ABSTRACT
-
Background/Aims
Cholangiocarcinoma (CCA) is a primary malignant neoplasm with an extremely poor prognosis. While combined chemoradiotherapy has been demonstrated to delay CCA progression to a certain extent, the absence of specific molecular biomarkers or targets significantly hinders the diagnosis and treatment of CCA.
-
Methods
Through cross-analysis of proteomics and ADMA modificationomics, we identified DDX1 overexpressed in CCA with elevated R602-ADMA modifications. HPLC-MS/MS identified PRMT1 as the methyltransferase and USP10 as the deubiquitinating enzyme for DDX1. Immunofluorescence and nuclear-cytoplasmic partitioning experiments confirmed DDX1’s nuclear localization. GO and KEGG analyses clarify the biological functions of DDX1 in response to hypoxia. RNA-seq transcriptomics analyzed key pathways influenced by DDX1. A hydrodynamic in situ CCA mouse model was established to validate the chemopreventive effects of the PRMT1-specific inhibitor GSK715 on CCA development.
-
Results
DDX1 promotes CCA progression both in vivo and in vitro and can be inhibited by GSK715. Mechanistically, PRMT1 mediates ADMA modification at position R602 of DDX1. This modification promotes DDX1 nuclear localization by recruiting USP10 to deubiquitinate DDX1, while simultaneously inhibiting PRMT1 degradation. DDX1 promotes the transcription of PRMT1 and USP10 by binding to the mRNA 3’UTR region, establishing a positive feedback regulatory pathway. This mechanism promotes the occurrence and development of CCA and can serve as a target for the inhibitor GSK715 to suppress CCA progression.
-
Conclusions
Our study identified DDX1-R602-ADMA modification as a novel ADMA modification in CCA. It further confirmed its pivotal role in CCA progression. Targeting the USP10-PRMT1-DDX1 axis may represent a significant therapeutic approach for CCA.
-
Keywords: Cholangiocarcinoma; Arginine asymmetric dimethylation; Ubiquitination; PRMT1; DDX1
Study Highlights
• We identified PRMT1 as the methyltransferase for DDX1 R602 in CCA, whose modification is enhanced under hypoxia.
• The USP10-PRMT1-DDX1 feedback loop is formed via ADMA modification-mediated deubiquitination and nuclear translocation.
• GSK3368715 inhibits CCA progression in a DDX1 R602-ADMA–dependent manner, supporting its therapeutic potential.
Graphical Abstract
INTRODUCTION
Cholangiocarcinoma (CCA) is a malignant neoplasm originating from the epithelial cells of the biliary system [
1]. Despite the presence of genetic heterogeneity, the common features of bile duct invasion and multipolar metastasis establish the biological commonality of the tumors [
2]. On a global scale, the morbidity and mortality rates of CCA are increasing annually [
3], and specific molecular markers or targets are absent for the diagnosis and treatment of CCA [
4]. Research has demonstrated a correlation between aberrant post-translational modifications (PTMs) of proteins and the development and progression of CCA [
5].
PTMs of non-histone proteins [
6,
7] play a critical role in various cellular processes [
8,
9]. Dysregulation of PTMs has been demonstrated to be a contributing factor to the development of cancer [
10,
11]. Recently, arginine methylation of non-histone proteins has been identified as a prevalent PTM catalyzed by arginine methyltransferases [
12,
13] and classified as monomethylation (MMA), symmetric dimethylation (SDMA), and asymmetric dimethylation (ADMA) [
14,
15]. PRMT1 mediates over 75% of protein ADMA modifications, exhibiting distinct catalytic activities and substrate specificities across different diseases. GSK3368715 (GSK715) is a potent, reversible, S-adenosylmethionine (SAM)-noncompetitive inhibitor that binds to the protein substrate-binding pocket of PRMT1 [
16]. Methylation frequently impacts the binding of ubiquitin ligases or deubiquitinating enzymes to the substrate, thereby functioning as a regulatory “on/off switch” that governs the ubiquitination of the substrate and affects a variety of biological functions [
17,
18]. The intricate regulatory network that PTMs in tumor cells form makes elucidating the specific mechanisms of interactions between different PTMs targeting the same substrate a key scientific issue for PTM therapies in tumors [
19-
21].
DDX1 is a DEAD-box RNA helicase and a key member of the RNA-binding protein (RBP) family [
22]. As a crucial regulator of RNA metabolism [
23], DDX1 plays a vital role in controlling transcription and translation initiation [
24], as well as providing genomic protection in various solid tumours [
25]. Although DDX1 is implicated in the pathogenesis of multiple cancers [
26], research on its role in CCA development remains limited to date.
This study provides a comprehensive elucidation of the mechanism by which ADMA of the R602 site on the DDX1 protein, facilitated by PRMT1 in CCA, promotes transcription of both PRMT1 and USP10 by recruiting USP10 to facilitate DDX1 nuclear translocation. USP10 enhances DDX1’s R602-ADMA modification by stabilizing the PRMT1 protein, thereby establishing a dual positive feedback regulation mechanism that amplifies the stem-like characteristics of CCA cells during cancer progression. According to data-independent acquisition (DIA) quantitative proteomics and ADMA modification profiling analyses, DDX1 expression is elevated in CCA tissues, and ADMA modification at the R602 site increases under hypoxic conditions. High-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) results indicate that DDX1’s R602-ADMA modification is mediated by PRMT1 and recruits USP10. RNA sequencing and enrichment analysis findings reveal that DDX1 activates stemness pathways in CCA cells. Notably, both in vivo and in vitro experiments have demonstrated that the PRMT1-specific inhibitor GSK3368715 suppresses DDX1 nuclear translocation by inhibiting R602-ADMA modification of DDX1. Consequently, this results in the attenuation of stemness characteristics in CCA cells and the subsequent inhibition of CCA progression. This therapeutic approach may offer a viable strategy for treating CCA.
MATERIALS AND METHODS
Patient samples
Human CCA and paracancerous samples were obtained from the Department of Biliopancreatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All studies related to clinical samples and data followed the principles of the Declaration of Helsinki and were approved by the Ethics Committee of Tongji Hospital (Approval No. SHYJS-CP-2001002). Patient details are presented in
Supplementary Table 1.
C57BL/6 mice and BALB/c nude mice were purchased from Spearfish Biotechnology Co. (Beijing, China). The Laboratory Animal Welfare and Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China, approved the animal studies (Approval No. TJH-202306014).
Mouse in situ CCA model
An in situ mouse CCA model was established using hydrodynamic tail vein injection and the Sleeping Beauty transposon [
27].
Subcutaneous xenograft tumor model
Six-week-old female BALB/c nude mice from Spearfish Biotechnology Co. were used. Cells were counted in advance according to the experimental grouping, 2×106 per mouse, and cells were resuspended in PBS.
Liver metastasis model
Six-week-old female BALB/c nude mice from Spearfish Biotechnology Co. were used. Cells were counted in advance according to the experimental grouping, 2×105 per mouse, and cells were resuspended in PBS. The following operations were performed in the operating room of the Laboratory Animal Center, and all instruments were sterilized. Ligation was performed in the center of the spleen, and the spleen was divided into proximal and distal ends with vascular tips. And 100 μL of cell suspension containing 2×105 cells was slowly injected into the proximal spleen. The mice were observed every two days, and their body weights were measured. When no weight gain was observed and the mice were depressed, the mice were executed, and the livers were fixed in 4% formaldehyde.
Plasmid construction, transfection, and lentivirus production
The total cDNA of CCA cells was used as the template, and the cDNA of DDX1, USP10, and PRMT1 was obtained by PCR. The polymerase used for PCR was 2×Phanta Flash Master Mix (P510, Vazyme, Nanjing, China).
CRISPR cas9-mediated gene knockout
To construct DDX1 as well as PRMT1 knockout cell lines, we designed single guide RNA (sgRNA) targeting human DDX1 or PRMT1 and ligated it into the Px459 vector.
Dual luciferase reporter assay
The 3’UTR, 5’UTR, and CDS sequences of USP10 and PRMT1 were obtained by PCR and cloned into a dual luciferase reporter vector. The following operations were performed in an environment protected from light using the Dual-Luciferase Reporter Assay Kit (DL101, Vazyme) according to the instructions.
Total RNA isolation, reverse transcription, and RT-qPCR
RNA extraction was performed using the TRIzol reagent (R701, Vazyme). Reverse transcription was performed using the reverse transcription kit HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) (R233-01, Vazyme). cDNA was obtained using 2 × Universal SYBR Green Fast qPCR Mix (RK21203, Abclonal, Wuhan, China) for RT-qPCR experiments as per instructions.
Cross-linking immunoprecipitation (CLIP)
Cells were irradiated with a UV crosslinker at 260 nm and then lysed with 5× Cell Lysis Buffer (G2002, Servicebio, Wuhan, China) and Protease Inhibi tor Cocktai l (20138ES05, Yeasen, Shanghai, China). After the addition of 10 mg/mL RNaseA (G3405, Servicebio), and digested at room temperature for 15 minutes, DDX1 antibody and A/G magnetic beads (HY-K0202, MedChemExpress, Monmouth Junction, NJ, USA) were added, and the cells were incubated at 4°C overnight. The immunocomplexes were eluted with 50 mM Tris-HCl solution (pH=7.8) at 60°C. Reverse transcription of the RNA to cDNA was performed and analyzed by RT-qPCR.
GST pull-down assay
Bacteria were lysed with GST lysis buffer (50 mM Tris-HCl, pH 7.4, 0.5% NP-40, 1 mM EDTA, 150 mM NaCl, and protease inhibitor cocktail) and sonicated to obtain the GST fusion proteins. The GST fusion proteins and cytosolic proteins were incubated with GST antibody and A/G magnetic beads at 4°C overnight. The next day, the cells were washed three times and boiled with 2× loading buffer for 15 minutes before being used for immunoblotting analysis.
Immunofluorescence staining
Cells were fixed in 4% formaldehyde, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 5% BSA. Primary antibodies were incubated with the cells overnight at 4°C. The following procedures were performed in a light-proof environment. The next day, coverslips were incubated with the secondary antibodies at 37°C for 2 hours. The nuclei were stained with DAPI.
Flow cytometry
Cells were digested with trypsin and obtained a cell suspension, which was incubated with a fluorescently labeled primary antibody for 30 minutes at room temperature. Cell viability was assessed by the Zombie Aqua Fixable Viability Kit (423101, Biolegend, San Diego, CA, USA). The stained cells were analyzed by flow cytometry using a BD FACSCelesta (423102, BD Biosciences, Franklin Lakes, NJ, USA) instrument and FlowJo software (TreeStar, Ashland, OR, USA). Antibodies used in this section are listed in Antibodies and Chemicals.
Tumor cell spheroid formation assay
CCA cells were inoculated in ultra-low adsorption dishes, and special medium for tumorsphere growth was prepared by adding B27 (1:50, Invitrogen, Carlsbad, CA, USA), human recombinant EGF (20 ng/mL, Sigma-Aldrich, St. Louis, MO, USA), bFGF (20 ng/mL, Sigma-Aldrich), and insulin (5 μg/mL, Sigma-Aldrich) to DMEM/F12 medium.
DIA relative quantitative proteomics analysis
After protein extraction and quantification, peptides were desalted using a C18 cartridge following FASP enzymatic digestion and then freeze-dried under vacuum. Each sample underwent DIA analysis with a screening threshold of Q value≤0.01. All mass spectrometry data were merged using Spectronaut software (version 19, Biognosys AG, Schlieren, Switzerland) to complete database searching of DIA mass spectrometry data and quantitative analysis of proteins via DIA. The database used was uniprotkb-Homo sapiens (Human) [9606]-205092-20250414.fasta, sourced from
https://www.uniprot.org/taxonomy/9606.
In addition to Section DIA Relative Quantitative Proteomics Analysis incorporates asymmetric dimethylated peptide enrichment and differential analysis of asymmetric dimethylated arginine sites.
HPLC-MS/MS
HPLC-MS/MS sequencing was conducted with the assistance of Shanghai Omicsspace Biotech Co., Ltd. (Shanghai, China). HPLC-MS/MS analysis was performed on an Orbitrap Exploris 480 mass spectrometer coupled to a nano-HPLC interface (Dionex UltiMate 3000 nano HPLC, Thermo Scientific, Bremen, Germany).
RNA sequencing and enrichment analysis
Enrichment analysis was based on the principle of hypergeometric distribution, and clusterProfiler software was used to perform Gene Ontology (GO) (
http://www.geneontology.org/), Kyoto Encyclopedia of Genes and Genomes (KEGG) (
http://www.kegg.jp/), and Reactome pathway enrichment analyses on the differential gene sets.
The following other small-molecule materials were used: GSK3368715 dihydrochloride (HY-128717A, MedChemExpress); AMI-1 free acid (HY-18962A, MedChemExpress).
Statistical analyses
Statistical analysis was performed using GraphPad Prism 10 (GraphPad Software, La Jolla, CA, USA). Each experiment was repeated at least three times. Data were expressed as mean±standard deviation (SD). Two-factor analysis of variance (ANOVA) or two-tailed unpaired t-test was used. Survival curves were analyzed using the Kaplan–Meier method and the log-rank test. P-values less than 0.05 were considered statistically significant, and P-values were expressed as follows: *P<0.05, **P<0.01, and ***P<0.001. On the contrary, P-values equal to or greater than 0.05 were not statistically significant.
RESULTS
ADMA modification of R602 in DDX1 is elevated in CCA and correlates with hypoxic environments
To identify proteins highly expressed in CCA, quantitative proteomic sequencing was performed on tissue samples from three pathologically confirmed CCA patients paired with adjacent non-cancerous tissue. Functional annotation and enrichment analysis revealed the response to hypoxia (
Supplementary Fig. 1A–
C). Furthermore, multiple studies have confirmed that hypoxia induction has been proven to be a critical intervention condition in cancer research [
28,
29]. In order to identify the key proteins involved in hypoxia-induced elevation of ADMA in CCA, TFK-1 cells were cultured under hypoxic conditions and then subjected to modified proteomics sequencing (
Fig. 1A). The cross-analysis of the 17 proteomics-upregulated results with the 9 modificationomics-upregulated results revealed asymmetric dimethylation at arginine 602 (R602) of the DDX1 peptide (
Fig. 1B,
C). A DDX1-R602-specific ADMA antibody (DDX1-R602-ADMA) was engineered and utilized to specifically detect DDX1-R602 ADMA through dot blotting (
Supplementary Fig. 1D). Analysis of the amino acid sequence homology revealed that R602 is highly conserved throughout evolution (
Fig. 1D). IHC staining of CCA tissue microarrays demonstrated that the expression of DDX1 and R602-ADMA was significantly elevated in CCA tissues compared with paracarcinoma tissues (
P<0.01) (
Fig. 1E,
Supplementary Fig. 1E). The protein and R602-ADMA levels of DDX1 can be increased in a hypoxic environment and in response to the increasing Ado-Met concentration or the arginine methylation inhibitor AMI-1 (
Supplementary Fig. 1F–
I). Upon converting all intracellular DDX1 to the R602K mutant, hypoxia no longer increased ADMA modification of DDX1 (
Fig. 1F,
Supplementary Fig. 1J). The above results confirm that ADMA modification at the R602 site of DDX1 is elevated in CCA and correlates with hypoxic environments.
To identify the arginine-modifying enzyme of DDX1, arginine methyltransferase 1 (PRMT1) was found after HPLCMS/MS (
Fig. 2A). Further experiments and molecular docking confirmed the direct combination of DDX1 and PRMT1 between the PRMT1d1 fragment and the DDX1F2 fragment (
Fig. 2B,
C,
Supplementary Fig. 2A–
C). Moreover, under hypoxic conditions, PRMT1 accumulates more DDX1 (
Supplementary Fig. 2D). Immunofluorescence staining revealed that DDX1 and PRMT1 exhibit binding interactions with each other within both the nucleus and cytoplasm (
Fig. 2D). R602-ADMA modification of DDX1 positively correlates with PRMT1 expression and correlates with its methyltransferase activity. This was confirmed in cell experiments using the PRMT1-specific inhibitor GSK3368715 (GSK715) and in PRMT1 knockout (PRMT1KO) cells (
Fig. 2E,
Supplementary Fig. 2E–
H). The hypoxia-induced expression of DDX1 in CCA and the elevated ADMA modification levels are similarly correlated with the methyl transfer activity of PRMT1 and the R602 site (
Fig. 2F,
Supplementary Fig. 2I–
L). It indicates that PRMT1-mediated ADMA modification at the DDX1R602 site is crucial for the elevated expression of DDX1 in CCA.
To investigate the impact of ADMA modification on the intracellular function of DDX1’s R602 residue, further GO and KEGG functional annotation and enrichment analysis were performed on the cross-results from proteomics and ADMA modification profiling. The results indicate that the majority of the enriched biological processes (BP) are associated with mRNA and nucleic acid metabolism within the nucleus (
Fig. 3A). Multiple studies indicate that protein methylation often interacts with other PTMs to jointly influence protein degradation or intracellular localization. Analysis of DDX1 HPLC-MS/MS results drew our attention to the deubiquitinating enzyme USP10 (
Fig. 3B). After validating the interaction between USP10 and DDX1 (
Supplementary Fig. 3A–
D), intracellular immunofluorescence colocalization analysis vrevealed that USP10 primarily associates with DDX1 in the cytoplasm (
Fig. 3C). Molecular docking was employed to simulate the binding of USP10 to the DDX1 protein (
Fig. 3D). USP10 catalytic inactivation plasmids (USP10C424A, USP10mut) confirmed the catalytic deubiquitination effect of USP10 on DDX1 (
Supplementary Fig. 3E). We further determine the relationship between ADMA modification and the deubiquitination of DDX1. R602-ADMA modification enhances the deubiquitination of DDX1 by USP10, whereas PRMT1 itself does not exert this effect (
Fig. 3E). Hypoxia also enhances the deubiquitinating activity of USP10, which correlates with the DDX1R602 site (
Fig. 3F,
Supplementary Fig. 3F,
G). The results demonstrate that hypoxia exerts a comparable effect on the recruitment of USP10, facilitated by ADMA modification at the DDX1R602 site.
USP10 mediates K63 deubiquitination of DDX1 (
Supplementary Fig. 4A), which is often associated with the intracellular localization of proteins. DDX1 is progressively transported into the nucleus as intracellular USP10 levels increase, and hypoxia enhances this process (
Fig. 4A,
Supplementary Fig. 4B–
D). PRMT1 and hypoxia both enhance USP10-mediated nuclear localization of DDX1 through R602 ADMA modification of DDX1 (
Fig. 4B,
Supplementary Fig. 4E,
F). In exploring the enhancing effect of PRMT1 on USP10-mediated deubiquitination of DDX1, we discovered that USP10 itself exhibits a certain degree of enhancing effect on ADMA modification of DDX1 R602 (
Fig. 3E). HPLC-MS/MS analysis of the PRMT1 and further experiments discovered that PRMT1 and USP10 interact within cells (
Fig. 4C,
Supplementary Fig. 5A–
F). USP10 attenuates PRMT1 ubiquitinylation via the K48 pathway (
Supplementary Fig. 5F,
G), not only inhibiting PRMT1 protein degradation but also enhancing PRMT1’s methyltransferase activity (
Fig. 4E,
F,
Supplementary Fig. 5H–
K).
We have thus identified a positive feedback loop involving PTMs of DDX1: PRMT1 catalyzes ADMA modification at the R602 site of DDX1, which in turn recruits USP10 to catalyze DDX1 deubiquitination. Concurrently, USP10 binds to PRMT1 and stabilizes the PRMT1 protein, thereby enhancing PRMT1’s R602-ADMA modification of DDX1 and recruiting additional USP10 molecules.
DDX1 promotes a positive feedback loop by binding to the 3’ UTR region of mRNA to facilitate PRMT1 and USP10 transcription
To elucidate the function of DDX1 upon nuclear localiza-tion in CCA, we performed RNA sequencing and revealed that both USP10 and PRMT1 were upregulated by DDX1 expression (
Fig. 5A,
Supplementary Fig. 6A). Reactome pathway annotation revealed strong associations with mRNA processing (
Fig. 5B). CLIP and dual luciferase reporter assays revealed that DDX1 exerted its effect on the 3′UTR region of mRNAs and that the signal intensity exhibited a positive correlation with DDX1 content (
Fig. 5C,
D,
Supplementary Fig. 6B,
C). PRMT1 could positively affect the USP10 content and correlate with its catalytic activity (
Fig. 6E,
Supplementary Fig. 6D). USP10 was unable to affect the mRNA or protein expression of USP10 in the ab-sence of ADMA modification of DDX1 (
Fig. F6).
Our experiments established a USP10-PRMT1-DDX1 positive feedback network. PRMT1 catalyzes ADMA modification of DDX1, which recruits USP10. USP10 simultaneously mediates DDX1 deubiquitination and nuclear translocation while deubiquitinating PRMT1 to enhance its protein stability, thereby further elevating DDX1’s ADMA modification levels. Upon entering the nucleus, DDX1 binds to the mRNA of PRMT1 and USP10, promoting transcription and thereby triggering positive feedback regulation of PRMT1 and USP10 protein levels.
GSK715 suppresses CCA stem cell characteristics and inhibits CCA progression by inhibiting DDX1R602’s ADMA modification through PRMT1 inhibition
Following the discovery of DDX1 overexpression in CCA through proteomics, we further investigated the impact of DDX1 on the development and progression of CCA. GO analysis on RNA-seq data identified epithelial cell differentiation-related pathways (
Fig. 6A). After confirming in vitro that TFK-1 cell proliferation rate positively correlates with DDX1 expression (
Supplementary Fig. 7A), xenograft ex-periments in nude mice demonstrated that DDX1 similarly promotes tumor cell proliferation in vivo (
Fig. 6B,
C). Transwell invasion and migration assays, along with liver metastasis experiments via spleen injection in nude mice, demonstrated that DDX1 enhances TFK-1 cell migration both in vivo and in vitro (
Supplementary Fig. 7B,
Fig. 6D). These experimental results suggest the potential of DDX1 as a therapeutic target for CCA. As DDX1 has been demonstrated to play a pivotal role in the development of CCA both in vivo and in vitro, we established wild-type and DDX1-overexpressing or knockdown in situ CCA models in C57BL/6 mice. The progression rate of liver cancer in mice is also positively correlated with DDX1 expression (
Supplementary Fig. 7C). This validated the role of DDX1 in CCA initiation and progression. As demonstrated in prior research, the elimination of DDX1 has been observed to impact the stem cell characteristics of cancerous cells [
30,
31]. The results demonstrated that TFK-1 cells overexpressing DDX1 formed tumor spheres with significantly increased diameters (
Fig. 6E) and led to a substantial increase in the expression levels of stem cell-associated nuclear transcription factors (EpCAM, CD24, CD44, SOX2, and OCT4) [
2,
32]. DDX1 overexpression significantly augmented cell membrane CD133 and Ep-CAM enrichment and increased the propor tion of CD24+CD44+ cells (
Fig. 6F).
Mechanistic experiments have demonstrated that PRMT1-mediated ADMA modification at R602 of DDX1 is critical for DDX1 function. Phenotypic studies further confirm that PRMT1-enhanced CCA cell stemness similarly depends on R602 ADMA modification (
Supplementary Fig. 8A,
B), while PRMT1 also promotes CCA progression both in vivo and in vitro (
Fig. 7A,
B,
Supplementary Fig. 8C–
E). We further investigated whether the PRMT1-specific inhibitor GSK715 could suppress CCA progression. The results demonstrated that GSK715 inhibited the formation of tumor spheres in the presence of the methylation site of DDX1 and reduced the membrane surface stemness markers and the proportion of CD24+CD44+ cells in CCA cells (
Supplementary Fig. 9A,
B). Following in vitro experiments confirming that GSK715 inhibits proliferation and invasion in TFK-1 cells (
Fig. 7C,
D), we generated mouse models of CCA with wild-type expression and with DDX1 or PRMT1 specifically inhibited. These models were then treated with intraperitoneal injections of GSK715 to evaluate its in vivo efficacy in suppressing CCA progression. Results showed that while GSK715 exhibited limited tumor progression inhibition in wild-type CCA mouse models, its therapeutic efficacy was significantly enhanced when DDX1 or PRMT1 expression was suppressed (
Fig. 7E). This finding provides a theoretical basis for incorporating DDX1 inhibitor combination therapy in subsequent experiments.
In summary, GSK715 has been demonstrated to impede the progression of CCA and mitigate liver injury to a certain extent by selectively inhibiting DDX1-R602ADMA.
DISCUSSION
PTM of proteins is a critical factor in tumor development [
33,
34], exerting its influence through multiple mechanisms [
35,
36]. The study of multiple PTMs targeting proteins that play key roles has emerged as a promising avenue for cancer therapy [
37,
38].
Our experimental findings elucidated the interplay between two PTMs of DDX1 in CCA, unveiling a positive feedback network. Cross-referencing proteomics and modificationomics results revealed the R602-ADMA modification of DDX1. HPLC-MS/MS analysis of DDX1-binding proteins revealed that PRMT1 mediates DDX1-R602-ADMA modification and USP10 mediates DDX1 deubiquitination and promotes DDX1 nuclear localization. The deubiquitination of DDX1 is not only enhanced by ADMA modification of DDX1-R602 but also correlates with hypoxic conditions. HPLC-MS/MS analysis of PRMT1-binding proteins revealed that USP10 stabilizes the PRMT1 protein, thereby amplifying ADMA modification and further intensifying USP10 recruitment. This positive feedback pathway confirms the central role of DDX1 R602 ADMA modification and elucidates the relationship between the two PTMs of DDX1 in detail.
To elucidate the biological function of DDX1 in CCA, RNA-seq was performed, and found that DDX1 upregulates the transcription of PRMT1 and USP10 by binding to the 3’ UTR region of mRNA. This elucidates a positive feedback relationship between DDX1 PTM and transcription. GO enrichment analysis of RNA-seq revealed that DDX1 upregulates the epithelial cell differentiation pathway. Inhibition of ADMA modification at DDX1R602 by GSK715 effectively weakened the stem-like characteristics of CCA cells and suppressed cancer progression in CCA mice. This finding provides a theoretical basis and feasible approach for targeted therapy in CCA.
In summary, we identified two PTM modifications of DDX1 under hypoxic conditions and established the central role of the R602-ADMA modification, which serves as a survival-related prognostic indicator for CCA and may represent a potential therapeutic target for its treatment.
FOOTNOTES
-
Authors’ contributions
W.L. and Y.L. designed the experiments; W.L., Y.L., and Y.K. performed most of the experiments with the assistance of X.G., J.C., J.L., J.S., X.Y., and J.Z.; W.L. and Y.C. wrote and edited the manuscript; Y.C. provided funding support for the project; X.G., J.C., J.L., J.S., X.Y., Y.K., S.H., Z.Z., B.W., F.P. and J.Z. performed data analysis and supervised the project. All authors read and approved the final paper.
-
Acknowledgements
The authors would like to express their sincerest gratitude to the Experimental Medicine Center of Tongji Hospital, Tongji Medical School, Huazhong University of Science and Technology, for providing invaluable equipment support for immunofluorescence experiments.
This work was supported by grants from the National Natural Science Foundation of China (Grant No. #82173069 and #82573911 to Y.C.), Key Research and Development Program of Hubei Province (Grant No. #2024BCB054 to Y.C.).
-
Conflicts of Interest
The authors have no conflicts to disclose.
SUPPLEMENTARY MATERIAL
Supplementary material is available at Clinical and Molecular Hepatology website (
http://www.e-cmh.org).
Supplementary Figure 1.
DDX1 is highly expressed in cholangiocarcinoma, and hypoxic induction enhances ADMA modification at the R602 site. (A) Proteome circular heatmap. (B) Proteomics GO analysis and display of the top 10 significant terms across three major branches. (C) GSEA analysis of ADMA-modified proteomics. (D) 10 μL of peptides of different concentrations (0.1–0.75 μg) were cross-linked with a PVDF membrane and then analyzed by IB using an R602-specific asymmetric dimethylated antibody (anti-DDX1R602-ADMA). (E) IHC staining of human CCA TMAs for DDX1. Representative images are shown in the right panel. Scoring of DDX1 expression based on CCA TMAs. Data were presented as means±SD. P-values were calculated by unpaired, two-tailed Student’s t-test. (F) Protein expression of DDX1 in the hypoxic environment in the TFK-1 cell line as well as the QBC939 cell line. (G) The Co-IP assay enriched DDX1 and detected changes in the levels of DDX1-R602-ADMA. (H) Co-IP assay enriched DDX1 in HEK293T cells and detected the ADMA modification level of DDX1 after Ado-Met treatment at different concentrations. (I) DDX1 was enriched by Co-IP assay and the ADMA modification level of DDX1 was detected after treatment with AMI-1. (J) Flag antibody-enriched DDX1 was used to restore DDX1 wild-type or R602K mutant expression in QBC939 cells following DDX1 knockout. R602-ADMA modification levels were then measured with or without hypoxia induction. **P<0.01. ADMA, asymmetric demethylation; CCA, cholangiocarcinoma; Co-IP, Co-inmunoprecipitation; ECC, Extrahepatic cholangiocarcinoma; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis; IB, Immunoblotting; IHC, immunohistochemistry; PVDF, Polyvinylidene Fluoride; TMAs, tissue microarrays. WT, wild-type.
Supplementary Figure 2.
PRMT1 acts as the arginine methyltransferase for DDX1, and ADMA mediates the increased expression of DDX1 in cholangiocarcinoma. (A) HEK293T cells were transfected with the indicated plasmids and subsequently immunoprecipitated with anti-Flag or anti-HA antibodies. (B) TFK-1 and QBC939 cell lysates were collected and subsequently immunoprecipitated with anti-PRMT1 or anti-DDX1 antibodies, respectively, and immunoblotted with anti-DDX1 or anti-PRMT1 antibodies. (C) HEK293T cells were transfected with either full-length or truncated mutants of PRMT1 or DDX1, and the cell lysates were collected and immunoprecipitated with anti-Flag or anti-HA antibodies to explore the PRMT1 and DDX1 binding region between PRMT1 and DDX1. (D) PRMT1 antibodies enriched DDX1 bound to CCA cell lines before and after hypoxic culture. (E) HEK293T cells were transfected with 0 μg, 2 μg, 4 μg, and 8 μg of the PRMT1-HA plasmid and equal amounts of DDX1-Flag plasmid. (F) PRMT1-HA plasmid as well as PRMT1mut-HA plasmid (left), and PRMT1-HA plasmid base was treated with GSK715 (right), and the cell lysates were collected, immunoprecipitated with anti-Flag antibody and immunoprecipitated with specific anti-DDX1R602-ADMAantibody for immunoblotting. (G) Replied PRMT1-HA as well as PRM-T1mut-HA in QBC939 cells from PRMT1KO and collected the cell lysates, immunoprecipitated with anti-DDX1 antibody, and immunoblotted with specific anti-DDX1R602-ADMA antibody. (H) DDX1-Flag and DDX1R602K-Flag were transfected in DDX1 knockdown stably transfected TFK-1 and QBC939 cell lines, and immunoblotting experiments were performed after collecting cell lysates and immunoprecipitating them with anti-Flag antibody followed by specific anti-DDX1R602-ADMA antibody. (I) PRMT1-HA or PRMT1mut-HA was re-expressed in PRMT1KO QBC939 cells, and cell lysates were collected, immunoprecipitated with anti-DDX1 antibody, and immunoblotted with specific anti-DDX1R602-ADMA antibody. (J) QBC939 cell line replied to in PRMT1KO PRMT1 or PRMT1mut, and then cultured under hypoxia, respectively, and immunoblotted with anti-DDX1 antibody. (K) HEK293T cells were transfected with the corresponding plasmid and then cultured in a hypoxic environment, cell lysates were collected and immunoprecipitated with anti-Flag antibody, followed by immunoblotting experiments with specific anti-DDX1R602-ADMA antibody. (L) After transfection of corresponding plasmids in QBC939 cell lines of DDX1KO and incubation in a hypoxic environment, cell lysates were collected and immunoprecipitated with anti-Flag antibody, followed by immunoblotting experiments with specific anti-DDX1R602-ADMA antibody. ADMA, asymmetric demethylation; CCA, cholangiocarcinoma; WT, wild-type.
Supplementary Figure 3.
USP10 binds to DDX1 and catalyzes deubiquitination, with increased deubiquitination levels observed when DDX1 R602 is modified by ADMA. (A) Cell lysates were collected after transfection of corresponding plasmids in HEK293T cells, and immunoprecipitation was performed with anti-Flag and anti-HA antibodies respectively followed by immunoblotting experiments with anti-HA and anti-Flag antibodies. (B) TFK-1 and QBC939 cell lysates were collected and immunoprecipitated with anti-DDX1 or anti-USP10 antibodies followed by immunoblotting experiments with anti-USP10 or anti-DDX1 antibodies. (C) Cell lysates from HEK293T cells were incubated with magnetic beads coupled with GST, GST-USP10, or GST-DDX1. Pull-down samples and whole-cell lysates were analyzed using immunoblotting and Caumas Brilliant Blue staining. (D) Schematic representation of full-length USP10 as well as DDX1 and their functional segmentation (top); HEK293T cells were transfected with either full-length or truncated mutants of USP10 or DDX1, and the cell lysates were collected and immunoprecipitated with anti-Flag or anti-HA antibodies to explore the USP10 and DDX1 binding region between USP10 and DDX1 (below). (E) HEK293T cells were transfected with DDX1-HA plasmid and USP10-Flag or USP10mut-Flag, along with MYC-ubiquitin, and cell lysates were collected and analyzed for ubiquitin levels by immunoprecipitation assay using anti-HA antibody and immunoblotting assay using anti-MYC antibody. (F) HEK293T cells were transfected with DDX1-HA or DDX1R602K-HA plasmid and USP10-Flag and cultured in a hypoxic environment while transfected with MYC-ubiquitin, and cell lysates were collected and analyzed for ubiquitin levels by immunoprecipitation assay using anti-HA antibody and immunoblotting assay using anti-MYC antibody. (G) DDX1KO QBC939 cells were transfected with DDX1-HA or DDX1R602K-HA plasmid, along with USP10-Flag plasmid and MYC-ubiquitin, and cultured in a hypoxic environment, and cell lysates were collected for immunoprecipitation experiments using anti-HA antibody and immunoblotting experiments using anti-MYC antibody. Ubiquitin levels were analyzed, and ADMA modification levels were analyzed by immunoblotting experiments using anti-DDX1R602-ADMA antibodies. ADMA, asymmetric demethylation; WT, wild-type.
Supplementary Figure 4.
USP10 mediates the deubiquitination of DDX1 via the K63 pathway and promotes DDX1 nuclear translocation. (A) HEK293T cells were transfected with DDX1-Flag and MYC-ubiquitin (wild-type [WT], K48O, K63O, K48R, and K63R) with or without USP10-HA cotransfection, immunoprecipitated using an anti-Flag antibody, and analyzed for ubiquitination by immunoblotting assay using anti-MYC antibody. (B) USP10-Flag plasmid was transfected in HEK293T cells in a gradient (0–6 μg), and nuclear and cytoplasmic proteins were extracted separately for immunoblotting experiments with anti-DDX1 antibody. (C) TFK-1 cells were transfected with USP10-Flag plasmid and cultured in an anoxic environment, and cytoplasmic and nuclear proteins were extracted separately for immunoblotting experiments with anti-DDX1 antibody. (D) Nuclei and cytoplasmic proteins were extracted from TFK-1 cell lines with stable overexpression or knockdown of USP10, respectively, and immunoblotting experiments were performed with anti-DDX1 antibody. (E) TFK-1 cells were transfected with DDX1-Flag or DDX1R602K-Flag in DDX1KO with or without USP10-HA cotransfection, respectively, and cytoplasmic and nuclear proteins were extracted separately for immunoblotting experiments with anti-DDX1 antibody. (F) PRMT1 was knocked down in USP10 overexpressing or knockdown TFK-1 cells with or without reporter PRMT1-HA, and cytoplasmic and cytoplasmic proteins were extracted, and immunoblotting experiments were performed with anti-DDX1 antibody, respectively.
Supplementary Figure 5.
USP10 binds to and mediates K48 deubiquitination of PRMT1 to stabilize the PRMT1 protein, thereby enhancing R602-ADMA modification of DDX1. (A) HEK293T cells were transfected with the corresponding plasmids, and cell lysates were collected for immunoprecipitation with anti-Flag or anti-HA antibodies and immunoblotting experiments with anti-HA or anti-Flag antibodies. (B) Cell lysates of TFK-1 and QBC939 were collected and immunoprecipitated with anti-PRMT1 or anti-USP10 antibodies, and immunoblotting experiments were performed with anti-USP10 or anti-PRMT1 antibodies. (C) Cell lysates from HEK293T cells were incubated with magnetic beads coupled with GST, GST-USP10, or GST-PRMT1. Pull-down samples and whole-cell lysates were analyzed using immunoblotting and Caumas Brilliant Blue staining. (D) Confocal microscopy observation of immunofluorescence staining of USP10 (green) and PRMT1 (red) in TFK-1 and QBC939 cells transfected with USP10-Flag and PRMT1-HA plasmids. Line intensity maps show co-localization of USP10 and DDX1. Images represent at least n=5 imaged cells. (E) Schematic representation of full-length USP10 and its functional segmentation (top); HEK293T cells were transfected with either full-length or truncated mutants of USP10, and the cell lysates were collected and immunoprecipitated with anti-Flag to explore the USP10 and PRMT1 binding region between USP10 and PRMT1 (below). (F) HEK293T cells were transfected with PRMT1-HA, co-transfected with USP10-Flag or USP10mut-Flag, and cell lysates were obtained after transfection with MYC-ubiquitin, and ubiquitin levels were analyzed by immunoprecipitation with anti-HA antibody followed by immunoblotting assay with anti-MYC antibody. (G) HEK293T cells were transfected with PRMT1-HA and MYC-ubiquitin (wild-type [WT], K48O, K63O, K48R, and K63R) with or without USP10-Flag cotransfection, immunoprecipitated using anti-HA antibody, and analyzed for ubiquitination by immunoblotting assay using anti-MYC antibody. (H) USP10-Flag plasmid was transfected in QBC939 cells in a gradient (0–6 μg), and immunoblotting experiments were performed with anti-PRMT1 antibody. (I) HEK293T cells were transfected with DDX1-Flag in HEK293T cells with or without PRMT1-His or USP10-HA cotransfection, and cell lysates were collected for immunoprecipitation with antiFlag antibody followed by immunoblotting experiments with anti-DDX1R602-ADMA antibody. (J, K) Lysates of USP10 stably overexpressing or knockdown TFK-1 (J) and QBC939 (K) cells were collected and immunoprecipitated with anti-DDX1 antibody, and immunoblotting experiments were performed with anti-DDX1R602-ADMA antibody and anti-PRMT1 antibody. ADMA, asymmetric demethylation.
Supplementary Figure 6.
DDX1 binds to USP10 and PRMT1 mRNA and promotes the transcription of USP10 and PRMT1. (A) RNA from DDX1 stably overexpressed or knockdown TFK-1 and QBC939 cell lines was extracted and RT-qPCR assay was performed to detect mRNA expression of USP10 and PRMT1. (B) RT-qPCR experiments with primers for USP10 and PRMT1 were performed after the CLIP test using an anti-DDX1 antibody or IgG-negative control antibody. (C) The luciferase reporter gene detects changes in fluorescence intensity upon overexpression or knockdown of DDX1 acting on the 3’UTR region of USP10 and PRMT1 mRNA. (D) RNA from TFK-1 and QBC939 cell lines with stable overexpression or knockdown of PRMT1 was extracted and RT-qPCR assay was performed to detect mRNA expression of USP10. CLIP, cross-linking immunoprecipitation. *P<0.05, **P<0.01, and ***P<0.001.
Supplementary Figure 7.
DDX1 promotes CCA progression. (A) CCK-8 assay and colony formation assay were used to evaluate the effect of DDX1 on CCA cell proliferation. (B) Transwell assays were conducted to evaluate the effects of DDX1 on the invasion and migration of CCA cells. (C) An overview, H&E staining, and DDX1 IHC staining of wild-type, DDX1 high-expressing and DDX1 knockdown in situ CCA models constructed in C57BL/6 mice at the 4th week. Statistical analysis of liver weight-to-body weight ratios, serum ALT and AST. *P<0.05, **P<0.01, and ***P<0.001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCA, cholangiocarcinoma; IHC, immunohistochemistry.
Supplementary Figure 8.
PRMT1 promotes CCA proliferation, invasion, and migration in vivo and in vitro. (A) The effects of PRMT1 on tumor cell spheroid formation and stemness markers, and the role of DDX1-R602 in this process. (B) The effect of PRMT1 on the positivity rate of surface dryness markers in CCA cells and the role of DDX1-R602 in this process. (C) CCK-8 assay and colony formation assay were used to evaluate the effect of DDX1-R602 on TFK-1 cell proliferation. (D) Validation of DDX1 and PRMT1 expression levels in xenograft models. (E) Transwell assays were conducted to evaluate the effects of DDX1-R602 on the invasion and migration of CCA cells. *P<0.05, **P<0.01, and ***P<0.001; ns, not significant. CCA, cholangiocarcinoma; WT, wild-type
Supplementary Figure 9.
GSK715 suppresses stem-like characteristics in CCA cells. (A, B) Effects of GSK715 on tumor cell spheroid formation and stemness markers, and the role of DDX1-R602 in this process. (C) Verification of successful modeling in mouse cholangiocarcinoma experiments. **P<0.01 and ***P<0.001; ns, not significant. CCA, cholangiocarcinoma; DMSO, dimethyl sulfoxide; WT, wild-type.
Figure 1.DDX1-R602-ADMA is hypoxically induced and overexpressed in CCA. (A) Flowchart of sample processing and data analysis for proteomics and ADMA-modified proteomics. (B) Proteomics volcano plot. ADMA modification proteomics heatmap. Venn diagram of proteins upregulated in proteomics and ADMA-modified proteomics, with heatmaps displayed after removal of proteomic background noise. (C) Secondary mass spectrum of the DDX1R602 ADMA. (D) Homology alignment of the DDX1-R602 across different species. (E) IHC staining of human CCA TMAs for DDX1-R602-ADMA and scoring. Data were presented as means±SD. (F) R602-ADMA modification levels were measured in DDX1 or DDX1-R602K cells with or without hypoxia induction. **P<0.01. ADMA, asymmetric demethylation; CCA, cholangiocarcinoma; DIA, data-independent acquisition; ECC, extrahepatic cholangiocarcinoma; IHC, Immunohistochemistry; TMAs, tissue microarrays; WT, wild-type.
Figure 2.PRMT1 acts as the arginine methyltransferase for DDX1, and ADMA mediates the increased expression of DDX1 in CCA. (A) Flowchart of sample processing for HPLC-MS/MS sequencing of DDX1-binding proteins and secondary mass spectrum of PRMT1. (B) Co-IP and GST pull-down of DDX1 and PRMT1. (C) Co-IP with truncated mutants of PRMT1 or DDX1. Computer-performed molecular docking simulation of DDX1 with PRMT1. (D) Immunofluorescence staining of PRMT1 (red) and DDX1 (green). Line intensity maps show co-localization of PRMT1 and DDX1 (n=5). (E) PRMT1 gradient increase, mutation, GSK715 detection of R602-ADMA modification level in DDX1. (F) Detection of the effects of hypoxia on PRMT1-mediated increased DDX1 expression and enhanced R602-ADMA modification. ADMA, asymmetric demethylation; CCA, cholangiocarcinoma; Co-IP, Co-immunoprecipitation; DMSO, dimethyl sulfoxide; HPLC-MS/MS, high-performance liquid chromatography-mass spectrometry; WT, wild-type.
Figure 3.USP10 binds to DDX1, and its deubiquitinating activity on DDX1 is enhanced by PRMT1-mediated DDX1 R602-ADMA modification. (A) After removing protein background through proteomics and ADMA modificationomics cross-analysis, all proteins underwent GO enrichment analysis. Functional enrichment analysis of annotation results was performed using Fisher’s exact test algorithm. Results are presented as P-values, with values below 0.05 indicating significant functional enrichment. Annotation and statistical analysis of cellular components (CC, subcellular localization) in the GO database. (B) Secondary mass spectrometry spectrum of the USP10 peptide segment binding to the DDX1 protein. (C) Confocal microscopy observation of immunofluorescence staining of USP10 (green) and DDX1 (red). Images represent at least n=5 imaged cells. (D) Computer-performed molecular docking simulation of DDX1 with PRMT1. (E) Detection of the enhancing effect of R602-ADMA modification on deubiquitination. (F) The role of ADMA modification in R602 in enhancing deubiquitination under hypoxia. ADMA, asymmetric demethylation; GO, Gene Ontology; WT, wild-type.
Figure 4.USP10 mediates DDX1 nuclear transport and enhances ADMA modification at the DDX1 R602 site by stabilizing the PRMT1 protein. (A) Immunofluorescence staining of DDX1 (red) and cytoplasmic marker α-Tubulin (green) was observed by confocal microscopy. Line intensity maps show co-localization of cytoplasmic and DDX1. Images represent at least n=5 imaged cells. (B) Detection of the role of PRMT1’s methyltransferase activity and DDX1-R602’s ADMA modification in USP10-mediated DDX1 nuclear translocation. (C) Secondary mass spectrometry spectrum of the USP10 peptide segment binding to the PRMT1 protein. (D) Computer-performed molecular docking simulation of DDX1 with PRMT1. (E) Effect of USP10 on PRMT1 protein levels. (F) The effect of USP10 on the level of R602-ADMA modification of DDX1 mediated by PRMT1. ADMA, asymmetric demethylation; WT, wild-type.
Figure 5.DDX1 binds to the 3’ UTR regions of PRMT1 and USP10 mRNA to promote transcription. (A) Radar chart of differential gene expression levels between DDX1OE and V101 in RNA-seq sequencing. (B) Differentially expressed genes reactome pathway analysis. (C) The luciferase reporter gene detects changes in fluorescence intensity when DDX1 acts on different structures of USP10 and PRMT1 mRNAs. (D) The luciferase reporter gene detects changes in fluorescence intensity upon overexpression or knockdown of DDX1 acting on the 3’UTR region of USP10 and PRMT1 mRNA. (E) Effect of PRMT1 on USP10 protein levels. (F) Detection of the role of the R602 site in PRMT1 on USP10 mRNA and protein levels. **P<0.01 and ***P<0.001; ns, not significant. WT, wild-type.
Figure 6.DDX1 promotes CCA progression. (A) Perform GO enrichment analysis on differentially expressed genes from RNA-seq to characterize their functions. (B, C) Subcutaneous xenograft tumor experiments, statistical analysis of volume and weight changes, and immunohistochemical Ki67 staining. (D) Spleen injection liver metastasis experiment and statistical analysis of the number of metastatic foci. (E) Representative images of tumor spheres formed by TFK-1 cells after DDX1 overexpression or knockdown and statistical plots of tumor sphere size (n=3). (F) CCA cell pluripotency markers after DDX1 overexpression or knockdown. (G) CCA cell surface stemness marker positivity rate after DDX1 overexpression or knockdown. *P<0.05, **P<0.01, and ***P<0.001. CCA, cholangiocarcinoma; GO, Gene Ontology.
Figure 7.GSK715 inhibits CCA progression. (A) Subcutaneous xenograft tumor experiments, statistical analysis of volume and weight changes, and immunohistochemical Ki67 staining. (B) Spleen injection liver metastasis experiment and statistical analysis of the number of metastatic foci. (C) CCK-8 assay and colony formation assay were used to evaluate the effect of GSK715 on TFK-1 cell proliferation. (D) Transwell assays were conducted to evaluate the effects of GSK715 on the invasion and migration of TFK-1 cells. (E) Evaluating the therapeutic efficacy of GSK715 against CCA in a mouse CCA model. *P<0.05, **P<0.01, and ***P<0.001; ns, not significant. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCA, cholangiocarcinoma; DMSO, dimethyl sulfoxide; WT, wild-type.
Abbreviations
cross-linking immunoprecipitation
data-independent acquisition
high-performance liquid chromatography-mass spectrometry
Kyoto Encyclopedia of Genes and Genomes
post-translational modifications
REFERENCES
- 1. Mizumoto M, Shibuya K, Terashima K, Murakami M, Murakami M, Shioyama Y, et al. Particle therapy for intrahepatic cholangiocarcinoma: a multicenter prospective registry study, systematic review and meta-analysis. Liver Cancer 2025;14:211-222.
- 2. Wang Q, Long T, Tang P, Xu C, Wang L, Liu J, et al. Metabolic reprogramming in cholangiocarcinoma cancer stem cells: emerging therapeutic paradigms. Cancer Lett 2025;622:217714.
- 3. Vithayathil M, Khan SA. Current epidemiology of cholangiocarcinoma in Western countries. J Hepatol 2022;77:1690-1698.
- 4. Niu M, Ding WX. Overcoming drug resistance by harnessing mitochondrial divisome for treating cholangiocarcinoma. J Hepatol 2025;83:835-837.
- 5. Yang L, Niu K, Wang J, Shen W, Jiang R, Liu L, et al. Nucleolin lactylation contributes to intrahepatic cholangiocarcinoma pathogenesis via RNA splicing regulation of MADD. J Hepatol 2024;81:651-666.
- 6. Masclef L, Ahmed O, Estavoyer B, Larrivée B, Labrecque N, Nijnik A, et al. Roles and mechanisms of BAP1 deubiquitinase in tumor suppression. Cell Death Differ 2021;28:606-625.
- 7. Guan C, Gao J, Zou X, Shi W, Hao Y, Ge Y, et al. A novel 167-amino acid protein encoded by CircPCSK6 inhibits intrahepatic cholangiocarcinoma progression via IKBα ubiquitination. Adv Sci (Weinh) 2025;12:e2409173.
- 8. Liu C, Wang X, Li Q, Gao X, Zeng K, Li B, et al. Apolipoprotein E promotes primary resistance to AR-targeted therapy via inducing TRIM25-mediated AR ubiquitination and sensitizes immunotherapy in prostate cancer. Theranostics 2025;15:5572-5591.
- 9. Yan Y, Yu J, Wang W, Xu Y, Tison K, Xiao R, et al. Palmitoylation prevents B7-H4 lysosomal degradation sustaining tumor immune evasion. Nat Commun 2025;16:4254.
- 10. Zhang N, Sun L, Zhou S, Ji C, Cui T, Chu Q, et al. Cholangiocarcinoma PDHA1 succinylation suppresses macrophage antigen presentation via alpha-ketoglutaric acid accumulation. Nat Commun 2025;16:3177.
- 11. Dai P, Sun Y, Huang Z, Liu YT, Gao M, Liu HM, et al. USP2 inhibition unleashes CD47-restrained phagocytosis and enhances anti-tumor immunity. Nat Commun 2025;16:4564.
- 12. Sun M, Qiao Z, Wei S, Liang Z, Zhang L, Jiang B, et al. Global methylation profiling by selective release of methylated sites from immobilized tryptic peptides. Anal Chem 2025;97:9620-9626.
- 13. Liu S, Zhang W, Liu W, Ding Z, Zhang R, Han Y, et al. PRMT1-mediated SWI/SNF complex recruitment via SMARCC1 drives IGF2BP2 transcription to enhance carboplatin resistance in head and neck squamous cell carcinoma. Adv Sci (Weinh) 2025;12:e2417460.
- 14. Walton J, Ng ASN, Arevalo K, Apostoli A, Meens J, Karamboulas C, et al. PRMT1 inhibition perturbs RNA metabolism and induces DNA damage in clear cell renal cell carcinoma. Nat Commun 2024;15:8232.
- 15. Wang X, Jin J, Yan H, Liu J, Huang S, Bai H, et al. The mRNA export pathway licenses viral mimicry response and antitumor immunity by actively exporting nuclear retroelement transcripts. Sci Transl Med 2025;17:eado4370.
- 16. Nicholson TB, Chen T, Richard S. The physiological and pathophysiological role of PRMT1-mediated protein arginine methylation. Pharmacol Res 2009;60:466-474.
- 17. Dong Z, She X, Ma J, Chen Q, Gao Y, Chen R, et al. The E3 ligase NEDD4L prevents colorectal cancer liver metastasis via degradation of PRMT5 to inhibit the AKT/mTOR signaling pathway. Adv Sci (Weinh) 2025;12:e2504704.
- 18. Wu Y, Qin X, Xiang M, Deng J. PRMT1 upregulates SIRT6 by enhancing arginine methylation of E2F7 to inhibit vascular smooth muscle cell senescence in aortic dissection. FASEB J 2025;39:e70579.
- 19. Yang X, Liu Y, Cao J, Wu C, Tang L, Bian W, et al. Targeting epigenetic and post-translational modifications of NRF2: key regulatory factors in disease treatment. Cell Death Discov 2025;11:189.
- 20. Ma HY, Sun R, Tian T, Zhou XJ, Chen ZH, Cao XC, et al. NFIX suppresses breast cancer cell proliferation by delaying mitosis through downregulation of CDK1 expression. Cell Death Discov 2025;11:77.
- 21. Oyon D, Lopez-Pascual A, Castello-Uribe B, Uriarte I, Orsi G, Llorente S, et al. Targeting of the G9a, DNMT1 and UHRF1 epigenetic complex as an effective strategy against pancreatic ductal adenocarcinoma. J Exp Clin Cancer Res 2025;44:13.
- 22. Wang Y, Yasmin L, Li L, Gao P, Xu X, Sun X, et al. DDX1 vesicles control calcium-dependent mitochondrial activity in mouse embryos. Nat Commun 2022;13:3794.
- 23. Suzuki T, Takagi S, Funada J, Egawa Y, Yamakawa M, Hara T, et al. DDX1 is required for non-spliceosomal splicing of tRNAs but not of XBP1 mRNA. Commun Biol 2025;8:92.
- 24. Li Z, Zhou M, Cai Z, Liu H, Zhong W, Hao Q, et al. RNA-binding protein DDX1 is responsible for fatty acid-mediated repression of insulin translation. Nucleic Acids Res 2018;46:12052-12066.
- 25. Zhu D, Liang H, Tong B, Du Z, Li G, Zhang W, et al. DDX1 methylation mediated MATR3 splicing regulates intervertebral disc degeneration by initiating chromatin reprogramming. Nat Commun 2025;16:6153.
- 26. Gao B, Li X, Li S, Wang S, Wu J, Li J, et al. Pan-cancer analysis identifies RNA helicase DDX1 as a prognostic marker. Phenomics 2022;2:33-49.
- 27. Di-Luoffo M, Pirenne S, Saandi T, Loriot A, Gérard C, Dauguet N, et al. A mouse model of cholangiocarcinoma uncovers a role for Tensin-4 in tumor progression. Hepatology 2021;74:1445-1460.
- 28. Collet L, Ardin M, Venet D, Berthet J, Ghamry-Barrin S, Treilleux I, et al. Unraveling the tumor microenvironment and PDL1 expression across tissue types in high-grade serous ovarian cancer in the NeoPembrOV/GINECO phase II randomized trial. Clin Cancer Res 2025;31:3317-3331.
- 29. Shepherd STC, Kelly-Morland C, Drage S, Brady AF, Macwana R, Pickering L, et al. Fatal intracranial haemorrhage shortly after belzutifan initiation in von Hippel-Lindau (VHL) disease-associated haemangioblastoma. ESMO Open 2025;10:105109.
- 30. Yang Q, Xu P, Liu Q, Hu F, Xie X, Jiang L, et al. Depleting DDX1 sensitizes non-small cell lung cancer cells to chemotherapy by attenuating cancer stem cell traits. Life Sci 2023;323:121592.
- 31. Wang F, He J, Liu S, Gao A, Yang L, Sun G, et al. A comprehensive RNA editome reveals that edited Azin1 partners with DDX1 to enable hematopoietic stem cell differentiation. Blood 2021;138:1939-1952.
- 32. Giron-Michel J, Padelli M, Oberlin E, Guenou H, Duclos-Vallée JC. State-of-the-art liver cancer organoids: modeling cancer stem cell heterogeneity for personalized treatment. Bio-Drugs 2025;39:237-260.
- 33. Ko PJ, Dixon SJ. Protein palmitoylation and cancer. EMBO Rep 2018;19:e46666.
- 34. Li Y, Zhang R, Hei H. Advances in post-translational modifications of proteins and cancer immunotherapy. Front Immunol 2023;14:1229397.
- 35. Wang S, Osgood AO, Chatterjee A. Uncovering post-translational modification-associated protein-protein interactions. Curr Opin Struct Biol 2022;74:102352.
- 36. Zamaraev AV, Kopeina GS, Prokhorova EA, Zhivotovsky B, Lavrik IN. Post-translational modification of caspases: the other side of apoptosis regulation. Trends Cell Biol 2017;27:322-339.
- 37. Wang WH, Yuan T, Qian MJ, Yan FJ, Yang L, He QJ, et al. Post-translational modification of KRAS: potential targets for cancer therapy. Acta Pharmacol Sin 2021;42:1201-1211.
- 38. Gong H, Zhong H, Cheng L, Li LP, Zhang DK. Post-translational protein lactylation modification in health and diseases: a double-edged sword. J Transl Med 2024;22:41.
, Bing Wang
