Squalene epoxidase-induced cholesteryl ester accumulation promotes nasopharyngeal carcinoma development by activating PI3K/AKT signaling.

Nasopharyngeal carcinoma (NPC) is a common malignant tumor and a major cause of mortality and morbidity in southern China. However, the mechanism is still elusive. Here, we focused on studying the role of squalene epoxidase (SQLE), a key enzyme of cholesterol biosynthesis, in the progression of NPC. Clinical study revealed that SQLE expression was significantly upregulated in NPC tissues compared to normal tissues from mRNA level and patients with high expression of SQLE showed a poor prognosis. In vitro experiments showed that SQLE overexpression led to a significant proliferation of cells whereas SQLE knockdown showed an opposite result. In vivo studies also showed that SQLE promoted tumor growth in nude mice. Further study revealed that SQLE promoted NPC proliferation by cholesteryl ester accumulation instead of cholesterol. Mechanism studies indicated that cholesteryl ester promoted NPC cell proliferation by activating the PI3K/AKT pathway and inhibition of this pathway in SQLE-overexpressed or cholesteryl ester-treated cells resulted in a significant reduction of NPC cell proliferation. These results indicate that the oncogenic effect of SQLE in NPC mainly resulted from cholesteryl ester accumulation and PI3K/AKT is a promising target for NPC with SQLE overexpression.

INTRODUCTION

Nasopharyngeal carcinoma (NPC) is a malignant head and neck cancer that associated with Epstein‐Barr virus (EBV) infection. In southern China, NPC is a major cause of mortality and morbidity, however, little is known about the mechanism of NPC. Studies revealed that body mass index (BMI) had a positive correlation with increased risk of NPC. It has also been reported that cholesterol is a predictor of poor survival in patients with NPC. , Moreover, BMI and cholesterol were positively correlated. So it is possible that genes related to cholesterol biosynthesis have an impact on the progression of NPC. Squalene epoxidase (SQLE) is a key rate‐limited enzyme in the biosynthesis of cholesterol. In several types of cancer, such as hepatocellular cancer, SQLE has been proved to be oncogenic and in colorectal cancer and prostate cancer, SQLE was shown to be significantly upregulated in tumor tissues compared to normal tissues, , , indicating it could affect multiple types of cancer. However, the role of SQLE in NPC is still largely unknown.

In this study, we investigate the impact of SQLE on NPC including its clinical value, the effect of SQLE on NPC cell growth, and the potential mechanism.

MATERIALS AND METHODS

Human samples

Nasopharyngeal carcinoma tissues and adjacent normal tissues were obtained from the pathology department of Shenzhen University General Hospital. Tissues without any therapeutic interventions were collected and clinical information was also available from the pathology department. Finally, 314 NPC samples and 58 normal tissues were analyzed, among which there were 5 paired samples. This project was approved by the ethics committee of Shenzhen University General Hospital.

The Cancer Genome Atlas (TCGA) data were downloaded (https://portal.gdc.cancer.gov/), and 44 normal samples and 519 NPC samples were analyzed after excluding patients with incomplete information.

Cell culture and treatment

Human NPC cell line HNE‐2 and HONE‐1 were obtained from Shenzhen University General Hospital. Cells were cultured in RPMI‐1640 medium (Sigma‐Aldrich) supplemented with 10% FBS (Gibco) and 100 U/mL penicillin and streptomycin (Invitrogen) and maintained in a humidified incubator at 37°C in the presence 5% CO2.

Establishment of SQLE stable cell lines

To establish the cell line stably overexpressing SQLE or knockdown of SQLE, pRL‐CMV (pCMV)‐SQLE (RC202008) and pCMV‐entry control plasmids, SQLE shRNA (TL309122V) and control shRNA (pGFP‐C‐shlenti) plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Puromycin was then added to cells for selection. Plasmids were ordered from Origene.

Small interfering RNA knockdown

Either siSOAT‐1 or siControl (50 nmol) (Santa Cruz Biotechnology) was transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

RNA extraction, semiquantitative RT‐PCR, and real‐time PCR analyses

Total RNA was extracted from cells and tissues using TRIzol reagent (Qiagen). Total RNA (1 μg) was used to synthesize cDNA using Transcriptor Reverse Transcriptase (Roche). Real‐time PCR was undertaken using a SYBR Green master mixture (Roche) on a LightCycler 480 instrument. Each sample was tested in triplicate. The ΔΔC t method was applied to determine the fold change in gene expression. The ΔC t method was applied to determine the relative expression of corresponding genes.

Western blot analysis

Protein of tissues of cells was extracted using Cytobuster Protein Extraction Reagent (Novagen). A total of 10 µg protein for each sample was loaded into 12% SDS‐PAGE and then transferred onto a nitrocellulose membrane followed with incubation of primary Abs. After finishing secondary Ab incubation, the signal of immune reactive bands was developed by electrochemiluminescent detection.

Colony formation assay

Cells (1000 per well) were plated in 6‐well plates. After culturing for 10 days, cells were fixed with 70% ethanol for 2 hours and stained with 0.5% crystal violet solution for 1 hour. Colonies with more than 50 cells per colony were counted. All experiments were carried out 3 times in triplicate.

Cell proliferation assay

For the MTT assay, 1000 cells were seeded into a 96‐well plate and cell viability was checked at different time points (0, 1, 2, 3, 4, and 5 days after seeding). Before reading, cells were stained with 20 μL MTT (0.5 mg/mL; Sigma‐Aldrich) for 4 hours followed by adding 100 μL DMSO (Sigma‐Aldrich). The absorbance was measured at 490 nm using a spectrophotometric plate reader (Tecan). All experiments were carried out 3 times in triplicate.

Cholesterol/cholesteryl ester concentrations

Cells (106) or tissues (2 mg) were used to detect the concentration of cholesterol and cholesteryl ester by a cholesterol/cholesteryl ester quantification kit (ab65359; Abcam) according to the manufacturer’s instructions. All experiments were carried out 3 times in triplicate.

Luciferase reporter assay

To investigate the signaling pathways modulated by SQLE, a series of signaling pathway luciferase reporters were examined in SQLE‐overexpressed HNE‐2 cells, including p53‐luc, AP1‐luc, WNT‐luc, and FOXO3‐luc. HNE‐2 cells were stably transfected with pCMV‐SQLE or pCMV‐vector in 24‐well plates and cotransfected with luciferase reporter plasmid (0.2 μg/well) and pCMV‐vector (2 ng/well) using Lipofectamine 2000 (Life Technologies). Cells were harvested 48 hours after transfection, and luciferase activity was analyzed by the Dual Luciferase Reporter Assay System (Promega).

Apoptosis assay

Cells were seeded into 24‐well plates. The TUNEL signal was assessed using the DeadEnd Colorimetric TUNEL System (Promega) according to the manufacturer’s instructions.

Apoptosis was also assessed using an annexin‐phycoerythrin/7‐AAD staining kit (BD Biosciences), according to instructions.

Ki‐67 staining

Paraffin slides of tumors collected from nude mice were used. Ki‐67 signal was assessed by an anti‐Ki‐67 Ab (ab833; Abcam). The proliferation index was determined by counting the number of positive staining cells as percentages of the total number of tumor cells.

Nude mice experiment

Cells (1 × 107) were s.c. injected into the right flank of 4‐week‐old specific pathogen‐free BALB/c nude mice. The volumes of the xenograft tumors were measured every week. The experiment lasted for 4 weeks.

For drug treatment, SQLE inhibitor terbinafine was gavaged with the dose of 80 mg/kg body weight daily. All animal studies were carried out in accordance with the guidelines approved by the Animal Experimentation Ethics Committee of Shenzhen University General Hospital.

Statistical analysis

All statistical tests were undertaken using SPSS or GraphPad Software. Data are presented as means ± SEM. Kaplan‐Meier survival curves and the log‐rank test were used to analyze survival. Student’s t test was used to compare the variables in 2 groups. The difference in cell viability and tumor growth rate between the 2 groups of nude mice was determined by repeated‐measures ANOVA. P values less than .05 were taken as being statistically significant.

RESULTS

Squalene epoxidase is upregulated in NPC tissues and associated with poor prognosis

To study the clinical value of SQLE, we compared the expression of SQLE in NPC tissues (n = 314) and normal tissues (n = 58). Using RT‐PCR, we found SQLE expression was significantly upregulated in NPC tissues compared to normal tissues (P < .001; Figure 1A). Western blot analysis revealed an upregulation of SQLE in 4 of 5 NPC tissues compared to their paired normal tissues (Figure 1B). Analysis of TCGA data also validated this finding (P < .001; Figure 1C). To analyze the relationship between SQLE and prognosis, we divided the 314 patients into 2 groups (High and Low) based on the relative expression of SQLE. We set the median value as the cut‐off value (High, median value or higher; Low, less than the median value). Patients’ clinical characteristics and their impact on SQLE expression are summarized in Table S1. Kaplan‐Meier curves showed that patients with high SQLE expression were associated with poor prognosis compared to patients with low SQLE expression (P < .001; Figure 1D), which is also validated from the survival analysis of TCGA database (P = .006; Figure 1E). Multivariate Cox proportional hazards regression analysis revealed that high SQLE was an independent prognostic factor associated with poor prognosis (P = .023; hazard ratio, 2.678; 95% confidence interval, 1.447 to 3.792; Tables S2 and S3).

Figure 1

Squalene epoxidase (SQLE) is upregulated in nasopharyngeal carcinoma (NPC) tissues compared to normal tissues. A, SQLE mRNA expression in 314 NPC patients and 58 normal tissues. B, SQLE protein expression in 5 paired samples. N, normal tissue; T, tumor tissue. C, SQLE mRNA expression in normal and NPC tissues of The Cancer Genome Atlas (TCGA) data. FPKM, D, The relationship between SQLE mRNA expression and overall survival of 314 NPC patients. E, Survival analysis of SQLE mRNA expression in TCGA data

Squalene epoxidase promotes cell proliferation, reduces apoptosis, and regulates cell cycle

To access the oncogenic effect of SQLE on NPC cells, we established HNE‐2 cells stably overexpressing SQLE cells (Figure 2A). Cell viability and colony formation assay showed that SQLE promoted cell growth, whereas knockdown of SQLE in HONE‐1 cells by shRNA led to suppressed cell growth compared with shControl cells (Figure 2A). Moreover, HNE‐2 cells overexpressing SQLE showed an increased S‐phase cell population and reduced G1 phase population (Figure 2B). In contrast, SQLE knockdown in HONE‐1 cells resulted in an opposite trend compared to SQLE overexpressing cells (Figure 2B), suggesting SQLE accelerated G1/S progression. Consistently, expression of proliferating cell nuclear antigen (PCNA) was also upregulated in SQLE overexpressed cells (Figure 2C). Apoptosis assay showed that SQLE decreased the percentage of apoptosis cells (Figure 2D) as well as the expression of cleaved caspase‐7 and cleaved caspase‐9 (Figure 2C). Those observations imply that SQLE promotes cell growth by activating cell cycle progression and suppressing cell apoptosis.

Figure 2

Squalene epoxidase (SQLE) promotes nasopharyngeal carcinoma (NPC) cell proliferation, reduces their apoptosis, and activates their cell cycle. A, SQLE overexpression promotes NPC cell growth; whole knockdown of SQLE suppresses their growth. B, SQLE overexpression activates the cell cycle in NPC cells, whereas knockdown of SQLE inhibits their cell cycle. C, SQLE activates cell proliferation‐related genes and inhibits cell‐apoptosis related genes. C‐, cleaved; PCNA, proliferating cell nuclear antigen. D, TUNEL staining for apoptosis analysis

Squalene epoxidase promotes tumor growth in nude mice

To further study the oncogenic effect of SQLE, we established a xenograft model. It showed that SQLE significantly promoted tumor growth in mice injected with SQLE‐overexpressed HNE‐2 cells compared to mice injected with control cells (Figure 3A). Ki‐67 staining showed that SQLE‐overexpressed tumors had significant higher levels of Ki‐67‐positive cells compared to control tumors (Figure 3B; P = .021). These observations are consistent with the cell line results that SQLE promotes NPC cell proliferation.

Figure 3

Squalene epoxidase (SQLE) promotes tumor growth in nude mice. A, SQLE‐overexpressed HNE‐2 nasopharyngeal carcinoma cells grows faster than normal HNE‐2 cells. B, SQLE‐overexpressed tumor has significantly higher levels of Ki‐67‐positive cells than normal tumor. Scale bar = 50 µm

Squalene epoxidase promotes cell growth by enhancing cholesteryl ester accumulation

Squalene epoxidase is a key enzyme in the biosynthesis of cholesterol, which plays a role in several types of cancer. We hypothesize that SQLE promotes cell growth by accumulation of cholesterol. First, we checked the level of cholesterol and cholesteryl ester in human NPC tissues and normal tissues. They all showed a significant upregulation in NPC tissues compared to normal tissues (Figure 4A; P < .001). The same observation was also seen in tumors collected from nude mice and SQLE‐overexpressed HNE‐2 cells (Figure 4B; P < .05). In contrast, their level was significantly downregulated in SQLE knockdown HONE‐1 cells (Figure 4B; P < .01). Next, we explored the impact of SQLE on the expression of proteins related to cholesterol transportation. Interestingly, low density lipoprotein receptor and scavenger receptor class B, type 1, the proteins responsible for importing low density lipoprotein and high density lipoprotein into tissues respectively, were upregulated in SQLE‐overexpressed HNE‐2 cells while ABCA1, the protein responsible for exporting cholesterol from tissues, was downregulated. In contrast, SOAT1, which is responsible for the synthesis of cholesteryl ester as a storage form of cholesterol, was upregulated (Figure 4C). Consistently, alteration of those proteins in paired NPC tissues showed a similar trend compared to normal tissues (Figure 4C). In contrast, knockdown of SQLE in HONE‐1 cells led to an opposite alteration (Figure 4C). These observations suggest that SQLE can increase the absorption of cholesterol into cancer tissues while suppressing its excretion from the tissue.

Figure 4

Squalene epoxidase (SQLE)‐induced cholesteryl ester, instead of cholesterol, contributes to nasopharyngeal carcinoma (NPC) cell proliferation. A, Cholesterol and cholesteryl ester level in NPC and normal tissues. B, Cholesterol and cholesteryl ester level in tumors collected from nude mice, SQLE‐overexpressed HNE‐2 cells, or SQLE‐knockdown HONE‐1 cells. C, Impact of SQLE on the expression of cholesterol imported and exported proteins. N, normal; T, tumor. D, Cholesteryl ester, instead of cholesterol, leads to NPC cell proliferation. E, Knockdown of SOAT1 leads to the increased cholesterol and decreased cholesteryl ester, abolishing SQLE‐mediated NPC cell proliferation

To further investigate the impact of altered cholesterol and cholesteryl ester on cell growth, cells were treated with cholesterol or cholesteryl ester. Interestingly, exogenous cholesterol did not show a promoted effect on cell growth whereas exogenous cholesteryl ester showed a significant promotion of cell growth (Figure 4D), indicating cholesteryl ester instead of cholesterol leads to the promoted cell growth. To further address this finding, we knocked down SOAT‐1, a key enzyme in cholesteryl ester synthesis, in SQLE‐overexpressed HNE‐2 cells and it showed a significantly decreased cholesteryl ester level and increased free cholesterol level (Figure 4E). Consistent with our finding, SOAT‐1 knockdown did not promote cell growth in the cell viability assay (Figure 4F). Collectively, these results indicate that SQLE‐mediated cholesteryl ester accumulation is responsible for the promoted cell growth in NPC cells.

Squalene epoxidase activates PI3K/AKT pathway to promote cell growth through cholesteryl ester accumulation

To study the molecular mechanism of the oncogenic impact of SQLE, cancer pathway luciferase reporter assays were carried out. Among several critical cancer‐related gene reporters, FOXO3 reporter (PI3K/AKT) activity was significantly inhibited in SQLE overexpressed or exogenous cholesteryl ester treated HNE‐2 cells (Figure 5A), indicating SQLE and cholesteryl ester could activate the PI3K/AKT pathway. Western blot analysis also revealed upregulated phosphorylated AKT (p‐AKT) and PI3K (p‐PI3K) in SQLE overexpressed or exogenous cholesteryl ester‐treated HNE‐2 cells and paired NPC tissues (Figure 5B,C). To analyze whether SQLE‐activated PI3K/AKT is dependent on cholesteryl ester accumulation, SOAT‐1 was knocked down in SQLE‐overexpressed HNE‐2 cells and it showed that upregulated p‐PI3K and p‐AKT were abolished (Figure 5D). These results show that SQLE activates PI3K/AKT through cholesteryl ester accumulation.

Figure 5

Mechanism study of squalene epoxidase (SQLE)‐mediated nasopharyngeal carcinoma (NPC) cell proliferation. A, Luciferase reporter assay of cancer pathways for SQLE and cholesteryl ester. B, Expression of phosphorylated (p‐)AKT in SQLE‐overexpressed HNE‐2 cells and cholesteryl ester‐treated HNE2 cells. C, Expression of p‐AKT in 5 paired NPC samples. N, normal tissue; T, tumor tissue. D, Inhibition of cholesteryl ester resulted in the reduction of p‐AKT. E, Inhibition of PI3k/AKT signaling leads to suppressed NPC cell proliferation

To further study the role of PI3K/AKT in SQLE‐promoted cell growth, we used PI3K inhibitor (BYL 719) to treat SQLE‐overexpressed HNE‐2 cells. It showed that cell growth was significantly suppressed with the inhibition of PI3K (Figure 5E). Collectively, these observations suggested that SQLE‐induced cholesterol accumulation activates the PI3K/AKT pathway to promote NPC cell growth.

Pharmacological inhibition of SQLE suppressed NPC cell growth

Given the important oncogenic impact of SQLE on NPC cells, we evaluated whether specific inhibition of SQLE can be repositioned for prevention or treatment of NPC. Different doses of SQLE specific inhibitor, terbinafine, were used to treat SQLE‐overexpressed HNE‐2 cells and HONE‐1 cells. Cell viability and colony formation assays showed that terbinafine significantly suppressed cell growth at 25 and 50 µM (Figure 6A). Western blot analysis revealed that terbinafine had no impact on SQLE expression and reduced PCNA expression while it enhanced apoptosis‐related genes, such as cleaved caspase‐7 and cleaved caspase‐9 (Figure 6B). Moreover, the expression of p‐AKT was also downregulated following treatment with terbinafine (Figure 6B). These results are consistent with the findings above.

Figure 6

Squalene epoxidase (SQLE) is a therapeutic target for nasopharyngeal carcinoma (NPC). A, SQLE inhibitor significantly inhibits NPC cell proliferation. B, SQLE inhibitor results in suppressed expression of proliferation‐related genes, phosphorylated (p‐)AKT, and increased expression of apoptosis‐related genes. PCNA, proliferating cell nuclear antigen. C, SQLE inhibitor leads to suppressed growth of xenograft tumors in nude mice. D, SQLE inhibitor prolongs nude mouse survival. E, SQLE inhibitor significantly reduces cholesteryl ester level. F, SQLE inhibitor significantly suppresses Ki‐67 staining. Scale bar = 50 µm

Next, we used the xenograft model to evaluate the efficacy of terbinafine and it showed that terbinafine markedly suppressed the growth of s.c. SQLE‐overexpressed xenografts (Figure 6C) in terms of tumor size and tumor weight. Survival analysis was also evaluated in the mice harboring SQLE‐overexpressed xenografts and we can see that SQLE inhibition significantly prolonged the survival of mice (Figure 6D). We further checked the cholesteryl ester level of the xenograft tumors and found that terbinafine markedly downregulated the cholesteryl ester level (Figure 6E). Ki‐67 staining revealed that cell proliferation was significantly inhibited by terbinafine (Figure 6F). These results indicate that SQLE is an effective target for the treatment of NCP.

DISCUSSION

Nasopharyngeal carcinoma is a malignant cancer and it is a major cause of mortality and morbidity in South China. , However, its mechanism is still unclear. It was previously reported that cholesterol is a marker of poor prognosis for patients with NPC. , Here, we studied the role of SQLE, a key enzyme in the biosynthesis of cholesterol, on the progression of NPC. Consistent with previous cholesterol findings, we also showed that SQLE is a marker for poor prognosis in the patients with NPC (Figure 1B,C) and the xenograft mouse model also showed SQLE overexpression was associated with shorter survival (Figure 6D). However, in the following functional analysis, we proved cholesteryl ester, instead of cholesterol, contributes to the oncogenic impact of SQLE (Figure 4C). Cholesterol has been proved to be associated with several types of cancer, such as hepatocellular, colorectal, and prostate cancer. , , However, recent studies showed that cholesteryl ester, but not cholesterol, is the main cause of the progression of cancer; for example, cholesteryl ester (not cholesterol) promotes nonalcoholic fatty liver disease (NAFLD)‐induced hepatocellular carcinoma (HCC), which is similar with our finding. As the main storage form of cholesterol in blood or tissues is cholesteryl ester and the free form of cholesterol is very rare, it totally makes sense that cholesteryl ester plays a major role in mediating cancer cell proliferation (Figure 4C‐E). Consistently, we also observed cholesteryl ester was markedly increased in human NPC tissues compared to normal tissues (Figure 4A).

A previous study showed that SQLE could activate the PI3K/AKT pathway to promote NAFLD‐HCC, which is similar with our finding in NPC cells, however, they did not directly prove the relationship between cholesterol and activated PI3K/AKT. We observed that SQLE activates PI3K/AKT, depending on cholesteryl ester accumulation, which directly activates the PI3K/AKT pathway (Figure 5B,C). Moreover, inhibition of cholesterol synthesis in SQLE overexpressed cells led to suppressed PI3K/AKT activation and NPC cell growth, indicating SQLE fulfills its function mainly through cholesteryl ester accumulation (Figure 5D,E). These observations revealed that SQLE is a potential therapeutic target for the treatment of NPC. Consistently, inhibition of SQLE using SQLE inhibitor (terbinafine) resulted in reduced NPC cell proliferation, increased apoptosis, and suppressed tumor growth (Figure 6A‐D).

In conclusion, our study proved that SQLE fulfills its oncogenic function by accumulating cholesteryl ester through activating the PI3K/AKT pathway.

CONFLICT OF INTEREST

The authors have no conflict of interest.

Table S1

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Table S2

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Table S3

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