ATP citrate lyase inhibitor triggers endoplasmic reticulum stress to induce hepatocellular carcinoma cell apoptosis via p‐eIF2α/ATF4/CHOP axis

Abstract ATP citrate lyase (ACLY), a key enzyme in the metabolic reprogramming of many cancers, is widely expressed in various mammalian tissues. This study aimed to evaluate the effects and mechanisms of ACLY and its inhibitor BMS‐303141 on hepatocellular carcinoma (HCC). In this study, ACLY was highly expressed in HCC tissues, especially in HepG2 and Huh7 cells, but was down‐regulated in Hep3B and HCC‐LM3 cells. Besides, ACLY knockdown inhibited HepG2 proliferation and clone formation, while opposite result was noticed in HCC‐LM3 cells with ACLY overexpression. Moreover, ACLY knockdown impeded the migration and invasion abilities of HepG2 cells. Similarly, BMS‐303141 suppressed HepG2 and Huh‐7 cell proliferation. The p‐eIF2α, ATF4, CHOP p‐IRE1α, sXBP1 and p‐PERK were activated in HepG2 cells stimulated by BMS‐303141. In cells where ER stress was induced, ATF4 was involved in BMS‐303141‐mediated cell death procession, and ATF4 knockdown reduced HCC cell apoptosis stimulated by BMS‐303141. In a mouse xenograft model, combined treatment with BMS‐303141 and sorafenib reduced HepG2 tumour volume and weight. In addition, ACLY expression was associated with HCC metastasis and tumour‐node‐metastases staging. Survival analysis and Cox proportional hazards regression model showed that overall survival was lower in HCC patients with high ACLY expression; AFP level, TNM staging, tumour size and ACLY expression level were independent risk factors affecting their overall survival. In conclusion, ACLY might represent a promising target in which BMS‐303141 could induce ER stress and activate p‐eIF2α/ATF4/CHOP axis to promote apoptosis of HCC cells, and synergized with sorafenib to enhance the efficacy of HCC treatment.


| INTRODUC TI ON
Primary liver cancer is a global refractory malignant tumour with high morbidity and high mortality, and more than 90% of primary liver cancer is hepatocellular carcinoma (HCC). 1 The occurrence of HCC is associated with chronic inflammation, non-alcoholic steatohepatitis, alcoholic steatohepatitis and chronic liver injury. 2 Therefore, in the process of abnormal differentiation of hepatocytes, these pathogenic factors induce endoplasmic reticulum (ER) stress through oxidative stress, inflammation and gene mutations and initiate related cascade reactions. 3 There are three ER stress sensors including inositol-requiring 1 (IRE1), activated transcription factor 6 (ATF6) and ER-resident PKR-like eIF2α kinase (PERK); these sensors detect the accumulation of unfolded or misfolded protein at the onset of ER stress and initiate the unfolded protein response (UPR). 4 Besides, the activations of inositol-requiring protein-1 α (IRE1α) and protein kinase RNA-like ER kinase (PERK) can also trigger UPR response.
PERK is a major transducer of the ER stress response and directly phosphorylate α-subunit of eukaryotic initiation factor 2 (eIF2α), 5 and phosphorylated eIF2α (p-eIF2α) promotes translation of activating transcription factor 4 (ATF4). 6 As a pivotal transcription factor in the ER stress pathway, ATF4 mediates the induction of pro-death transcriptional regulator CCAAT enhancer-binding protein homologous protein (CHOP). 7 Previous study has suggested that ER stress and its mediated apoptosis are closely related to the occurrence and development of HCC. 8 In addition, a recent research has revealed that one of the essential factors in the regulation of tumorigenesis and progression is reprogramming of tumour cell metabolism. 9 Metabolic reprogramming of tumour cells redefines the flux and flow of nutrients in the metabolic network of tumour cells, so as to meet the specific needs of tumour cell material metabolism and energy metabolism, thereby ensuring the survival and proliferation of tumour cells and maintaining the survival advantage of tumour cells under adverse conditions. Therefore, the analysis of crucial enzymes in reprogramming of tumour cell metabolism will help to understand the mechanism of tumorigenesis and progression, thus finding a promising target for targeted therapy.
ATP citrate lyase (ACLY), a critical enzyme in the metabolic reprogramming of many cancers, is the first rate-limiting enzyme in the de novo synthesis of fatty acids and is widely expressed in a variety of mammalian tissues. 10 Citric acid produced by sugar metabolism in cytoplasm can be catalytically cracked by ACLY to oxaloacetate and acetyl-CoA, which are the basic raw materials for the synthesis of fatty acids and cholesterol; acetyl-CoA can also be used for protein modification, such as histone acetylation, which is crucial for the transcription and activation of target genes. 11 It is well documented that ACLY overexpression can maintain the malignant proliferation of various tumour cells and promote their malignant evolution. Furthermore, ACLY is up-regulated to different degrees in osteosarcoma, renal, cervical, breast, prostate and lung cancers. [12][13][14] Additionally, Chen et al 15 have believed the possibility of ACLY as a potential and independent biomarker for the recurrence prediction in breast cancer patients. However, the role and molecular mechanism of ACLY and its inhibitor BMS-303141 in liver cancer are still unclear.
This study was designed to test the hypothesis that ACLY can promote the proliferation, migration and invasion of HCC, while its inhibitor BMS-303141 alleviates this effect by triggering ER stress through activation of p-eIF2α/ATF4/CHOP axis; moreover, ACLY in combination with sorafenib can improve the efficacy of HCC therapy.

| The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases
To investigate the ACLY expression profile in HCC and non-tumour liver tissue samples, we used the RNA sequencing and microarray gene profiling data from GEO (GSE14520) and TCGA. There were 374 existing liver cancer sequencing data in TCGA database (September 2016). Among them, 50 paired liver cancer samples and matched tumour-adjacent normal samples were included in the differential expression analysis, and 370 complete clinical data of survival data were extracted for follow-up survival analysis (3 cases with recurrent tumour tissues and 1 case without survival time data were excluded). The sequencing data were obtained from Illumina HiSeq 2000 sequencing platform. GSE14520 dataset was downloaded from Gene Expression Omnibus (GEO) was enrolled in survival analysis.

| Patient samples and cell culture
The first group of liver cancer tissue samples were from 105 patients All cell lines were routinely incubated in Dulbecco's Modified Eagle Medium (DMEM) with high glucose supplemented with 10% FBS, penicillin G (10 000 U/mL) and streptomycin (10 000 µg/mL) at 37°C in a humidified 5% CO 2 incubator. When the density of cultured cells reached 80%, they were washed twice in PBS, followed by addition of an appropriate amount of trypsin to digest the adherent cells. Next, the cells were put back into the incubator and let them stand for 3 minutes until the cells showed a discrete state, and then gently tapped the side wall of the culture bottle to make the cells fall off the bottom. Finally, the cells were stopped digesting by adding an appropriate amount of pre-warmed complete medium.

| Western blot
Total protein from 4 pairs of newly operated HCC and normal control tissues, as well as normal liver cell line (LO2) and human HCC cells (Hep3B, HepG2, HCC-LM3, Huh7), was extracted by RIPA histiocyte rapid lysate (JRDUN, Shanghai, China), supplemented with protease inhibitor of Cocktail (Sigma-Aldrich). Protein concentrations were measured using BCA protein assay kit (Pierce). Samples containing 20-30 µg of protein were mixed with SDS sample buffer and boiled for 5 minutes. Proteins were separated by SDS-polyacrylamide gel electrophoresis using 10% tris-glycine polyacrylamide gradient gel and transferred to polyvinylidene difluoride membrane (PVDF; Applygen Technologies Inc, Beijing, China). The membranes were blocked with 5% (wt/vol) skimmed milk powder in TBS for 1 hour at room temperature and then incubated with the primary antibodies against ACLY, p-eIF2α, eIF2α, ATF4, CHOP, PERK, phosphorylated PERK (p-PERK), IRE1α, phosphorylated IRE1α (p-IRE1α) and splices X-box binding protein (sXBP1) (Affinity Biosciences) and GAPDH (Cell Signaling Technology) overnight at 4°C. After 3 washes in TBST, the membrane was then incubated with HRP-conjugated secondary antibody. Next, the bands were visualized through Gel Doc™ EZ Imager (Bio-Rad Laboratories). Finally, the density of each target protein in the Western blot was normalized according to the GAPDH loading control to get semi-quantification of the target protein in each lane.

| Overexpression or knockdown system
The full-length cDNA of ACLY was cloned from human HepG2 cells by RT-PCR and cloned into mammalian expression vector GV230-EGFP (GENE, Shanghai, China) to construct overexpression plasmids, and the empty vector (vector) was used as a negative control. The two siRNAs of ACLY (siRNA1-2) and nonsense RNAi (si-NC) were designed and synthesized by Shanghai Biotech. The primers and siRNA sequences were list in Table 1 Transfection efficiency of PARP-1 siRNA or PARP-1 plasmid was detected by Western blot.

| Cell proliferation assay
The effect of ACLY on the proliferation of HepG2 and HCC-

| Colony formation assay
Colony formation assay was adopted to evaluate the effects of ACLY on HepG2, HCC-LM3 and Huh-7 cells' proliferation and tumorigenicity in vitro. Cells were plated in a 6-well plate containing 1 × 10 4 cells per well in triplicate and divided into two groups.
After 2 weeks, the cells were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet. The visible colonies were counted.

| Transwell migration and invasion assays
Transwell assays were performed to examine the effects of ACLY on cell migration and invasion. Cells in serum-free medium were seeded into the upper chamber in triplicate. The transwell filter chambers were pre-coated with or without Matrigel (BD Biosciences), and the cell medium was supplemented with 10% FBS placed in the lower chamber. After 36-48 hours of incubation, migrated cells were fixed in 4% paraformaldehyde, stained with 0.1% crystal violet, imaged and counted.

| Statistical analysis
Experimental statistical analysis was performed using SPSS 23.0 statistical software. ANOVA analysis was applied to assess interactions between groups and differences between means. The cumulative survival rate of survival data was calculated by Kaplan-Meier. Logrank was used to compare the survival rate between groups. Cox regression analysis was stepwise regression method. A P value of <.05 was considered to be statistically significant.

| ACLY is highly expressed in HCC samples
According to TCGA and GEO databases, ACLY was found to exhibit significantly higher expression levels in HCC tissues than that in normal liver tissues (both P < .01) ( Figure 1A-B). We also took 50 HCC samples and 50 normal liver samples from TCGA data, and the ACLY expression was highly expressed in HCC tissues ( Figure 1C). To further determined the expression of ACLY in HCC, we selected 4 pairs of newly operated HCC and adjacent normal tissue specimens from patients. As shown in Figure 1D, ACLY was up-regulated in HCC tissues compared with that in the adjacent normal tissues. In addition, the mRNA level of ACLY was increased in 15 HCC tissues as shown by RT-PCR ( Figure 1E)

| Up-regulated ACLY expression is strongly related with disease progression
The correlations between ACLY expression and clinicopathologic  (Table 3). In addition, stratification of ACLY mRNA showed a strong association between ACLY and overall survival rate of patients with HCC. As shown in Figure 2, patients with high ACLY expression had shorter overall survival rate than patients with low ACLY expression.

| ACLY promotes proliferation, migration and invasion of HCC cells
To investigate the biological function of ACLY in pathogenesis of HCC, we constructed HCC-LM3 cells with ACLY overexpression and HepG2 cells with ACLY knockdown using siRNAs of ACLY (siRNA-1 and siRNA-2). As shown in Figure 3A, lentivirus-mediated overexpression or knockdown systems were established and noticed that siRNA-2 had a better effect in knocking down ACLY than siRNA-1.

| ACLY inhibitor suppresses HCC cell viability and colony number
To identify the effect of BMS-303141 on the proliferation of HepG2 and Huh7 cells with different up-regulation of ACLY, MTT and colony formation assays were performed. From Figure 4, stimulation of BMS-303141 at 10 μmol/L or 20 μmol/L significantly suppressed the proliferation and colony number of HepG2 and Huh7 cells.

| ACLY inhibitor triggers ER stress and activate p-eIF2α/ATF4/CHOP axis in vitro
Unfolded protein response is an important cellular that responses to ER stress. To find out whether ER stress can be triggered after BMS-303141 exposure, ER stress-related proteins (eIF2α, p-eIF2α, ATF4 and CHOP) and UPR signal transduction molecules (p-PERK, PERK, p-IRE1α, IRE1α and sXBP1) were measured via Western blot. As shown in Figure 5A, p-eIF2α, ATF4 and CHOP were obviously elevated in a when compared with control cells, suggesting that BMS-303141 activated UPR, which might probably due to ER stress ( Figure 5B).
Considering the key role of ATF4 in cell apoptosis, siRNA1 was used to knock down ATF4 expression. The transfection of si-ATF4 reduced the ATF4 expression in HepG2 cells compared with control group ( Figure 5C). Subsequently, Annexin V-FITC/PI double-staining and flow cytometry assay was used to evaluate the apoptotic rate of HepG2 cells after lentivirus-mediated ATF4 knockdown. As expected, the apoptosis of ATF4 knockdown cells was notably reduced after BMS-303141 administration ( Figure 5D). These findings indicated that BMS-303141 triggered ER stress and induced HepG2 cell apoptosis via the activation of p-eIF2α/ATF4/CHOP axis.

| ACLY inhibitor amplifies the therapeutic effect of sorafenib in the treatment of HCC in vivo
To evaluate the synergetic effect of sorafenib and BMS-303141 in vivo, we injected HepG2 cells in athymic nu/nu mice. When the tumours grew to about 100 mm 3 , mice were treated with indicated compounds.
As shown in Figure 6A-C, treatment of sorafenib alone inhibited HCC cells growth in mice (P < .01). Moreover, combined treatment with BMS-303141 and sorafenib markedly reduced HepG2 tumour volume and weight compared to sorafenib alone group (P < .05). Notably, there was no significant difference of bodyweight in mice among the control and combined treatment groups ( Figure 6D). Moreover, Ki-67 immunostaining result showed that combined treatment with BMS-303141 and sorafenib remarkably inhibited Ki-67 expression ( Figure 6E). These findings indicated that BMS-303141 and sorafenib could synergistically reduce HCC cells of HepG2 tumour in vivo.

| D ISCUSS I ON
The most notable finding of this study was that ACLY could enhance the proliferation, invasion and migration of HCC cells, while ACLY inhibitor BMS-303141 triggered ER stress to induce apoptosis of HCC cells via activation of p-eIF2α/ATF4/CHOP axis. This finding fills the gap in the impact of ACLY and BMS-303141 on hepatocellular carcinoma. We first analysed the expression of ACLY in the TCGA and GEO database, and found that ACLY expression in HCC tissues was significantly higher than that in adjacent tissues. In addition, this trend was further testified in clinical pathology specimens by using Western blot and RT-PCR.  Apoptosis can be induced when the UPR fails to compete with severe and persistent external stimuli, resulting in ER stress. 22 Maurel et al 23  Cox proportional hazards regression analysis indicated that AFP level, TNM staging, tumour size and ACLY expression level were independent risk factors affecting the overall survival rate of HCC patients. Besides, the overall survival rate was significantly higher in patients with low ACLY expression than that with high ACLY expression. AFP is a glycoprotein, which is mainly synthesized in the foetal liver and reaches its peak in the third month of gestation, and then gradually decline. 32,33 However, HCC resumes the function of producing AFP when liver cells become cancerous, which is a good indicator of HCC screening and postoperative follow-up. 34 In our study, AFP and ACLY expression abundance were both independent risk factors affecting the overall survival of patients, but AFP was not associated with ACLY expression, which requires further study in the future.

| CON CLUS ION
In conclusion, the present study reveal that ACLY may represent a promising target in which its inhibition BMS-303141 can induce ER stress and activate p-eIF2α/ATF4/CHOP axis to promote apoptosis of HCC cells, and enhance the efficacy of HCC treatment through synergy with sorafenib. However, there are several limitations in this study, such as the lack of the assessment of ER stress in HCC cell lines with high and low expression of ACLY, and the evaluation of lipotoxic stress, which require further in-depth research in the future.

ACK N OWLED G EM ENTS
This study was supported by Zhejiang Provincial Natural Science

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest. data curation (lead); investigation (supporting).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the finding of this study are available from the corresponding author upon reasonable request.