Abstract

   The histone methyltransferase EZH2 silences gene expression via H3
   lysine 27 trimethylation and has been recognized as an important
   antitumour therapeutic target. However, the clinical application of
   existing EZH2 inhibitors is not satisfactory for the treatment of solid
   tumours. To discover novel strategies against head and neck squamous
   cell carcinoma (HNSCC), we performed genomics, metabolomics and RNA
   omics studies in HNSCC cells treated with EZH2 inhibitors. It was found
   that EZH2 inhibitors strongly induced the expression of genes in
   cholesterol synthesis. Through extensive drug screening we found that
   inhibition of squalene epoxidase (a key enzyme of endogenous
   cholesterol synthesis) synergistically increased the squalene content
   and enhanced the sensitivity of HNSCC cells to EZH2 inhibitors. Our
   findings provide an experimental and theoretical basis for the
   development of new combinations of EZH2 inhibitors to treat HNSCC.

   Subject terms: Cancer metabolism, Translational research

Introduction

   As a core component of the polycomb protein family, histone
   methyltransferase EZH2 and its binding partner proteins form a
   multisubunit complex, which can specifically catalyse the
   trimethylation of histone H3 lysine 27 (H3K27me3) and silence target
   genes in embryonic development and regulate cell proliferation^[46]1.
   EZH2 has been found to be highly expressed in many tumours, such as
   prostate cancer, breast cancer, colorectal cancer, hepatocellular
   carcinoma, lymphoma and head and neck squamous cell carcinoma
   (HNSCC)^[47]2. Overexpression of EZH2 is closely related to tumour
   progression and poor prognosis^[48]3. In HNSCC tissues, the
   upregulation of EZH2 is positively correlated with the overexpression
   of cyclin D1 and poor clinical prognosis^[49]2. EZH2 also regulates the
   STAT3/HOTAIR axis^[50]4, disrupts differentiation^[51]5 and promotes
   oral cancer metastasis by mediating ROS1 oncogene^[52]6. Currently,
   several small molecule compounds (EPZ6438, GSK126 and GSK343) have been
   developed as EZH2 inhibitors^[53]7. Among them, EPZ6438 has been
   approved for adults and paediatric patients aged 16 years and older
   with metastatic or locally advanced epithelioid sarcoma not eligible
   for complete resection. EPZ6438 has also been approved for adult
   patients with relapsed or refractory follicular lymphoma whose tumours
   are positive for an EZH2 mutation as detected by an FDA-approved test
   and who have received at least 2 prior systemic therapies, and for
   adult patients with R/R FL who have no satisfactory alternative
   treatment options^[54]8. However, the treatment of solid tumours with
   EZH2 inhibitors is not satisfactory, which limits their clinical
   application range. Therefore, discovering novel drug combinations of
   EZH2 inhibitors with other clinical drugs will greatly improve the
   value of EZH2 inhibitors.

   Increasing scientific evidence supports the close relationship between
   the disorder of cholesterol metabolism and cancer^[55]9. Dysregulation
   at different stages of cholesterol metabolism promotes tumour
   development and resistance to therapeutic drugs^[56]10,[57]11. On the
   other hand, some intermediates of cholesterol synthesis and metabolism
   show obvious antitumour properties. For example, squalene has been
   reported to block the cell cycle in the G[0]/G[1] phase^[58]12.
   Squalene monooxygenase (SQLE), the key enzyme of the endogenous
   cholesterol synthesis pathway, catalyses the first oxidation step of
   cholesterol biosynthesis, converting squalene into 2,3-oxo
   squalene^[59]13. Terbinafine and butenafine are used as antifungal
   agents to inhibit SQLE in fungi^[60]14,[61]15. However, few studies
   have reported the antitumour effects of SQLE inhibitors on HNSCC.

   In this study, we utilized multiple omics to validate that EZH2 can
   modulate endogenous cholesterol synthesis and that blocking the
   endogenous cholesterol synthesis with SQLE inhibitors can largely
   enhance the sensitivity of HNSCC cells to EZH2 inhibitors.

Methods and materials

Cell culture

   The human head and neck squamous cell lines HN4, HN6, HN30, CAL27, SCC9
   and SCC25 were obtained from the NIH. These cells were cultured in
   high-glucose medium (L110KJ, Basal Media, China), which contained 10%
   fetal bovine serum (SA311.02, CellMax, China) 100 U/ml penicillin, and
   100 μg/ml streptomycin (C125C5, NCM, China). All cell lines were
   cultured in incubators with humidified air at 37 °C and 5% CO[2].
   Authentication by STR profiling, population doubling time and cell
   morphology were checked as well to confirm the genotype.

Lentivirus transfection and screening of stable cell lines

   The lentivirus (sh-nc, sh-FDFT1-1, sh-FDFT1-2) were constructed by
   ZORIN Company (Shanghai, China). According to the manufacturer’s
   instruction, CAL27 cells were transfected with lentivirus (10^6 TU/ml)
   at 40% density. After 72 h in the cell incubator, the cells were
   cultured in the medium containing 5 μg/ml puromycin for one generation.
   Then the cells were cultured in the medium containing 10 μg/ml
   puromycin for two generations. The sequences of sh-nc, sh-FDFT1-1 and
   sh-FDFT1-2 are showed below: 5ʹ-GCAGUUUCGCAGCUGUUAUTT-3ʹ;
   5ʹ-AUAACAGCUGCGAAACUGCTT-3ʹ 5ʹ-GCAGUGCCUGAAUGAAACUUTT-3ʹ;
   5ʹ-AAGUUCAUUCAGGCACUGCTT-3ʹ.

Chemical compounds

   The following compounds were purchased from commercial vendors: GSK126
   (S7061, Selleck, China), GSK343 (S7164, Selleck, China), EPZ6438
   (S7168, Selleck, China), NB-598 (HY-16343C, MCE, China), Atorvastatin
   (HY-17379, MCE, China), Ro48-8071 (HY-18630A, MCE, China), Terbinafine
   (HY-17395, MCE, China), Butenafine (HY-17396, MCE, China), Cholesterol
   (C3045, Sigma, China), Desmosterol (H130206, Aladdin, China),
   Lathosterol (HY-17395, MCE, China), Squalene (S3626, Sigma, China).

Cell growth and survival assays

   Cells were seeded into 96-well plates at a density of 2000 cells per
   well, each group has three replicates, and then different inhibitors
   were added to the plates. Then, 10 μl MTT reagent (Do Jindo, Kumamoto,
   Japan) was added to 100 μl of culture medium at 72 h. The cells were
   then incubated at 37 °C for 4 h. After the supernatant was aspirated,
   150 μl DMSO was added, and the optical density was measured at 490 nm
   using a multi-functional microplate reader instrument (Spectra Max i3,
   Molecular Devices, USA).

Colony formation

   The cells indicated in this study were seeded in 6-well plate (1000 per
   well) or 24-well plate (300 per well), then cultured with different
   treatments for 7 days. After fixing with 4% paraformaldehyde for
   15 min, the cells were stained with crystal violet for 30 min. Then we
   use scanners for flat-panel scanning and count the colony numbers by
   ImageJ.

Plasmids

   The human SQLE Gene ORF cDNA clone expression plasmid was purchased
   from Sino Biological, China (HG21419-CF), and Pcmv3-c-FLAG was used as
   the vector.

qRT-PCR analysis

   Total RNA was isolated with the RNAiso Plus kit (TAKARA, China), and
   reverse transcription of RNA into cDNA using the Prime Script RT
   Reagent Kit (TAKARA, China). Quantitative PCR was performed using the
   TB Green Premix Ex Taq reagent kit (TAKARA, China) according to the
   introduction of ABI Step One Real-Time PCR System (Life Technologies,
   USA). The sequences of the primers indicated are shown in Supplementary
   Table [62]S1.

Immunoblotting analysis

   The process of protein extraction and SDS gel electrophoresis is
   referred to the previous study^[63]16. The antibodies are listed in
   Supplementary Table [64]S2.

Chromatin immunoprecipitation (ChIP) assays

   ChIP was performed using the Simple Chip Plus Enzymatic Chromatin IP
   Kit (Magnetic Beads) (9005S, Cell Signalling Technology, USA) with
   CAL27 cells, and all procedures were in accordance with the
   manufacturer’s instructions.

ChIP-Seq

   The procedures of ChIP were performed as the previous study^[65]16.

   In order to remove technical sequences, including adaptors, polymerase
   chain reaction (PCR) primers or fragments thereof, and quality of bases
   lower than 20, pass filter data of fastq format were processed by
   Cutadapt^[66]17 (version 1.9.1) to be high-quality clean data.

   The reference genome sequences and gene model annotation files of
   relative species are downloaded from genome website, such as UCSC, NCBI
   and ENSEMBL. The Bowtie2-build is used to index reference genome
   sequence and clean data are aligned to reference genome via software
   Bowtie2 (version 2.2.6). MACS2^[67]18 is used to analyse peaks’ quality
   control, peaks’ calling and peaks’ annotation. GO and KEGG enrichment
   analysis were performed as previously described in RNA-Seq analysis.

RNA interference and transfection

   The indicated siRNAs (Supplementary Table [68]S3) were synthesized by
   GenePharma, China. All siRNAs were transfected into cells using
   Lipofectamine 3000 (Invitrogen) according to the manufacturer’s
   instructions.

Flow cytometric analysis for detecting the cell cycle and cellular apoptosis

   CAL27 cells were treated with different drugs for the indicated times.
   The pretreatment was performed according to the manufacturer’s
   instructions for PI/RNase Staining Buffer (BD Biosciences, 550825).
   Then, the cells were analysed on a BD FACSCalibur^TM Flow Cytometer
   (Becton Dickinson, BD Biosciences, USA).

   For the apoptosis experiment, the cells treated as described above were
   washed three times with PBS and centrifuged at 1000 rpm for 3 min.
   Then, the cells were resuspended in 1× binding buffer and fixed for
   30 min. The cells were resuspended with Annexin V and PI dye in binding
   buffer at a ratio of 1:1:50, placed on ice for 15 min, and then tested
   on a BD FACSCalibur^TM Flow Cytometer (Becton Dickinson, BD
   Biosciences, USA).

MS analysis of non-targeted metabolomics

   In total, 31 samples of CAL27 cells were collected for non-targeted
   metabolomics. There were four groups of cells treated with DMSO,
   EPZ6438, terbinafine or EPZ6438/terbinafine for 72 h. Then 1.5 ml
   methanol water solution (1:1, V/V) was added to the cell culture dish,
   and the cell suspension was transferred to the centrifuge tube with
   cell scrapers. The process of sample processing and computer operation
   is referred to the published article^[69]19.

GC/MS data analysis

   Metabolite identification was performed by ChromaTOF software combined
   with National Institute of Standards and Technology mass spectral
   libraries (NIST 17), LECO/Fiehn Metabolite mass spectral library
   (Version 1.00) and our in-house library.

   Based on the retention time of a series of fatty acid methyl esters
   (C6–C28), retention index was calculated by Retention Index Method
   function in ChromaTOF software^[70]20. In the meantime, the acquired MS
   files from GC-TOFMS analysis were processed by ChromaTOF software
   (v5.50, Leco Co., CA). The data of pretreatment procedures included
   baseline correction, denoising, time-window splitting, deconvolution,
   peak filtering and multivariate curve resolution. It is further
   designed to align and combine peaks from the multiple table files
   (.csv) obtained from the ChromaTOF software. The resulting
   three-dimensional data set includes sample information, peak retention
   time and peak areas. The peak areas were normalized to the area of the
   internal standards (IS), mean-centred, and then treated by unit
   variance scaling for further statistical analysis. Finally, IS and any
   known artificial peaks, such as peaks caused by noise, column bleed and
   BSTFA derivatization procedure, were removed from the data set^[71]21.

   According to the instructions and Xia and Wishart^[72]22, the online
   processing of web page was used to calculate the data expression
   profile and analyse the function enrichment [73]www.metaboanalyst.ca.

Detection of squalene monooxygenase activity

   The activity of squalene monooxygenase was detected by the kit
   purchased from Haling Biological Technology Company ([74]GMS50782.1,
   Shanghai). CAL27 cells were treated with vehicle, EPZ6438, terbinafine
   and EPZ6438 + terbinafine for 72 h, respectively, according to the
   manufacture’s instruction. After a series of reactions, the OD values
   of the standard and the sample was detected at 400 nm by
   multi-functional microplate reader (Spectra Max i3, Molecular Devices,
   USA). Finally, the enzyme activity of the samples were calculated
   according to the instructions.

Animal experiments

   Three-week-old male nude mice were purchased from Shanghai SLAC
   Laboratory Animal Company and housed in ventilated cages for 1 week.
   CAL27 cells, sh-FDFT1 CAL27 cells and sh-nc CAL27 cells (resuspended in
   DMEM without FBS) were used to establish xenograft models by
   subcutaneously injecting 100 µl of cells at a concentration of 10^6 per
   position in the right and left flanks of each mouse. After 1 week, mice
   were randomized into four groups based on random number table: vehicle
   (0.5% CMCNa + 0.1% Tween 80 + ddH[2]O), EPZ6438 (200 mg/kg),
   terbinafine (60 mg/kg) and EPZ6438 (200 mg/kg) + terbinafine
   (60 mg/kg). Each group (N = 6) received treatment once daily via oral
   administration. Mouse weight was measured once a day, while tumour
   volume was measured once every 2 days by callipers. Tumour volume was
   calculated using the formula V = (L × W^2)/2. At the end of the study,
   mice were sacrificed, and tumours were collected and stored at −80 °C
   until analysis.

Immunohistochemistry assay and immunofluorescence staining

   The paraffin slices were baked in a 65 °C oven for 1 h, soaked in
   xylene for 10 min, and then soaked in 100% ethanol, 95% ethanol and 75%
   ethanol for 5 min, respectively. Then, they were boiled in sodium
   citrate antigen retrieval solution heated to boiling in advance for
   30 min to complete the antigen retrieval. A drop of peroxygenase
   blocking solution was added to each slice, incubated at room
   temperature for 10 min, and then washed with PBS three times, each time
   for 10 min. Then, a drop of goat serum was added to each section and
   incubated at room temperature for 10 min. After removing the serum,
   100 μl of primary antibody (1:100) was added to each slide. After
   incubation at 4 °C overnight, each slice was washed with PBS three
   times for 10 min each time and then covered with a drop of
   biotin-labelled secondary antibody and incubated at room temperature
   for 1 h.

BODIPY staining

   Cell lines were plated in 24-well plates and treated with the indicated
   pharmacological inhibitors for 48 h. The cells were fixed with 4%
   paraformaldehyde, washed twice with 1× PBS, and incubated with 1 ml of
   BODIPY 493/503 (D3922, Thermo Fisher) at 37 °C for 30 min. Then, the
   cells were washed three times with 1× PBS for 5 min. After DAPI
   staining for 5 min, the cells were washed three times with 1× PBS for
   5 min each time. Images were captured using an inverted fluorescence
   microscope (AXIO ZEISS, USA).

RNA-Seq

   According to the previous methods^[75]23, CAL27 cells plated in 10 cm
   dishes were treated with vehicle or 10 μM EPZ6438 for 72 h. Then, the
   cells were washed twice with PBS and lysed with an RNAiso Plus kit
   (TAKARA) according to the manufacturer’s protocol. The cDNA libraries
   were then constructed for each pooled RNA sample using the VAHTSTM
   Total RNA-Seq (H/M/R). Differentially expressed mRNAs were then
   identified through fold change, and p values were calculated using
   t-tests. The thresholds set for up- and downregulated genes were fold
   change ≥ 2.0 and p value ≤ 0.05.

RNA-Seq analysis

   The transcriptome sequencing and analysis were conducted by OE Biotech
   Co., Ltd. (Shanghai, China). Raw data (raw reads) were processed using
   Trimmomatic^[76]24. The reads containing ploy-N and the low-quality
   reads were removed to obtain the clean reads. Then the clean reads were
   mapped to reference genome using HISAT2^[77]25.

   FPKM^[78]26 value of each gene was calculated using cufflinks^[79]27,
   and the read counts of each gene were obtained by HTSeq-count^[80]28.
   DEGs were identified using the DESeq R package function^[81]29 to
   estimate Size Factors and nbinom Test. p value < 0.05 and fold change
   > 2 or <0.5 was set as the threshold for significantly differential
   expression. Hierarchical cluster analysis of DEGs was performed to
   explore genes expression pattern. GO enrichment and KEGG^[82]30 pathway
   enrichment analysis of DEGs were, respectively, performed using R based
   on the hypergeometric distribution.

Statistical analysis

   The investigators were always blinded to the group allocation during
   the experiments of the study. Statistical analysis was carried out
   using or GraphPad Prism 6. Data were presented as the mean ± SD. The
   correlation was determined by Pearson correlation analysis. One-way
   analysis of variance (ANOVA) were performed to assess the significance
   of the differences. Student’s t-test was used for pairwise comparisons
   between groups. A p value (two-sided) <0.05 was considered to be
   statistically significant. Samples sizes were chosen according to the
   basis of previous publications without prior power analysis.

Results

EZH2 inhibitors upregulate the gene expression in the endogenous cholesterol
synthesis pathway

   To understand the influence of EZH2 inhibitors on the growth of HNSCC,
   we first tested the sensitivity of different HNSCC cell lines to EZH2
   inhibitors. We found that the IC[50] (half-inhibition concentration)
   values of most HNSCC cells (HN4, HN6, HN30, CAL27, SCC9 and SCC25) to
   EZH2 inhibitors (GSK343, GSK126 and EPZ6438) was higher than 10 μM
   (Fig. [83]1A). In the RNA-Seq analysis, the EZH2 inhibitors (GSK126 and
   EPZ6438) had the greatest effect on inducing genes expression of the
   cholesterol synthesis pathway (Fig. [84]1B and Supplementary Fig.
   [85]1A). The bubble map of pathway enrichment analysis indicated that
   steroid biosynthesis have the highest enrichment score (Supplementary
   Fig. [86]1B, C). The more detailed comparison of enriched genes between
   the EZH2i group and vehicle group is shown in Fig. [87]1C and
   Supplementary Fig. [88]1D. According to GSEA analysis of the RNA-Seq
   data, we found that genes in cholesterol biosynthesis process were
   statistically enriched (false discovery rate [FDR] q < 0.05) in the
   cells treated with EZH2i than in the vehicle group (Fig. [89]1D and
   Supplementary Fig. [90]1E). The expression of SQLE was increased over
   3-fold (Fig. [91]1E and Supplementary Fig. [92]1F). Next, we selected
   three small molecular compounds to incubate different HNSCC cells. All
   the inhibitors significantly upregulated the mRNA level of endogenous
   cholesterol synthesis-related enzymes, including HMGCS1, FDFT1, SQLE,
   LSS, CYP51A1, DHCR7, DHCR24 and HMGCR (Fig. [93]1F, G and Supplementary
   Fig. [94]1G). The protein levels were also significantly increased
   (Fig. [95]1H). Moreover, the mRNA and protein levels of the target
   genes were further verified in the cells that were knocked down of EZH2
   by specific siRNAs compared to the scramble siRNA (Fig. [96]1I, J).
   These data suggest that the inhibition of EZH2 significantly
   upregulates the gene expression of the endogenous cholesterol synthesis
   pathway in HNSCC cells.

Fig. 1. Inhibition of EZH2 promotes the cholesterol synthesis pathway.

   [97]Fig. 1
   [98]Open in a new tab

   A IC[50] of three EZH2 inhibitors in different HNSCC cell lines. B KEGG
   enrichment of RNA-Seq data to analyse the significantly 2-fold changed
   genes in the CAL27 cell line treated with the EPZ6438 compared to the
   vehicle. C The most changed genes in the cholesterol synthesis pathway.
   D GSEA was performed to analyse the expression pattern. E Multiple
   changes in the endogenous enzyme cholesterol synthesis pathway after
   HNSCC cells were treated with EPZ6438. F Real-time PCR assays to detect
   the EPZ6438-induced expression of cholesterol synthesis enzymes in
   various HNSCC cell lines. N = 3 replicates. E10 (EPZ6438-10 μM), E20
   (EPZ6438-20 μM). G Real-time PCR assays to detect the GSK126-induced
   expression of cholesterol synthesis enzymes in various HNSCC cell
   lines. N = 3 replicates. G1 (GSK126-1 μM), G5 (GSK126-5 μM). H
   Immunoblotting analysis to examine the protein levels of cholesterol
   synthesis enzymes in multiple HNSCC cell lines treated with increasing
   concentrations of EPZ6438. I Real-time PCR assays to detect the
   expression of target genes in CAL27 cells. N = 3 replicates, t-test
   analysis was used to assess the statistical significance, **p < 0.01,
   ***p < 0.001. J Immunoblotting analysis was performed to examine the
   cholesterol synthesis enzymes after cells were treated with
   EZH2-specific siRNAs and scrambled siRNA.

EZH2 inhibitor promotes the cholesterol synthesis pathway by H3K27me3
modification

   To examine whether EZH2 can specifically regulate the expression of the
   cholesterol synthesis pathway by modulating H3K27me3 levels, HNSCC
   cells were incubated with the EZH2-specific inhibitor EPZ6438. EPZ6438
   selectively reduced H3K27me3, rather than other modifications (Fig.
   [99]2A). Next, we used H3K27me3 antibody to enrich target genes in the
   ChIP assay (Fig. [100]2B). Compared to the vehicle control group,
   H3K27me3 modification were enriched at the loci of SQLE promoter and
   other genes in cholesterol synthesis (Fig. [101]2C and Supplementary
   Fig. [102]2A, B). The H3K27me3 levels of SREBF2 and SQLE decreased
   significantly after EZH2 inhibitor treatment, but the levels of other
   genes involved in cholesterol metabolism did not change significantly
   (Fig. [103]2D). These data suggest that EZH2 regulates the expression
   of related metabolic enzymes in the cholesterol synthesis pathway at
   least partially through H3K27me3.

Fig. 2. EZH2 regulates cholesterol synthesis through H3K27me3.

   [104]Fig. 2
   [105]Open in a new tab

   A Immunoblotting analysis to examine the methylation modification of
   different histone residues by the EZH2 inhibitor EPZ6438. B Heatmaps of
   H3K27me3 ChIP-Seq signals at the TSS in vehicle- and EPZ6438-treated
   CAL27 cells. C Genome browser view of normalized ChIP-seq signals of
   H3K27me3 at the SQLE locus in vehicle and EPZ6438-treated CAL27 cells.
   D ChIP-PCR analysis to examine the level of H3K27me3 modification in
   the promoter regions of different target genes after CAL27 cells were
   treated with vehicle or EPZ6438. N = 3 replicates, t-test analysis was
   used to assess the statistical significance, *p < 0.05, **p < 0.01,
   ***p < 0.001, ****p < 0.0001.

Inhibiting the cholesterol metabolic pathway enhances the sensitivity of
HNSCC to EZH2 inhibitors

   Next, we incubated CAL27 cells with the combination of EZH2 inhibitors
   (GSK126, GSK343 and EPZ6438) and inhibitors of different key enzymes in
   the cholesterol synthesis (the SQLE inhibitor NB-598, the HMGCR
   inhibitor atorvastatin and the LSS inhibitor Ro48-8071). All the
   combination indexes (CI) of EZH2 inhibitors plus NB-598 were lower than
   those of other formulas and indicated a potent synergistic effect
   (CI < 0.6) (Fig. [106]3A). Furthermore, we analysed the combination
   effect of EPZ6438 and terbinafine through orthogonal test. EPZ6438 and
   terbinafine have a strong inhibitory effect on CAL27 cells
   (Supplementary Fig. [107]3B). Except for CAL27 cells, terbinafine and
   EPZ6438 indicated potent synergistic effects on a variety of cell lines
   (HN4, HN6, HN30, CAL27, SCC9 and SCC25) (Fig. [108]3B). Another
   evaluation in the Bliss model indicated that the combination scores of
   EPZ6438 and terbinafine are lower than 0.3 (Fig. [109]3C). The
   cholesterol metabolizing enzyme genes such as SQLE, FDFT1, LSS,
   CYP51A1, DHCR7 and HMGCR were silenced with siRNAs and verified
   efficiency with real-time PCR assays (Supplementary Fig. [110]3A). The
   sensitivity of HNSCC cells to EPZ6438 increased significantly when the
   expression of SQLE was silenced, rather than other genes (Fig. [111]3D
   and Supplementary Fig. [112]3C). EPZ6438 substantially enhances the
   enzymatic activity of SQLE in CAL27 cells, which was completely
   opposite to terbinafine. There was no significant difference between
   the combination group and the vehicle group (Fig. [113]3E). The colony
   number in the combination group was significantly lower than that in
   the control group (Fig. [114]3F). And EPZ6438 treatment also
   synergistically inhibited cell viability with the specific siRNA
   against SQLE (Supplementary Fig. [115]3D). Compared with the vehicle
   group, both mRNA and protein expressions of the cholesterol synthesis
   enzymes were upregulated in the combination group in HN6 and CAL27 cell
   lines (Fig. [116]3G, H). According to the results of flow cytometry,
   the CAL27 and HN6 cells were significantly blocked in G[1] phase after
   the treatment with the combination (Fig. [117]3I and Supplementary Fig.
   [118]3E). Meanwhile, the combination also promoted the apoptosis of
   CAL27 and HN6 cells (Fig. [119]3J and Supplementary Fig. [120]3F).
   These data suggest that inhibiting SQLE and EZH2 could arrest the
   tumour cells in G[1] phase and reduce the growth of tumour cells.

Fig. 3. Dual pharmacological inhibition of EZH2 and SQLE synergistically
decreases the proliferation of HNSCC.

   [121]Fig. 3
   [122]Open in a new tab

   A The combination index (CI) of EZH2 inhibitors and key enzyme
   inhibitors against cholesterol synthesis in CAL27 cells. N = 3
   replicates. B The viability of CAL27 cells was detected by an MTT assay
   after EPZ6438 incubation and silencing of the corresponding gene with
   siRNAs. C The CI value of EPZ6438 combined with NB-598 or terbinafine
   in different cells. D The Bliss model was used to calculate the
   combination effect of EPZ6438 and terbinafine. E The enzyme activity of
   SQLE in the CAL27 cell with indicated treatment. N = 3 replicates,
   one-way ANOVA analysis was used to assess the statistical significance,
   ****p < 0.0001, EPZ vs vehicle; ^###p < 0.001, TB vs vehicle;
   ^$$$$p < 0.0001, ET vs EPZ. F Representative images and quantitative
   analysis of colony formation to indicate the potent synergistic
   anti-proliferative effect of EPZ6438 and terbinafine on HN6 and CAL27
   cells. N = 3 replicates, one-way ANOVA analysis was used to assess the
   statistical significance, ***p < 0.001, ****p < 0.0001. G Real-time PCR
   detection to examine the expression of cholesterol synthesis-related
   genes in CAL27 and HN6 cell lines with different treatment. EPZ (single
   EPZ6438), TB (single terbinafine), ET (EPZ6438 + terbinafine), ATOR
   (atorvastatin), EA (EPZ6438 + atorvastatin), RO (Ro-488071), ER
   (EPZ6438 + Ro-488071), N = 3 replicates. H Immunoblotting assays to
   detect the protein levels of cholesterol synthesis-related genes in
   CAL27 and HN6 cell lines. I Flow cytometry examination to detect the
   cell cycle of CAL27 cells with different treatments for 48 h. J Flow
   cytometry examination to detect the apoptosis of CAL27 cells with
   different treatments for 72 h.

Accumulation of squalene in HNSCC cells inhibits cell growth

   Upon morphological examination, the tumour cells turned slender by the
   co-treatment with EPZ6438 and terbinafine contained many vacuoles in
   the cellular plasma (Fig. [123]4A). As indicated in the BODIPY staining
   assay, more lipid drops accumulated in the cellular plasma of
   combination group than those of other groups (Fig. [124]4A). To examine
   the possible accumulated lipids, the comparative detection of 297
   metabolites were performed with mass spectrometry. Although some lipid
   metabolites of EPZ6438 or TB-treated cells were slightly elevated, the
   content of lipid metabolites in the drug combination group was
   different from that in the control group (Fig. [125]4B). The top 10
   changed metabolites were enriched. In particular, the contents of
   1-hexadecanol, octadecanol, 9-octadecen-1-ol, phytol, squalene and
   1-heptadecanol increased after drug combination. Through joint pathway
   analysis with RNA-Seq, the steroid biosynthesis was the pathway with
   highest impact score in cells treated with the combination compared to
   the vehicle group (Fig. [126]4C). Four metabolic substances accumulated
   in the co-treatment group, including squalene, lathosterol, zymosterol
   and desmosterol (Fig. [127]4D).

Fig. 4. Double inhibition of EZH2 and SQLE promotes lipid accumulation in
HNSCC cells.

   [128]Fig. 4
   [129]Open in a new tab

   A Morphological changes and BODIPY staining of neutral lipids in HN6
   cells in different treatment groups. Cells were treated with vehicle,
   EPZ6438, terbinafine or the combination for 48 h. Bar = 100 μm. B
   Heatmap of the top 10 metabolites with significant changes among 289
   metabolites detected by GC/MS/MS in CAL27 cells after treatment for
   72 h. N ≥ 7 replicates. C The combined analysis of gene expression and
   metabolism profile. The sterol biosynthesis pathway was enriched with
   the highest score by the combination of EZH2 inhibitor and SQLE
   inhibitor. D Squalene is one of the four changed metabolites involved
   in sterol biosynthesis.

   According to the mass spectrometry analysis, the single EPZ6438
   treatment significantly decreased the cellular content of squalene,
   while the co-treatment of EPZ6438 and terbinafine accumulated squalene
   content (Fig. [130]5A). Although lanosterol and 24-dehydrocholesterol
   were also accumulated in the combination group, there was no
   significant change in the cholesterol content among the different
   groups (Fig. [131]5A). To verify the effect of metabolites on HNSCC
   cells, we supplemented the cholesterol, squalene, lanosterol and
   desmosterol to the culture medium. The number of colony formation in
   squalene-treated group was dose-dependently reduced, while other
   metabolites had no obvious effects on colony formation (Fig. [132]5B).
   After the squalene synthase FDFT1 was silenced by specific siRNAs, the
   combination of EPZ6438 and terbinafine inhibited less cell viability in
   the si-FDFT1 cells than in the si-NC cells (Fig. [133]5C). To further
   examine the role of FDFT1, the CAL27 cells were infected with sh-FDFT1
   lentivirus and established as stable cell lines (Fig. [134]5D, E). The
   proliferation of sh-FDFT1 cells was much higher than that of the sh-NC
   cells (Fig. [135]5F, G), suggesting that the production of squalene is
   negatively related to the cell proliferation.

Fig. 5. Squalene accumulation potently inhibits the proliferation of HNSCC
cells.

   [136]Fig. 5
   [137]Open in a new tab

   A Changes in four cholesterol metabolites in different treatment groups
   detected by GC/MS/MS analysis. N ≥ 7 replicates, one-way ANOVA analysis
   was used to assess the statistical significance, *p < 0.05, **p < 0.01,
   ***p < 0.001. B Colony formation of CAL27 cells was detected after the
   cholesterol metabolites were supplemented in cell culture medium. C The
   viability of CAL27 cells was detected with an MTT assay after blocking
   endogenous squalene production by knockdown of FDFT1 with specific
   siRNAs. D The real-time PCR assays to detect the protein levels of
   FDFT1 in CAL27 cells treated with sh-NC or sh-FDFT1 lentivirus. E The
   immunoblotting assays to detect the protein levels of FDFT1 in CAL27
   cells treated with sh-NC or sh-FDFT1 lentivirus. F The cell
   proliferation of CAL27 cells treated with sh-NC or sh-FDFT1 lentivirus.
   G The colony formation of CAL27 cells treated with sh-NC or sh-FDFT1
   lentivirus. H Expression of cholesterol synthesis-related enzymes in
   HNSCC cell lines and oral mucosa epithelial cells. I Expression of SQLE
   and EZH2 in HNSCC (N = 25). J There was a negative correlation between
   the expression of EZH2 and SQLE in HNSCC (N = 25). K Expression of SQLE
   in HNSCC and normal tissues in TCGA database. L, M Relationship between
   expression level of SQLE in TCGA database and survival rate and
   clinical stage.

   The expression level of SQLE in HNSCC cell lines was significantly
   higher than that in normal human oral epithelial cells (HOEC) (Fig.
   [138]5H and Supplementary Fig. [139]4). Although the mRNA expression of
   EZH2 and SQLE were highly expressed in HNSCC samples (Fig. [140]5I),
   the expression of EZH2 was negatively correlated with the expression of
   SQLE in HNSCC tissues (Fig. [141]5J). Moreover, the expression of SQLE
   in HNSCC tissues of TCGA library was significantly higher than that in
   normal tissues (Fig. [142]5K). The expression level of SQLE mRNA was
   negatively correlated with the survival of patients, but not with the
   tumour stage (Fig. [143]5L, M).

   The above results indicate that the simultaneous inhibition with EZH2
   and terbinafine cause accumulation of squalene and inhibition of tumour
   cell growth.

The combination of EZH2 inhibitor and SQLE inhibitor reduces HNSCC growth in
vivo

   Next, we established the xenograft models with CAL27 and HN6 cells to
   examine the effect of the combination in vivo. The co-treatment with
   EPZ6438 and terbinafine significantly inhibited tumour growth compared
   with the vehicle group (Fig. [144]6A–D and Supplementary Fig. [145]5A,
   B). The co-treated tumour weight was significantly lower than that of
   the vehicle group (Supplementary Fig. [146]5C). There was no
   statistical significance in the weight change of mice between the four
   groups (Supplementary Fig. [147]5D). Based on the real-time PCR
   detection in tumour tissue, EPZ6438 significantly upregulated the
   expression of cholesterol synthesis-related genes, but the combination
   increased the expressions much higher than any other groups (Fig.
   [148]6D and Supplementary Fig. [149]5E). Meanwhile, in the quantitative
   analysis of mass spectrometry, the content of squalene in the tumour
   tissues of combination group were significantly higher than that in the
   vehicle group (Fig. [150]6E). Furthermore, we detected the subcutaneous
   tumorigenesis using sh-FDFT1 and sh-NC CAL27 cells. In the tumours of
   sh-FDFT1 group, the combination of EPZ6438 and terbinafine failed to
   inhibit tumour growth, indicating that the necessity of squalene
   production (Fig. [151]6F and Supplementary Fig. [152]5F). In the images
   of tumour tissues stained by polychromatic immunofluorescence, H3K27me3
   levels were reduced by EPZ6438, while the expression of SQLE increased
   significantly upon EPZ6438, terbinafine and combination treatment in
   the identical area of tumour tissue. In addition, compared with the
   vehicle group, EPZ6438 combined with terbinafine significantly reduced
   Ki67 signal in the same area (Fig. [153]6G and Supplementary Fig.
   [154]5G). Together, these data suggest that simultaneously targeting of
   EZH2 and SQLE could significantly inhibit the tumour growth of HNSCC.
   Together, these data suggest that simultaneously targeting of EZH2 and
   SQLE could significantly inhibit the tumour growth of HNSCC (Fig.
   [155]7).

Fig. 6. Double inhibition of EZH2 and SQLE decreases HNSCC tumour growth in
vivo.

   [156]Fig. 6
   [157]Open in a new tab

   A Relative tumour (CAL27 cells) volume showed that EZH2 and SQLE
   inhibitors significantly inhibited tumour growth. N = 6 replicates,
   two-way ANOVA analysis was used to assess the statistical significance,
   ***p < 0.001, ****p < 0.0001. B Representative image of the tumour
   (CAL27 cells). C Relative tumour (HN6 cells) volume showed that EZH2
   and SQLE inhibitors significantly inhibited tumour growth. N = 6
   replicates, ***p < 0.001, ****p < 0.0001. D Representative image of the
   tumour (HN6 cells). E Detection of cholesterol metabolizing
   enzyme-related genes in tumour tissues by real-time PCR. N = 3
   replicates, one-way ANOVA analysis was used to assess the statistical
   significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs
   vehicle. F Quantitative concentrations of squalene in vehicle or ET
   combination-treated CAL27 tumours. N = 3 replicates, two-way ANOVA
   analysis was used to assess the statistical significance,
   ****p < 0.0001. G Relative tumour volume showed that combination of
   EZH2 and SQLE inhibitors failed to inhibit tumour growth of
   sh-FDFT1-treated CAL27 xenograft compared to the sh-NC group. H The
   expression levels of SQLE, H3K27me3 and Ki67 in tumour tissues of
   different treatment groups were detected by immunofluorescence
   staining.

Fig. 7. Simultaneously targeting of EZH2 and SQLE significantly inhibits the
tumour growth of HNSCC.

   Fig. 7
   [158]Open in a new tab

   A schematic illustration of the proposed model in which EZH2 inhibition
   sensitizes SQLE inhibitors to inhibit HNSCC growth.

Discussion

   EZH2 has been widely studied as an important antitumor target. In most
   solid tumours, EZH2 is present in an overexpressed wild-type form that
   bears H3K27me catalysing activity. Therefore, more research on the
   combination of EZH2 inhibitors and tumour therapy has emerged. It has
   been reported that EZH2i and BRD4i regimens or triple combinations that
   include MAPK inhibitors can be used for EZH2-positive tumour
   patients^[159]31. EZH2 inhibitors and PARP inhibitors can
   synergistically inhibit the growth of ovarian cancer^[160]32. It
   remains unclear, however, whether overexpressing EZH2 could also
   functionally result in growth dependency of endogenous cholesterol
   synthesis. Insight into this problem may expand the therapeutic
   benefits of EZH2 inhibitors to a broad spectrum of solid tumours. In
   the current study, we discovered a novel combination strategy of EZH2
   inhibitors based on cholesterol metabolism in HNSCC.

   Cholesterol is a unique lipid that is essential in the structure of
   bilayer phospholipids, so it plays an important role in biofilm
   formation, cell proliferation and cell differentiation. In addition,
   cholesterol regulates the functions of different receptors,
   participates in molecular and vesicular transport and is also the
   precursor of lipid-soluble vitamins, bile acids and steroid
   hormones^[161]11. Tumour cells support cell survival and rapid
   proliferation by reprogramming the cholesterol metabolism
   pathway^[162]33. Under normal circumstances, to maintain the
   appropriate cholesterol balance in cells, multiple pathways strictly
   control cholesterol homeostasis^[163]34. However, there are a few
   studies on the regulation of cholesterol metabolism at the epigenetic
   level of cells. Our data suggest that the transcription factor SREBF2
   and its target gene SQLE are directly regulated by EZH2-mediated
   H3K27me3 modification in HNSCC cells.

   SQLE is highly expressed in the liver, nervous tissue, digestive tract
   and skin^[164]35. High expression of SQLE and high copy number of the
   SQLE gene are closely related to poor prognosis of many
   tumours^[165]36. Like almost all genes encoding cholesterol synthase,
   SQLE is a target gene of SREBPs. SREBPs are activated when the
   concentration of sterol in cells is reduced, and the binding of SREBPs
   with the SRE sequence leads to the transcription of the SQLE
   gene^[166]37. The high copy number (CN) of the SQLE gene has been
   reported in a variety of tumours, including breast cancer, prostate
   cancer, lung cancer and pancreatic cancer^[167]38. Knockdown of SQLE or
   the use of SQLE inhibitors can inhibit the growth of breast cancer
   cells and NAFLD-HCC^[168]39, and the copy number of the SQLE gene in
   tumour cells is positively correlated with the sensitivity of SQLE
   inhibitors. Further studies should examine the relationship between
   high copy number and the efficacy of combination with EZH2i plus SQLEi.

   The EPZ6438 induced the mRNA expression of HMGCS, SQLE and LSS through
   decreasing the H3K27me3 at the promoter region of these genes as an
   epigenetic modification. We observed the single TB treatment also
   increased the SQLE mRNA. Some enzyme inhibitors could induce the mRNA
   of the enzyme through the transcript factors at the transcriptional
   regulation^[169]40. TB is the activity inhibitor of SQLE. Therefore, we
   detected the activity of SQLE, and concluded that the effect of
   terbinafine was to inhibit the enzyme activity of SQLE. When the two
   drugs are used in combination, the expression of HMGCS, SQLE and LSS is
   increased above the single agents alone, indicating that mRNA
   expressions are separately modulated by epigenetic and transcriptional
   mechanism.

   SQLE inhibitors have been extensively investigated for a long time for
   their antifungal properties^[170]41. Notably, several studies conducted
   in recent years demonstrated that the fungal SE inhibitor terbinafine
   decreased cell number and viability in various cultured human malignant
   cells in a dose-dependent manner, arresting the cell cycle at the
   G[0]/G[1] transition^[171]42–[172]44. Accordingly, we chose terbinafine
   as the agent to inhibit SQLE in HNSCC cells for potential further
   clinical research in combination with EPZ6438. The IC[50] values of
   terbinafine for mammalian SQLE are an order of magnitude higher than
   that for the fungal enzyme. Hence, during the anti-cancer treatment,
   further potent SQLE inhibitors will produce more synergistic effects
   than terbinafine. Currently, there are several NB-598 derivatives
   targeting SQLE in human cancer cells^[173]45,[174]46. Our study will
   provide a potential option for combined treatment with terbinafine and
   EPZ6438 in HNSCC patients.

   The core of the study is to examine the certain relationship between
   EZH2 and SQLE, and the combined drug treatment has produced a certain
   anti-cancer effect. The anti-cancer effect of ET combination is
   dependent on the presence of endogenous expression of FDFT1, while the
   FDFT1 is the key enzyme to produce squalene. However, still a lot of
   work should be performed in the further studies to discover the
   detailed mechanism. We still could not exclude that other potential
   pathways are involved in the anti-cancer effect of ET combination.

   In conclusion, EZH2 reprogramed the endogenous cholesterol synthesis
   via modulating H3K27me3 levels; dual inhibition of EZH2 and SQLE caused
   cellular accumulation of squalene and synergistically inhibited HNSCC
   growth. Our study may provide novel insight showing that solid tumours
   are not sensitive to EZH2 inhibitors.

Supplementary information

   [175]Supplementary Files.^ (3MB, doc)

Acknowledgements