Abstract Background Protein palmitoylation is a reversible fatty acyl modification that undertakes important functions in multiple physiological processes. Dysregulated palmitoylations are frequently associated with the formation of cancer. How palmitoyltransferases for S-palmitoylation are involved in the occurrence and development of hepatocellular carcinoma (HCC) is largely unknown. Methods Chemical carcinogen diethylnitrosamine (DEN)-induced and DEN combined CCl[4] HCC models were used in the zinc finger DHHC-type palmitoyltransferase 20 (ZDHHC20) knockout mice to investigate the role of ZDHHC20 in HCC tumourigenesis. Palmitoylation liquid chromatography-mass spectrometry analysis, acyl-biotin exchange assay, co-immunoprecipitation, ubiquitination assays, protein half-life assays and immunofluorescence microscopy were conducted to explore the downstream regulators and corresponding mechanisms of ZDHHC20 in HCC. Results Knocking out of ZDHHC20 significantly reduced hepatocarcinogenesis induced by chemical agents in the two HCC mouse models in vivo. 97 proteins with 123 cysteine sites were found to be palmitoylated in a ZDHHC20-dependent manner. Among these, fatty acid synthase (FASN) was palmitoylated at cysteines 1471 and 1881 by ZDHHC20. The genetic knockout or pharmacological inhibition of ZDHHC20, as well as the mutation of the critical cysteine sites of FASN (C1471S/C1881S) accelerated the degradation of FASN. Furthermore, ZDHHC20-mediated FASN palmitoylation competed against the ubiquitin-proteasome pathway via the E3 ubiquitin ligase complex SNX8-TRIM28. Conclusions Our findings demonstrate the critical role of ZDHHC20 in promoting hepatocarcinogenesis, and a mechanism underlying a mutual restricting mode for protein palmitoylation and ubiquitination modifications. Supplementary Information The online version contains supplementary material available at 10.1186/s12943-024-02195-5. Keywords: Hepatocarcinogenesis, S-palmitoylation, ZDHHC20, FASN Background Protein post-translational modifications (PTMs) are chemical modifications of translated protein amino acid residues to regulate protein activities, localization, folding, and interactions between proteins and other biological macromolecules. To date, hundreds of PTMs have been discovered including phosphorylation, methylation, acetylation, ubiquitination, glycosylation, and acylation. Among them, protein palmitoylation, an acylation modification, is accompanied by dynamic reversible characteristics [[38]1]. S-palmitoylation, the major type of palmitoylation, signifies the connection of 16-carbon fatty acids with cysteine sulfhydryl groups through thioester bonds, thereby affecting protein structure, localization, stability, maturation, transportation, and function to regulate membrane receptors, ion channels and other (patho)physiological processes [[39]2]. In mammalian cells, S-palmitoylation is mediated by a family of 23 zinc finger aspartate-histidine-histidine-cysteine (ZDHHC) protein acyltransferases (PATs) [[40]2, [41]3]. A growing number of studies have demonstrated that altered S-palmitoylation is closely linked to tumours [[42]4–[43]8]. Hundreds of proteins have been verified as palmitoylation substrates which are crucial driving factors involved in the development of many diseases especially neurological disorders and cancers [[44]9–[45]12]. For example, ZDHHC7 and ZDHHC21 play a vital role in the proliferation of breast cancer cells by mediating the palmitoylation of ERα [[46]13, [47]14]. The expression of ZDHHC18 and ZDHHC23 is upregulated in glioma cells and promotes glioma survival in the tumour microenvironment by mediating proto-oncogene BMI1(polycomb ring finger) palmitoylation [[48]15]. Palmitoylation catalyzed by ZDHHC5 may be associated with a poor prognosis of p53 mutation [[49]16]. Hepatocellular carcinoma (HCC) is the most aggressive and major type of primary liver cancer, with high global morbidity and case fatality rates due to its complex pathogenesis and limited diagnostic tools and treatments [[50]17, [51]18]. HCC carcinogenesis is primarily caused by repeated cycles of liver injury, repetitive inflammation, hepatic fibrosis, and compensatory hepatocyte proliferation [[52]19]. Despite recent advancements in pathophysiological mechanisms and curative treatment of HCC, the therapeutic effect remains insufficient and the overall prognosis remains unsatisfied [[53]20]. Therefore, elucidating the molecular background of hepatocarcinogenesis will provide insights into early-screening biomarkers and effective therapeutic targets for HCC. Many studies have shown that the occurrence, metastasis, and invasion of HCC are closely related to abnormal PTM activities. For instance, protein kinase C-delta (PKCδ) activates the insulin-like growth factor-1 receptor (IGF1R), and extracellular regulated protein kinases1/2 (ERK1/2) activity is enhanced to accelerate the growth of HCC [[54]21]. However, the properties of S-palmitoylation in HCC are currently unclear, and a thoroughly exploration of the key ZDHHC family and critical substrates is imperative. ZDHHC20 is a member of the ZDHHC family which have been reported to play roles in different physiological processes. In 2018, Rana et al. analyzed the three-dimensional structure of ZDHHC20 and confirmed its two active sites in the palmitoylation modification process [[55]22]. ZDHHC20 is highly expressed in multiple tumours and inhibiting its activity can achieve the purpose of cancer treatment. Research in breast and lung cancer have suggested that epidermal growth factor receptor (EGFR) palmitoylated by ZDHHC20 and that blocking EGFR palmitoylation by inhibiting ZDHHC20 may suppress KRAS-mutant primary lung adenocarcinoma formation [[56]4, [57]23]. Nevertheless, in melanoma, ZDHHC20 was found to mediate the melanoma cell adhesion molecule (MCAM) palmitoylation and to promote polarized localization and cell invasion [[58]24]. Similarly, several studies have identified many substrates of ZDHHC20, including YTHDF3, NCAM1 and VAMP7, and that their palmitoylation could promote the progression and metastatic of pancreatic cancer [[59]25, [60]26]. Feng et al. investigated the function of ZDHHC20 in the metastasis and apoptosis of liver hepatocellular carcinoma (LIHC), and put forward that ZDHHC20 exhibits significant clinical and immune relevance in LIHC [[61]27]. The integration and mutual regulation between carcinogenic signals and lipid metabolism promote the growth, survival, proliferation, migration, invasion, and metastasis of cancer cells [[62]28–[63]35]. Fatty acid synthase (FASN) is an essential enzyme for the synthesis of mammalian endogenous fatty acids in the body. The dysfunction of enzyme activity or the abnormal expression of FASN can play important roles in the survival of tumour cells, which can provide the energy and structural substances required for tumour cell survival [[64]36, [65]37]. Recent studies exploring the effects of PTMs on FASN expression and protein levels have proved that FASN is a therapeutic target for many diseases related to lipid metabolism (e.g., obesity, non-alcoholic fatty liver disease (NAFLD), and HCC) [[66]38–[67]41]. Previous studies have proved strongly that the acetyl-CoA carboxylase (ACC) /FASN axis plays a crucial role in hepatocarcinogenesis. Li et al.’s study highlighted that FASN is necessary for AKT-driven hepatocarcinogenesis [[68]42]. Hu et al. elucidated that the activation of AKT together with c-Met in accelerating HCC development through the mTORC1/FASN pathway [[69]43]. Another study indicated a significant role for ACC enzymes in terms of redox regulation and cell survival [[70]44]. According to the studies mentioned above, blocking FASN pharmacologically could become a novel and useful therapeutic strategy for HCC. Many PTMs of FASN have been identified, such as ubiquitination, SUMOylation, and acetylation. Among them, ubiquitination is an important way to regulate the level of the FASN protein. The degradation of FASN is regulated by the E3 ligase tripartite motif containing 28 (TRIM28) [[71]45]. Hu. et al. revealed that sorting nexin 8 (SNX8) mediated FASN protein degradation by recruiting TRIM28 and enhancing the TRIM28-FASN interaction, thereby, acting as a key suppressor of NAFLD [[72]45]. It follows that the signal pathways involving FASN have an irreplaceable impact on the biological processes and activities of liver diseases. Further exploration of the tumour-specific regulation of FASN could help explore new treatment opportunities of cancer. In this study, we demonstrate that ZDHHC20 knock-out in mice leading to a decrease in chemical-induced hepatocarcinogenesis. FASN is S-palmitoylated at cystine 1471 and 1881 residues by ZDHHC20, which promotes its stabilization by blocking FASN ubiquitin-proteasome-related degradation induced by the SNX8-TRIM28 complex. The disturbance of ZDHHC20-mediated palmitoylation of FASN mitigates HCC formation in mouse models, indicating that ZDHHC20 plays an essential role in the tumourigenesis and progression of HCC. These findings also indicate that ZDHHC20 may be a promising biomarker and therapeutic target for the treatment of HCC. Methods Mice All animal experiments, including plans and protocols were reviewed and approved by the Stamp of the Animal Ethical and Welfare Committee of Tianjin Medical University Cancer Institute and Hospital (LLSP2019001) and Laboratory Animal Use Management and Welfare Ethics Committee of Chongqing University Cancer Hospital (CQCH-LAE-A0000202015). All mice used in the study were maintained under specific pathogen-free conditions and housed 5 per cage with a room temperature of 25 °C and humidity of 50%±10% under 12-h light/12-h dark cycles. The light cycle began at 8 am. All mice had free access to water and food. Zdhhc20^−/− genetically engineered mice were designed by the Shanghai Model Organisms Center, Inc. (Shanghai, China) and the CRISPR/Cas9 gene-editing technique was used. The construction design strategies and genetic identification results are presented in the Supplementary Fig. [73]1. Treatment of mice For the DEN-induced HCC model, 2-week-old Zdhhc20^+/+ or Zdhhc20^−/− mice were injected with DEN (40 mg/kg) intraperitoneally once and sacrificed at 40-weeks-old [[74]46]. For the DEN + CCl[4] induced HCC model, 2-week-old Zdhhc20^+/+ or Zdhhc20^−/− mice were first injected with DEN (20 mg/kg) intraperitoneally once, and then the mice were injected with CCl[4] (5 µl/g body weight, diluted with olive oil to 20% concentration) twice a week lasting until the end of the experiment. The mice were sacrificed at 20-weeks-old (male littermates) or 22-weeks-old (female littermates) according to procedures described previously [[75]47, [76]48]. Histopathological analysis Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) were performed according to a standard protocol [[77]49]. Tissues were preserved using a 4% paraformaldehyde solution, followed by a progressive dehydration process using ethanol (70-100%). Finally, the tissues were embedded in paraffin and then cut into 4 μm-thick sections using a rotary microtome for H&E staining and oil red O staining (Sigma). The proliferation factor was assessed using IHC labeled with the Ki-67 antibody (Servicebio, [78]GB111499). Cell lines and cell culture HEK293T, Huh7, HCCLM3, Hep3B and HepG2 were obtained from the American Type Culture Collection (ATCC). All the cell lines were authenticated by (STR) profiling and tested for mycoplasma regularly. HEK293T, Huh7, HCCLM3 and Hep3B cell lines were cultured in Dulbecco’s modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) plus 100 U/ml penicillin, and 100 mg/ml streptomycin) at 37℃in an atmosphere of 5% CO[2]. HepG2 cell line was cultured in minimum essential medium with non-essential amino acid (MEM with NEAA) with 10% FBS plus 100 U/ml penicillin and 100 mg/ml streptomycin at 37℃ in an atmosphere of 5% CO[2]. Construction of plasmids and establishment of stable cell lines MIAOLING Biology (Wuhan, China) was the source of the plasmids Flag-Vector, Flag-FASN, HA-Vector, HA-FASN, Flag-ZDHHC20, HA-ZDHHC20, Myc-SNX8, His-TRIM28, and Flag-TRIM28. GENEWIZ (Suzhou, China) constructed plasmids negative control shRNA, shRNAZDHHC20-1, shRNAZDHHC20-2, and shRNAZDHHC20-3. Plasmids that encode the C1471S, C1881S, and C1471/1881S mutants of FASN were produced by mutating Flag-FASN at certain sites. For the mutagenesis, the following primers were used: for C1471S mutant, GGGAACCGCCTCCGGTCTGTGCTGCTCTCC (F) and GTTGGAGAGCAGCACAGACCGGAGGCGGTTC(R); for C1881S mutant, CATCTCCAAGACCTTCAGTCCGGCCCACAAG (F) and TGTAGCTCTTGTGGGCCGGACTGAAGGTCTTGG(R). The site-directed mutagenesis was carried out using the Mut Express II Fast Mutagenesis Kit V2 (Vazyme). Briefly, the target plasmids were amplified using Phanta Max Super-Fidelity DNA Polymerase. The amplification products were then subjected to DpnI digestion to remove the methylated template plasmid. 1 µl of DpnI was added and the mixture was incubated at 37 °C for 1–2 h. The purified digestion products were recovered afterwards. The amplified digestion products were then catalyzed by Exnase II, which enables efficient recombination of the site to be mutated and in vitro cyclization of linear DNA. The DpnI digestion product, 5 × CE II Buffer, and Exnase II were mixed well and incubated at 37 °C for 30 min, followed by immediate cooling on ice. Finally, 10 µl of the recombinant product was transformed into 100 µl of DH5α Competent Cell for transformation. DNA sequencing was used to confirm each build and mutation. As directed by the manufacturer, plasmid transfection experiments were carried out utilizing Lipofectamine 2000 (Life Technologies). HEK293T cells were transfected with lentiviral transfer plasmids (plv-FLAG-FASN, plv-FLAG-FASN C1471S, plv-FLAG-FASN C1881S, and plv-FLAG-FASN C1471/1881S), packaging plasmids pMDLg/pRRE and pRSV-Rev in a 1:1 ratio, and the envelope plasmid pMD2.G using polyethyleneimine to produce lentiviral particles. Afterwardt, the lentiviral particle-containing medium was kept in storage at 4 °C. HCC cells were infected with lentiviruses by treating them with the lentiviral particle solution. The cells were selected and passed aging under puromycin-containing medium for a period of 4–7 days. As a result, the cells that produced stable FASN expression were made available for additional research. Cell transfection Cells were seeded at a confluency of 60-70% and allowed to adhere overnight to start transient transfection. Using Lipofectamine 2000 (Invitrogen), the transfection procedure was carried out in accordance with the manufacturer’s instructions. Western blot or quantitative real-time polymerase chain reaction analysis (qRT-PCR) was used to confirm the transfection’s effectiveness. RNA isolation, reverse transcription, and qRT-PCR TRIzol Reagent (Life Technologies) was used to begin the RNA extraction procedure, following the manufacturer’s instructions. After extraction, Oligo d (T) and SuperScript II reverse transcriptase (Life Technologies) were used to assist in the reverse transcription of the RNA into cDNA. The resultant cDNA was submitted to real-time PCR using the StepOnePlus Real-Time PCR System (Applied Biosystems) with gene-specific primers and SYBR Green PCR master mix. All qRT-PCR primers can be searched in the supporting information. Sample preparation and palmitoylation liquid chromatograph-mass spectrometer (LC-MS) analysis Tissue samples were weighed, then washed with ice-cold PBS 2 times and ground into powder using liquid nitrogen. Approximately 50 mg of powder were resuspended in 200 µl lysis buffer (4% SDS, 150 mM Tris-HCl pH 8.0). Cell samples were dissolved with 200 µl lysis buffer (4% SDS, 150 mM Tris-HCl pH 8.0). All samples were sonicated for 2 min on cold water bath. Then proteins were extracted by centrifugation and determined the concentration by BCA method. Protein (3 mg) from each sample was precipitated with pre-cooled acetone and the free cysteines of proteins were blocked by mixing for 12 h at 4 °C in 5 ml total PBS containing 0.5% SDS, 1% Triton X-100 (Sigma-Aldrich), protease inhibitors (Thermo Scientific), 5 mM EDTA and 25 mM N-ethylmaleimide (NEM, Thermo Scientific). The samples were precipitated using chloroform/methanol to remove excess NEM. Proteins were resuspended in 1.5 ml of resuspension buffer (4% SDS, protease inhibitors (Thermo Scientific), 5 mM EDTA in PBS pH 7.4), and mixed with 3 ml of 1 M hydroxylamine (pH 7.4 with NaOH, Thermo Scientific), and 500 µl of 4 mM Biotin-HPDP (Thermo Scientific) in DMSO, and then incubated at 25 °C for 2 h with gentle mixing. Proteins were precipitated again and resuspended in 6 M urea and then diluted six-fold with 50mM ammonium bicarbonate, and then digested with trypsin at the enzyme-to-protein ratio of 1:50 into the sample and incubated at 37℃ for 20 h. The tryptic digested peptides were first desalted with C18 spin column (Thermo Scientific). The dried peptides were resuspended with 200 ul loading buffer (0.2% SDS, 0.2% Triton X-100 and 500 mM NaCl) and mixed with 100 µl of high-capacity streptavidin beads (Thermo Scientific) for 2 h at room temperature. Beads were washed three times with 5 ml PBS containing 0.2% SDS, 0.2% Triton X-100 and 500 mM NaCl and then collected after wash with 1 ml PBS twice. The beads were then incubated with 0.2 ml elution buffer (50 mM NH[4]HCO[3], 10 mM TCEP) for 2 h at room temperature. The eluted peptides were collected and mixed with 50 mM iodoacetamide to block reduced cysteine residues, which indicated the palmitoylation sites. Finally, the peptides were desalted with C18 Stage Tips and prepared for further LC-MS/MS analysis. Liquid chromatography-mass spectrometry (nanoLC-MS/MS) was performed on a Q Exactive HF-X (Thermo Scientific) coupled with Easy nLC 1200 system for chromatographic separation. Peptides were loaded onto a C18 column (20 cm long, 75 μm ID, 2 μm, Dr. Maisch GmbH, Ammerbuch, Germany)) in buffer A (2% ACN and 0.1% FA) and separated with a linear gradient of buffer B (90% ACN and 0.1% FA) at a flow rate of 300 nl/min over 120 min. The linear gradient was set as follows: 0–2 min, linear gradient from 2 to 5% buffer B; 2–82 min, linear gradient from 5 to 20% buffer B; 82–100 min, linear gradient from 20 to 35% buffer B; 100–112 min, linear gradient from 35 to 90% buffer B; 112–120 min, buffer B maintained at 90%. Mass spectrometry was performed on a nano electrospray ion source (nESI). The spry voltage was set as 2.1 kv. For MS data acquisition, the full MS scans were surveyed from m/z 350 to m/z 1800 at a resolution of 60,000 at m/z 200 with an AGC target values of 3e6 and a maximum injection time 50 ms. A lock mass of 445.120025 Da was used as internal standard for mass calibration. Then data-dependent top 20 MS/MS scans were applied by higher-energy collision dissociation (HCD) with normalized energy 28 at a resolution of 15,000 at m/z 200 with an AGC target values of 1e5 and a maximum injection time 50 ms. The isolation window was set to 1.6 Th and dynamic exclusion duration was 30 s. Acyl-biotin exchange assay(ABE) The ABE assay was employed to identify the palmitoylation of the FASN protein, using the previously established protocol [[79]50]. Briefly, cells were used to extract total proteins, which were then treated with NEM at a temperature of 4 °C for 1 h while being vigorously mixed. Following purification of the target protein using a particular antibody and magnetic beads, the purified protein was treated with 1 M HAM (pH 7.2) and incubated at room temperature for 1 h. The target protein was then exposed to a concentration of 5 µM biotin-BMCC at a temperature of 4 °C for a duration of 2 h. The beads and target protein complex were suspended again in 40 µl of Sodium dodecyl sulfate (SDS) sample buffer, subjected to boiling at 100 °C for 10 min, and thereafter examined using Western blot analysis. Protein extraction and Western blot analysis Protein from cells was extracted using RIPA lysis buffer (Beyotime, P0013K) accompanied with Phenylmethanesulfonyl fluoride (PMSF) and protease inhibitors on ice for 30 min after washing with ice-cold PBS three times. After ultrasonic crushing, all lysates were centrifuged at 12,000 g (4℃, 20 min). The supernatant protein concentration was examined following the manufacturer’s protocols of the BCA Protein Assay kit (Pierce, 23227). A total amount of 30–50 µg of protein in each sample was applied to the following Western blot experiments. Western blot was carried out in accordance with the standard SDS-polyacryamide gel electrophoresis (SDS-PAGE) procedures and the UltraSignal supersensitive ECL chemiluminescence substrate (4 A Biotech, 4AW011-1000). All antibodies applied are listed in the supporting information. Immunoprecipitation (IP) assay For immunoprecipitation and co-immunoprecipitation (co-IP) assays, following declared transfections and treatments, cells were washed with ice-cold PBS three times and then lysed with Western/IP lysis buffer (Beyotime, P0013J) containing PMSF and protease inhibitors on ice for 30 min. After ultrasonic crushing, all lysates were centrifuged at 12,000 g (4℃, 20 min). The supernatant protein concentration was examined f following the manufacturer’s protocols of the BCA Protein Assay kit (Pierce, 23227), and 150 µg of protein was removed as “input”. Equal mass protein lysate was added to protein A/G magnetic beads (Pierce, 26162) pre-incubated with the desired antibody. After at least 8 h of enrichment, the beads were washed with lysis buffer three times, and the total bound protein was eluted in 1 × SDS-PAGE loading buffer. The following Western blot analysis was conducted in accordance with described steps. Immunofluorescence microscopy Briefly, 60,000 cells were evenly distributed on a cover slide and incubated overnight in an environment with 5% CO2 at a temperature of 37℃. Following the declared transfections or treatments, the cells were washed with ice-cold PBS three times. The cells were subsequently treated with 4% paraformaldehyde to fix them and then permeabilized with 0.3% Triton X-100 in PBS to allow substances to pass through their membranes. Following the blocking step with 5% bovine serum albumin in PBS, the cells were subsequently exposed to primary and secondary antibodies. Finally, the nucleus of the cells was dyed with DAPI andthe sheet was sealed with ProLong Diamond anti-quenching tablets (Invitrogen, [80]P36971). The images were acquired using confocal microscopy (Leica, STELLARIS 5). Ubiquitination assays Cells at 80% confluence were transfected with the specified constructs. Forty-eight hours after transfection, the cells were exposed to 10 µM MG132 (MedChemExpress, HY-13259) for a duration of 6 h. Subsequently, the cells were lysed using RIPA buffer. Following sonication, all the lysates were subjected to centrifugation at 12,000 g (4℃, 20 min). The supernatant protein concentration was examined following the manufacturer’s protocols of the BCA Protein Assay kit (Pierce, 23227), and 150 µg of protein was removed as “input”. Equal mass protein lysate was added to protein A/G magnetic beads (Pierce, 26162) pre-incubated with the desired antibody overnight. The pull-down proteins were separated using 1 × SDS-PAGE loading buffer and prepared for immunoblotting. Protein half-life assay To conduct half-life tests, the medium was supplemented with 100 µg/ml cycloheximide (CHX) (Abmole, M4879). Subsequently, the cells were collected at certain time intervals and the protein levels were determined using immunoblot analysis. Protein-protein docking The amino acid sequences of human ZDHHC20 (ID: [81]Q5W0Z9) and FASN (ID: [82]P49327) were derived from the Uniprot database. HDOCK was used to conduct docking studies on ZDHHC0 and FASN proteins. The prepared ZDHHC20 and FASN proteins were imported into HDOCK for rigid molecular docking. Global docking was adopted, and the other parameters were kept default. The lowest energy construct was selected, and the protein-protein interaction was visualized using pymol. CCK-8 assay CCK8 assay was performed according to the instruction to assess the proliferation ability. Specifically, the corresponding cells in the logarithmic phase were counted and seeded in 96-well plates overnight (2000 cells in 200 µl medium /well). Then cells were incubated with CCK8 solution at 37℃ for 2 h at designated time points, according to the manufacturer’s instructions. Finally, the optical absorbance at 450 nm was monitored using a microplate reader in a dark environment. Colony formation assay Colony formation assay was conducted to detect cell viability. The corresponding cells in logarithmic phase were counted and seeded in 6-well plates (500 cells in 2 ml medium /well). After culturing for 10–14 days, the colonies were then fixed with methanol for 20 min, stained with 1% crystal violet for another 15 min, washed four times with water, and then dried flat at last. Colonies with more than 50 cells were regarded as surviving colonies. Statistical analysis Statistical analysis was performed using software SPSS 26.0 and GraphPad Prism 8. The results are presented as the mean ± SD (standard deviation). Unpaired two-tailed Student’s t-tests and the non-parametric Mann-Whitney test were applied to analyze the significant differences between two experimental collections. Values of different levels less than 0.05(* p < 0.05 and **p < 0.01) were all deemed statistically significant. Results ZDHHC20 is highly expressed in HCC tissues and negatively correlated with prognosis The aim of this study was to evaluate the expression profile of all known palmitoyltransferases (ZDHHC family) in HCC and identify the predominant one, as well as to examine how palmitoylation is involved in the development of HCC. We screened the ZDHHC family members by combining the mRNA and protein expression of Human Protein Atlas database (HPA database), and the prognostic correlation in HCC cases of the Cancer Genome Atlas database (TCGA database) through the Gene Expression Profiling Interactive Analysis (GEPIA) web server. By detecting the mRNA expression levels of the ZDHHC family in the HCCLM3, Hep3B and Huh7 cell lines, the top five were found to be ZDHHC6, ZDHHC4, ZDHHC20, ZDHHC21, and ZDHHC5 (Supplementary Fig. [83]1A). We also evaluated the expression of all ZDHHC enzymes in HCC based on the immunohistochemistry results from the HPA database. The ZDHHC family proteins were divided into two groups based on expression levels: weak and strong. The top five with stronger expression were ZDHHC12, ZDHHC9, ZDHHC21, ZDHHC20, and ZDHHC15 (Supplementary Fig. [84]1B). The prognostic survival analysis based on the TCGA database showed that the top five most significantly correlated with overall survival rates in HCC were ZDHHC20, ZDHHC7, ZDHHC16, ZDHHC9, and ZDHHC15 (Supplementary Fig. [85]1C-G). By cross-comparing these three groups of candidate proteins into a Venn diagram, we found that ZDHHC20 had the closest relationship with HCC (Supplementary Fig. [86]1H). We also observed a significant upregulation of ZDHHC20 mRNA in human HCC samples compared with normal liver specimens in the TCGA database (Supplementary Fig. [87]1I). Patients with higher ZDHHC20 protein levels exhibited lower disease-free survival rates (Supplementary Fig. [88]1J) and disease progression survival rates (Supplementary Fig. [89]1K). Based on these findings, we have a greater interest in investigating the regulatory role and characteristics of ZDHHC20 in HCC. The colony formation assay and CCK8 assay results suggested that ZDHHC20 overexpression profoundly increased the proliferation of HCC cells, while ZDHHC20 silencing decreased HCC cells proliferation (Supplementary Fig. [90]2A-D). In addition, stable overexpression of ZDHHC20 in HCC cells facilitated tumour growth in vivo by employing a cell-derived xenograft (CDX) model (Supplementary Fig. [91]2E-J). These data indicate that ZDHHC20 plays a key role in promoting HCC development and is an effective poor prognostic factor for HCC patients. ZDHHC20^−/− mice show reduced HCC formation induced by chemical agents Given the abnormal expression and unfavorable prognosis correlation of ZDHHC20 in HCC, we next explored the biological function of ZDHHC20 in HCC. Using the CRISPR/Cas9 technology, we genetically edited the third exon of the Zdhhc20 gene in C57BL/6J mice (deleting a 2268 bp fragment) (Supplementary Fig. [92]3A). After two generations of breeding, Zdhhc20 knockout mice were obtained (Supplementary Fig. [93]3B). Through the design of two pairs of PCR primers, genotyping was confirmed on three types of mice (Zdhhc20^+/+, Zdhhc20^+/−, Zdhhc20^−/−) (Supplementary Fig. [94]3C). Liver tissue verification for the mRNA levels was also confirmed (Supplementary Fig. [95]3D). Subsequently, we generated two mouse models of HCC using the chemical carcinogen DEN only, or in combination with CCl[4] (DEN + CCl[4]) (Fig. [96]1A). Both male and female mouse HCC models were induced in the DEN model. The liver tumour occurrence is summarized in Supplementary Fig. [97]4A. The gross appearance of the mouse livers is shown in Fig. [98]1B and F, Supplementary Fig. [99]4B, and Supplementary Fig. [100]4C. H&E staining was utilized, and the microscope revealed a disorganized histological structure. It was manifested as hepatocyte edema degeneration and necrosis to varying extents, a substantial infiltration of lymphocytes or atypical hyperplasia of hepatocytes, a partial remodeling of the hepatic lobular architecture, and certain pathologies in the peripheral lobules. The cell arrangement structure was disordered, the chromatin was obviously thickened, the cell size and shape were different, and pathological nuclear divisions can be seen (Fig. [101]1C and Supplementary Fig. [102]4D). The tumour incidence was significantly reduced in Zdhhc20^−/− mice (Fig. [103]1D and Supplementary Fig. [104]4E). Statistical analysis of the tumour nodule sizes revealed that Zdhhc20^+/+ mice developed more HCC with larger nodules than Zdhhc20^−/− mice (Fig. [105]1F). The biochemical markers of liver injury, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels in Zdhhc20^−/− mice were also significantly lower than those in Zdhhc20^+/+ mice (Supplementary Fig. [106]4G, H). The levels of inflammatory markers interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) were also lower than those of the wild-type (Supplementary Fig. [107]4I, J). These results indicate that ZDHHC20 knockout leads to reduced liver cell damage and a smaller liver inflammatory response to DEN. Fig. 1. [108]Fig. 1 [109]Open in a new tab Zdhhc20^−/− mice developed fewer liver tumours in chemical induced-HCC models. (A) Experimental scheme of DEN single and DEN + CCl[4]-induced HCC mouse models. (B, C) Representative images showing gross morphology(B) and H&E staining(C) of livers harboring DEN-induced tumours from the indicated groups. The red arrows manifested the HCC nodules. Scale bar = 200 μm–50 μm. (D, E) With(w/) and without(w/o) tumour mouse number(D) and nodule size distribution (0, ≤ 2, >2, >5 mm) (E) in DEN-induced HCC model were shown in the indicated male groups. (F-L) Representative liver images(F), H&E staining(G) and RF staining of HCC nodules, nodule mean diameter(H), maximum nodule diameter(I), liver/body weight ratio(J), nodule size distribution (≤ 2, >2, >5 mm) (K) and tumour numbers(L) in DEN + CCl4 model were shown in the indicated male groups. Scale bar = 200 μm–50 μm. DEN, diethylnitrosamine. Data are presented as mean ± SD. ns, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired two-tailed Student’s t-tests and non-parametric Mann-Whitney test were used In the DEN + CCl[4] model, which is closely related to the pathophysiological process of human HCC (Fig. [110]1A), Zdhhc20^−/− mice exhibited significantly reduced HCC development (Fig. [111]1F, Supplementary Fig. [112]5A, and Supplementary Fig. [113]5C). H&E staining and reticular fiber (RF) staining suggested a higher degree of malignancy and hyperplasia of fiber tissue in the liver of Zdhhc20^+/+ mice, as reflected in the damage or disappearance of hepatic lobular structure, increased fiber deposition around the central vein and in the junction area, infiltration of a large number of inflammatory cells and fibroblasts in the fibrous septa, varying cell sizes, edema, degeneration and necrosis of hepatocytes in various forms, large and deeply stained nuclei, frequent mitotic images, and pathological nuclear division (Fig. [114]1G, Supplementary Fig. [115]5D). Ki-67 immuno-histochemical staining demonstrated higher tumour cell proliferation activity in Zdhhc20^+/+ mice (Supplementary Fig. [116]5B). The quantitation of tumour nodules is summarized in Supplemental Tables [117]1 and [118]2. Specifically, in male mice, there were significant statistical differences in several indices, including the average nodule diameter (Fig. [119]1H), the maximum nodule diameter (Fig. [120]1I), the liver/body weight ratio (Fig. [121]1J), and the number of nodules with less than 2 mm, greater than 2 mm, and greater than 5 mm in diameter (Fig. [122]1K). The total number of nodules did not differ between Zdhhc20^−/− and Zdhhc20^+/+ mice (Fig. [123]1L). While in female mice, significant statistical differences were observed in the total number of nodules (Supplementary Fig. [124]5E), the maximum nodule diameter (Supplementary Fig. [125]5F), the liver/body weight ratio (Supplementary Fig. [126]5G), and the number of nodules with less than 2 mm, greater than 2 mm, and greater than 5 mm in diameter (Supplementary Fig. [127]5H). There was no significant difference in the average nodule diameter between Zdhhc20^−/− and Zdhhc20^+/+ mice (Supplementary Fig. [128]5I). These results indicate that knockout of ZDHHC20 leads to the alleviation of liver injury in mice, ultimately resulting in reduced HCC development. To further investigate whether ZDHHC20 knockout having effects on the early stages of HCC, we also employed an acute liver injury model that is highly relevant to the inflammatory responses in HCC. Mice were injected intraperitoneally with a high dose of 100 mg/kg DEN at 8 weeks of age and sacrificed 48 h later (Supplementary Fig. [129]4A). Significant elevations in ALT and AST levels were observed in Zdhhc20^+/+ mice compared with Zdhhc20^−/− mice (Supplementary Fig. [130]4B, C). Oil Red O staining revealed more severe damage in Zdhhc20^+/+ mice (Supplementary Fig. [131]4D), and Zdhhc20^+/+ mice exhibited significantly reduced body weight (Supplementary Fig. [132]4E). However, there was no significant difference in the liver/body weight ratio between the two groups (Supplementary Fig. [133]4F). These findings indicate that knockout of ZDHHC20 effectively protects mice from DEN-induced liver toxicity and inflammatory responses, as well as CCl[4]-induced liver injury and fibrosis accumulation. Collectively, our results demonstrate that ZDHHC20 plays a critical role in promoting chemical-induced HCC development. Proteomic analysis of palmitoylation reveals the fatty acid metabolism pathway and substrates of ZDHHC20 To identify the palmitoylated protein targets of ZDHHC20 in the progression of HCC, we performed palmitoylation liquid chromatography-mass spectrometry (LC-MS/MS) analysis on mouse HCC tissues (Fig. [134]2A). Compared with the Zdhhc20^+/+ group, the palmitoylation level of 97 proteins with 123 cysteine sites was significantly decreased in the Zdhhc20^−/− group. (Fig. [135]2B, C, Supplemental Table [136]3). The GO enrichment analysis bubble chart of the top 20 biological processes and KEGG signaling pathway enrichment analysis showed that fatty acid metabolism was significantly affected by ZDHHC20 knockout (Fig. [137]2D, E). Supplementary Fig. [138]7A illustrates a schematic diagram of the interactions and pathways involved in fatty acid metabolism. To gain insights into the role of ZDHHC20 in fatty acid metabolism, we analyzed the correlation of transcriptional expression between ZDHHC20 and fatty acid metabolism-related factors in HCC using th GEPIA database. We found a positive correlation between ZDHHC20 and SREBP1, GPAT, ACLY, ACC, LPIN1, ELOVL1, and FADS2, however, no significant correlation was found between ZDHHC20 and DGAT1, FASN mRNA expression (Supplementary Fig. [139]7B-J). Similarly, RT-qPCR analysis of the DEN + CCl[4] mouse liver tissues also revealed that knocking out of ZDHHC20 decreased the mRNA levels of Srebp1, Gpat, Acly, Acc, Lpin1, Elovl1, and Fads2,but did not affect Fasn mRNA level (Fig. [140]2F). We next examined the mRNA expression levels of the key components involved in cholesterol synthesis and efflux, fatty acid uptake, and fatty acid synthesis. Our results revealed a significant upregulation of CYP7A1, FABP-1, CD36, SREBP1, ACC1, and ACC2 upon overexpression of ZDHHC20 in HCC cells. However, no significant changes were observed in the expression of HMGCR, FATP1, and FASN (Fig. [141]2G). We also found that overexpression of ZDHHC20 led to increased levels of total cholesterol and triglycerides, which are fatty acid metabolites (Fig. [142]2H, I), and vice versa (Fig. [143]2J, K). We further explored the correlation between ZDHHC20 and lipid metabolism-related molecules at the protein level. Overexpression of ZDHHC20 increased the expression of ACC and SREBP1 (Supplementary Fig. [144]7K), and vice versa (Supplementary Fig. [145]7L). Furthermore, we carried out a metabolic mouse model by feeding mice with high-fat and high-sugar diet starting from eight-weeks-old (Supplementary Fig. [146]8A). The weight gain of Zdhhc20^−/− mice was slower than that of Zdhhc20^+/+ mice (Supplementary Fig. [147]8B). These findings support the role of ZDHHC20 in fatty acid metabolism, thereby contributing to the development of HCC. Fig. 2. [148]Fig. 2 [149]Open in a new tab Functional annotation of altered palmitoylation proteome in response to ZDHHC20 knockout. (A) Flow path of palmitoylation LC-MS analysis. (n = 5 biological replicates). (B, C) Heatmap and volcano plot of changed palmitoylation protein-sites were shown. (D, E) GO (Gene Ontology) analysis(D), KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis(E) of palmitoylation proteins in mouse HCC tissues. BP: Biological Process, CC: Cellular Component, MF: Molecular Function. (F) Relative mRNA expression levels of hepatic de novo lipogenesis pathway genes were measured in mouse HCC tissues. (G) Relative mRNA expression levels of several lipid metabolism-related genes were measured in HepG2 cells transfected with vector or Flag-tagged ZDHHC20 plasmid. (H, I) Total cholesterol(H) and triglycerides levels(I) were tested in HepG2 cells transfected with vector or Flag-tagged ZDHHC20 plasmid. (J, K) Total cholesterol(J) and triglycerides levels(K) were tested in HepG2 cells transfected with shNC or shZDHHC20 plasmid Fig. 3. [150]Fig. 3 [151]Open in a new tab Palmitoyltransferase ZDHHC20 interacts with FASN and mediates FASN palmitoylation. (A) Cell lysates of Huh7 cells were transfected with Flag-tagged FASN and HA-tagged ZDHHC20 plasmids and immunoprecipitated with Flag, HA and IgG antibodies and immunoblotted using indicated antibodies to detect protein interactions. (B) Cell lysates of Huh7 cells were transfected with Flag-ZDHHC20 plasmids and immunoprecipitated with Flag and IgG antibodies and immunoblotted using indicated antibodies to detect protein interactions. (C) FASN and Flag-tagged ZDHHC20 were stained by immunofluorescence microscopy in HCCLM3 cells. DAPI, nucleus. Scale bar = 10 μm. (D) Exogenous FASN and ZDHHC20 were stained by immunofluorescence assay in HCCLM3 and Huh7 cells. DAPI, nucleus. Scale bar = 10 μm. (E, F) The palmitoylation of endogenous FASN and Flag-tagged FASN in Huh7 cells was analyzed by ABE assay and analyzed by immunoblotting for streptavidin-HRP antibody. HAM+: with HAM, HAM-: without HAM. (G) Huh7 cells were treated with 2-bromopalmitate (2-BP) 50µM for 8 h. Cells were subjected to ABE assay. (H) The palmitoylation of endogenous FASN in Huh7 cells after transfected with shNC or shZDHHC20 plasmid was analyzed by ABE assay. (I) The palmitoylation of endogenous FASN in Huh7 cells after transfected with vector or Flag-tagged ZDHHC20 plasmid was analyzed by the ABE assay ZDHHC20 interacts with FASN and mediates FASN palmitoylation As the liver is the most important metabolic organ in the body and a crucial site for fatty acid metabolism, we hypothesized that ZDHHC20 promotes the development of HCC by palmitoylating key molecules in fatty acid metabolism. Normalization and quantitative palmitoylation LC-MS/MS analysis revealed 123 significantly downregulated palmitoylation sites (in 97 proteins) in the ZDHHC20 knockout group (Supplemental Table [152]3). Among them, FASN underwent the greatest change in palmitoylation proteins involved in fatty acid metabolism. Thus, we aimed to validate the interaction between ZDHHC20 and FASN. To achieve this goal, we co-transfected exogenous ZDHHC20 and FASN into three HCC cell lines. Co-IP assays revealed an interaction between ZDHHC20 and FASN (Fig. [153]3A and Supplementary Fig. [154]9A). Additionally, endogenous FASN was also shown to be associated with exogenous ZDHHC20 (Fig. [155]3B). Immunofluorescence experiments further showed co-localization of endogenous and/or exogenous ZDHHC20 and FASN proteins in HCC cells (Fig. [156]3C, D and Supplementary Fig. [157]9B). To determine whether FASN is palmitoylated in HCC cells, we used the IP-ABE method and demonstrated that endogenous FASN and exogenous Flag-FASN showed strong palmitoylation signals (Fig. [158]3E, F). Treatment with the broad-spectrum palmitoylation inhibitor 2-bromopalmitate (2-BP) significantly decreased the palmitoylation of FASN, thus indicating that FASN possesses S-palmitoylation through thioester bonds (Fig. [159]3G). Furthermore, IP-ABE experiments showed that knockdown of ZDHHC20 significantly reduced the palmitoylation of FASN (Fig. [160]3H), while overexpression of ZDHHC20 markedly increased the palmitoylation signal (Fig. [161]3I). These experiments provide the evidence that ZDHHC20 interacts with FASN and serves as a palmitoyltransferase for FASN. ZDHHC20 palmitoylates FASN at cysteines 1471 and 1881 The secondary mass spectrometry results showed that cysteines 1464 and C1874 of FASN in mice were palmitoylated (Fig. [162]4A, B), and that the palmitoylation of these two sites significantly decreased in Zdhhc20^−/− mice (Fig. [163]4C). We used CSS-Palm, a palmitoylation research software, and found that both sites in human and mouse FASN have a strong palmitoylation score (Fig. [164]4D). Protein-protein docking analysis revealed a direct protein-protein interaction between ZDHHC20 and FASN (Fig. [165]4E). It should be noted that C1471 and C1881 are highly conserved across different species (Fig. [166]4F). We then mutated the cysteine sites to serine (Fig. [167]4G) and found that the mutation of single or double cysteines led to a significantly reduced palmitoylation signal of FASN compared with that of the wild-type (Fig. [168]4H, I). Taken together, we prove that FASN is a palmitoylated substrate of ZDHHC20, with cysteines 1471 and 1881, serving as critical sites for palmitoylation. Fig. 4. [169]Fig. 4 [170]Open in a new tab FASN palmitoylation at cysteines 1471 and 1881 is ZDHHC20 dependent. (A, B) The changed palmitoylation sites of FASN detected by LC-MS in mouse HCC tissues. (C) The significative palmitoylation sites of FASN were identified by LC-MS. (D) The changed palmitoylation sites of FASN were verified by CSS-Palm software. (E) Protein-protein docking analysis indicated the structure of ZDHHC20 (in green) bound with FASN (in blue). (F) Alignment of the similarity of FASN sequences in different vertebrate orthologs. (G) Schematic diagram of human full-length FASN domain and cysteine mutation at position 1471 and 1881 in FASN. (H, I) Cells transfected with HA-Vector/ HA-tagged FASN/ C1471S/ C1881S/ C1471S + C1881S respectively were collected and subjected to ABE assay Palmitoylation of FASN competes with its ubiquitination modification To study the functional significance of ZDHHC20-mediated FASN palmitoylation in HCC, we first treated cells with 2-BP and found no significant change in FASNsubcellular localization. However, the fluorescence intensity of FASN decreased significantly (Supplementary Fig. [171]10A). It should be noted that 2-BP exposure significantly decreased FASN protein expression (Supplementary Fig. [172]10B, C). Conversely, the FASN protein levels were significantly increased by the depalmitoylation inhibitors ML348 or palmostatin B (Palm B) (Supplementary Fig. [173]10D, E). Furthermore, CHX experiments demonstrated that 2-BP decreased (Supplementary Fig. [174]10F), while ML348 or Palm B increased, FASN protein stability (Supplementary Fig. [175]10G). To determine the primary protein degradation pathway of FASN, we treated HCCLM3 cells with the proteasome pathway inhibitor MG132 and lysosomal pathway inhibitors chloroquine and NH[4]Cl. MG132 rescued the effect of CHX and enhanced FASN protein stability, while chloroquine and NH[4]Cl had no significant effect on CHX (Fig. [176]5A, B). These results indicate that the proteasome pathway is the primary degradation route for FASN in HCC. Upon inhibition of palmitoylation by 2-BP, we observed a significant increase in ubiquitinated degradation of endogenous and exogenous FASN when ubiquitin signals were accumulated using MG132 (Fig. [177]5C, D). These findings reflect that FASN palmitoylation positively regulates protein stability and competes with ubiquitination. We then hypothesized that this phenomenon was dependent on ZDHHC20 for FASN. We observed that overexpression of ZDHHC20 led to a significant increase in the FASN protein level detected by Western blot (Fig. [178]5E) and immunofluorescence microscopy (Fig. [179]5F). Conversely, knockdown of ZDHHC20 resulted in a marked decrease in the FASN protein level (Supplementary Fig. [180]11A). The CHX experiments confirmed that knockdown of ZDHHC20 reduced FASN protein stability, while overexpression of ZDHHC20 increased it (Fig. [181]5G, H). Correspondingly, we detected enhanced ubiquitination signals for FASN upon knockdown of ZDHHC20 (Fig. [182]5I) and reduced ubiquitination signals upon its overexpression (Supplementary Fig. [183]11B). Taken together, our results indicate that ZDHHC20-dependent FASN palmitoylation competes with its ubiquitination modification. This competition likely plays a crucial role in FASN stability and function in HCC. Fig. 5. [184]Fig. 5 [185]Open in a new tab Palmitoylation of FASN competes with its ubiquitination modification. (A, B) Protein levels of Flag-tagged FASN were measured in the presence of cycloheximide (CHX,100 µg/ml), chloroquine (CQ,20µM), NH[4]Cl(10µM) or MG132(10µM) for 6 h as indicated. (C, D) Endogenous FASN or Flag-tagged FASN ubiquitination levels were measured in Huh7 cells after treatment with or without 2-bromopalmitate (2-BP) 50µM for 24 h followed by MG132(10µM) treatment for 8 h. (E) FASN protein levels were detected in Huh7 cells transfected with vector or Flag-tagged ZDHHC20 plasmid. (F) FASN and Flag were stained by immunofluorescence assay in Huh7 cells transfected with vector or Flag-tagged ZDHHC20 plasmid. DAPI, nucleus. Scale bar = 10 μm. (G-H) Endogenous FASN or Flag-tagged FASN ubiquitination levels protein levels with CHX (100 µg/ml) treatment in indicated groups. (I) Flag-tagged FASN ubiquitination levels were measured in HCCLM3 cells after transfected with the shZDHHC20 plasmid ZDHHC20-mediated FASN palmitoylation attenuates FASN degradation by competing with SNX8-TRIM28 A previous study reported that the SNX8-TRIM28 complex interacted with FASN and regulated its expression in HCC [[186]45]. To further elucidate the competitive mechanism between the two PTMs (protein palmitoylation and ubiquitination) of FASN, we hypothesized that ZDHHC20-mediated palmitoylation interferes with the interaction between FASN and TRIM28, resulting in the enhanced stability of FASN. To test this hypothesis, we co-expressed Myc-tagged SNX8 and Flag-tagged FASN into Huh7 cells in the presence or absence of HA-tagged ZDHHC20. FASN protein levels decreased when Myc-tagged SNX8 was expressed. However, in the presence of HA-tagged ZDHHC20, the suppressive effect of SNX8 on FASN was reduced, as shown by the returned level of Flag-tagged FASN (Fig. [187]6A). The same is true for His-tagged TRIM28 expression, as HA-tagged ZDHHC20 can diminish the TRIM28-induced inhibition of Flag-tagged FASN expression (Fig. [188]6B). Co-IP experiments showed that the presence of ZDHHC20 attenuated the interaction between SNX8 (Fig. [189]6C) or TRIM28 (Fig. [190]6D) with FASN. In addition, 2-BP enhanced the degradation effect of TRIM28 or TRIM28 on FASN (Fig. [191]6E, F), while Palm B had the opposite effect (Supplementary Fig. [192]12A, B). Collectively, these results indicate that ZDHHC20-mediated FASN palmitoylation is competitive to its ubiquitination mediated by the SNX8-TRIM28 complex (Fig. [193]6G). Fig. 6. [194]Fig. 6 [195]Open in a new tab ZDHHC20 increases FASN protein stability by competing with the interaction between FASN and SNX8-TRIM28 complex. (A) The protein levels of Flag-tagged FASN in Huh7 cells transected with Myc-tagged SNX8 in the presence and absence of HA-tagged ZDHHC20 were analysed by Western blot. (B) The protein levels of Flag-tagged FASN in Huh7 cells transected with His-tagged TRIM28 in the presence and absence of HA-tagged ZDHHC20 were analysed by Western blot. (C) The interaction levels of SNX8 and FASN in Huh7 cells in the presence and absence of ZDHHC20 followed by MG132(10µM) treatment for 8 h were evaluated by Co-IP assay. (D) The interaction levels of TRIM28 and FASN in Huh7 cells in the presence or absence of ZDHHC20 followed by MG132(10µM) treatment for 8 h were evaluated by Co-IP assay. (E, F) FASN ubiquitination levels were measured in HCCLM3 cells that stably express HA-vector, or HA-tagged FASN plasmid after transfected with Flag-tagged TRIM28 (E) or Myc-tagged SNX8 (F) and treatment with or without 2BP 50µM for 24 h followed by MG132(10µM) treatment for 8 h. (G) Schematic diagram showed that FASN-TRIM28-SNX8 interaction competes with FASN-ZDHHC20 interaction FASN cysteine 1471/1881 palmitoylation increases protein stability To further investigate the functional role of FASN palmitoylation in C1471/1881, we expressed Flag-tagged C1471S, C1881S and C1471/1881S plasmids into HCC cells. The CHX experiment indicated that mutating the cysteines led to decreased protein stability compared with the wild-type protein (Fig. [196]7A). Immunofluorescence microscopy further confirmed these findings, showing a marked decrease in the FASN fluorescence signal in the mutant forms compared with the wild-type (Fig. [197]7B). Consistently, the ubiquitination of FASN significantly increased in the mutant forms, particularly in the double mutant (Fig. [198]7C). Based on the positive effect of ZDHHC20 on HCC progression and regulation of ZDHHC20 on palmitoylation of FASN, we then explored whether the ZDHHC20-FASN axis plays a key role in this process. To this end, we performed the CCK8 assay and clone formation assay of HCC cells expressing HA-tagged FASN WT, the C1471S mutant, the C1881S mutant and the C1471S + C1881S mutant, and found decreased proliferation of HCC cells after mutant the FASN palmitoylation sites (Fig. [199]7D-E). We also found that a stable overexpression of mutant FASN in HCC cells exhibited lesser bearing tumour growth than FASN WT in vivo by employing a cell-derived xenograft (CDX) model (Supplementary Fig. [200]13A-C). Additionally, co-IP experiments revealed that mutating the palmitoylation sites increased the interaction between FASN and TRIM28 compared with the wild-type (Supplementary Fig. [201]13D, E). These results provide further evidence that ZDHHC20-mediated palmitoylation of FASN in C1471 and C1881 competes with SNX8-TRIM28 complex-mediated ubiquitination. Moreover, we verified the protein expression levels of ZDHHC20 and FASN in 10 HCC tissues and paired normal tissues adjacent to the tumour (NATs) collected from our hospital. Our data exhibited higher protein levels of ZDHHC20 and FASN in HCC tissues than in NATs (Supplementary Fig. [202]14). Fig. 7. [203]Fig. 7 [204]Open in a new tab Blocking FASN cysteine 1471/1881 palmitoylation accelerates FASN degradation. (A) FASN protein levels with cycloheximide (CHX, 100 µg/ml) treatment after transfected with Flag-tagged FASN/ C1471S/ C1881S/ C1471S + C1881S plasmid in Huh7 cells. (B) HA were stained by immunofluorescence assay in HCCLM3 cells that stably express HA-Vector/ HA-tagged FASN/ C1471S/ C1881S/ C1471S + C1881S. DAPI, nucleus. Scale bar = 10 μm. (C) FASN ubiqutination levels were measured in HCCLM3 cells that stably express HA-Vector/ HA-tagged FASN/ C1471S/ C1881S/ C1471S + C1881S plasmid followed by MG132(10µM) treatment for 8 h. (D, E) HCCLM3 cells infected with lentivirus vectors expressing HA-tagged FASN/ C1471S/ C1881S/ C1471S + C1881S were harvested for CCK-8 assay n = 3 biologically independent experiments, two-way ANOVA (D); colony formation assay, n = 3 biologically independent experiments, one-way ANOVA(E) Discussion Intracellular fatty acids are used as building blocks to produce membrane lipids, signal molecules, and modify groups [[205]29, [206]30]. They also serve as an alternative source of energy for tumour growth. In recent years, significant advancements have been made in our understanding of palmitoylation and its potential effects on fatty-acid-associated diseases. However, the specific molecular processes by which palmitoylation contributes to hepatocarcinogenesis have not yet been fully characterized. In our study, we demonstrate that the ZDHHC20-dependent palmitoylation of FASN results in the inhibition of FASN ubiquitination. When ZDHHC20 is absent, the production of lipids and the development of liver cancer are significantly reduced in both in vitro and in vivo models (schematic in Fig. [207]8). These findings reveal a novel mechanism mediated by ZDHHC20 that plays a critical role in HCC carcinogenesis. Fig. 8. [208]Fig. 8 [209]Open in a new tab Schematic diagram displaying the overall process and mechanism of this study Mechanistically, ZDHHC20-mediated FASN palmitoylation in C1471 and C1881 competes with its interaction with the SNX8-TRIM28 complex. This mutual crosstalk is critical for FASN’s stability in HCC carcinogenesis. Inhibiting FASN’s palmitoylation alteration enhances the interaction between FASN and SNX8-TRIM28, offering insight into how palmitoylation regulates FASN expression. Our findings indicate that characterizing novel regulating molecules for FASN expression may reveal novel processes underlying disease etiology. Furthermore, understanding the dynamic balance between ZDHHC20-mediated FASN palmitoylation and the SNX8-TRIM28 complex interaction may provide a basis for developing strategies to disrupt the oncogenic signaling pathways in HCC and improve patient prognosis. FASN is involved in the S-palmitoylation of key proteins such as GSDMD in macrophages, endothelial nitric-oxide synthase (eNOS) in endothelial cells, and MYD88 in neutrophils [[210]51, [211]52]. FASN catalyzes palmitate production and is therefore necessary for protein palmitoylation. Growing evidence has shown that interactions between FASN and palmitoylated substrates are necessary for protein palmitoylation. Our findings indicate that FASN can also be palmitoylated. However, it is still unclear whether protein palmitoylation involving FASN requires itself to undergo palmitoylation. The palmitoylation-based proteomics analysis in this study revealed a list of palmitoylated proteins that were reduced in ZDHHC20 knockout mice. We discovered possible palmitoylation substrates for ZDHHC20 and identified potential palmitoylation sites. Two palmitoylation sites (C1471 and C1881) of FASN were validated in various experiments. However, it is not known which site of FASN palmitoylation is more important for HCC. In-depth research is needed to determine whether ZDHHC20 palmitoylates both sites concurrently or as part of a cascade process. Recent studies have shown that ZDHHC20 is highly expressed in breast cancer, lung cancer, and pancreatic cancer; therefore, blocking its activity may help with cancer treatment [[212]25, [213]53, [214]54]. Besides the influence on the oncology field, more and more studies have confirmed ZDHHC20’s richer and diverse character in other fields such as immunity, inflammation, virus infection, and cell behavior. Chopard et al. revealed that ZDHHC20 palmitoylated HIV-Tat and consequently increased Tat affinity for PI (4,5) P2 in uninfected cells [[215]55]. Another study showed that the fusogenic activity of Spike depends on its palmitoylation which is regulated by ZDHHC20 and could easy for SARS-CoV-2 infection [[216]56]. Besides, ZDHHC20 strengthens the palmitoylation of interferon-induced transmembrane protein3 (IFITM3) and then plays an antiviral role [[217]57]. A study carried out by Lu et al. demonstrated the S-palmitoylation of CD80 mediated by ZDHHC20 as a novel mechanism for its regulation in T cell activation [[218]58]. Remarkably, Bu et al. eclosed the regulation of AKT by PA-ZDHHC17/24 mediated palmitoylation and the activization of AKT in a PIP3- independent manner, leading to NASH and liver tumourigenesis [[219]59]. This study implies us a novel HCC therapeutic strategy for target the PA-ZDHHC-AKT axis by taking PA-restricted diets, limiting PA synthesis, or directly targeting AKT palmitoylation [[220]59]. More structural insights and mechanistic details provided by these studies have helped us understand how ZDHHC20 recognizes and modifies its substrates. We assume that the interesting and diverse palmitoylation-associated cell behaviors may be related to various substrates profiles of palmitoyltransferases under different tissue environment. Our current data prove the carcinogenic effect of ZDHHC20 in HCC as well as its positive regulatory role in fatty acid metabolism, indicating that the ZDHHC20-FASN axis could be an anti-tumour target. Nevertheless, our results demonstrate that ZDHHC20 plays an important role in hepatocarcinogenesis through FASN stabilization and could be a therapeutic intervention for HCC. Conclusion Taken together, we demonstrate the role of ZDHHC20 in promoting hepatocarcinogenesis and identify that FASN is palmitoylated by ZDHHC20 at specific cysteine sites (1471 and 1881). Furthermore, we reveal that FASN palmitoylation mediated by ZDHHC20 competes with the ubiquitin-proteasome pathway through the E3 ubiquitin ligase complex SNX8-TRIM28. Our study provides a significant step toward understanding HCC pathogenesis and pinpoints the potential targets for HCC precise molecular therapy. Electronic supplementary material Below is the link to the electronic supplementary material. [221]Supplementary Material 1^ (9.8KB, xlsx) [222]Supplementary Material 2^ (9.7KB, xlsx) [223]Supplementary Material 3^ (883.7KB, pdf) [224]Supplementary Material 4^ (3.2MB, xlsx) [225]Supplementary Material 5^ (2MB, pdf) Author contributions Bo Xu and Mingming Xiao contributed to the conception of the study; Yaqi Mo, Yamei Han, Qing Li, Yang Chen, and Zhuang Liu performed the experiments; Yaqi Mo, Yamei Han, Bo Xu, and Mingming Xiao performed the data analyses and wrote the manuscript; Bo Xu and Mingming Xiao helped perform the analysis with constructive discussions. All authors read and approved the final manuscript. Funding This work was supported by grants from the National Natural Science Foundation of China [81974464, 82272754 and 82003237] and Chongqing Technology Innovation and Application Development Special Project [CSTB2023TIAD-KPX0050]. Data availability No datasets were generated or analysed during the current study. Declarations Ethical approval All animal experiments including plans and protocols were reviewed and approved by the Stamp of The Animal Ethical and Welfare Committee of Tianjin Medical University Cancer Institute and Hospital (LLSP2019001). and Laboratory Animal Use Management and Welfare Ethics Committee of Chongqing University Cancer Hospital (CQCH-LAE-A0000202015). Human HCC samples used in this study was approved by the Chongqing University Cancer Hospital of Medicine Institutional Review Board. Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Yaqi Mo and Yamei Han contributed equally to this work. Contributor Information Mingming Xiao, Email: xiaomingming@fudanpci.org. Bo Xu, Email: xubo731@cqu.edu.cn. References