Expression profiling microarray data of human lung adenocarcinoma clinical specimens, which was published between 2005 and 2015, was collected from the National Center for Biotechnology Information Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) (10). Microarray data from cell lines or small sample sizes (<10 samples) were excluded. Six microarray datasets including GSE2514 (11), GSE7670 (12), GSE10072 (13), GSE31210 (14), GSE32863 (15), and GSE43458 (16) were collected and the relative expression levels of fatty acyl-CoA metabolic enzymes between adjacent non-tumor lung tissue and lung adenocarcinoma were analyzed in these microarray datasets. The expression value was analyzed using the GEO2R interface (http://www.ncbi.nlm.nih.gov/geo/geo2r/).

The survival analysis in lung adenocarcinoma patients with different expression levels of ACOT11 and ACOT13 was performed using the KM-Plotter database (17). The prognostic value of each gene was analyzed by splitting patient samples into two groups according to median expression. After the subtype of lung cancer was restricted (‘Histology: adenocarcinoma’) and survival rate was analyzed through the ‘2015 version’ database and ‘excluded biased array’, 720 patients were analyzed.

Dimethyl sulfoxide (DMSO), puromycin, myristic acid, palmitic acid and stearic acid were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Myristic acid, and palmitic acid and stearic acid were dissolved in DMSO.

Human lung adenocarcinoma cell lines CL1-0 and CL1-5 were provided by Dr Pan-Chyr Yang (Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan) and were cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (all Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified incubator at 37°C with 5% CO₂ (18,19).

shRNA targeting ACOT11 (shACOT11; TRCN0000048912; targeting sequence: 5′-GTCTCCTCCTTGAAGATGCT-3′), ACOT13 (shACOT13-1; TRCN0000048954; targeting sequence: 5′-CGATATGAACATAACGTACAT-3′; shACOT13-2; targeting sequence: TRCN0000048956; targeting sequence: 5′-GAAGAGCATACCAATGCAATA-3′) and a negative control construct (luciferase shRNA, shLuc) were obtained from the National Core Facility for Manipulation of Gene Function by RNAi, miRNA, miRNA sponges, and CRISPR/Genomic Research Center, Academia Sinica (Taipei, Taiwan). CL1-0 and CL1-5 cells were transfected with each shRNA plasmid using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Transfected cells were selected and maintained in medium containing 2 µg/ml puromycin.

Total RNA was extracted from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. Complementary DNA (cDNA) was reverse transcribed from mRNA using the PrimeScript RT reagent kit (Clontech Laboratories, Inc., Mountainview, CA, USA) according to the manufacturer's protocol. PCR was performed using the following primers: ACOT13 forward, 5′-TCTGCTATGCACGGAAAGGG-3′ and reverse, 5′-TTTCCTGTGGCCTTGTTGGT-3′; ACOT11 forward, 5′- GCG ATC TGG AGA GCA GAG AC-3′ and reverse, 5′-GTGGCCACATTCTCCATCCA-3′; GAPDH forward, 5′-GAGTCAACGGATTTGGTCGT-3′ and reverse, 5′-TTGATTTTGGAGGGATCTCG-3′. PCR was performed on a StepOne Plus Real-Time PCT System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the Fast SYBR Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following settings were used: 1 cycle of 95°C for 20 sec, and 40 cycles of 95°C for 3 sec and 60°C for 30 sec. The mRNA expression levels were normalized to the expression level of GAPDH using the 2^−∆∆Cq method (20).

Cells were lysed in radioimmunoprecipitation assay buffer (EMD Millipore, Billerica, MA, USA) and the total cell lysate was collected by centrifugation at 4°C, 12,000 × g for 15 min. Quantification of protein concentration was performed using a BCA protein assay kit (EMD Millipore). A total of 30 µg/lane protein was loaded, subjected to 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore). Membranes were blocked with 5% dried skimmed milk in Tris-buffered saline with Tween-20 buffer and then incubated with the primary antibodies overnight at 4°C. Protein expression was detected using the following primary antibodies: Anti-ACOT11 (dilution, 1:3,000; cat. no. ab153835), anti-ACOT13 (dilution, 1:1,000; cat. no. ab166684; both Abcam, Cambridge, UK) and anti-GAPDH (dilution, 1:6,000; cat. no. MAB374; EMD Millipore). Each membrane was then incubated with secondary andibodies, including peroxidase conjugated goat anti-rabbit IgG (dilution, 1:5,000; cat. no. AP132P) and peroxidase conjugated goat anti-mouse IgG, (dilution, 1:5,000; cat. no. AP124P; both EMD Millipore) at room temperature for 1 h. Immunoreactive signals were detected with Immobilon horseradish peroxidase substrate enhanced chemiluminescence reagents (EMD Millipore) and analyzed with the Alpha Innotech FluorChem FC2 imaging system (ProteinSimple; Bio-Techne, Minneapolis, MN, USA).

For cell proliferation measurements, WST-1 (Clontech Laboratories, Inc.) was used. A total of 5×10³ CL1-0 or 2.5×10³ CL1-5 cells were seeded in 96-well plates. The proliferation rate was determined at a wavelength of 450 nm on a microplate spectrophotometer (PowerWave X340, BioTek Instruments, Inc., Winooski, VT, USA).

A total of 1×10⁶ CL1-0 cells were seeded into 10 cm dishes with 8 ml of culture medium. After 48 h, culture medium and cells were collected for free fatty acid quantification. Free fatty acids released into the culture medium and intracellular free fatty acid were quantified using a Free Fatty Acid Quantification Colorimetric/Fluorometric kit (BioVision, Inc., Milpitas, CA, USA) according to the manufacturer's protocol. The results were determined on a fluorescent microplate reader at excitation/emission=485/590 nm (FLX800 Microplate Fluorescence Reader; BioTek Instruments, Inc.).

All statistical analyses were performed using GraphPad Prism software (version 5.03; GraphPad Software, Inc., La Jolla, CA, USA). All error bars in the figures represent standard error of the mean. The differences between two independent groups were analyzed using a Student's t-test. A log-rank test was performed to assess differences in survival rate. P<0.05 was considered to indicate a statistically significant difference.

To investigate whether the expression of ACSs and ACOTs was associated with lung adenocarcinoma, microarray datasets with lung adenocarcinoma specimens were collected from the GEO database. Following the exclusion of microarray data regarding cell line studies and small sample sizes (<10 samples), six microarrays that were performed by five different array platforms were selected for the current study (Fig. 1). A number of enzymes were significantly differentially expressed in lung adenocarcinoma tissue compared with non-tumor lung tissue (Tables I and II). ACOT11 and ACOT13 overexpression was observed in lung adenocarcinoma tissue across all selected microarray datasets (Table I). A KM-Plotter database was used to further investigate the association between the expression of ACOT11 and ACOT13 and clinical outcome. Poor overall survival rate of patients with lung adenocarcinoma was associated with high expression of ACOT11 and ACOT13 (P<0.001; Fig. 2). These results suggest that ACOT11 and ACOT13 serve roles in the development of lung adenocarcinoma.

The CL1-0 and CL1-5 cell lines were established from a patient with lung adenocarcinoma and exhibit different invasive and metastatic properties (19). Similar protein levels of ACOT11 and ACOT13, and similar levels of intracellular and medium free fatty acid, were observed in the two cell lines (Fig. 3A and B). In order to determine the role of ACOT11 and ACOT13, shRNAs targeting ACOT11 (shACOT11) and ACOT13 (shACOT13-1 and shACOT13-2) were transfected into CL1-0 and CL1-5 cells. Cells treated with shACOT11 exhibited significantly decreased expression of ACOT11 (P<0.05; Fig. 4A). Similarly, cells treated with shACOT13-1/−2 exhibited significantly decreased expression of ACOT13 (P<0.01 and P<0.001, respectively; Fig. 4A). These changes were also exhibited at the protein level (Fig. 4B). Although decreasing expression of ACOT11 and ACOT13 did not result in a significant change in total free fatty acids (Fig. 4C), significantly decreased proliferation rates were observed in CL1-0 and CL1-5 cells treated with shACOT11 compared with the control (P<0.001 and P<0.01, respectively; Fig. 4D). In addition, CL1-0 cells treated with shACOT13-2, and CL1-5 cells treated with ahACOT13-1 exhibited significantly decreased proliferation rates compared with the control (P<0.001 and P<0.05, respectively; Fig. 4D). These results suggest that ACOT11 and ACOT13 are critical for proliferation in lung adenocarcinoma cell lines.

ACOT11 and ACOT13 are members of the Type II ACOT family (21). The substrates of ACOT11 and ACOT13 include medium (6–12 carbon) to long (13–21 carbon) chain acyl CoA (22). It was hypothesized that the observed decreased proliferation rates following ACOT11/13 knockdown were associated with insufficient availability of free fatty acids. Therefore ACOT11/13 knockdown CL1-0 cells were treated with a fatty acid mixture, including myristic (C14:0), palmitic (C16:0) and stearic acid (C18:0). The cell proliferation rate was significantly decreased in the control groups (medium and vehicle) following treatment with shACOT11/13-2 compared with the control luciferase shRNA group (P<0.05; Fig. 5); however, treatment with the fatty acid mixture (0.1–10 µM) restored shACOT11 and shACOT13-mediated growth inhibition. However, this effect was abolished following the addition of mixed free fatty acids at the highest dose (100 µM), which may indicate a negative feedback mechanism. The results suggest that the proliferation of lung adenocarcinoma cells is dependent on ACOT11 and ACOT13-mediated metabolic products.