ZDHHC20 mediated S-palmitoylation of fatty acid synthase (FASN) promotes hepatocarcinogenesis

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 CCl₄ 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.

Keywords: FASN; Hepatocarcinogenesis; S-palmitoylation; ZDHHC20.

Fig. 1

Zdhhc20^−/− mice developed fewer liver tumours in chemical induced-HCC models. (A) Experimental scheme of DEN single and DEN + CCl₄-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

Fig. 2

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

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

Fig. 4

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

Fig. 5

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), NH₄Cl(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

Fig. 6

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

Fig. 7

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)

Fig. 8

Schematic diagram displaying the overall process and mechanism of this study