Global crotonylome reveals hypoxia-mediated lamin A crotonylation regulated by HDAC6 in liver cancer

Abstract

Lysine crotonylation is a recently discovered post-translation modification involved in transcription regulation, cell signal transduction, and other processes. Scientists have identified several crotonylases and decrotonylases of histones, including P300/CBP, HDACs, and SIRTs. However, the regulation of non-histone protein crotonylation remains unclear. In the current study, we verified that crotonylation was upregulated in hypoxia and promoted liver cancer cell growth. We performed TMT-labeled quantitative lysine crotonylome analysis in 12 pairs of hepatocellular carcinoma and adjacent liver tissue and identified 3,793 lysine crotonylation sites in 1,428 proteins. We showed that crotonylation of lamin A at the site of K265/270 maintains its subcellular position, promotes liver cancer cell proliferation, and prevents cellular senescence. Our data indicate that HDAC6 is the decrotonylase of lamin A and downregulated in response to hypoxia, resulting in lamin A K265/270cr. Taken together, our study reveals the lamin A crotonylation in liver cancer progression and fills the research gap in non-histone protein crotonylation function.

Fig. 1. Lysine crotonylation associated with hypoxia contributes to liver cancer cell proliferation.

a, b Kcr level of liver cancer tissue (T) and adjacent liver tissue (N) was measured by Western blot with pan-Kcr. Image J was applied to quantify the blots. Beta-tubulin served as an internal reference. ***,P < 0.001. c Western blot analysis of Kcr level in PLC/PRF/5 cells treated with NaCr at indicated concentration. d CCK-8 analysis of HepG2, Huh7, PLC/PFR/5, and SK-Hep1 cells treated with 10 mM NaCr or vehicle. Data presented as the mean ± SEM. N = 3, ***, P < 0.001. e–g Subcutaneous xenograft assay of PLC/PFR/5 cells treated with 12 mmol/kg body wt NaCr or vehicle (N = 6). Data presented as the mean ± SEM of three independent experiments. **, P < 0.01; ***, P < 0.001. h–j Western blot analyzed Kcr level of PLC/PRF/5 cells treated with indicated stimulus. Cells were cultured in 1% O₂, cultured in glucose-free DMEM, or treated with 100 mM ethanol for 12 h. k HIF1α expression and Kcr level were detected with IHC in a tissue microarray. Typical image of IHC is shown in (k). l Spearman correlation analysis of IHC score was performed. P values are indicated.

Fig. 2. Identification of the proteins with Kcr in liver cancer.

a A workflow schematic illustration showing TMT-labeled LC-MS/MS analysis of Kcr proteins in hepatocellular carcinoma and adjacent liver tissue. b The number of identified Kcr sites of each protein shown in pie chart. c Motif analysis of all indicated Kcr peptide. d Amino acid preference on both sides of the crotonylated lysine. e, f Gene Ontology and KEGG analysis of identified Kcr proteins. g The number of Kcr proteins identified in PLC/PRF/5 cells treated with hypoxia and normoxia. h Intracellular pathway enrichment analysis of the identified Kcr proteins in both groups.

Fig. 3. Characterization of crotonylation of lamin A.

a Immunoprecipitation and Western blot analysis of crotonylation of lamin A with or without NaCr incubation. b, c Immunoprecipitation and Western blot analysis were performed to detect the crotonylation of endogenous lamin A. d Crotonylation of lamin A in hypoxia. e CCK-8 assays in PLC/PRF/5, sk-Hep1,PLC/PRF/5 LMNA^–/– and sk-Hep1 LMNA^–/– cells with or without NaCr incubation. Data presented as the mean ± SEM. N = 3, ns, not significant; ***, P < 0.001. f Subcutaneous xenograft assay of PLC/PRF/5 cells and PLC/PRF/5 LMNA^–/– cells treated with or without NaCr (N = 6). Data presented as the mean ± SEM. Ns, not significant; ***, P < 0.001. g Tumor weight of the subcutaneous xenograft at day 21 after inoculation. h Tumor volume of subcutaneous xenograft shown in line graph. i Schematic representation of lamin A motif and the recombinant of lamin A. j The Kcr of recombinant lamin A was analyzed by immunoprecipitation and Western blot. k 241–308 amino acids sequence of lamin A. l Immunofluorescence assay using anti-mCherry antibodies. NaCr was used at a concentration of 10 mM for in vitro assay and 12 mmol/kg body wt for in vivo assay.

Fig. 4. The crotonylation of lamin A promotes the proliferation of liver cancer cells in vivo and in vitro.

Colony formation (a, b) and CCK-8 (c) assay in PLC/PRF/5 LMNA^–/– and sk-Hep1 LMNA^–/– cells with overexpression of lamin A WT or K265/270 R mutant. Data presented as the mean ± SEM. N = 3, ***, P < 0.001. d Ki-67 expression was analyzed by flow cytometry. Data presented as the mean ± SEM. N = 3, ***, P < 0.001. e Subcutaneous xenograft assay of PLC/PRF/5 LMNA^–/– cells with overexpression of lamin A WT or K265/270 R mutant (N = 5). f Tumor weight of the subcutaneous xenograft at day 30 after inoculation (N = 5). Data presented as the mean ± SEM. **, P < 0.01. g Tumor volume of subcutaneous xenograft shown in line graph (N = 5). Data presented as the mean ± SEM. ***, P < 0.001. h HE stain of the xenograft tumor tissue. i Ki-67 expression was detected by IHC. Data presented as the mean ± SEM. N = 5, ***, P < 0.001.

Fig. 5. HDAC6 was associated with the crotonylation of Lamin A.

a Kcr level of PLC/PRF/5 cells treated with small molecular inhibitors and NaCr (10 mM) was measured by Western blot. The crotonylation of lamin A with indicated concentration of Nexturastat A (b), knock down of HDAC6 (c) or overexpression HDAC6 (d) was detected by IP followed with Western blot. e–g The exogenous and endogenous interaction of HDAC6 and lamin A was determined by co-immunoprecipitation (Co-IP). h HDAC6 expression in hypoxia and normoxia was detected by Western blot. i The crotonylation of exogenous WT or mutant lamin A in LMNA-KO PLC/PRF/5 cells with or without knock-down of HDAC6. CCK-8 assay (j) AND Ki-67 expression (k) in LMNA-KO PLC/PRF/5 cells with overexpression of WT of mutant lamin A and HDAC6. Data presented as the mean ± SEM. N = 3, ns, not significant; **, P < 0.01; ***, P < 0.001.

Fig. 6. HDAC6/lamin A complex regulated the senescence of liver cancer cells.

a PE/annexin-V staining of PLC/PRF/5 LMNA^–/– cells with the overexpression of WT or mutant lamin A. Data presented as the mean ± SEM. N = 3, ns, not significant. b SA-β-Gal staining of PLC/PRF/5 LMNA^–/– cells with the overexpression of WT or mutant lamin A. Data presented as the mean ± SEM. N = 3, **, P < 0.01. c Relative mRNA levels of p21, p16, IL-6, and IL-8 were quantified with qPCR. Data presented as the mean ± SEM. N = 3, **, P < 0.01; ***, P < 0.001. d The expression of p21 and p16 was detected by Western blot. e SA-β-Gal staining of subcutaneous xenograft tumor tissue. f SA-β-Gal staining of sk-Hep1 LMNA^–/– and PLC/PRF/5 LMNA^–/– cells with overexpression of WT or mutant lamin A and HDAC6. Data presented as the mean ± SEM. N = 3, ns, not significant; ***, P < 0.001. g The relative mRNA levels of p21, p16, IL-6, and IL-8 were quantified by qPCR. Data presented as the mean ± SEM. N = 3, ns, not significant; ***, P < 0.001. h The expression of p21 and p16 was detected with Western blot. i A hypothetical model showing that lamin A crotonylation was upregulated in hypoxia due to the downregulation of HDAC, leading to liver cancer senescence bypassing and proliferation.