, 2016; DOI: 10.1158/0008-5472.CAN-15-2601 Cancer Molecular and Cellular Pathobiology Research Decreased Expression of Fructose-1,6- bisphosphatase Associates with Glucose Metabolism and Tumor Progression in Hepatocellular Carcinoma Hidenari Hirata1,2,3, Keishi Sugimachi1, Hisateru Komatsu1, Masami Ueda1,Takaaki Masuda1, Ryutaro Uchi1, Shotaro Sakimura1, Sho Nambara1, Tomoko Saito1, Yoshiaki Shinden1, Tomohiro Iguchi1, Hidetoshi Eguchi1, Shuhei Ito1, Kotaro Terashima2, Katsumi Sakamoto2, Masakazu Hirakawa2, Hiroshi Honda3, and Koshi Mimori1 Abstract Fructose-1,6-bisphosphatase (FBP1), the rate-limiting enzyme promoter methylation and copy-number loss of FBP1 were inde- in gluconeogenesis, is reduced in expression in certain cancers pendently associated with decreased FBP1 expression. Similarly, where it has been hypothesized to act as a tumor suppressor, FBP1 downregulation in HCC cell lines was also associated with including in hepatocellular carcinoma (HCC). Here, we report copy-number loss. HCC specimens exhibiting low expression of functional evidence supporting this hypothesis, providing a pre- FBP1 had a highly malignant phenotype, including large tumor clinical rationale to develop FBP1 as a therapeutic target for HCC size, poor differentiation, impaired gluconeogenesis, and enhanced treatment. Three independent cohorts totaling 594 cases of HCC aerobic glycolysis. The effects of FBP1 expression on prognosis and were analyzed to address clinical significance. Lower FBP1 expres- glucose metabolism were confirmed by gene set enrichment anal- sion associated with advanced tumor stage, poor overall survival, ysis. Overall, our findings established that FBP1 downregulation in and higher tumor recurrence rates. In HCC cell lines, where HCC contributed to tumor progression and poor prognosis by endogenous FBP1 expression is low, engineering its ectopic over- altering glucose metabolism, and they rationalize further study of expression inhibited tumor growth and intracellular glucose FBP1 as a prognostic biomarker and therapeutic target in HCC uptake by reducing aerobic glycolysis. In patient specimens, patients. Cancer Res; 76(11); 3265–76. Ó2016 AACR. Introduction cytes (3). A recent study reported the loss of gluconeogenic capacity in HCC (4). Moreover, the switch from aerobic glycolysis Hepatocellular carcinoma (HCC) is one of the most prevalent to gluconeogenesis may be an effective treatment in patients with cancers worldwide and is associated with a high mortality rate (1). HCC (5). Nevertheless, the role of impaired gluconeogenesis in Therefore, identification of useful biomarkers predicting clinical HCC progression is still poorly understood. outcomes and molecular targets for treatment is required. Altered The tumor-suppressive function of the fructose-1,6-bisphospha- glucose metabolism, particularly aerobic glycolysis, is one of the tase (FBP1) gene, which encodes a rate-limiting gluconeogenic hallmarks of cancers, including HCC (2), supporting the notion enzyme, has been reported in certain cancers. Loss of FBP1 that the molecules involved in altered glucose metabolism may be expression has been shown to promote tumor progression by potential biomarkers and therapeutic targets. Many studies have enhancing aerobic glycolysis, thereby resulting in poor prognosis examined aerobic glycolysis in various cancers; however, gluco- in patients with clear cell renal cell carcinoma (ccRCC; ref. 6) and neogenesis has not been studied extensively. Gluconeogenesis is breast cancer (7). With regard to HCC, in vitro experiments have also thought to play an important role in tumor progression in shown that FBP1 expression is silenced by promoter methylation HCC because gluconeogenesis mainly occurs in normal hepato- and that ectopic FBP1 expression inhibits anchorage-dependent growth of a cell line through cell-cycle arrest (8). However, the role 1Department of Surgery, Kyushu University Beppu Hospital, Beppu, of FBP1 in altered glucose metabolism has not been assessed in Japan. 2Department of Radiology, Kyushu University Beppu Hospital, HCC. Furthermore, no studies have investigated whether aberrant 3 Beppu, Japan. Department of Clinical Radiology, Graduate School of FBP1 expression affects clinical outcomes in patients with HCC. Medical Sciences, Kyushu University, Fukuoka, Japan. Therefore, the objective of this study was to examine the Note: Supplementary data for this article are available at Cancer Research function and clinical significance of FBP1 expression in HCC. Online (http://cancerres.aacrjournals.org/). Through analysis of three independent large cohorts of HCC Corresponding Author: Koshi Mimori, Kyushu University Beppu Hospital, 4546 cases, the present study evaluated whether aberrant FBP1 expres- Tsurumihara, Beppu 8740838, Japan. Phone: 81-977-27-1650; Fax: 81-977-27- sion was associated with tumor progression and prognosis in 1651, E-mail: [email protected] patients with HCC. We also elucidated the genetic and epigenetic doi: 10.1158/0008-5472.CAN-15-2601 mechanisms regulating FBP1 expression and investigated the role Ó2016 American Association for Cancer Research. of FBP1 expression in altered glucose metabolism. www.aacrjournals.org 3265 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst April 6, 2016; DOI: 10.1158/0008-5472.CAN-15-2601 Hirata et al. Materials and Methods Encyclopedia (CCLE; ref. 10) from cBioPortal For Cancer Geno- mics (http://www.cbioportal.org/index.do; ref. 11). Of the 967 Patients and sample collection human cancer cell lines in the CCLE database, FBP1 expression One hundred eighteen patients with HCC who underwent profiles were available for 27 liver cancer cell lines. A z-score hepatic resection at Kyushu University Beppu Hospital and affil- threshold of 2 was used as the cutoff value. For the analysis of iated hospitals between 2000 and 2004 were enrolled in this copy-number alterations, data were available for 23 liver cancer study. Resected tumor tissues and paired normal liver tissues were cell lines (Supplementary Table S1). Moreover, FBP1 promoter immediately stored in RNAlater (Ambion), frozen in liquid methylation status was determined using the results of methyl- nitrogen, and kept at –80C until RNA extraction. A 5-year ation-specific PCR, as described in a previous report (8), or follow-up was conducted after operation. The median follow-up methylation array data from GEO database accession numbers for the 118 patients was 59.5 months (range, 3.0–60 months). GSE60753 (12) and GSE67485 (13). Data on promoter methyl- Patients were staged according to the seventh edition of the ation profiles were available for 7 liver cancer cell lines (Supple- International Union against Cancer TNM classification system. mentary Table S2). Of the 118 HCC patients, paired normal liver tissues were avail- able for 115 patients. All protocols were approved by the Ethics Western blotting analysis and Indications Committee of Kyushu University. Written Proteins were detected using antibodies against FBP1 informed consent was obtained from all patients. (SAB1405798; Sigma-Aldrich), thioredoxin-interacting protein (TXNIP; K0204-3; Medical & Biological Laboratories), and b-actin Cell culture (Santa Cruz Biotechnology). The methods are described in detail HuH7 and HepG2 cells authenticated by short tandem repeat in the Supplementary Materials and Methods. profiling using the PowerPlex 1.2 System (Promega) were obtained from the cell bank of RIKEN BioResource Center (Tsu- Sphere formation assays kuba, Japan) in 2015. Cells were used within 6 months of Detailed methods are provided in the Supplementary Materials culturing after resuscitation. Details are provided in the Supple- and Methods. mentary Materials and Methods. Xenograft studies Construction of an FBP1 expression lentiviral vector Five-week-old female BALB/c nu/nu mice were obtained from FBP1 fi A full-length cDNA insert of human was ampli ed by SLC, Inc. and maintained under specific pathogen-free condi- PCR. Lentiviruses were generated by transfection of HEK293T cells tions. All animal procedures were performed in compliance with with pCMV-VSV-G-RSV-Rev, pCAG-HIVgp, and either CSII-CMV- the Guidelines for the Care and Use of Experimental Animals 0 Nhe 0 FBP1 or CSII-CMV-MCS (empty) plasmid DNAs (5 I and 3 established by the Committee for Animal Experimentation of Xba I sites) using Lipofectamine 2000 (Invitrogen) following the Kyushu University. A total of 5  106 cells (empty vector control manufacturer's protocol. Forty-eight hours after transfection, the cells or HuH7 cells stably expressing FBP1) in 150 mL of phos- lentivirus-containing supernatant was collected and passed phate-buffered saline were injected subcutaneously into both m fi through a 0.45- m lter. Infections were then carried out by flanks of the mice. Tumor size was measured every 3 to 4 days incubating cells with medium containing the lentiviral superna- with a digital caliper and calculated using the following formula: tant for 48 hours. tumor volume ¼ length  width2  0.5. RNA preparation and quantitative real-time RT-PCR Quantification of metabolites Detailed methods are provided in the Supplementary Materials Intracellular glucose uptake was determined by 2-(N-[7-nitro- and Methods. benz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxy-D-glucose (2-NBDG), a fluorescently-tagged glucose derivative, using a Glucose Uptake Analysis of public clinical datasets and gene set