2674 ONCOLOGY REPORTS 40: 2674-2682, 2018 Regulatory mechanism of fatty acid‑CoA metabolic enzymes under endoplasmic reticulum stress in lung cancer KUAN-TING LIU1-3, I-JENG YEH3, SHIH-KAI CHOU4, MENG-CHI YEN1,3 and PO-LIN KUO1,5 1Graduate Institute of Clinical Medicine and 2School of Medicine, College of Medicine, Kaohsiung Medical University; 3Department of Emergency Medicine, Kaohsiung Medical University Hospital; 4Graduate Institute of Medicine, College of Medicine and 5Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung 807, Taiwan, R.O.C. Received March 23, 2018; Accepted August 10, 2018 DOI: 10.3892/or.2018.6664 Abstract. The endoplasmic reticulum (ER) is an organelle enzymes, the biological effects of decreased enzyme expres- involved in various physiological processes such as lipid sion levels, possible regulatory elements, and the interaction metabolism, protein synthesis and folding, and cellular network involved in responses to ER stress in lung cancer calcium storage. In a physiological tumor microenvironment, hypoxia, nutrient deprivation, and calcium dysregulation cause Introduction accumulation of unfolded and misfolded proteins. Such accu- mulation induces ER stress and unfolded protein responses Lung cancer is one of the most common cancers in the (UPRs). Increased UPR signaling pathways are associated world (1), and non-small cell lung cancer (including adenocar- with multiple types of cancer. The influence of ER stress cinoma and squamous cell carcinoma) accounts for most of the on acyl-CoA metabolic enzymes is not well understood. diagnosed cases of lung cancer (2). Tumor progression mecha- Evaluation of PRECOG and Kaplan-Meier plotter databases in nisms and therapeutic strategies remain crucial subjects for the present study suggested that high expression of acyl-CoA investigation. Dysregulation of metabolic pathways such as the thioesterase (ACOT)7, ACOT11, ACOT13, soluble carrier tricarboxylic acid cycle and the metabolic pathways of serine, family 27 member A4 (SLC27A4) and SLC27A5 was asso- glycine, and glutamine play a critical role in tumor progres- ciated with poor clinical outcomes. In addition, expression sion (3-5). Lipid metabolic pathways are involved in various levels of ACOT7, ACOT11, SLC27A4 and SLC27A5 were not physiological functions. Multiple lipid metabolic enzymes altered after induction of ER stress. By contrast, expression regulate the behavior of lung cancer cells. High expression of some enzymes was decreased, such as those of long-chain of fatty acid synthase is associated with relatively high risk acyl-CoA synthetase (ACSL)3, ACSL4 and SLC27A2. Fatty of lung carcinoma recurrence (6). In lung adenocarcinoma, acid uptake capacity was suppressed in lung cancer cell lines high expression of stearoyl CoA desaturase 1 (SCD) leads to A549 and CL1-0 after thapsigargin treatment but intracellular enhanced cell migration and invasion capacity in lung cancer reactive oxygen species levels were not suppressed. Gene cells and is associated with poor prognosis in patients (7). enrichment and regulatory element analysis were performed; Furthermore, the plasma levels of some saturated and unsatu- the results provided potential targets for further investigation. rated fatty acids, including arachidonic, palmitic, linoleic, and On the whole, our findings demonstrate the potential regula- oleic acids, in patients with lung adenocarcinoma are higher tory mechanism of high-expression of acyl-CoA metabolic than those in healthy people (8‑10). These findings suggest that lipid metabolic enzymes and fatty acid transporters are affected in the progression of lung cancer. The endoplasmic reticulum (ER) is an organelle that Correspondence to: Dr Meng-Chi Yen, Department of Emergency performs protein folding and posttranslational modifica- Medicine, Kaohsiung Medical University Hospital, 100 Tz-You tion and is involved in the processes of energy metabolism, 1st Road, Kaohsiung 807, Taiwan, R.O.C. lipid biosynthesis, and homeostasis of intracellular calcium 2+ E-mail: [email protected] ions (Ca ) (11). Certain physiological conditions interfere with protein folding, including calcium depletion, nutrient Professor Po-Lin Kuo, Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, 100 Shih-Chuan deprivation and DNA damage. Accumulation of unfolded and 1st Road, Kaohsiung 807, Taiwan, R.O.C. misfolded proteins in ER lumen causes ER stress (12). In a E-mail: [email protected] tumor microenvironment, hypoxia, nutrient deprivation, and calcium dysregulation induce ER stress in tumor cells (12). Key words: lung cancer, unfolded protein response, ER stress, lipid Generally, unresolved ER stress leads to apoptosis (13). metabolic enzyme, acyl-CoA synthetase, acyl-CoA thioesterase Unfolded protein responses (UPRs) can protect cells from apoptosis. Thus, UPR activation serves as a prognostic marker for several human cancers (14). UPR signaling affects sterol LIU et al: fatty acid-CoA metabolic enZymes UNDER ER STRESS 2675 regulatory element binding proteins (SREBPs), which are were respectively maintained in RPMI-1640 medium and upstream regulators of lipid biosynthesis (15). In liver cells, Ham's F12K medium. Both media consisted of 10% fetal activation of UPR signaling results in increased lipid accu- bovine serum and 1% penicillin-streptomycin-amphotericin B mulation (16). Furthermore, silencing of SREBPs decreases (Lonza, Walkersville, MD, USA), and the cultures were stored the desaturation of fatty acids and increases accumulation of in an incubator at 37˚C with a 5% CO2 environment. reactive oxygen species (ROS) (17). Thus, ER stress and lipid metabolism are highly regulated in human physiology. Western blot analysis. The cells were lysed in radioimmu- Fatty acyl-CoA is a critical metabolite in the lipid metabolic noprecipitation lysis buffer (RIPA) with a protease inhibitor pathway and is involved in various physiological processes, cocktail at a 100:1 ratio at 24 h after vehicle control [dimethyl including β-oxidation and triacylglycerol and phospholipid sulfoxide (DMSO)] or thapsigargin (Tg; dissolved in DMSO; synthesis (18). Fatty acyl-CoA formation and degradation Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) treat- respectively occur in two enzyme families: acyl-CoA synthe- ment. A bicinchoninic acid (BCA) protein assay kit (Thermo tases (ACSs) and acyl-CoA thioesterases (ACOTs) (19). Human Fisher Scientific, Inc., Rockford, IL, USA) was used to physiology involves more than 26 ACS enzymes and 12 ACOT determine protein concentration. Equal amounts (30 µg) of enzymes (19). Recent studies have demonstrated the critical protein were loaded and separated using 12% sodium dodecyl role of these enzymes in lung cancer. An increased risk of sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). lymph node metastasis in lung adenocarcinoma is associated After electrophoresis, the proteins were transferred onto poly- with high ACOT8 expression (20). Our previous study revealed vinylidene difluoride (PVDF) membranes (EMD Millipore, that high expression of ACOT11 and ACOT13 in patients Billerica, MA, USA). After 1 h of blocking in 5% skim milk with lung adenocarcinoma was associated with cell prolif- and Tris-buffered saline solution with Tween-20 (TBST) buffer, eration and poor prognosis (21). Regulation of other ACS and the PVDF membranes were incubated with the following ACOT enzymes under the condition of ER stress is not fully primary antibodies: anti-ACOT11 (1:3,000; cat. no. ab153835; understood. The present study investigated these phenomena Abcam, Cambridge, UK), anti-ACSL1 (1:1,000, cat. no. 4047; with a view to identifying the relevant mechanisms through Cell Signaling Technology, Danvers, MA, USA), anti-ACSL4 bioinformatic and experimental approaches. (1:1,000, cat. no. ab155282; Abcam) and anti-GAPDH (dilution, 1:5,000; cat. no. MAB374; EMD Millipore). Materials and methods After TBST washing, the membranes were incubated with secondary antibodies, including peroxidase-conjugated goat Bioinformatic analysis. Cancer gene expression and clinical anti-rabbit immunoglobulin G (IgG; 1:5,000; cat. no. AP132P; outcomes for each type of lung cancer were evaluated using EMD Millipore) and peroxidase-conjugated goat anti-mouse IgG PREdiction of Clinical Outcomes from Genomic Profiles (1:5,000; cat. no. AP124P; EMD Millipore) at room temperature (PRECOG; https://precog.stanford.edu) (22). Z‑scores were for 1 h. The results were acquired using an imaging capturing obtained from the PRECOG website and a heatmap was system (Alpha Innotech FluorChem FC2 imaging system, drawn using Morpheus (https://software.broadinstitute.org/ ProteinSimple; Bio-Techne, Minneapolis, MN, USA). Protein morpheus/). Overall survival in lung cancer patients was expression was quantified using ImageJ software (version 1.51; evaluated using the Kaplan-Meier (KM) plotter (http://kmplot National Institutes of Health, Bethesda, MD, USA). .com/; 23). For KM plotter analysis, the studied lung cancer patients were divided into high- and low-expression groups Fatty acid uptake assay. Fatty acid uptake was determined according to median gene expression. To evaluate gene using the Free Fatty Acid Uptake Assay Kit (Fluorometric) expression in lung cancer cell line A549 under ER stress, a according to the manufacturer's instructions (cat. no. ab176768; microarray dataset was obtained from the National Center Abcam). Before fatty acid uptake assay, 2x104 A549 and for Biotechnology