Cell lines and cell culture
Cultured Huh7, SMMC-7721, and 293T cells were purchased from the Cell Bank of Type Culture Collection (Chinese Academy of Sciences, Beijing, China). SMMC-7721 and Huh7 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich Corporation, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), while 293T cells were maintained in high-glucose Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich) supplemented with 10% FBS. All cell lines were cultured in an incubator at 37 °C under an atmosphere of 5% CO2/95% air. Short tandem repeats of four liver-specific genes in SMMC-7721 cells and the cycle threshold values obtained by quantitative real-time polymerase chain reaction (qRT-PCR) are provided in the supplemental materials. Sorafenib-resistant (SR) cell lines were cultured in accordance with standard methods, as previously described . The resultant Huh7SR and 7721SR cells were continuously cultured in RPMI 1640 complete medium containing 2 µg/mL of sorafenib.
Drugs and chemicals
Sorafenib and orlistat were purchased from MedChemExpress (catalog nos. HY-10,201 and HY-B0218, respectively; Monmouth Junction, NJ, USA). The following inhibitors and activators (all from Shandong Topscience Biotech Co., Ltd., Rizhao, China) were used in this study: erastin (T1765) at 10 µM, RSL3 (T3646) at 1 µM, ferrostatin-1 (T6500) at 10 µM, Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone) (T6013) at 10 µM, belnacasan (T6090) at 10 µM, necrostatin-1 (T1847) at 0.5 µM, and YC-1 (T4381) at 10 µM. All required co-incubation with the indicated cells for 24 h before sorafenib treatment. Hypoxic cells were treated with 250 µM CoCl2 (Selleck Chemicals, Houston, TX, USA) for 12 h.
Antibodies against the following proteins were used for western blot analyses: SLC7A11 (ab175186 and ab216876; Abcam, Cambridge, MA, USA), hypoxia-inducible factor 1-alpha (HIF1α; 66730-1-Ig; Proteintech, Rosemont, IL, USA), FASN (A0461; ABclonal Technology, Woburn, MA, USA), and β-actin (AC026; ABclonal Technology).
Human full-length FASN, HIF1α, and SLC7A11 were used for overexpression studies with empty plasmids as controls. Small interfering RNA (siRNA) was used to knockdown expression of FASN. For short-term knockdown or overexpression, HCC and HCC-SR cells were transfected with siRNA or plasmids using Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s instructions.
Cell-counting kit-8 (CCK-8) assay
The specified cells were seeded in wells of 96-well plates at approximately 2000 cells/100 µL per well and monitored for 5 days or at approximately 5000 cells/100 µL per well, then incubated with a specific drug for 24 h. Afterward, 10 µL of CCK-8 reagent (CK04; Dojindo Laboratories Co., Ltd., Kumamoto, Japan) were added to each well and the cells were incubated for an additional 2 h. Finally, the optical density at 450 nm was measured using a microplate reader.
GSH and malondialdehyde (MDA) assays
The relative concentration of MDA in at least 1 × 107 cells was measured using the MDA assay kit (M496; Dojindo Laboratories), while the concentration of GSH was measured using the GSSG assay kit (S0053; Beyotime Institute of Biotechnology, Shanghai, China) in accordance with the manufacturers’ instructions.
Lipid peroxidation measurement
Lipid peroxidation were assessed using the fluorescent probe C11-BODIPY (581/591) (GC40165; GlpBio Technology, Montclair, CA, USA) according to the manufacturer’s protocol. Briefly, cells were treated as indicated, then collected and incubated with C11-BODIPY (581/591) at a final concentration of 2.5 µM for 30 min. The cells were then washed twice with phosphate-buffered saline (PBS) to remove residual C11-BODIPY. Lipid ROS of the cell suspensions were measured with a flow cytometer (LSR II; BD Biosciences, San Jose, CA, USA).
Human specimens and immunohistochemical (IHC) staining
Tissue microarrays of human HCC tumor tissues and adjacent tissues were obtained from the First Affiliated Hospital of Anhui Medical University under institutional approvals. Paraffin-embedded tissue samples were cut into 4 μm-thick slices, which were dewaxed and dehydrated. After antigen repair, the slices were incubated with antibodies against SLC7A11 (dilution, 1:500; TD12509; Abmart, Shanghai, China), HIF1α (dilution, 1:100; 66730-1-Ig; Proteintech), FASN (dilution, 1:100; A0461; ABclonal Technology), and Ki-67 (dilution, 1:500; GB11141; Wuhan Servicebio Technology, Wuhan, China), followed by an anti-goat secondary antibody (Wuhan Servicebio Technology). Subsequently, the tissue slices were stained with 3,3′-diaminobenzidine and hematoxylin. Images were acquired under an optical microscope.
RNA extraction and RT-qPCR
Total RNA was isolated from cells using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions and then reverse transcribed into complementary DNA using the PrimeScript™ RT kit (Takara Bio, Shiga, Japan) with Hieff® qPCR SYBR Green Master Mix (Low Rox Plus) (11202ES03; Shanghai Yeasen Biotechnology, Shanghai, China), the primers listed in Table S1, and the QuantStudio Dx PCR system (Thermo Fisher Scientific). Relative mRNA expression was calculated using the 2−ΔΔCt method and normalized against expression of β-actin as an internal control.
Dual luciferase reporter gene assay
The CDS region of SLC7A11 was searched by NCBI, and nucleotides containing the SLC7A11 promoter (-2000 to 100 of the human SLC7A11 locus) were used to predict potential transcription factor binding sites. Potential binding sites for HIF1α to the SLC7A11 promoter region were predicted from the JASPAR database (https://jaspar.genereg.net/). The entire nucleic acid sequence of the promoter of SLC7A11 was used as the wild-type (WT). Nucleotides containing the WT SLC7A11 promoter fragment or binding site truncated mutant (MUT) SLC7A11 (SLC7A11-WT, SLC7A11-MUT1, and SLC7A11-MUT2) were cloned into pGL4.10 vector by IBSBIO (Shanghai, China). HEK 293T cells were seeded into 24-well plates and then cotransfected with HIF1α plasmid, pRL TK plasmid (renilla luciferase reporter vector), and pGL4.10 vector plasmids (or SLC7A11-WT, SLC7A11-MUT1, SLC7A11-MUT2 plasmids). After 24 h, the reporter gene cell lysate was added and the luciferase and renilla luciferase activities were measured using a dual luciferase reporter gene assay (RG027; Beyotime Institute of Biotechnology).
Western blot and co-immunoprecipitation (Co-IP) analyses
Treated cells were lysed with radioimmunoprecipitation assay buffer (P0013B; Beyotime Institute of Biotechnology) supplemented with protease and phosphatase inhibitors on ice for 30 min and centrifuged at 12,000 rpm for 15 min at 4 °C. The total protein content was quantified using a bicinchoninic acid assay kit (20201ES76; Shanghai Yeasen Biotechnology) and equivalent amounts of proteins were separated by electrophoresis and then transferred using transfer buffer (WB4600; NCM Biotech, Suzhou, China) to polyvinylidene fluoride membranes, which were blocked with 5% milk in Tris-buffered saline with Tween® 20 detergent (TBST) and then incubated with primary antibodies at 4 °C overnight followed by appropriate secondary antibodies at room temperature for 1 h. Immunoreactive protein bands were visualized with an enhanced chemiluminescence system (Amersham™ Imager 600; GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) and an Odyssey® M Infrared Imaging System ( LI-COR Biosciences, Lincoln, NE, USA).
Interactions between FASN and HIF1α were detected using the Pierce™ Classic Magnetic IP/Co-IP Kit (Thermo Fisher Scientific). In brief, cells were lysed on ice with IP lysis buffer containing protease and phosphorylation inhibitors and centrifuged at 12,000 × g for 10 min at 4 °C. After discarding the supernatant, the pelleted cells were incubated with antibody-coupled beads, washed three times with TBST, and then boiled in loading buffer containing sodium dodecyl sulfate for western blot analysis.
Detection of intracellular Fe2+
Cells were inoculated in confocal dishes and incubated with 1 µM FerroOrange (F374; Dojindo Laboratories Co., Ltd.) working solution at 37 °C for 30 min. Then, intracellular Fe2+ was observed under a confocal microscope (LSM 780; Carl Zeiss AG, Jena, Germany).
Immunofluorescence (IF) assay
Treated cells were inoculated on fibronectin-coated glass coverslips in 24-well culture plates. After attachment, the cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 10 min, washed twice with phosphate-buffered saline, and fixed with 10% goat serum for 1 h. Afterward, the cells were incubated with primary antibodies at 4 °C overnight followed by the appropriate secondary antibodies at room temperature for 1 h. Meanwhile, the nuclei were stained with 4′,6-diamidino-2-phenylindole for 15 min. Finally, the cells were imaged under a confocal microscope (LSM 780; Carl Zeiss AG).
Colony formation assay
Huh7SR and 7721SR cells were seeded in the wells of six-well plates at 1000 cells/well and continuously cultured for 10–14 days. The culture medium was replaced every 3 days. After the colonies had formed, the medium was removed and the cells were fixed with 4% paraformaldehyde for 15 min and then stained with 0.1% crystalline violet. Images were obtained with a camera. Each colony consisted of at least 50 cells.
Protein half-life assay
Cycloheximide can inhibit the synthesis of new proteins and, therefore, can be used to detect the half-life of protein degradation. In brief, cells were treated with 100 µg/mL of cycloheximide for 0, 5, 30, 60, 90, or 120 min before protein collection. Protein levels were measured by western blot analysis.
Nuclear and cytoplasmic protein extraction
Nuclear and cytoplasmic proteins were extracted from cultured cells using the Nuclear and Cytoplasmic Protein Extraction kit (P0027; Beyotime Institute of Biotechnology) in accordance with the manufacturer’s instructions.
The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data analysis
HCC gene expression data and relevant clinical information were retrieved from TCGA and GEO databases. The results are presented as box or violin plots. Genetic correlations were identified with Spearman’s rank correlation test and statistical significance was assessed with the Wilcoxon test.
Function enrichment analysis
Gene Set Enrichment Analysis (GSEA) enrichment analysis was performed using normalized RNA-Seq data obtained from the TCGA database. Gene ontology terms and pathways in the Kyoto Encyclopedia of Genes and Genomes were assessed to determine the biological functions of FASN. Enrichment results with a false discovery rate (FDR) < 0.25 and adjusted p < 0.05 were considered statistically significant.
The study protocols involving animals were approved by the Animal Care and Use Committee of the Shanghai Tenth People’s Hospital. Four-week-old male nude mice were purchased from Shanghai Super-B&K Laboratory Animal (Shanghai, China) and subcutaneously injected into the right side with 1 × 107 7721SR cells. When the volume of the subcutaneous tumors reached approximately 150–250 mm3, the mice were randomly assigned to one of four designated groups, which included a negative control (NC) group (equal amount of saline, gavage, 5 days/week), sorafenib group (20 mg/kg, gavage, 5 days/week), orlistat group (240 mg/kg, gavage, 5 days/week), or combination treatment group (20 mg/kg of sorafenib + 240 mg/kg of orlistat, gavage, 5 days/week). The length and width of the tumors were measured every 3 days with calipers, and the tumor volume (V) was calculated as V (mm3) = length (mm) × width2 (mm2)/2. On day 21, the mice were euthanized and the tumors were resected and weighed.
All statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA). The two-sided unpaired t-test was used to analyze parametric data and the Mann–Whitney U-test for nonparametric data. One-way analysis of variance was performed to assess differences among multiple groups. Spearman’s rank correlation coefficient was employed to identify correlations between the expression levels of different genes. Data are presented as mean ± SD. p-values less than 0.05 were regarded as statistically significant (* p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001).