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

Protein posttranslational modifications (PTMs) such as phosphorylation, acetylation, ubiquitinylation, methylation, and various short-chain lysine acylations (including crotonylation) diversify cellular functions and enable adaptation to stimuli; dysregulation can lead to carcinogenesis and metastasis. Crotonylation was first identified on histone lysines, enriched on sex chromosomes and implicated in germ cell differentiation, DNA damage and repair, stem cell self-renewal and differentiation, HIV latency, cardiac homeostasis, and carcinogenesis by affecting protein structure, stability, localization, and interactions. Kcr can occur enzymatically or non-enzymatically: elevated crotonyl-CoA increases Kcr; histone acetyltransferases (p300/CBP, MOF, PCAF) can act as crotonyltransferases, and HDAC1/2/3 and SIRT1/2/3 exhibit decrotonylase activity. However, environmental stimuli driving protein crotonylation are not fully defined. Hypoxia, a hallmark of solid tumors due to high oxygen demand, reprograms cancer cell behavior via metabolic alterations, angiogenesis, therapy resistance, and immune suppression, and may influence protein turnover and interactions. How hypoxia affects Kcr in liver cancer is unclear. This study identifies hypoxia as an inducer of crotonylation in liver cancer, positively correlated with HIF1α expression, and provides a global quantitative crotonylome in HCC vs adjacent liver. Lamin A is identified as crotonylated at K265/270, with functional significance in maintaining subcellular localization, promoting proliferation, and preventing senescence; HDAC6 is shown as its decrotonylase, downregulated by hypoxia, leading to increased lamin A K265/270 crotonylation.

Study design included discovery and functional validation. Quantitative crotonylome profiling used TMT-labeled LC-MS/MS on 12 paired hepatocellular carcinoma (HCC) and adjacent liver tissues to identify and quantify lysine crotonylation (Kcr) sites and proteins, followed by GO/KEGG enrichment and motif analyses. Additional IP-MS proteomics identified Kcr proteins in PLC/PRF/5 cells under hypoxia (1% O2, 5% CO2, 94% N2 for 12 h) versus normoxia to assess hypoxia-responsive crotonylation. Cell culture: PLC/PRF/5, HepG2, Huh7, and SK-HEP1 lines authenticated by STR were maintained in DMEM with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C, 5% CO2. Treatments included sodium crotonate (NaCr; 10 mM in vitro; 12 mmol/kg body weight in mice), glucose starvation (glucose-free DMEM), ethanol (100 mM, 12 h), and small-molecule inhibitors including the HDAC6 inhibitor Nexturastat A; various HDAC and HAT inhibitors were screened. Protein extraction used RIPA buffer with protease inhibitors; lysates were processed for SDS-PAGE and Western blotting using antibodies against pan-Kcr, β-tubulin, HIF1α, Flag, lamin A/C, Ki-67, HDAC6, 6×HIS, p21, p16, and HRP-conjugated secondary antibodies. Molecular cloning: human LMNA cloned into pcDNA3.1-3xFlag and pmCherry-C1 vectors; truncations (pre-lamin A; residues 1–385, 1–308, 1–241) generated; site-directed mutants including K→R substitutions at K260, K261, K265, K270 and double K265/270R (also K265/270Q) constructed. HDAC6 was cloned into pcDNA3.1-6xHIS. Plasmids were prepared with standard kits. Transfections used NEOFECT DNA reagent for plasmids and ABclonal HighGene for siRNA. RNAi used custom siRNA targeting HDAC6 (3'-GGACAACATGGAGGAGGACAATGTA-5'). CRISPR/Cas9: gRNAs targeting LMNA exon 2 were cloned into lentiCRISPR-v2; lentiviral particles produced in 293T cells (co-transfection with pVSVg and psPAX2), concentrated, and used to infect PLC/PRF/5 cells with polybrene (8 μg/mL); selection with puromycin, single-cell cloning, and validation by Western blotting and sequencing. Immunoprecipitation (IP) and co-IP were performed using anti-Flag, anti-lamin A/C, or anti-Kcr antibodies to assess lamin A crotonylation and interactions with HDAC6. Immunofluorescence used anti-mCherry to assess lamin A localization. Functional assays: cell proliferation measured by CCK-8 over time; colony formation assays; Ki-67 staining by flow cytometry; apoptosis by PE/Annexin-V staining; senescence by SA-β-Gal staining; qPCR for CDKN1A (p21), CDKN2A (p16), IL6, CXCL8 (IL-8); Western blot for p21 and p16. In vivo studies: female BALB/c nude mice received subcutaneous injections of 1×10^6 cells (control or genetically modified) into the right axilla; tumor volume measured as 0.5×length×width^2; endpoint tumor weight recorded; tissues processed for HE and IHC (ServiceBio). For orthotopic in situ assays, 2×10^6 cells in 50 μL PBS injected into liver lobes under isoflurane anesthesia. Mice received intraperitoneal NaCr (12 mmol/kg) or saline every 72 h as indicated. Statistical analysis: data are mean ± SEM from at least three replicates; unpaired Student’s t-test for two-group comparisons; significance at P<0.05 with * P<0.05, ** P<0.01, *** P<0.001. Ethical approvals were obtained, and data are available upon request.

• Kcr levels were higher in HCC tissues versus adjacent non-tumor liver in 10 paired samples by Western blot (P<0.001). In PLC/PRF/5 cells, NaCr dose-dependently increased global Kcr.
• NaCr (10 mM) significantly promoted proliferation of HepG2, Huh7, PLC/PRF/5, and SK-HEP1 cells in vitro (N=3, P<0.001). In vivo, NaCr (12 mmol/kg) enhanced tumor growth in subcutaneous xenografts (N=6; increased tumor volume and weight; P<0.01 to P<0.001).
• Hypoxia (1% O2, 12 h) increased Kcr in PLC/PRF/5 cells; tissue microarray IHC from liver cancer patients (n=32) showed Kcr positively correlated with HIF1α (Spearman r=0.578, P<0.001).
• TMT-labeled LC-MS/MS crotonylome (12 HCC pairs) identified 3,793 Kcr sites in 1,428 proteins; 2,229 sites in 921 proteins were quantified. 51.05% of proteins had a single Kcr site; 10.22% had >6 sites. Motif analysis revealed enriched hotspots EKXXXXXR, XXXKEXXX, and XXXEKXXX flanking Kcr lysines. GO/KEGG indicated involvement in signaling, metabolism, translation, acylation, and carcinogenesis.
• Quantitative comparison (HCC vs adjacent) with fold-change thresholds >1.5 or <0.67 showed 222 Kcr sites (114 proteins) upregulated and 94 sites (68 proteins) downregulated. Upregulated Kcr proteins enriched in metabolism and chemical carcinogenesis; downregulated associated with infection-related pathways.
• IP-MS in PLC/PRF/5 under normoxia vs hypoxia identified 59 Kcr proteins (normoxia) and 136 (hypoxia), with 48 overlapping; enriched processes included mRNA splicing, RNA metabolism, and oxidative stress response. Intersection with tissue crotonylome highlighted CAT, S100A8, EEF2, and LMNA.
• Lamin A crotonylation: NaCr increased Kcr of exogenous and endogenous lamin A; hypoxia also increased lamin A Kcr. Mapping using truncations localized Kcr to residues 241–308, containing K260, K261, K265, K270. Site mutants showed K265/270R markedly reduced lamin A crotonylation, identifying K265 and K270 as Kcr sites.
• Functional impact: In LMNA knockout cells reconstituted with lamin A WT vs K265/270R, the K265/270R mutant reduced colony formation and CCK-8 proliferation in PLC/PRF/5 and SK-HEP1 (N=3, P<0.001), decreased Ki-67, and in vivo reduced xenograft growth (N=5; lower tumor volume and weight; P<0.01 to P<0.001). K265/270R increased senescence (SA-β-Gal) and upregulated p21, p16, IL6, and IL8 at mRNA and protein levels; apoptosis was unchanged.
• HDAC6 is a lamin A decrotonylase: HDAC6 inhibitor Nexturastat A increased global and lamin A Kcr dose-dependently. HDAC6 knockdown elevated, whereas overexpression reduced, lamin A Kcr. HDAC6 and lamin A physically interacted (co-IP exogenous and endogenous). Hypoxia downregulated HDAC6 protein in PLC/PRF/5 and SK-HEP1. HDAC6 silencing specifically enhanced Kcr at lamin A K265/270. HDAC6 overexpression attenuated the proliferation and Ki-67 increases driven by lamin A Kcr.
• Mechanism model: Hypoxia downregulates HDAC6, increasing lamin A K265/270 crotonylation, maintaining lamin A localization, bypassing senescence (via reduced p21/p16), and promoting liver cancer proliferation.

This study demonstrates that hypoxia is a physiological stimulus that elevates protein crotonylation in liver cancer, correlating with HIF1α expression. A comprehensive crotonylome of HCC and adjacent liver tissues reveals thousands of Kcr sites and characteristic sequence motifs, indicating broad involvement in cellular processes. Integrating tissue crotonylome with hypoxia-responsive Kcr proteomics identifies lamin A as a critical non-histone Kcr target. Lamin A crotonylation at K265/270 maintains its appropriate subcellular positioning and promotes proliferation by suppressing senescence pathways, as evidenced by reduced p21/p16 and senescence markers when crotonylated; loss of crotonylation (K265/270R) induces senescence and suppresses growth without affecting apoptosis. Mechanistically, HDAC6 functions as a lamin A decrotonylase: pharmacologic inhibition or knockdown increases lamin A Kcr, while overexpression reduces it; HDAC6 interacts with lamin A and is downregulated under hypoxia, linking the hypoxic microenvironment to enhanced lamin A Kcr and tumor growth. These findings extend the understanding of non-histone crotonylation in cancer biology and position the HDAC6–lamin A axis as a mediator of hypoxia-driven proliferation via senescence bypass. The results suggest potential therapeutic strategies targeting HDAC6 activity or lamin A crotonylation to modulate tumor growth, while also highlighting the need to explore metabolic contributions (e.g., crotonyl-CoA flux) to hypoxia-induced crotonylation.

Protein crotonylation is upregulated in hypoxia and contributes to liver cancer progression. A global crotonylome of HCC identified 3,793 Kcr sites across 1,428 proteins. Lamin A is crotonylated at K265/270, and this modification maintains proper localization, suppresses senescence, and promotes proliferation. HDAC6 serves as a decrotonylase for lamin A and is downregulated under hypoxia, thereby increasing lamin A Kcr and driving tumor growth. Future research should identify the crotonyltransferase(s) responsible for lamin A Kcr, define downstream pathways linking lamin A crotonylation to p21/p16 regulation, determine whether other hypoxia-induced crotonylated non-histone proteins contribute to tumor progression, and elucidate how metabolic pathways and crotonyl-CoA availability under hypoxia regulate global crotonylation.

• The study focuses functionally on lamin A; it remains unclear whether other hypoxia-induced crotonylated proteins (e.g., CAT, S100A8, EEF2) also contribute to liver cancer progression.
• The crotonyltransferase(s) responsible for lamin A K265/270 crotonylation were not identified.
• The precise signaling mechanisms by which lamin A Kcr modulates p21/p16 and senescence require further elucidation.
• While hypoxia increased global crotonylation and downregulated HDAC6, the role of metabolic flux (crotonyl-CoA production) in mediating hypoxia-induced crotonylation was not directly tested.
• Generalizability beyond the studied cell lines and mouse models and across diverse HCC patient populations remains to be established.