The role of acetyl-coA carboxylase2 in head and neck squamous cell carcinoma

Patients and tissue specimen preparation

All tissue samples (carcinoma and para-carcinoma tissues) from patients diagnosed as having laryngocarcinoma were collected at the First Affiliated Hospital of Anhui Medical University from 2012 to 2013. No distant metastases were found in any patient before surgery, and these patients were followed-up for 5 years after surgery to determine the survival rate (Table 1). Specimens were collected after receiving written informed consent from the participating patient. This study was approved by the Ethics Committee of Anhui Medical University (Ethical Application Ref: 20150192). The procedures were performed in accordance with the Declaration of Helsinki and good clinical practice.

Cell culture, transfection, and regents

The FaDu cells and NP69 cells were purchased from American Type Culture Collection. The FaDu cells or NP69 cells were cultured in minimum essential medium Eagle’s (Mod.) or 1,640 medium (Wisent, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and antibiotics (100 KU/L penicillin and 100 mg/L streptomycin) in an incubator at 37 °C with 5% CO₂. The FaDu and NP69 cells were transiently transfected with ACC2 siRNA (sense, CCUGCCUACUUUCUUCUAUTT; antisense, AUAGAAGAAAGUAGGCAGGTT) (GenePharma, Shanghai, China) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions and were cultured for another 48 h before being used in experiments.

Western blot assay

The protein levels in FaDu cells or NP69 cells or in specimens were assessed by western blot assay. Briefly, the cells from different treatment groups or specimens were treated with RIPA lysis buffer (strong; Sigma, Ronkonkoma, NY, USA) on ice. The samples were then centrifuged at 4 °C and 12,000×g for 20 min. The protein was extracted from the supernatant, and the remaining supernatant was mixed with loading buffer using a ratio of 4:1 at 100 °C for 10 min. An equal amount of protein (30 μg) was loaded onto a gel for sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated in 5% nonfat milk containing primary antibodies at 4 °C overnight. The membranes were incubated with the secondary antibody for 1 h at room temperature. Visualization of the secondary antibody was accomplished using an ECL detection system (Shanghai Peiqing Technology Co., Ltd, Shanghai, China). The optical density (OD) of each protein band was measured using the free software Image J (National Institutes of Health, Bethesda, MD, USA) and was normalized to β-tubulin (Biosharp, Hefei, China), which had been located in the same lane. The following primary antibodies were used in this study: rabbit anti-ACC2, rabbit anti-Bax, rabbit anti-Bcl-2, and rabbit anti-Caspase 3 (all antibodies were from Cell Signaling Technology, Danvers, MA, USA).

Immunohistochemistry

Human laryngeal carcinoma tissues from patients were obtained during clinical surgery. Specimens were fixed with 4% paraformaldehyde and embedded in paraffin. The specimens were cut into five μm-thick sections, deparaffinized, and dehydrated. Antigen retrieval was accomplished by heating the sections in citrate buffer in a microwave oven for 15 min. The sections were then incubated with hydrogen peroxide (3%) for 30 min to destroy endogenous peroxidase activity. Subsequently, the sections were incubated with primary antibody to ACC2 (#3661; Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C prior to incubation with an anti-rabbit secondary antibody (Biosharp, Hefei, China). After being treated first with horseradish peroxidase and then with 3,3′-diaminobenzidine, the sections were counterstained with hematoxylin, dehydrated, cleared, and mounted. For the negative control group, the primary antibody was omitted. Images of stained sections were captured using a light microscope and analyzed with Image Pro Plus 5.1 (Media Cybernetics, Rockville, MD, USA) software.

TUNEL analysis

The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was conducted using a TUNEL kit (Vazyme, Najing, China) following the manufacturer’s instructions. Images were captured using fluorescence microscopy and analyzed with Image J software.

Cell proliferation assay

The proliferation of FaDu and NP69 cells was tested by CCK8 Cell Counting Kit (Santa Cruz Biotechnology, Dallas, TX, USA). The cells were seeded on 96-well plates and transfected with ACC2 siRNA and scrambled siRNA, respectively. Incubated for 48 h in a 37 °C 5% CO₂ incubator. Then, 10 μL of CCK8 solution was added to each well. After following incubation for 2 h, the absorbance of each well was measured at 450 nm. The data were expressed as OD.

Lipids oil red stain

The intracellular lipids stain is following the manufacturer’s instructions of Oil Red O staining kit (Jiancheng Biotech, Nanjing, People’s Republic of China). All specimens were washed with 40% isopropyl alcohol and distilled water. Images were captured using an light microscope and analyzed with Image J software.

Statistical analysis

SigmaPlot software was used to analyze all data. Data are expressed as means ± SEM. Two-tailed, unpaired Student’s t-tests were used to compare the results between groups. Values of P < 0.05 were considered statistically significant.

Increased ACC2 expression in patients with laryngocarcinoma

We demonstrated that the p-ACC expression level was significantly higher in laryngocarcinoma tissue than that in the adjacent tissue (Fig. S1), which was consistent with Su et al. (2014) Findings. Importantly, we used immunohistochemical analysis to evaluate ACC2 expression levels in laryngocarcinoma tissues obtained during surgery as clinical specimens from patients with laryngocarcinoma (Table 1). The ACC2 expression pattern differed between laryngocarcinoma and the adjacent normal tissues (Figs. 1A and 1B). In addition, the ACC2 expression level, represented as an integrated optical density (IOD) value, was significantly higher in laryngocarcinoma tissue than that in the adjacent tissue (Fig. 1C; Table 1). The results of western blot analyses of these tissues were consistent with this finding, that is, ACC2 protein expression levels in laryngocarcinoma tissue were significantly than higher than those in the adjacent normal tissue (Figs. 1F and 1G).

The laryngocarcinoma specimens used in immunohistochemical analysis were then analyzed to determine whether an association existed between the level of ACC2 expression and the clinical cancer stage or the degree of cell differentiation. Each patient’s clinical stage was classified as one of three stages (I, II, or III, with III being more advanced) based on the tumor-lymph node-metastasis (TNM) classification system. As shown in Fig. 1D, the ACC2 expression level for patients with stage III laryngocarcinoma was significantly higher (represented as a higher IOD value) than that for patients with stage I and II. Regarding a relationship between the degree of cell differentiation and the level of ACC2 expression, our results indicated that the ACC2 expression level was inversely associated with the degree of differentiation of the laryngocarcinoma cells, that is, the more poorly differentiated the cancer cells, the higher the ACC2 expression level (Fig. 1E). In summary, our findings indicated that ACC2 protein was highly expressed in laryngocarcinoma tissue and that the ACC2 expression level was significantly higher in patients with a more advance clinical stage and was significantly lower in patients having well-differentiated laryngocarcinoma cells.

ACC2 expression level association with patient survival

Immunohistochemistry was performed to determine the ACC2 expression level in laryngocarcinoma tissue. The patients were divided into two groups, low ACC2 and high ACC2, based on the level of ACC2 expression. A total of 5-year patient survival data were obtained and the Kaplan–Meier method was used to investigate the association of survival time with ACC2 expression. As shown in Fig. 2, patients in the low ACC2 expression group survived longer than those in high ACC2 expression group. This result that ACC2 overexpression is associated with reduced 5-year survival in patients is consistent with our finding that ACC2 is higher in tissue from patients with a more advanced stage of laryngocarcinoma.

Knockdown of ACC2 decreases intracellular lipids levels and enhances FaDu cell apoptosis

Because we demonstrated in the present study that ACC2 is highly expressed in laryngocarcinoma and the prognosis for patients with laryngocarcinoma is poor owing to its rapid metastasis and growth, we next explored whether ACC2 affects apoptosis and proliferation in HNSCC. The FaDu cell line was derived from a hypopharyngeal carcinoma, a type of HNSCC. The NP69 cell line was derived from human nasopharyngeal epithelial cells, which was used as control cell line. Western blot analyses results showed that the expression level of ACC2 in FaDu cells was significantly higher than that in NP69 cells (Figs. 3A and 3B). Next, we used western blot analyses to detect protein expression levels for ACC2, bax, caspase 3, and bcl-2 in FaDu cells transfected with scrambled or ACC2 siRNAs. Our results indicated that the expression levels of ACC2 and bcl-2 proteins were markedly and significantly decreased, whereas those for bax and caspase 3 proteins were significantly increased, in FaDu cells transfected with ACC2 siRNA compared with those transfected with scrambled siRNA (Figs. 3C, 3D and 4A–4D). To examine apoptosis, we performed a TUNEL staining experiment. The results showed that more green fluorescence (representing apoptotic cells) was observed in FaDu cells transfected with ACC2 siRNA than in FaDu cells transfected with scrambled siRNA (Fig. 4E). Knockdown of ACC2 markedly increased apoptosis rates in FaDu and NP69 cells (Fig. 4F; Fig. S2). Additionally, we tested the effect of knockdown of ACC2 on the proliferation in Fadu and NP69 cells via ACC2 siRNA. Our results showed that ACC2 siRNA did not significantly affect the proliferation of FaDu and NP69 cells (Figs. 3C–3F, 5A and 5B). To investigate the cause of FaDu cell apoptosis induced by ACC2 knockdown, intracellular lipids levels were detected. The results showed that the lipids levels of cells transfected with ACC2 siRNA were significantly reduced, compared to those of cells transfected with scrambled siRNA (Fig. 6). Therefore, we demonstrated that knockdown of ACC2 accelerated FaDu cell apoptosis, possibly by interfering with intracellular lipids synthesis.