The Role And Mechanism Of Protein Lactylation Underlying The Progression And Targeted Therapy Resistance In Primary Liver Cancer

Background and ObjectiveIn the year 2022,the global landscape of liver cancer revealed a staggering 865,273 newly diagnosed cases and 757,906 patients succumbed to this malignancy.Liver cancer is characterized by its insidious onset and aggressive biological behavior,with a dismal fiveyear survival rate of merely 14.1%for patients.Currently,surgical resection remains the principal avenue for long-term survival for HCC patients.Nevertheless,the hypervascular nature of the liver predisposes these patients to a heightened susceptibility to recurrence and metastasis.Additionally,owing to a typically late-stage diagnosis,the majority of HCC patients are left with systemic treatment as their only recourse.Primary liver cancer(PLC)exhibits notable insensitivity to conventional radiotherapy and chemotherapy.Although molecular targeted therapy has demonstrated some advantages in the therapeutic landscape of liver cancer,its overall clinical efficacy remains suboptimal.Drug resistance plays a pivotal role in determining the efficacy of molecular targeted agents,yet the precise mechanisms underlying the resistance have yet to be fully elucidated.Hence,an intensive exploration into the molecular mechanisms underlying PLC progression and targeted therapy resistance is mandated.Such an endeavor holds significant promise for the identification of novel therapeutic targets and the enhancement of the efficacy of targeted therapy in PLC.The Warburg effect elucidates that tumor cells predominantly rely on glycolysis for energy metabolism,even under conditions of ample oxygen availability,leading to the generation of ATP concomitant with a significant accumulation of lactate.Within the tumor microenvironment(TME),lactate assumes multifaceted roles:it is not only pivotal in maintaining the acidic milieu of the TME but also implicated in immune reprogramming and the modulation of various tumor-associated biological processes,including tumor cell proliferation,stemness,invasion,and metastasis.However,the role and molecular mechanisms of lactate in conferring resistance to targeted therapy in PLC remain underexplored.Post-translational modifications(PTMs)are critical for the functional diversity of proteins.Beyond the canonical PTMs such as methylation,acetylation,phosphorylation,and ubiquitination,a spectrum of novel acyl modifications,including lactylation,have been identified in recent years.Lactylation can be broadly categorized into histone lactylation and non-histone lactylation,depending on the protein types.Histone lactylation predominantly exerts its biological functions by regulating gene transcription.The histone H3K18,one of the most common sites for lactylation,has been documented to participate in regulating the biological properties of various cancers.However,the biological functions and underlying mechanisms of lactylation at other common histone sites are yet to be fully explored.The process of histone lactylation and delactylation can be catalyzed by specific regulatory enzymes.Currently,P300 stands out as a well-recognized lactyltransferase,whereas reports on enzymes responsible for histone delactylation are considerably scarce.Elucidating the regulatory enzyme system governing histone lactylation is of paramount importance for deciphering its biological implications,meriting extensive research.Furthermore,nonhistone lactylation exhibits a wide range of complex biological effects,primarily studied in metabolic enzymes.The biological role and clinical significance of lactylation in transcription factors,however,remains enigmatic.Yes-associated protein(YAP),a transcription co-activator,can regulate gene expression through nuclear translocation and subsequently affect tumor development,stemness,metastasis,and drug resistance.Whether YAP undergoes lactylation and if such modification impacts its subcellular localization or biological functions in tumors has yet to be reported.In summary,the present study is intended to investigate the patterns and characteristics of protein lactylation in PLC,elucidating its clinical significance.We will elucidate the role and molecular mechanisms of protein lactylation underlying the progression of PLC and in the resistance to targeted therapy,from the perspectives of histone and non-histone proteins.A deeper exploration into the regulatory enzymes that modulate lactylation at key histone sites will be undertaken.This exploration aspired to identify novel targets for therapeutic intervention and is poised to contribute substantially to propel the trajectory of precision medicine for PLC.Methods1.Tissue microarrays derived from PLC patients were employed to analyze the tissue localization of pan-lysine lactylation(pan-Kla),H4K12 lactylation(H4K 121a),and YAPK90 lactylation(YAP-K901a)signals.Subsequently,their correlations with overall survival,disease recurrence rate,clinicopathological characteristics,and the efficacy of sorafenib in PLC patients were investigated.2.Hepatoma cells were treated with exogenous lactate,sodium lactate,or glycometabolic regulators,the changes in intracellular lactate levels were measured,and the regulatory effects of lactate anions on pan-Kla or H4K121a levels were assessed using Western blot analysis.3.The effects of lactate anions or RAPGEF3 on hepatoma cell proliferation,migration,cell death,and sensitivity to targeted drugs were evaluated using EdU staining,Wound healing assays,Transwell assays,and flow cytometry.4.The effect of RAPGEF3 on the growth of HCC was assessed via an in vivo experiment in nude mice bearing subcutaneous HCCLM3.5.4-Dimensional label-free quantitative proteomics combined with lactylation analysis was employed to identify histone lactylation site H4K12 and non-histone lactylation site YAP-K90 in hepatoma cells,and the patterns as well as characteristics of lactylation were explored in PLC.6.Hepatoma cells were treated with small interfering RNA(siRNA)or small molecule inhibitors,and the regulatory effects of P300 and HDAC11 on H4K121a levels were assessed using Western blot analysis.7.In HDAC11-knockdown hepatoma cell lines,the regulatory effects of H4K121a on cell migration and sensitivity to targeted drugs were analyzed using Wound healing assays.8.The cleavage under targets and tagmentation(CUT&Tag)technique integrated with the assay for transposase accessible chromatin with high throughput sequencing(ATAC-seq)was employed to screen for ADCY7/RAPGEF3/RAP1 signaling pathway regulated by H4K121a,and the effects of exogenous lactate on ADCY7 and RAPGEF3 expression levels,alongside the activity of RAP 1 were verified.9.The prognostic impact of ADCY7 expression levels on PLC patients in the TCGA database was analyzed,the correlation between ADCY7 expression levels and the sensitivity of PLC patients to sorafenib was assessed in the GDSC database and GEO datasets,and the differences in ADCY7 expression levels between parental hepatoma cells and lenvatinib-resistant hepatoma cells were compared.10.Immunofluorescence(IF)was used to analyze the effects of K90 lactylation on the subcellular localization of YAP.11.Co-Immunoprecipitation(Co-IP)was employed to analyze the effects of K90 lactylation on YAP binding to KPNA1/2/7 or CRM1.12.Molecular docking analysis was performed to examine the spatial relationship between the YAP-K90 site and CRM1.13.The effects of YAP-K901a on cell proliferation and the stemness of hepatoma cells were assessed using CCK-8 assays and spheroid formation assays.Results1.The intranuclear levels of pan-Kla,H4K121a,and YAP-K90la in PLC tissues were significantly elevated compared to normal liver tissues and were associated with poor prognosis and low response rates to sorafenib in PLC patients.Elevated levels of panKla and H4K12la were also correlated with a higher risk of recurrence in PLC patients,and H4K121ahigh could serve as an independent prognostic risk factor for PLC patients receiving sorafenib.Furthermore,elevated H4K121a levels were also associated with malignant clinicopathological features such as portal vein tumor thrombosis,satellite lesions,and incomplete tumor capsule.2.Lactate anions could regulate pan-Kla and H4K121a levels in hepatoma cells,promoting cell proliferation and migration,inhibiting cell death,and reducing sensitivity of hepatoma cells to targeted drugs.3.Upregulation of histone H4K121a levels promoted hepatoma cell migration and reduced sensitivity of hepatoma cells to targeted drugs.4.The level of H4K12la in hepatoma cells could be regulated by HDAC11 or P300.5.H4K121a regulated the transcriptional expression of ADCY7 and RAPGEF3 and exerted biological effects in PLC by activating the ADCY7/RAPGEF3/RAP1 signaling pathway.6.High level ofADCY7 was associated with poor prognosis and targeted therapy resistance in PLC patients.7.RAPGEF3 promoted the growth and migration of HCC,inhibited hepatoma cell death,and reduced the sensitivity of hepatoma cells to targeted drugs.While,inhibition of RAPGEF3 significantly enhanced the killing effect of targeted drugs on hepatoma cells.8.YAP-K901a did not affect the binding of YAP to KPNA1/2/7.9.YAP-K901a impeded the binding of YAP to CRM1,inhibiting YAP nuclear export and leading to YAP retention in the nucleus of hepatoma cells.10.YAP-K901a promoted cell proliferation and induced the stemness of hepatoma cells.ConclusionIn this study,we found that lactate anions could promote the aggressive progression of PLC and induce resistance to targeted therapy in hepatoma cells.Mechanistically,lactate anions exert biological effects by upregulating H4K121a level,which promotes the transcription of ADCY7 and RAPGEF3,leading to the further activation of the RAP1 signaling pathway.Furthermore,the level of H4K12la in hepatoma cells could be regulated by HDAC11 or P300.Clinically,elevated levels of H4K121a could serve as an independent prognostic risk factor for PLC patients receiving sorafenib treatment,and high levels of H4K121a were suggestive of sorafenib resistance in PLC.On another note,elevated levels of YAP-K901a also suggested a poor prognosis and resistance to sorafenib in PLC patients,and YAP-K901a could promote cell proliferation as well as the stemness of hepatoma cells.At the molecular level,YAP-K901a impedes the binding of YAP to CRM1,thus inhibiting the CRM1-mediated nuclear export of YAP,resulting in its retention within the nucleus of hepatoma cells,where it may act as a transcriptional co-activator to exert biological effects.The present study elucidated the underlying mechanisms of the progression and targeted therapy resistance in PLC from the perspective of a novel PTM—lactylation,which holds promise for providing new therapeutic targets in PLC and advancing the personalized medicine in the management of PLC.