Interfering with lipid metabolism through targeting CES1 sensitizes hepatocellular carcinoma for chemotherapy

Hepatocellular carcinoma (HCC) is the most common lethal form of liver cancer. Apart from surgical removal and transplantation, other treatments have not yet been well established for patients with HCC. In this study, we found that carboxylesterase 1 (CES1) is expressed at various levels in HCC. We further revealed that blockage of CES1 by pharmacological and genetical approaches leads to altered lipid profiles that are directly linked to impaired mitochondrial function. Mechanistically, lipidomic analyses indicated that lipid signaling molecules, including polyunsaturated fatty acids (PUFAs), which activate PPARα/γ, were dramatically reduced upon CES1 inhibition. As a result, the expression of SCD, a PPARα/γ target gene involved in tumor progression and chemoresistance, was significantly downregulated. Clinical analysis demonstrated a strong correlation between the protein levels of CES1 and SCD in HCC. Interference with lipid signaling by targeting the CES1-PPARα/γ-SCD axis sensitized HCC cells to cisplatin treatment. As a result, the growth of HCC xenograft tumors in NU/J mice was potently slowed by coadministration of cisplatin and CES1 inhibition. Our results, thus, suggest that CES1 is a promising therapeutic target for HCC treatment.


Generation of CES1 KO cell lines
To generate CES1 KO HepG2 cell lines, gRNA1(5'-GGCCACTCTCTCTGCTTCCG-3') and gRNA2(5'-TGCCTTTATCCTGGCCACTC-3') targeting CES1 were cloned into the BbsI site of the CRISPR/Cas9 vector (pSpCas9n(BB)-2A-Puro, Addgene #62988). Upon verification of the sequence, the plasmids were co-transfected into HepG2 cells. Thirty-six hours after transfection, the cells were treated with 7 ng/ml puromycin to select the transfected cells. Three days later, the cells were trypsinized and seeded into 100 mm 2 plates (100 cells/plate) to achieve single-cell colonies. The CES1 KO efficiency of each colony was determined by western blotting with an anti-CES1 antibody. In the end, two colonies with complete CES1 KO were selected for the study.

Transfection of plasmids by electroporation
The plasmid of RFP-CES1d was constructed as follow: the cDNA of mouse CES1d was amplified from eGFP.CES1d by PCR using Pfu DNA polymerase and ligated into the pcDNA3-mRFP (Addgene #13032) between EcoRI and NotI restriction sites. The cloned construct was verified by Sanger sequencing (Integrated DNA Technologies).
Cells were transfected by electroporation with GenePulser Xcell system (Bio-Rad) following the manufacturer's protocol. Briefly, 2 x 10 6 suspended HepG2 cells in OPTI-MEM were electroporated at 160V and 600 µF capacitance with 20 µg indicated plasmids or 10 µmol siRNA in a 0.2 mm cuvette. The transfected cells were then reseeded into well plate and cultured for 48 hours before harvest for further analysis. siPPARα (sc-36307), siPPAR (sc-29455), or scrambled siRNA were used in this study. Each biological replicate was multiple-well analysis at the same time. Meanwhile, the related experiments were repeated for three times.

Cell viability assay
The cell viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates at the density of 5,000-10,000 cells per well for 24 h followed by indicated treatments for another 48 h. After the incubation period, MTT labeling reagent was added into the wells at a final concentration of 0.5 mg/ml and incubated for 4 h. MTT solvent (4 mM HCl, 0.1% NP40 in isopropanol) was then added into each well and incubated for 15 min at room temperature. The absorbance was measured at 590 nm. Each biological replicate was multiple-well analysis at the same time.
Meanwhile, the related experiments were repeated for three times.

ROS analysis
The production of ROS was estimated with the fluorescent dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (Thermo Fisher Scientific, #D399). After treatments as indicated, the cells were detached and incubated in 10 mM cell-permeant H2DCFDA for 60 min at 37°C. Afterwards, the cells were washed 3 times with PBS to remove the free fluorescent dye and then subjected to flow cytometry analysis. Data were acquired using a BD FACS ARIA II SORP flow cytometry (BD, Biosciences) and analyzed using FlowJo software.

Apoptosis analysis
Cell apoptosis was tested by Annexin V staining. After treatments as indicated, the cells were stained with Annexin V-FITC (BD Pharmingen, #560931) for 15 min and washed with PBS to remove free dye. The stained cells were counted by flow cytometry analysis.

Mitochondrial function assays
Oxygen consumption rates (OCR) of the mitochondria in HepG2 cells were measured by a Seahorse XFe24 Analyzer (Agilent Technologies). Briefly, cells were first seeded and cultured in XFe24 cell culture plates.
Upon experiment, the medium was replaced with pre-warmed assay medium (Seahorse base medium supplemented with 1 mM pyruvate, 10 mM glucose, and 2 mM glutamine, pH = 7.4) and the plate were incubated at 37°C in a non-CO2 incubator for 1 h. Next, the plate was transferred to the Seahorse XFe24 to record the OCR of the cells at different time windows. The following compounds were subsequently injected into assay medium to assess cell mitochondrial function: 5 μM oligomycin, 1 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and a mixture of 5 μM rotenone and 15 μM antimycin A.
10 µM WWL229 was added through port A 15 min prior to the oligomycin. The key parameters of mitochondrial respiration including basal respiration, proton leak, and maximal respiration were calculated within the Seahorse Wave software. The OCR readings were normalized to the cell numbers in each well.
Fatty Acid Oxidation (FAO) was measured with a FAO assay kit from Assay Genie (Cat #: BR00001, Dublin, Ireland).

MPB.
A Thermo Fisher Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer with heated electrospray ionization source was operated in data dependent acquisition mode, in both positive and negative ionization modes, with scan ranges of 150 -827 and 825 -1500 m/z. An Orbitrap resolution of 120,000 (FWHM) was used for MS1 acquisition and a spray voltage of 3,600 and -2900 V were used for positive and negative ionization modes, respectively. Vaporizer and ion transfer tube temperatures were set at 275 and 300 °C, respectively. The sheath, auxiliary and sweep gas pressures were 35, 10, and 0 (arbitrary units), respectively. For MS 2 and MS 3 fragmentation a hybridized HCD/CID approach was used. Each sample was analyzed using four injections making use of the two aforementioned scan ranges, in both ionization modes.
Lipid data were processed and annotated using Thermo Scientific LipidSearch software (version 5.0) and analyzed using R scripts written in house.

Quantification of Free Fatty Acids (FFA)
Free fatty acids (FFAs) profiling was performed at the Metabolomics Core at MD Anderson Cancer Center, using a chemical derivatization approach. An internal standard mixture consisted of 12.5 µg/mL of (1, 2, 3, 4, 5, 6-13 C6) 22:0 and 50 µg/mL 13 C-labeled 14:0, 16:1n7c, 16:0, 17:0, 18:2n6, 18:1n9c and 18:0 in ethanol (Cambridge Isotope Laboratories, Tewksbury, MA, USA). To each cell sample in glass tube (prewashed with methanol), 32 µL of internal standard mixture, 1 µL of 10 mM butylated hydroxytoluene in methanol and 1.5 mL of ice-cold methanol were added. The tubes were vortexed for 10 min and sat on iced for 10 min. Following centrifugation at 3,500 rpm at 4°C for 10 min, the supernatants were transferred to 2 mL vials with Teflon caps and dried under nitrogen. Extracted FFAs were converted to acyl chloride intermediates by treatment with 300 µL of 2M oxalyl chloride in dichloromethane at 65°C for 5 min. The solutions were then dried and samples were derivatized by adding 225 µL of 1% (v/v) 3-picolylamine in acetonitrile. After sitting in room temperature for 5 min, the solutions were dried and derivatization products were reconstituted in 100 µL ethanol. Injection volume was 10 µL. Mobile phase A (MPA) was 0.1% formic acid in water, and mobile phase B (MPB) was 0.1% formic acid in acetonitrile. The chromatographic method included a Thermo Fisher Scientific Accucore C30 column (2.6 µm, 150 x 2.1mm) and the following gradient elution: 0-5 min, 5% MPB; 5-45 min, 95% MPB; 45-85 min, 95% MPB; 85.1-90 min, 5% MPB. Flow rate was set at 0.5 mL/min. A Thermo Fisher Scientific Orbitrap Fusion Tribrid mass spectrometer with heated electrospray ionization source was operated in data dependent acquisition mode with a scan range of 150-550m/z. Orbitrap resolutions of 120,000 (FWHM) and 30,000 for MS1 and MS2 were used, respectively. The instrument was operated in positive ionization mode with a spray voltage of 3,600 V, and vaporizer and capillary temperatures set at 350 and 325 °C, respectively. The sheath, auxiliary and sweep gas pressures were 50, 10, and 1 (arbitrary units), respectively. Ions were fragmented using assisted HCD with stepped collision energies of 25, 30, and 35%.

Western blotting
Protein samples were lysed in NENT buffer (100 mM NaCl, 20 mM Tris-Cl (pH 8.0), 0.5 mM EDTA, 0.5% (v/v) NP-40) with protease inhibitors and DTT, except that the normal human liver lysates were directly ordered from Santa Cruz Biotechnology (Santa Cruz, California). The samples were then spun at 12,000 x g for 10 minutes at 4 °C and the supernatant was collected and subsequently heated for the protein SDS PAGE analysis. Specifically, for the SCD proteins, the samples were incubated at room temperature for 10 minutes upon mixing with SDS loading buffer (without boiling). Protein samples were separated by the mini SDS gel and transferred to polyvinylidene difluoride (PVDF) membranes where they were then blocked in 5% fat free milk. Afterward, the PVDF membranes were incubated with primary antibodies at 4°C overnight or room temperature (RT) for 3 hours and then washed with PBST (0.1% Tween 20 in 1 x PBS, pH = 7.4) for 3 times (5 min/each wash). Following this, the membranes were incubated with IRDye 800 CW or 680 RD secondary antibodies (LI-COR) at RT for 1 hour. After being washed with PBST 3 times (5 min/each wash), the blots were imaged by Odyssey software (LI-COR Biosciences) and the band densities were analyzed by ImageJ software. For the Western blotting, the following primary antibodies

Quantitative PCR (qPCR)
Total RNAs from HepG2 cells were isolated using Trizol reagent (Thermo Fisher Scientific, #15596018) following the manufacturer's instructions. For qPCR, cDNAs were obtained by reverse-transcribing 1 μg of total RNAs with RevertAid Reverse Transcription Kit (Thermo Fisher Scientific, #K1691). qPCR reactions were carried out on Bio-Rad CFX96 system (Bio-Rad Laboratories). Results were normalized by β-actin and calculated using the 2-ΔΔCt method. The primers were listed in Supplementary Table 1. Each biological replicate was multiple-well analysis at the same time. Meanwhile, the related experiments were repeated for three times.

Fluorescent labeling of lipid droplets and immunofluorescence (IF) staining of the cells
To label the lipid droplets in cells, 5 µg/ml of BODIPY 493/503 (Thermo Fisher Scientific, #D3922) was added to the cell culture media and incubated for 1 hour. The cells were then washed with warm 1 x PBS 3 times and fixed by 4% paraformaldehyde for 1 h. They were then imaged using a Leica TCS SP5 Confocal Laser Scanning Microscope. For immunofluorescence staining, the cells were washed with PBS three times and fixed in 4% paraformaldehyde for 30 min. After being washed by 1 x PBST for 3 times (5 min/wash) and blocked in 5% bovine serum albumin for 1 h, the cells were then stained with indicated primary antibodies at 4°C for overnight. The cells were then washed with 1 x PBST for 3 times (5 min/wash) and incubated with indicated secondary antibodies at RT for 1 h. After being washed with 1 x PBST for 3 times (5 min/wash), the cells were mounted and images were acquired with a Leica TCS SP5 Confocal Laser Scanning Microscope.