International Journal of Molecular Sciences Article Prognostic Value of the Overexpression of Fatty Acid Metabolism-Related Enzymes in Squamous Cell Carcinoma of the Head and Neck Ying-Wen Su 1, Pao-Shu Wu 2,3,* , Sheng-Hsiang Lin 4,5, Wen-Yu Huang 6, Yu-Shao Kuo 7 and Hung-Pin Lin 3,8,* 1 Division of Hematology and Medical Oncology, Department of Internal Medicine, MacKay Memorial Hospital, Taipei 10449, Taiwan; [email protected] 2 Department of Pathology, MacKay Memorial Hospital, Tamsui Branch, New Taipei City 25160, Taiwan 3 MacKay Junior College of Medicine, Nursing, and Management, Taipei 25245, Taiwan 4 Institute of Clinical Medicine and Department of Public Health, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan; [email protected] 5 Biostatistics Consulting Center, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan 6 Laboratory of Good Clinical Research Center, MacKay Memorial Hospital, Tamsui Branch, New Taipei City 25160, Taiwan; [email protected] 7 School of Chinese Medicine, China Medical University, Taichung 40402, Taiwan; [email protected] 8 Division of Oral Pathology, Department of Stomatology, MacKay Memorial Hospital, Taipei 10449, Taiwan * Correspondence: [email protected] (P.-S.W.); [email protected] (H.-P.L.) Received: 28 June 2020; Accepted: 16 September 2020; Published: 18 September 2020 Abstract: Reprogramming of cellular energy metabolism, such as lipid metabolism, is a hallmark of squamous cell carcinoma of the head and neck (SCCHN). However, whether protein expression related to fatty acid oxidation (FAO) affects survival in SCCHN remains unclear. We aimed to investigate FAO-related enzyme expression and determine its correlation with clinicopathological variables in SCCHN patients. Immunohistochemical analysis (IHC) of FAO-related protein expression, including carnitine palmitoyltransferase 1 (CPT1), the acyl-CoA dehydrogenase family, and fatty acid synthase (FAS), was performed using tissue microarrays from 102 resected SCCHN tumors. Expressions were categorized according to IHC scores, and the statistical association with clinicopathological factors was determined. Moderate-to-high expression of long-chain acyl-CoA dehydrogenase (LCAD) had a protective role against cancer-related death (adjusted hazard ratio (HR), 0.2; 95% confidence interval (CI), 0.05–0.87) after covariate adjustment. Age and clinical stage remained independent predictors of survival (adjusted HR, 1.75; 95% CI, 1.22–2.49 for age; adjusted HR, 14.33; 95% CI, 1.89–108.60 for stage III/IV disease). Overexpression of medium-chain acyl-CoA dehydrogenase and FAS correlated with advanced tumor stage (T3/T4); however, none of these factors were independent predictors of survival. Several FAO-related enzymes were upregulated and LCAD overexpression had a protective effect on overall survival in advanced SCCHN patients. FAO-related-enzyme expression might have a prognostic impact on survival outcomes in SCCHN. Keywords: fatty acid metabolism; head and neck; squamous cell carcinoma; long-chain acyl-CoA dehydrogenase 1. Introduction Squamous cell carcinoma of the head and neck (SCCHN) is one of the leading causes of cancer-related death globally [1]. One of the hallmarks of SCCHN is the reprogramming of cellular Int. J. Mol. Sci. 2020, 21, 6851; doi:10.3390/ijms21186851 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 6851 2 of 13 energy metabolism [2,3]. SCCHN tumors expressing a high level of phosphorylated acetyl-CoA carboxylase 1 (pACC1) or ACC2 are associated with decreased survival [4,5]. The major cellular function of ACC is to produce malonyl-CoA from acetyl-CoA for fatty acid synthesis [6]. Phosphorylation of ACC inhibits catabolic functions such as lipid storage and causes cellular metabolism to switch to fatty acid oxidation (FAO) [7]. FAO is a catabolic process by which fatty acid molecules are broken down enzymatically. In mammalian cells, FAO primarily occurs in mitochondria except for the oxidation of very-long-chain fatty acids, which occurs in peroxisomes [8]. The major enzymes involved in FAO include carnitine palmitoyltransferase (CPT), the acyl-CoA dehydrogenase family, and hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (HADHA). The acyl-CoA dehydrogenase (ACAD) family includes very-long-chain, long-chain, medium-chain, and short-chain acyl-CoA dehydrogenases (VLCAD, LCAD, MCAD, and SCAD, respectively) [9]. In addition to useful intermediates, the final product of FAO is acetyl-CoA, which enters the citric acid cycle in the mitochondrion for energy generation. Despite the negative correlation of pACC1 or ACC2 expression with clinical outcome [4,5], it remains unclear whether the expression of the enzymes involved in FAO is associated with cancer progression in SCCHN. Therefore, this study aimed to retrospectively examine the expressions of major enzymes involved in FAO in patients with resected SCCHN and determine the correlation with clinicopathological variables. 2. Results 2.1. Incidence of Expression of Fao-Related Proteins in Scchn Tumors and Patient Characteristics According to Survival Status, Age, and Clinical Stage A total of 102 patients with SCCHN were included in the study between 1 January 2011, and 31 December 2012. The median follow-up time was 5.1 years (interquartile range (IQR), 2.6–6.1). Twenty-nine patients died during the study period. The clinicopathological characteristics of the patients are listed in Table1. Table 1. Characteristics of patients (n = 102). Patient Characteristics Number (%) Age, median (range) 51.96 (30.3–86.5) <60 years 74 (72.5%) >60 years 28 (27.5%) Gender Female 7 (6.9%) Male 95 (93.1%) Adjuvant treatment None 17 (16.7%) Yes 85 (83.3%) Site Non-oral cavity 13 (12.7%) Oral cavity 89 (87.3%) T 1 13 (12.7%) 2 31 (30.4%) 3 23 (22.5%) 4 35 (34.3%) Int. J. Mol. Sci. 2020, 21, 6851 3 of 13 Table 1. Cont. Patient Characteristics Number (%) N 0 49 (48.0%) 1 19 (18.6%) 2 31 (30.4%) 3 3 (2.9%) Stage I 8 (7.84%) II 15 (14.71%) III 25 (24.51%) IV 54 (52.94%) HPV P16 Positive 13 (12.7%) Negative 89 (87.3%) T: tumor; N: node; HPV: Human papillomavirus. Compared with patients who died during the study period, patients who survived were significantly younger (50.64 vs. 56.09 years, p = 0.026, Mann–Whitney U test), did not have lymph node involvement (54.79% vs. 31.03%, p = 0.032, χ2 test), and were more likely to experience stage I/II disease (30.14% vs. 3.45%, p = 0.004, χ2 test) (Table2). Table 2. Correlations between the survival status and patients’ characteristics (n = 102). Factor Alive (n = 73) Dead (n = 29) p-Value a n (%) n (%) Age median (IQR) 50.64 (45.13, 57.02) 56.09 (48.61, 67.05) 0.026 * Pretreatment fasting sugar <100 mg/dL 42 (57.53) 15 (51.72) 0.755 100 mg/dL 31 (42.47) 14 (48.28) ≥ median (IQR) 97.00 (91.00, 105.00) 96.00 (87.00, 118.00) 0.807 Pretreatment cholesterol <200 mg/dL 49 (67.12) 20 (68.97) 1.000 200 mg/dL 24 (32.88) 9 (31.03) ≥ 183.00 (156.00, 172.00 (150.00, median (IQR) 0.404 208.00) 202.00) Pretreatment triglyceride <150 mg/dL 58 (79.45) 22 (75.86) 0.896 150 mg/dL 15 (20.55) 7 (24.14) ≥ median (IQR) 109.00 (84.00, 132.00) 98.00 (84.00, 129.00) 0.722 Stage I/II 22 (30.14) 1 (3.45) 0.004 * III/IV 51 (69.86) 28 (96.55) T 1–2 35 (47.95) 9 (31.03) 0.120 3–4 38 (52.05) 20 (68.97) N 0 40 (54.79) 9 (31.03) 0.032 * 1–3 33 (45.21) 20 (68.97) FAS Negative or low expression 61 (83.56) 27 (93.10) 0.207 (0~1+) Moderate or strong 12 (16.44) 2 (6.90) expression (2~3+) CPT1 Negative to moderate 63 (86.30) 25 (86.21) 0.990 expression (0~2+) Strong expression (3+) 10 (13.70) 4 (13.79) Int. J. Mol. Sci. 2020, 21, 6851 4 of 13 Table 2. Cont. Factor Alive (n = 73) Dead (n = 29) p-Value a n (%) n (%) MCAD Negative to moderate 51 (69.86) 16 (55.17) 0.159 expression (0~2+) Strong expression (3+) 22 (30.14) 13 (44.83) LCAD Negative or low expression 59 (80.82) 27 (93.10) 0.124 (0~1+) Moderate or strong 14 (19.18) 2 (6.90) expression (2~3+) VLCAD Negative or low expression 65 (89.04) 23 (79.31) 0.198 (0~1+) Moderate or strong 8 (10.96) 6 (20.69) expression (2~3+) HADHA Negative to moderate 66 (90.41) 24 (82.76) 0.279 expression (0~2+) Strong expression (3+) 7 (9.59) 5 (17.24) a χ2 test or Fisher’s exact test for categorical variables/Mann–Whitney U test for continuous variables. * p < 0.05; n: number; IQR: interquartile range; T: tumor; N: node; FAS: fatty acid synthase; CPT1: carnitine palmitoyl transferase 1; MCAD: medium-chain acyl-CoA dehydrogenase; LCAD: long-chain acyl-CoA dehydrogenase; VLCAD: very-long-chain acyl-CoA dehydrogenase; HADHA: hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase. We next examined the expressions of FAO-related proteins according to age. None (0%) of the 28 patients aged 60 years showed a high expression of CPT1A (3+ score), whereas 14 (18.92%) out ≥ of 74 patients aged <60 years exhibited a high expression of CPT1A (3+ score) (p = 0.01, Fischer’s exact test). However, the negative correlation between CPT1A and age was not further demonstrated by the Spearman correlation analysis (Spearman coefficient for CPT1A and age: 0.09, p = 0.35).