Acyl-CoA thioesterase 8 is a specific protein related to nodal metastasis and prognosis of lung adenocarcinoma

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Original article

Acyl-CoA thioesterase 8 is a specific protein related to nodal metastasis and prognosis of lung adenocarcinoma Woon Yong Jung, Young Hye Kim, Young Joon Ryu, Baek-Hui Kim, Bong Kyung Shin, Aeree Kim, Han Kyeom Kim ∗ Department of Pathology, Korea University College of Medicine, Seoul, South Korea

a r t i c l e

i n f o

Article history: Received 27 August 2012 Received in revised form 23 January 2013 Accepted 7 February 2013 Keywords: Lung Adenocarcinoma Neoplasm metastasis Prognosis Proteomics

a b s t r a c t Metastasis is a major cause of cancer recurrence or death. This study attempted to quantitatively identify different proteins in metastatic lung adenocarcinoma. The N/T quotient [number of metastatic lymph nodes (n)/tumor diameter (cm)] was used to select samples with an extreme metastatic phenotype. Among the six fresh frozen lung adenocarcinoma specimens, the three showing the highest N/T quotient represented the metastatic group, and others with the greatest tumor diameters without metastasis represented the non-metastatic group. After 2-dimensional electrophoresis, the significantly different protein spots were selected by image analysis and analyzed with MALDI-TOF mass spectrometry. AcylCoA thioesterase 8 isoform c (ACOT8) was one of most overexpressed proteins in the metastatic group, and it was validated by Western blot and immunohistochemical staining on 108 paraffin-embedded tumor samples. High ACOT8 expression was correlated with lymph node metastasis (p = 0.002), recurrence (p = 0.034), predominant histologic subtypes (p = 0.007), and higher stage (p = 0.005). In multivariate analysis, high ACOT8 expression was significantly associated with increased risks of lymph node metastasis (p = 0.009) and cancer-related death (p = 0.030), independent of clinical factors. ACOT8 may be a candidate prognostic biomarker and therapeutic target of lung adenocarcinoma. © 2013 Elsevier GmbH. All rights reserved.

Introduction The incidence and mortality of lung cancer is increasing every year, and lung cancer is the leading cause of cancer-related deaths. In Korea, lung cancer death accounts for 21.4% of cancer deaths. Generally, the prognosis of cancer patients depends upon primary tumor size and metastases to lymph nodes or remote organs. Since metastasis is the most important cause of cancer recurrence and cancer-related death, efforts to identify proteins related to metastasis are critical for estimating patient prognoses or discovering targets for new therapies. Several proteomic studies have shown that several proteins are associated with lung cancer metastasis, including proApolipoprotein A1, 14-kDa phosphohistidine phosphatase, annexin A3, hsp27, 14-3-3␴, and CK19. However, currently these are not clinically used as metastatic or prognostic markers [19,21,23,28,33,36]. Adenocarcinoma is the most common lung cancer that tends to metastasize early and shows poor prognosis. Usually, adenocarcinoma patients undergo chemotherapy after surgical resection

∗ Corresponding author at: Department of Pathology, Korea University Guro Hospital, 97, Gurodong-Gil, Guro-Gu, Seoul 152-703, South Korea. Tel.: +82 2 2626 1485; fax: +82 2 2626 1486. E-mail address: [email protected] (H.K. Kim).

to control metastasis, but the efficacy of postoperative adjuvant chemotherapy is controversial. Since not all the lung cancer patients respond to chemotherapy, it is important to select candidates for chemotherapy using biomarkers. Recently, based on the finding that certain proteins expressed in primary tumors may be related to patient prognosis, several studies selecting candidates for chemotherapy by identifying specific marker proteins expressed in surgical specimens were performed. For example, a group of stage I lung adenocarcinoma patients with tumors that did not express myosin IIA or vimentin were shown to have a good prognosis regardless of postoperative adjuvant chemotherapy [22]. Two-dimensional polyacrylamide gel electrophoresis (2DPAGE) is a simple and widely used protein separation method for parallel quantitative expression profiling of complex mixtures of proteins. Depending on the gel size, pH gradient, and staining method, 2D-PAGE can resolve more than 5000 proteins simultaneously, and can detect < 1 ng protein per spot [11]. In the present study, we analyzed six fresh-frozen human lung adenocarcinoma tissues by 2D-PAGE to identify specific, differentially expressed proteins. We identified three proteins, acyl-CoA thioesterase 8 isoform c (ACOT8), enoyl-CoA hydratase, and complement component C3, which were overexpressed in lung adenocarcinoma with an extreme metastatic phenotype. Of these three proteins, ACOT8 showed a strong correlation with increased risks for lymph node metastasis and poorer prognosis.

0344-0338/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.prp.2013.02.008

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Table 1 Clinical information on 108 patients with lung adenocarcinoma.

approved by the Institutional Review Board of the Korea University Guro Hospital (KUGGR-2009-018).

No. Patients (%) 59.2

Mean age (yr, range) Sex Male Female

(37–81)

60 48

(55.6) (44.4)

Pathologic T stage T1 T2 T3

40 59 9

(37.0) (54.6) (8.3)

Lymph node metastasis N0 N1 N2

71 19 18

(65.7) (17.6) (16.7)

Stage I II III

65 24 19

(60.2) (22.2) (17.6)

Materials and methods Study samples Among the samples surgically resected and deposited in the Korea Lung Tissue Bank, six fresh frozen lung adenocarcinoma tissues were used for proteomic analysis and validation by western blotting. All of the six patients underwent lobectomy plus hilar and mediastinal lymph node dissection. The total numbers of dissected lymph nodes were more than 15 in all cases. After surgical resection, the tumor tissues were immediately frozen in liquid nitrogen and stored at −80 ◦ C until further use. One hundred and eight patients who had been diagnosed with primary lung adenocarcinoma and had undergone surgical treatment at Korea University Guro Hospital, Seoul from January 1996 to June 2009 were subjected to this study for clinical validation. The follow-up times for these patients ranged between 1 and 143 months, with a median follow-up time of 28 months. The pathological stages were re-evaluated according to the American Joint Committee on Cancer (AJCC) Staging System 7th edition (Table 1) [31]. To select samples with an extreme metastatic phenotype, we used a numerical value, the N/T quotient, which was defined as follows: N/T quotient (arbitrary unit) = metastatic lymph node number (n)/tumor diameter (cm) [6]. A high N/T quotient for a primary tumor indicates a small tumor size with a high number of lymph node metastases. Three frozen tissue samples with the highest N/T quotients were selected as representatives of the metastatic group for proteomic analysis and western blotting. Three samples with the largest tumor diameters but no metastatic lymph nodes represented the non-metastatic group (Table 2). Written informed consent for the collection and use of tissues for research purposes was obtained from the patients prior to surgery. This research was

2D PAGE and image analysis To extract proteins, tissue samples approximately 8 mm3 were sliced to a thickness of 10 ␮m by a cryocutter at −20 ◦ C and immediately immersed in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% DTT, 2% IPG buffer (pH 3–10), 10 U DNase, and 10 mM protease inhibitor cocktail). After extraction, protein concentration was determined by a modified Bradford protein assay (Bio-Rad, Richmond, CA, USA). One milligram of protein was diluted to 450 ␮L with rehydration buffer (7 M urea, 2 M thiourea, 1% CHAPS, 0.4% DTT, 0.5% IPG buffer (pH 3–10), and trace bromophenol blue). Following rehydration of 24-cm IPG strips (pH 3–10) in the diluted samples for 12 h at room temperature, isoelectric focusing was performed for 52 kVh. The strips were equilibrated for 15 min in 50 mM Tris–HCl buffer (pH 3–10) containing 6 M urea, 30% glycerol, 2% SDS, 0.01% bromophenol blue, and 1% DTT, then treated for 15 min in the same buffer containing 4% iodoacetamide in place of DTT. The strips were then transferred to 12.5% SDS-PAGE gels (230 mm × 200 mm × 1 mm) for 2-dimensional separation. The protein spots were stained with Coomassie blue G250 for 6 h. The stained gels were scanned, and the images were analyzed using ImageMaster 2D Platinum 6.0 software (Amersham Biosciences, Uppsala, Sweden). Protein identification by mass spectrometry (MS) The protein spots of interest were manually excised and subjected to trypsin digestion. Through desalting and concentration using porous resin, the extracted peptides were applied to a MALDI plate and analyzed with a 4800 MALDI TOF Analyzer (Applied Biosystems, Framingham, MA, USA). The mass spectra were processed by Data Explorer software (v4.4, Applied Biosystems, Framingham, MA, USA) to generate a peak list. The resulting peak list was interpreted by MASCOT software (http://www.matrixscience.com/) and the NCBI database (NCBInr 20090509, selected for Homo sapiens, 115818 sequences). Western blotting Protein lysates (50 ␮g/lane) were electrophoresed on a 15% SDSPAGE gel and transferred to PVDF membranes. Membranes were then blocked by incubation in 5% BSA for 30 min and washed three times with TBS buffer. The primary antibodies were diluted in 4% BSA TBS solution and applied to membranes at 4 ◦ C for 12 h. The primary antibody used was acyl-coenzyme A thioesterase 8 (mouse polyclonal, diluted in 4% BSA solution, 1:500; Abcam, Cambridge, UK). After washing with TBS-T buffer three times, the membranes were incubated with agitation for 1 h with secondary antibodies diluted in blocking solution. Thermo Scientific blotting detection reagents (Thermo Fisher Scientific, Rockford, IL, USA) were used to visualize the target protein. The developed films were scanned, and

Table 2 Clinicopathologic information on patients selected for proteomic analysis. Sex/age

Histologic type

Size (cm)

MLN

TLN

N/T ratio

Metastatic group

M1 M2 M3

M/59 F/68 M/65

Adenocarcinoma, papillary and solid Adenocarcinoma, acinar Adenocarcinoma, solid

3.7 1.6 1.3

25 6 5

27 23 59

6.76 3.75 3.84

Non-metastatic group

T1 T2 T3

F/54 M/55 M/62

Adenocarcinoma, solid Adenosquamous carcinoma Adenocarcinoma, acinar and solid

4.0 4.0 5.0

0 0 0

16 18 26

0 0 0

MLN: metastatic lymph node; TLN: total lymph node.

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band intensities were measured by the ImageJ program (National Institute of Health, MD, USA). The relative band intensities were calculated by dividing the signal intensity of the proteins of interest by that of ␤-actin.

2, KIAA0990 protein, and protein disulfide isomerase-associated 4 were downregulated in metastatic tumor tissue (Table 3).

Tissue microarray (TMA) and immunohistochemistry

Among proteins identified by MALDI-TOF-MS and database searches, expression of the protein ACOT8, which showed the greatest difference in expression between the metastatic and nonmetastatic groups, was analyzed by western blotting. Although the expression levels of ACOT8 tended to be higher in the metastatic group, the difference was not statistically significant (p = 0.127) (Fig. 2).

TMAs were made from the formalin-fixed, paraffin-embedded tissues of 108 patients. Two tissue cores of 2 mm in diameter were taken from central and peripheral areas and embedded in recipient paraffin blocks. Immunohistochemical staining was performed using the standard streptavidin–biotin–peroxidase complex method with an automated staining system (Autostainer Plus, Dako, Denmark). The tissue sections on the slides were deparaffinized and rehydrated. For antigen retrieval, the slides were heated in a microwave oven for 15 min in 10 mM citrate buffer (pH 6.0). Endogenous peroxidase in the tissue sections was blocked with 3% hydrogen peroxide for 20 min. The slides were then incubated with diluted primary antibody (polyclonal rabbit antihuman acyl-coenzyme A thioesterase 8, 1:400, Abcam) for 30 min at room temperature. After washing with TBS, tissue sections were incubated with biotinylated secondary antibody and then with DAB substrate provided in a DAKO Envision kit (DAKO, Glostrup, Denmark). Nuclei were counterstained with hematoxylin. A pathologist examined the slides twice. Immunostain was interpreted as high ACOT8 expression when the tumor showed a granular cytoplasmic staining pattern at least in a core. When the tumor showed weak staining intensity in both cores, it was interpreted as low ACOT8 expression. Statistical analysis Statistical analysis was performed using Pearson’s 2 test, Fisher’s exact test, and the Mann–Whitney U-test. Survival was estimated using the Kaplan–Meier method, and the resulting curves were compared via a log-rank test. Multivariate analysis using the Cox proportional hazards regression model and the logistic regression model was performed to calculate the independent prognostic significance. SPSS for Windows version 16.0 (Chicago, IL, USA) was used for statistical calculations, and p values less than 0.05 were considered statistically significant. Results

Validation of protein spot using western blot analysis

Immunohistochemical staining of ACOT8 on independent tumor samples In the normal lung tissue, ACOT8 was homogeneously expressed in the cytoplasm of various cellular components, including alveolar macrophages, type II pneumocytes, bronchial epithelium, and smooth muscle cells. Eight whole sections of tumor tissue were examined prior to the evaluation of tissue microarrays (TMAs). ACOT8 staining revealed a heterogeneous granular pattern in the cytoplasm (Fig. 3). High ACOT8 expression was significantly associated with lymph node metastasis (p = 0.002), recurrence (p = 0.034), predominant histologic pattern (p = 0.007), and higher anatomic stage (p = 0.005). The proportion of the solid or micropapillary subtype was significantly larger in the high ACOT8 expression group. The higher anatomic stage seemed to be due to a higher pathological N stage rather than a higher pathological T stage (Table 4). In multivariate analysis using a logistic regression model, the high expression level of ACOT8 was associated with an increased risk of lymph node metastasis independent of pathological T stage and lymphovascular invasion (p = 0.009, Table 5) The group with low ACOT8 expression showed significantly better prognoses (p < 0.001). Among 65 cases of stage I adenocarcinoma, 26 cases (40%) presented low ACOT8 expression, and they also showed better prognoses (p = 0.02, Fig. 4). In univariate analysis, lymph node metastasis and high ACOT8 expression increased the risk of cancerrelated death. Multivariate analysis using the Cox regression model has demonstrated that the ACOT8 expression level is associated with an increased risk of cancer-related death independent of clinical factors such as age, sex, pathologic T and N stage (p = 0.030, Table 6).

Clinicopathologic features Discussion The average size of tumors was 3.3 ± 1.6 cm, and 37 cases had lymph node metastasis. In the metastatic group, the median and average N/T quotients were 0.54 and 1.75, respectively, and about 80% of the cases had N/T quotients less than 2. The N/T quotients were greater than 2 in 9 cases (8%). Among the metastatic group, this subgroup had more frequent lymphovascular invasion (p = 0.002), but the overall survival was not significantly different. Identification of proteins by 2D PAGE and MALDI-TOF-MS The number of protein spots separated by 2D PAGE was 1039 to 1887. The number of spots showing differential expression between the metastatic and non-metastatic groups was 121. Among these, seven spots showing an expression ratio of at least ±2.0 were selected for MALDI-TOF-MS (Fig. 1). The identification data of these spots are presented in Supplemental Fig. 1. Peroxisomal acyl-CoA thioesterase 1 (acyl-coenzyme A thioesterase 8, ACOT8) isoform c, enoyl-CoA hydratase, and complement component C3 were upregulated, while internexin neuronal intermediate filament protein-␣, eukaryotic translation elongation factor 1 ␤

For identification of significant differences in protein expression, it is most important to select samples showing extremely different physiological characteristics. The N stage of samples could be used to examine the metastatic tendency of tumor cells. However, it is unsuitable for evaluating metastatic tendencies of the tumor itself because the N stage is related to the site of metastasis regardless of the number of metastatic lymph nodes [7]. The number of metastatic lymph nodes alone is also unsuitable for analysis because it usually increases as the tumor grows [4]. Therefore, the N/T quotient was suitable for comparing the degree of lymph node metastasis regardless of tumor size [6]. In our study, this was significantly correlated with lymphovascular invasion. Several studies have examined altered lipid metabolism in cancers. For example, after the identification of OA-519 in 1994, a molecule associated with poor prognosis of breast cancer, overexpression of fatty acid synthase was observed in various cancers and became a new target for chemotherapy [18,20]. De novo fatty acid synthesis is indispensable to cancer cells for membrane formation, energy production through ␤-oxidation, and lipid modification of

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Fig. 1. (A) Representative 2D PAGE gels using 24 cm IPG strips (pI 3–10). Large gels (24 cm × 20 cm) were used to analyze protein profiles, and protein spots were visualized by Coomassie blue staining. (B) The magnified images of a square area of each gel showed differently expressed spots between the metastatic and non-metastatic groups. (C) MALDI-TOF-MS analysis of protein spot 1 using MASCOT software. The mass spectrum of spot 1 was obtained by MALDI-TOF-MS, and the peak data were entered to a search program. Carbamidomethyl (C) and oxidation (M) were selected as fixed and variable modifications, respectively. Among 254 amino acids, 112 were matched (shown in bold) and the sequence coverage was 44%.

proteins [24]. Although ACOTs are related to fatty acid metabolism in another enzyme group, the relationship between ACOTs and cancer has not been reported. There is only one report dealing with the activity of ACOT in tumor cells. In this study, the authors cultured placental choriocarcinoma (BeWo) cell with fatty acid and found an increase in ACOT activity and expression of PPAR␥ by conjugated linoleic acid and gamma linolenic acid; however, there was no mention of tumorigenesis or metastatic ability of the tumor cells [9].

The present study represents the first report on the relationship between ACOT8 and lymph node metastasis or prognosis of lung adenocarcinoma. At present, it is not clear whether the changes in ACOT8 expression are a cause or merely a result of differences in the physiological character of the tumor cells. The normal physiological functions of ACOT have been studied in in vitro experiments. ACOTs are a group of enzymes promoting hydrolysis of acyl-CoA into free fatty acid and coenzyme A. They are widely distributed in cells of various animal organs, yeasts,

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Table 3 Differentially expressed proteins in metastatic tumors compared with non-metastatic tumors. No.

NCBI accession no.

Protein name

MASCOT score

Mr (kDa)

pI

Mean intensity

Expression ratioa

1 2 3 4 5 6 7

gi|34577073 gi|1922287 gi|179665 gi|14249342 gi|4503477 gi|40789012 gi|4758304

Acyl-CoA thioesterase 8 isoform c Enoyl-CoA hydratase Complement component C3 Internexin neuronal intermediate filament protein, ␣ Eukaryotic translation elongation factor 1 ␤ 2 KIAA0990 protein Protein disulfide isomerase-associated 4

64 66 99 127 94 64 169

28.53 31.81 188.59 55.53 24.92 100.96 73.23

8.83 8.34 6.02 5.34 4.5 9.85 4.96

0.076 0.049 0.023 0.008 0.128 0.024 0.037

7.75 7.03 4.02 –b −2.38 –b −5.26

a b

Expression ratio refers to metastatic group per non-metastatic group. Spot numbers 4 and 6 were not expressed in the metastatic group.

Fig. 2. (A) Western blot analysis of ACOT8 in metastatic lung cancer tissue (M; n = 3) and paired non-metastatic lung cancer tissue (T; n = 3). The same blots were reprobed with a monoclonal anti-␤-actin antibody as a loading control. (B) The intensity of the ACOT8 bands was compared to that of ␤-actin. The average relative intensity tended to be higher in the metastatic group, however it was not statistically significant (p = 0.127).

and microorganisms. In cells, these enzymes localize in various organelles such as mitochondria, peroxisome, cytosol, and microsomes. Among ACOTs, ACOT8 is present in peroxisomes, where it is known as peroxisomal acyl-CoA thioesterase [12]. According

Table 4 Relationship between adenocarcinoma.

clinical

features

and

ACOT8

expression

ACOT8 Low (n = 32) Age (yr) <60 ≥60 Sex Male Female a N/T quotient <2 ≥2 Predominant histologic pattern Lepidic Papillary Acinar Solid/micropapillary Recurrence Absent Present LV invasion Absent Present pT stage T1 T2 + T3 pN stage N0 N1 + N2 TNM stage I II + III a

in

lung

p value High (n = 76) 0.833

15 17

(46.9%) (53.1%)

39 (51.3%) 37 (48.7%)

19 13

(59.4%) (40.6%)

41 (53.9%) 35 (46.1%)

32 (100.0%) 0 (0.0%)

67 (88.2%) 9 (11.8%)

2 5 23 2

1 (1.3%) 4 (5.3%) 45 (59.2%) 26 (34.2%)

0.674

0.055

0.007 (6.3%) (15.6%) (71.9%) (6.3%)

0.034 23 9

(71.9%) (28.1%)

37 (48.7%) 39 (51.3%)

30 2

(93.8%) (6.2%)

62 (81.6%) 14 (18.4%)

0.141

(40.6%) (59.4%)

27 (35.5%) 49 (64.5%) 0.002

28 4

(87.5%) (12.5%)

43 (56.6%) 33 (43.3%) 0.005

26 6

(81.3%) (18.7%)

Table 5 Univariate and multivariate analysis for lymph node metastasis in lung adenocarcinoma. Univariate OR

0.666 13 19

to the experiments on animals, ACOT8 is strongly activated by peroxisome proliferators including hypolipidemic drugs such as clofibrate, bezafibrate, and gemfibrozil; industrial plasticizer such as DEHP; and sulfur-substituted fatty acids [1,2,5,10,14–17,26,30]. The effects of these chemicals involved in lipid metabolism are transmitted through peroxisome proliferator-activated receptors (PPARs). Although there is no report on the direct relationship between ACOT8 gene expression and PPARs, the control of ACOT1 expression by PPAR␥, and the relationship between PPARs and tumorigenesis, metastasis and neovascularization are becoming topics of interest, and several studies attempting targeted therapy using PPAR ligands suggest a possible relationship between ACOT8 and PPARs [3,8,13,25,27,29,32,35]. Following studies dealing with signaling pathway controlling ACOT8 expression and revealing a mechanism for affecting patient prognosis are necessary. Another factor possibly affecting ACOT8 expression is eicosanoids. ACOT is involved in the process of ␤-oxidation of eicosanoids, such as prostaglandin, leukotrienes and thromboxane, to free fatty acid [34]. Considering that eicosanoids are involved in dilatation or constriction of blood vessels and inflammation, it is necessary to study

39 (51.3%) 37 (48.7%)

N/T quotient refers to number of metastatic lymph nodes (n) per tumor diameter (cm). LV, lymphovascular

pT stage T1 1.000 T2 + T3 2.411 LV invasion 1.000 Absent Present 5.585 ACOT8 1.000 Low 5.372 High

95.0% CI

Multivariate p value

OR

95.0% CI

p value

0.995–5.845

0.051

1.000 2.324

0.893–6.050

0.084

1.768–17.645

0.003

1.000 4.476

1.349–14.852

0.014

1.715–16.825

0.004

1.000 4.887

1.491–16.014

0.009

OR: odds ratio estimated by logistic regression model; CI: confidence interval of the estimated OR.

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Fig. 3. Representative immunohistochemistry images of ACOT8 in pulmonary adenocarcinoma specimens; (A) a representative image of TMA, (B) weak or perinuclear dot pattern staining (low expression) (100×), (C) granular cytoplasmic staining (high expression) (100×). Table 6 Univariate and multivariate analysis for overall survival in lung adenocarcinoma. Univariate HR

Multivariate 95.0% CI

p value

HR

95.0% CI

p value

Age (yr) <60 ≥60

1.000 1.486

0.701–3.152

0.301

1.000 1.790

0.824–3.890

0.141

Sex Female Male

1.000 1.871

0.900–3.892

0.094

1.000 2.483

1.146–5.378

0.021

pT stage T1 T2 T3

1.000 1.226 2.316

0.540–2.781 0.710–7.559

0.627 0.164

1.000 0.931 4.182

0.399–2.171 0.610–7.143

0.863 0.241

pN stage N0 N1 N2

1.000 5.494 3.306

2.279–13.244 1.209–9.036

<0.001 0.020

1.000 5.470 3.746

2.132–14.030 1.302–10.774

<0.001 0.014

1.886–102.077

0.010

1.000 9.390

1.242–70.968

0.030

ACOT8 Low High

1.000 13.876

HR: hazard ratio (HR) estimated by Cox proportional hazard regression model; CI: confidence interval of the estimated HR.

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Further studies to elucidate the molecular mechanisms and potential clinical applications of ACOT8 are warranted. Taken together, we found the association of ACOT8 overexpression with lymph node metastasis and poorer prognosis in lung adenocarcinoma, and the result suggests that ACOT8 could be a prognostic biomarker and therapeutic target. Acknowledgements Tissue samples were provided by the Korea Lung Tissue Bank through the Infrastructure Project for Basic Science of the Ministry of Education, Science and Technology (MEST), South Korea. Mass spectrometry and protein identification were technically supported by the Yonsei Proteome Research Center. The authors thank Ie-Hwan Bae for performing the immunohistochemical staining. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prp.2013.02.008. References

Fig. 4. Kaplan–Meier curves for overall survival of pulmonary adenocarcinoma. (A) Cases were classified by the ACOT8 expression level. The low ACOT8 expression group showed better prognoses (p = 0.001). (B) In stage I cases, the low ACOT8 expression group showed good prognoses (p = 0.006).

the relationship between the amount of eicosanoids in peritumoral tissue and lymph node metastasis, lymphatic tumor emboli, and ACOT8 expression. Although ACOT8 is of interesting clinical relevance, the results of the present study have some limitations due to the small number of samples included in the identification and validation sets and the short median follow-up time, as well as the lack of previous reports concerning the role of ACOT8 protein in lung adenocarcinoma.

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Please cite this article in press as: W.Y. Jung, et al., Acyl-CoA thioesterase 8 is a specific protein related to nodal metastasis and prognosis of lung adenocarcinoma, Pathol. – Res. Pract (2013), http://dx.doi.org/10.1016/j.prp.2013.02.008