Mechanism of GLG1 driving malignant progression of hepatocellular carcinoma and development of targeted compound YHB-5

Mechanism of GLG1 driving malignant progression of hepatocellular carcinoma and development of targeted compound YHB-5 | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 15 January 2026 V1 Latest version Share on Mechanism of GLG1 driving malignant progression of hepatocellular carcinoma and development of targeted compound YHB-5 Authors : Jiayao Lu , Qiuju Bu , Haibo Yue , Mingming Gao , Danyan Huang , Yongqi Lv , Ziwe Zhang , Xiaohui Zhu , Zhi Ren , and Shasha Song [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176848048.85869925/v1 188 views 75 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose: Hepatocellular carcinoma (HCC) poses a significant threat to human life due to its high mortality and recurrence rates. Human Golgi complex Glycoprotein 1 (GLG1) serves as a biomarker for various cancers, but its role in HCC remains unreported. This study aimed to investigate the function and mechanism of GLG1 in the malignant progression of HCC. Experimental Approach: GLG1 expression in HCC was analyzed using clinical databases, qRT-PCR, and Western blot. The potential roles of GLG1 in proliferation, migration, and invasion were determined via CCK-8, colony formation, wound healing, and Transwell assays. Downstream pathways were identified by gene enrichment analysis and Western blot. Potential GLG1-binding compounds were predicted using the DGldb database, leading to the synthesis and optimization of YHB-5, which was tested for its anti-tumor activity. Key Results: Knockdown of GLG1 significantly inhibited the proliferation, migration, and invasion of Huh-7 and MHCC97H cells. Mechanistically, GLG1 was found to exert its oncogenic role by regulating the P53/P21 pathway. Furthermore, the novel small molecule compound YHB-5 demonstrated potent anti-tumor activity against multiple HCC cell lines. Conclusion and Implications: Our findings reveal that GLG1 promotes HCC progression via the GLG1/P53/P21 axis. The newly synthesized GLG1-targeting compound, YHB-5, represents a promising candidate drug, highlighting the potential of GLG1 as a therapeutic target for the diagnosis and treatment of hepatocellular carcinoma. Mechanism of GLG1 driving malignant progression of hepatocellular carcinoma and development of targeted compound YHB-5 Jiayao Lu 1,2 *, Qiuju Bu 1,2 *, Haibo Yue 1,2 , Mingming Gao 1 , Danyan Huang 3 , Yongqi Lv 4 , Ziwe Zhang 5 , Xiaohui Zhu 1 # , Zhi Ren 1 # , Shasha Song 1, # 1 College of Pharmacy, Shenzhen Technology University, Shenzhen 518118, Guangdong, China 2 College of Pharmacy, Shenzhen University Medical School, Shenzhen University, Shenzhen 518055, Guangdong, China 3 Institute of Applied Higher Education Research, Shenzhen Technology University, Shenzhen 518118, Guangdong, China 4 School of Pharmacy, Jiangsu University, Zhenjiang 212013, Jiangsu, China 5 Pharmacy Department, Shenzhen Second People’s Hospital, Shenzhen 518035, Guangdong, China *These authors contributed equally to this work. # Corresponding author: Shasha Song ( [email protected] ), Zhi Ren (renzhi@)sztu.edu.cn), Xiaohui Zhu ( [email protected] ). Abstract: Background and Purpose: Hepatocellular carcinoma (HCC) poses a significant threat to human life due to its high mortality and recurrence rates. Human Golgi complex Glycoprotein 1 (GLG1) serves as a biomarker for various cancers, but its role in HCC remains unreported. This study aimed to investigate the function and mechanism of GLG1 in the malignant progression of HCC. Experimental Approach: GLG1 expression in HCC was analyzed using clinical databases, qRT-PCR, and Western blot. The potential roles of GLG1 in proliferation, migration, and invasion were determined via CCK-8, colony formation, wound healing, and Transwell assays. Downstream pathways were identified by gene enrichment analysis and Western blot. Potential GLG1-binding compounds were predicted using the DGldb database, leading to the synthesis and optimization of YHB-5, which was tested for its anti-tumor activity. Key Results: Knockdown of GLG1 significantly inhibited the proliferation, migration, and invasion of Huh-7 and MHCC97H cells. Mechanistically, GLG1 was found to exert its oncogenic role by regulating the P53/P21 pathway. Furthermore, the novel small molecule compound YHB-5 demonstrated potent anti-tumor activity against multiple HCC cell lines. Conclusion and Implications: Our findings reveal that GLG1 promotes HCC progression via the GLG1/P53/P21 axis. The newly synthesized GLG1-targeting compound, YHB-5, represents a promising candidate drug, highlighting the potential of GLG1 as a therapeutic target for the diagnosis and treatment of hepatocellular carcinoma. Key words： GLG1, HCC, Bioinformatics, P53/P21 Pathway Bullet point summary What is already known? GLG1 is an established diagnostic biomarker for multiple cancer types. What does this study add? GLG1 drives HCC progression via the P53/P21 signaling pathway. A novel GLG1-targeting compound, YHB-5, exhibits potent anti-HCC activity. What is the clinical significance? YHB-5 is a promising candidate drug for hepatocellular carcinoma treatment. 1.Introduction Hepatocellular carcinoma (HCC) is the main form of primary liver cancer, making up 75% to 85% of cases. It’s also the sixth most common cancer worldwide, with 906,000 new cases each year. Additionally, it’s the third leading cause of cancer-related deaths, responsible for 830,000 deaths annually (Sung et al., 2021; Bray et al., 2024). In China, HCC is the second leading cause of cancer-related deaths, accounting for 47.12% of global HCC deaths. Its high mortality rate is associated with challenges in early diagnosis, chemotherapy resistance, multiple lesions, and high rates of metastasis and recurrence (a postoperative five-year recurrence rate of >70%) (Vogel et al., 2022; Sidali et al., 2022; Galle et al., 2018; Hoffman & Mehta, 2021). In current treatment strategies, the median survival of 30% of patients can only be extended to 10.7-13.6 months by targeted therapies such as sorafenib, and resistance often develops (Wang et al., 2015). Additionally, the objective response rate (ORR) of PD-1/PD-L1 inhibitors (such as pembrolizumab) as monotherapy is less than 20% (Cheng et al., 2009; Sangro et al., 2020; Kudo et al., 2018; El-Khoueiry et al., 2017), therefore there is an urgent need to explore new molecular targets and intervention strategies. The P53/P21 pathway serves as a core tumor suppressor mechanism. When cells undergo stress responses, such as hypoxia, DNA damage, or oncogene activation, P53 undergoes stable activation through phosphorylation at Ser15/20 by ATM/ATR kinases. The activated P53 then activates the cell cycle regulatory factor P21 (CDKN1A) (Levine & Oren, 2009; Bunz et al., 1998), DNA repair-related factors such as GADD45 and XPC, and pro-apoptotic genes such as Bax and PUMA (Kulesza et al., 2019), thereby participating in the regulation of biological processes such as the cell cycle, DNA damage repair, and apoptosis. However, >50% of tumors harbor TP53 mutations (Joerger & Fersht, 2016), leading to the inactivation of the P53/P21 pathway, abnormal proliferation, treatment resistance, and immune escape (Wang et al., 2024; Hobor et al., 2024). Therefore, restoring P53/P21 function may be key to improving HCC treatment (Swisher et al., 1999; Miyachi et al., 2009; Wiman, 2006). Human Golgi protein 1 (GLG1) is a transmembrane protein located on chromosome 16q22-q23 (Mourelatos et al., 1995), which produces different subtypes through alternative splicing. Membrane-localized GLG1 mediates E-selectin-dependent tumor metastasis (Yamaguchi et al., 2022; Esposito et al., 2019), and Golgi-retarded GLG2 regulates intracellular storage of FGF and processing of TGF-β precursors (Zuber et al., 1997; Yang et al., 2010). GLG1 can serve as a biomarker for diagnosing Ewing sarcoma with 97.5% specificity when co-expressed with BCL11B (Orth et al., 2020) and for predicting prostate cancer bone metastasis (AUC = 0.79 (Yasmin-Karim et al., 2014)), as well as promoting the proliferation and migration of gastric cancer (Cuijuan et al., 2020), colorectal cancer (Hao et al., 2020), prostate cancer (Guo et al., 2023), and breast cancer bone metastasis cells (Esposito et al., 2019), the mechanism of GLG1’s action in HCC remains unclear. GOLM1, another Golgi apparatus protein, is significantly upregulated in HCC. It plays a crucial role in the early diagnosis of hepatocellular carcinoma, the assessment of postoperative recurrence, and poor prognosis (Zhang et al., 2023; Mao et al., 2010; Ye et al., 2016) Additionally, GOLM1 activates the AKT/mTOR signaling pathway, which phosphorylates MDM2 and increases P53 ubiquitination and degradation. GOLM1 also regulates the TGF-β1/Smad-MMP2 axis, inducing EMT and driving the malignant progression of HCC (Chen et al., 2015; Spano & Colanzi, 2022). This suggests that GLG1 may regulate the P53 pathway through a similar mechanism. Studies have shown that, in lung cancer, GOLM1 inhibits p53 tetramer formation by inducing S315 phosphorylation. This leads to reduced p53 stability and weakened tumor suppressor function (Song et al., 2021). However, research on the interaction between GLG1 and the p53/p21 pathway in HCC is lacking. The expression pattern and clinical significance of GLG1 in HCC were analyzed by our research system, and the mechanism by which GLG1 influences the malignant phenotype of HCC mediated by P21 through regulating the stability of P53 and the phosphorylation of Ser15 was further elucidated. Additionally, based on virtual screening, we developed a small-molecule compound, YHB-5, that targets GLG1 and validated its efficacy in inhibiting HCC by restoring the P53/P21 pathway. This study provides new strategies for targeted therapy in HCC and deepens our understanding of the interaction network between Golgi proteins and tumor suppressor pathways. 2.Materials and methods Bioinformatics data analysis STAR-counts data and corresponding clinical information for normal liver tissue, HCC, and adjacent tumor tissue were obtained from the TCGA database(https://portal.gdc.cancer.gov), GEO database (Barrett et al., 2013)(https://www.ncbi.nlm.nih.gov/geo/), and GTEx database (GTEx, 2013)(https://gtexportal.org/home/datasets). The data were then extra-cted in TPM format and normalized using log2(TPM+1). Subsequently, sample-s with both RNA-seq data and clinical information were retained for further analysis. We performed a differential analysis of mRNA and protein expression levels, a correlation analysis of clinical and pathological features, a survival analysis, a prognostic correlation analysis, an immune-related analysis, a biologic-al function and pathway correlation analysis, a co-expressed genes analysis and a drug screening using various databases, including UALCAN, HPA, Kaplan-Meier Plotter, GEPIA2, TIMER, LinkedOmics, DAVID and DGLdb. Cell lines and cell culture MIHA cells (Hunan Fenghui Biotechnology Co., Ltd.) were cultured in RPMI-1640 complete medium containing 10% FBS and 1% Penicillin-Streptomycin Solution at 37°C, 95% O2, and 5% CO2 in a constant temperature and humidity cell culture incubator. MHCC-97H (Wuhan Savier Biotechnology Co., Ltd.), HUH-7 (Dalian Meilun Biotechnology Co., Ltd.), HCC-LM3 (Wuhan Savier Biotechnology Co., Ltd.), and HLF (Nanjing Kebai Biotechnology Co., Ltd.). Ltd.), and HEPG2 cells (Shanghai Institute of Life Sciences, Chinese Academy of Sciences Cell Bank) were cultured in DMEM high-glucose complete medium containing 10% FBS and 1% Penicillin-Streptomycin Solution. The cells were maintained at 37°C, 95% O₂, and 5% CO₂ in a constant temperature and humidity cell culture incubator. All cells in question have undergone rigorous sterility, mycoplasma, and STR testing. Real-time fluorescence quantitative PCR Total RNA was obtained from HCCs using TRIzol reagent(15596026CN,Invitrogen) and its concentration and purity was assessed by microplate reader(Biotek).Subsequently cDNA was gained by reverse transcription following the procedure of PrimeScript TM RT Master Mix(RR036A,Takara).The PCR reaction solution containing SYBR Green 1 dye was prepared according to the TB Green Premix Ex Taq II kit(RR820A,Takara) and placed on a QuantStudio 5 Realtime PCR instrument(Applied Biosystems) for PCR amplification. An invariant mRNA of GAPDH was used as an internal control. The primers sequences used are listed as follows: GLG1(Gene ID:2734),sense:5´-TGG AGT TAC GCA GCA AAG G-3´,and antisense:5´-ACC TGT ATT GTA GCC TGT CCT T-3´; P21(Gene ID:1026),as follows:sense:5´- TCC AGC GAC CTT CCT CAT CCA C-3´,and antisense:5´- TCC ATA GCC TCT ACT GCC ACC ATC-3´; P53(Gene ID:7157),as follows:sense:5´-ATG AGC CGC CTG AGG TTG G-3´,and antisense:5´-CAG TGT GAT GAT GGT GAG GAT GG-3´. Western Blotting Analysis Proteins from HCC cells line and MIHA were solubilized and extract with cold lysis buffer which was contained RIPA(MA0151, Meilunbio), Protease Inhibitor Cocktail 1mM(MB2678, Meilunbio), Protease Phosphatase Inhibitor Cocktail 1mM(MB12707, Meilunbio), and the supernatants were collected after ultrasonication and centrifugation.BCA Protein Assay Kit(MA0082,Meilunbio) was used for protein quantification.Then the total protein mixing with loading buffer was heated for 5 min at 100℃.Equivalent amounts of proteins(20-30μg) were separated through 10% or 12.5% SDS-PAGE and transferred onto PVDF membrane(Immobilon-P,Ireland).After blocked with PBST Buffer containing 5% non-Fat milk for 2h,membranes were incubated with primary antibodies at indicated dilutions overnight at 4℃ and respective secondary antibodies for 2h at room temperature subsequently.Enhanced chemiluminescence reagents(MA0186-2,Meilunbio) were used to visualized the protein bands and the densities of the band were quantified by a Bio-Rad gel scanner. The following antibodies were applied: Anti-GLG1 antibody (Rabbit monoclonal [EPR24347-15], 1:1000, abcam, Cat#ab271182) Ati-GAPDH Monoclonal antibody (1E6D9, 1:50000, proteintech, Cat#60004-1-Ig, RRID: AB_2107436) HRP-conjugated Goat Anti-Rabbit IgG(H+L) (1:5000, proteintech, Cat#SA00001-2, RRID: AB_2722564) HRP-conjugated Goat Anti-Mouse IgG(H+L)(1:5000, proteintech, Cat#SA00001-1, RRID: AB_2722565) Ant-PCNA Monoclonal antibody(10D10E11, 1:10000, proteintech, Cat#60097-1-Ig, RRID: AB_2236728) Anti-E-cadherin Polyclonal antibody(1:50000, proteintech, Cat#20874-1-AP, RRID: AB_10697811) Anti-MMP7 Rabbit pAb(1:500, Wanleibio, Cat#WL04679, RRID:AB_3697612) Anti-P21 Polyclonal antibody(1:2000, proteintech, Cat#10355-1-AP, RRID: AB_2077682) Anti-P53 Polyclonal antibody(1:10000, proteintech, Cat#10442-1-AP, RRID: AB_2206609) Anti-P53 Monoclonal antibody(6C4B6, 1:10000, proteintech, Cat#60283-2-Ig, RRID: AB_2881401) Anti-Phospho-P53(Ser15) Antibody(1:1000, Cell Signaling Technology, Cat#9284, RRID:AB_331464) SiRNA,ShRNA Design and Transfection Plasmid and SiRNA against GLG1 and siNC were designed by Genechem(Shanghai,China).Non-target control siRNA(siNC) and shRNA(shCon) were used as the negative control.They were trans The sequence of siRNA and shRNA were as follows: SiGLG1#1(sense:5´-GCU GUG UUC UCU UGU UUA UTT-3´,and antisense:5´-AUA AAC AAG AGA ACA CAG CTT-3´); SiGLG1#2(sense:5´-GGG AGC AUC UGU AUA UUG UTT-3´,and antisense:5´-ACA AUA UAC AGA UGC UCC CTT-3´); ShGLG1(CCT GTA AAG CTG ACA TTC CTA); and siNC(sense:5´-GCG ACG AUC UGC CUA AGA UdTdT-3´,and antisense:5´-AUC UUA GGC AGA UCG UCG CdTdT-3´); ShCon(TTC TCC GAA CGT GTC ACG T) Huh-7 and MHCC97H were cultured until cell confluence was 30%-60%.Then cells were transfected according to the transfection protocol of transfection reagent Lipofectamine 3000 reagent(L3000015, thermo fisher scientific) and Lipofectamine 2000 reagent(11668019, thermo fisher scientific) Cell proliferation assay In order to perform a comprehensive assessment of the proliferation capacity of HCC cell lines following GLG1 knockdown and drug treatment, this study employed a combination of the Cell Counting Kit-8 (CCK-8)(MA0218, Meilunbio) assay and the cell colony formation assay. For the CCK-8 assay, 3 × 10³ cells were added to each well of a 96-well plate, with 3–6 replicates per group, and then cultured in an incubator for 6–24 hours. Subsequent measurements were obtained on days 2, 3, 4, and 5. The conventional medium was aspirated, and 100 µl of fresh medium containing 10% CCK-8 reagent was added to each well. After an incubation of 1–2 hours, the absorbance was measured at 450 nm using a microplate reader. To investigate the short-term effects of drugs on HCC cell proliferation, HCC cell lines were exposed to different concentration gradients (final concentration 0–100 μM). CCK-8 assays were performed at 24, 48, and 72 hours post-drug treatment. For the colony formation assay, HCC cells were seeded at low density (2000 cells/well for Huh-7, 1000 cells/well for MHCC97H, HCCLM3, and HLF) into 12-well plates. The medium was changed every three days, and the cell status was observed. The cultivation process was terminated after a period of 14 days or when the majority of individual clones contained more than 50 cells. To investigate the long-term effects of the drug on HCC cell proliferation, after HCC cells adhered to the culture surface, they were treated with drug-containing medium for 48 hours. Then, the medium was replaced with drug-free medium, and cultivation continued until termination. After colony formation, the colonies were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 30–60 minutes, stained with crystal violet for 30 minutes, rinsed with running water, dried, photographed, and counted using ImageJ software for statistical analysis. Wound-Healing Assay Transfected or untransfected cells were seeded at a density of 4x10 4 cells/well in a 12-well plate and allowed to adhere overnight. A 200 μl pipette tip was utilized to create a scratch perpendicular to the cell layer. Subsequently, the cells were washed twice with DMEM to remove non-adherent cells, thereby rendering the gaps left by the scratch clearly visible. Thereafter, fresh medium containing 2% serum or a medium containing the drug was added. Observations and photographs were recorded at 24-hour intervals until the cells in the control group demonstrated nearly complete healing, at which point the recording process was halted. Finally, the ImageJ image processing software was employed to calculate the migration area for statistical analysis. Transwell assay Transfected Huh-7 and MHCC97H cells or normal Huh-7 and HLF cells were digested with trypsin and subsequently subjected to centrifugation (1000 rpm, 3 min). Thereafter, the cells were washed twice with PBS and resuspended in serum-free DMEM medium or serum-free drug-containing DMEM medium, adjusting the density (Huh-7: 1.2 × 10⁵ cells/150 μl; MHCC97H: 2.5 × 10⁵ cells/150 μl; HLF: 8 × 10⁴ cells/150 μl). The lower chamber was added with 600 μl of complete medium containing 20% FBS or drug-containing medium, and the upper chamber was injected with 150 μl of cell suspension. The cells were cultured for 36 h (Huh-7) or 72 h (MHCC97H, HLF). After removing the chamber, rinse twice with PBS, fix with 4% paraformaldehyde for 30 min, and stain with 0.1% crystal violet for 30 min. The unmigrated cells must be meticulously removed from the upper chamber using a cotton swab, followed by thorough drying. A random selection of three fields of view (accommodated by the 10× objective lens) is then imaged under an inverted microscope. The number of migrated cells was then quantified using ImageJ software. Tumor formation experiments in mice BALB/c-nu mice (4–6 weeks old) were obtained from Zhuhai Baitong Biotechnology Co., Ltd. All animals involved in the experiment were approved by the Animal Ethics Committee. BALB/c-nu mice were divided into two groups of eight mice each . MHCC97H cells were transfected with ShCon or ShGLG1 for 48 hours, then washed and resuspended. The cells were inoculated at a dose of 5 × 10 6 cells per mouse into the left axillary region of mice. Tumor length and width were measured at 4-day intervals beginning on day 7 post-inoculation, and tumor volume was calculated and plotted on a growth curve. Cycloheximide (CHX) Chase Assay Add 100 μg/ml CHX solution to Huh-7 cells transfecting siRNA for 48 hours, and extract cell proteins at various differenttime points (0, 4, 8, 12 hours) after the addition of the CHX solution. These proteins will then be used in subsequent Western blot experiments. Data and Statistical Analysis The analysis, processing, and visualization of all data were conducted using statistical software ImageJ and GraphPad Prism 8.0. The final data are presented as mean ± standard deviation (mean ± SD).Student’s t-test was used to compare two independent samples. For comparisons involving multiple groups of data, Dunnett’s multiple comparisons test following one-way ANOVA or Sidak’s multiple comparisons test following two-way ANOVA was employed. The statistical data were obtained from three or more independent replicate experiments. A p-value < 0.05 was considered statistically significant. 3.Results 3.1 GLG1 is upregulated in HCC and correlates with survival, prognosis, and immune status. To predict GLG1 expression in human HCC tissues, we analyzed mRNA expression data from the TCGA database (371 tumors [T] vs. 50 normal [N]), GEO dataset GSE36376 (240 T vs. 193 N), and GETx database (226 normal samples). Results demonstrated that GLG1 mRNA was significantly upregulated in HCC tumor tissues compared to normal tissues in both TCGA (Fig 1A-C) and GSE36376 (Fig 1D) . Consistent with mRNA findings, protein expression analysis using UALCAN (Fig 1E) and HPA (Fig 1F) databases confirmed elevated GLG1 protein levels in HCC. Logistic regression analysis revealed that high GLG1 expression significantly correlated with adverse clinicopathological features, including advanced T stage (T2&T3&T4 vs. T1, P = 0.013), pathological stage (III & IV vs. I & II, P = 0.022), tumor status (tumor vs. tumor-free, P = 0.044), higher histological grade (G3 & G4 vs. G1 & G2, P = 0.003), as well as gender (P = 0.012), race (P = 0.006), age (P = 0.037), and liver function grade (B & C vs. A, P = 0.028) (Fig 1G) . These findings indicate that HCC patients with elevated GLG1 expression are more prone to advanced stage, higher grade, and poor differentiation compared to those with low GLG1 expression. Given the established link between TP53 mutation and HCC, we investigated its association with GLG1. A significant positive correlation was observed, with markedly increased GLG1 expression in HCC patients harboring TP53 mutations (Fig 1H) . Kaplan-Meier survival analysis via the Kaplan-Meier Plotter database (using optimal cutoff values) demonstrated that high GLG1 expression was significantly associated with poorer overall survival (OS) (Fig 1I) and shorter recurrence-free survival (RFS) (Fig 1J) in HCC patients. Log-rank tests confirmed statistical significance. Univariate Cox regression analysis identified T stage, M stage, pathological stage, tumor status, and GLG1 expression level as significant risk factors (P < 0.05) for shorter OS in HCC patients (Fig 1K) . Multivariate Cox analysis incorporating variables with P < 0.2 from the univariate analysis further confirmed that advanced pathological stage, recurrent tumor status, and high GLG1 expression were independent prognostic indicators for poor OS (Fig 1L) . Analysis using the TIMER database revealed significant positive correlations between GLG1 expression levels and the abundance of tumor-infiltrating immune cells, including B cells (r = 0.35, P < 0.0001), CD8⁺ T cells (r = 0.349, P < 0.0001), CD4⁺ T cells (r = 0.378, P < 0.0001), macrophages (r = 0.48, P < 0.0001), and neutrophils (r = 0.472, P < 0.0001) (Fig 1M) . These results suggest a potential role for GLG1 in modulating the tumor immune microenvironment in HCC. (A–D) GLG1 mRNA expression in normal/adjacent tissue and hepatocellular carcinoma (HCC) tissue from the UALCAN database, combined TCGA and GETx databases, and GSE36376. (E) UALCAN database analysis of GLG1 protein expression in normal tissue and HCC tissue from TCGA. (F) HPA database analysis of GLG1 protein expression in normal tissue and HCC tissue from TCGA. (G) Logistic regression analysis of the correlation between GLG1 expression and clinical pathological characteristics. (H) UALCAN database analysis of GLG1 expression in normal tissue and HCC patient tissue with TP53 mutations and non-mutations. (I-J) Kaplan-Meier Plotter database analysis of overall survival (OS) and recurrence-free survival (RFS) in patients with high and low GLG1 expression. (K-L) Forest plots of univariate and multivariate COX regression analyses in hepatocellular carcinoma patients. (M) TIMER database analysis of the correlation between GLG1 expression and immune cell infiltration levels. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. 3.2 Biological function of GLG1 and GESA enrichment analysis Using the LinkedOmics database (filtering criteria: |Pearson’s r| ≥ 0.2, FDR < 0.01,P< 0.05), we identified 5218 genes significantly co-expressed with GLG1 (3880 positively correlated, 1338 negatively correlated) in HCC (Fig 2A) . Differential gene expression analysis (|log₂FC| ≥ 1, padj < 0.05) between GLG1-high and GLG1-low HCC groups (stratified by median expression in TCGA) using DEseq2 identified 2411 significantly dysregulated genes (1857 upregulated, 554 downregulated) (Fig 2B) . Intersection analysis of co-expressed genes and differentially expressed genes (DEGs) yielded 770 overlapping genes highly associated with GLG1 (Fig 2C) . Gene Ontology (GO) enrichment analysis of these 770 genes via DAVID revealed significant enrichment in specific biological processes (BP), cellular components (CC), and molecular functions (MF). Top enriched BPs included extracellular matrix organization, regulation of cell proliferation, epithelial to mesenchymal transition, and cell-matrix adhesion. Enriched CC terms involved protein complexes in cell-substrate adhesion, basement membrane, Golgi lumen, and Golgi membrane. Key enriched MFs included transcription factor activity, calcium channel activity, WNT-activated receptor activity, G protein-coupled peptide receptor activity, and epidermal growth factor receptor binding (Fig 2D) . Thereafter, Gene Set Enrichment Analysis (GSEA) based on KEGG and Hallmark gene sets was performed to elucidate potential molecular mechanisms regulated by GLG1. KEGG analysis showed significant enrichment of DEGs in pathways crucial for cancer progression, including WNT signaling, pathways in cancer, cell cycle, TGF-β signaling, and PPAR signaling. Hallmark analysis revealed enrichment in P53 pathway, E2F targets, PI3K-AKT-mTOR signaling, and DNA repair pathways (Fig 2E) . Notably, GLG1-high tumors exhibited activation of key genes in the TGF-β family (TGF-β1, TGF-β2, TGF-β3), CDK family (CDK1, CDK2, CDK4, CDK6), E2F pathway (E2F3, E2F4, E2F5), CREBBP, BCL-2, TP53A1P1, and THBS1 (Fig 2F) . This suggests GLG1 may promote HCC proliferation by influencing cell cycle regulation and P53 pathway activity, leading us to focus on the P53/P21 signaling axis. Fig 2 Biological function of GLG1 and GESA enrichment analysis. (A) Volcano plot and heatmap of co-expressed genes of GLG1 in the LinkedOmics database. The heatmap shows the top 50 positively and negatively correlated genes. (B) Volcano plot of differentially expressed genes between high- and low-expression groups of GLG1. (C) Venn diagram of the intersection between the selected differentially expressed genes (DEGs) and co-expressed genes (Co). (D) GO-BP, CC, and MF biological function enrichment analysis of the intersecting genes in the DAVID database. (E) GSEA enrichment analysis of the differentially expressed genes in the high- and low-expression groups of GLG1. (F) Visualization results of the GSEA enrichment analysis for the Cell Cycle and P53 pathways. 3.3 Knockdown of GLG1 inhibits proliferation of HCC cells in vitro and in vivo. Consistent with database results, GLG1 mRNA and protein levels were significantly elevated in human HCC cell lines (HepG2, Huh-7, HCCLM3, MHCC97H, HLF) compared to the normal human hepatocyte line MIHA (Fig 3A, B) . To investigate GLG1’s functional role, we successfully knocked down GLG1 expression in Huh-7 and MHCC97H cells using siRNA (siGLG1#1, siGLG1#2) (Fig 3C-F) . CCK-8 assays demonstrated that GLG1 knockdown significantly impaired the proliferation capacity of both Huh-7 (Fig 3G) and MHCC97H (Fig 3H) cells compared to negative control (siNC) (P< 0.001). Similarly, colony formation assays revealed a significant reduction in the number of colonies formed by siGLG1-transfected cells (Fig 3I-J) (P< 0.01). Western blot analysis further showed decreased expression of the proliferation marker PCNA upon GLG1 knockdown (Fig 3K-L) (P< 0.05). These results collectively indicate that Knockdown of GLG1 promotes the in vitro proliferation of HCC cells. Subcutaneous injection of MHCC97H cells stably expressing shGLG1 or shCon into BALB/c-nu mice demonstrated that GLG1 knockdown significantly inhibited tumor growth in vivo, resulting in smaller tumor volumes and reduced tumor weights compared to the shCon group (Fig. 3M-N). Above findings confirm that GLG1 expression enhances HCC proliferative capacity. Fig 3 Knockdown of GLG1 inhibits proliferation of HCC cells in vitro and in vivo. (A–B) mRNA and protein expression levels of GLG1 in normal human liver cells (MIHA) and various HCC cell lines. (C–F) Validation of small interfering RNA transfection efficiency in Huh-7 and MHCC97H cells. (G-H) CCK-8 proliferation assay to detect the effect of GLG1 knockdown on the in vitro proliferation capacity of HuH-7 and MHCC97H cells. (I-J) Colony formation assay to validate the effect of GLG1 knockdown on the colony formation capacity of HuH-7 and MHCC97H cells. (K-L) Western blot assay to validate PCNA expression and quantitative analysis. (M) Growth curve of tumors in BALB/c-nu after injection of transfected MHCC97H cells. (N) Tumor morphology and volume at the end of the in vivo experiment. *, p <0.05; **, p <0.01; ***, p <0.001; ****, p <0.0001. 3.4 Knockdown of GLG1 inhibits migration and invasion of HCC cells. Wound healing assays showed that GLG1 knockdown significantly attenuated the migratory ability of both Huh-7 (Fig 4A) and MHCC97H (Fig 4B) cells compared to siNC. Transwell migration assays corroborated these findings, revealing a significant decrease in the number of HCC cells passing through the polyester membrane (Fig 4C,D) . Western blot analysis indicated that GLG1 knockdown led to decreased expression of the pro-invasive protein MMP7 and increased expression of the epithelial marker E-cadherin in both cell lines (Fig 4E). These data demonstrate that GLG1 promotes the migratory and invasive potential of HCC cells in vitro. Fig. 4. Knockdown of GLG1 inhibits migration and invasion of HCC cells. (A-B) Scratch assay to validate the in vitro migration ability of HuH-7 and MHCC97H cells after GLG1 knockdown.(C-D) Transwell assay to validate the in vitro migration ability of HuH-7 and MHCC97H cells after GLG1 knockdown.(E) Western blot to detect MMP7 and E-cadherin protein expression and quantitative data analysis.*， p <0.05；**， p <0.01；***， p <0.001. 3.5 GLG1 promotes HCC cells proliferation by suppressing the P53/P21 pathway. RT-qPCR analysis showed that GLG1 knockdown in Huh-7 (Fig 5A) and MHCC97H (Fig 5B) cells significantly increased the mRNA levels of P53 and P21. Consistently, Western blot analysis revealed elevated protein levels of total P53, P21, and notably, phosphorylated P53 (Ser15) in siGLG1-transfected cells compared to siNC (Fig 5C, D) . Since phosphorylation at Ser15 is associated with P53 stabilization, we performed protein degradation kinetics analysis. Results demonstrated that GLG1 knockdown slowed the degradation rate of P53 protein in Huh-7 cells (Fig 5E) . These findings suggest that GLG1 promotes HCC proliferation by inhibiting P53 phosphorylation (Ser15), thereby destabilizing P53 and suppressing the P53/P21 signaling pathway. Fig 5 GLG1 promotes HCC cells proliferation by suppressing the P53/P21 pathway. (A-B) RT-qPCR experiments were conducted to detect the mRNA expression levels of P53 and P21 in HuH-7 and MHCC97H cells treated with siRNA transfection. (C-D) Western blot experiments were performed to detect the protein expression levels of p-p53 (Ser 15), P53, and P21 in SiRNA-transfected HuH-7 and MHCC97H cells. (E) Western blot analysis was performed to detect and quantify the protein expression levels of P53 in siRNA-transfected HuH-7 cells treated with CHX. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. 3.6 Prediction, optimization, and anticancer activity of small molecule compounds with potential GLG1 binding activity. Screening the DGIdb database identified 9 compounds with potential GLG1 binding activity (Fig 6A) . TTK21, a more chemical accessible candidate, was selected for initial evaluation. Anticancer activity assessment over 24 h, 48 h, and 72 h revealed that TTK21 exhibited cytotoxic effects only in Huh-7 and HCCLM3 cells among the four HCC lines tested (Huh-7, MHCC97H, HCCLM3, HLF) (Fig 6B) . The IC₅₀ values for TTK21 in Huh-7 cells were 59.30 μM (48 h) and 21.01 μM (72 h), and 32.18 μM (72 h) in HCCLM3 cells. Keeping the N-phenylbenzamide core of TTK21 as a scaffold, we synthesized seven derivatives (YHB-1 to YHB-7) (Fig 6C) . CCK-8 assays in MHCC97H cells identified YHB-5, YHB-6, and YHB-7 as having superior anticancer activity compared to TTK21 (Fig 6D) . Further evaluation across Huh-7, HLF, and HCCLM3 cell lines confirmed that YHB-5 possessed the most potent anticancer activity (Fig 6E-G) . Consequently, YHB-5 was selected for subsequent studies. Compared to TTK21, YHB-5 demonstrated significantly enhanced cytotoxicity at 72 h across all tested HCC cell lines, with the most pronounced effect observed in HLF cells (Fig 6H) , indicating successful structural optimization. Fig 6 Prediction, optimization, and anticancer activity of small molecule compounds with potential GLG1 binding activity. (A) Screening results from DGIdb and structural representation of TTK21. (B) CCK-8 assay to assess the anticancer activity of TTK21. (C) Structures of the optimized compounds of TTK21. (D) Evaluation of the anticancer activity of each compound. (E–G) CCK-8 assay to assess the effects of YHB-5 (E), YHB-6 (F), and YHB-7 (G) on the viability of Huh-7, HLF, and HCCLM3 cells. (H) Comparison of the IC50 values of YHB-5 and TTK21 in various HCC cell lines after 72 hours.*， p <0.05；**， p <0.01；***， p <0.001；****， p <0.0001 . 3.7 YHB-5 inhibits HCC cells proliferation and migration by suppressing GLG1 and activating the P53/P21 pathway. Colony formation assays demonstrated that YHB-5 treatment concentration-dependently suppressed the proliferation of MHCC97H (Fig 7A) , Huh-7 (Fig 7B) , HLF (Fig 7C) , and HCCLM3 (Fig 7D) cells, significantly reducing colony numbers. Combined with the data of CCK-8 assay, these results confirm the potent anti-proliferative effect of YHB-5 in HCC cells. Furthermore, wound healing (Fig 7E,F) and Transwell migration assays (Fig 7G,H) revealed that YHB-5 concentration-dependently inhibited the migration of Huh-7 and HLF cells, significantly decreasing the number of migrated cells. Furthermore, molecular docking simulations using AutoDock Vina and PyMol predicted a binding affinity between YHB-5 and GLG1 with a binding energy of -5.15 kcal/mol. Key interaction residues included ALA-626, THR-653, TYR-534, and ARG-695, forming hydrogen bonds of lengths 3.1 Å, 2.7 Å, 3.2 Å, and 2.3 Å, respectively (Fig 7I) . Based on the CCK-8 assay and cell plate cloning results, the antiproliferative effect of YHB-5 on Huh-7 cells is evident. Therefore, we detected PCNA protein expression by Western blot, and the results confirmed that YHB-5 treatment concentration-dependently downregulated GLG1 expression and the proliferation marker PCNA in Huh-7 cells (Fig 7J,K) . Crucially, YHB-5 activated the P53/P21 pathway: higher concentrations (5 μM) increased p-P53 (Ser15) levels, moderate to high concentrations (3.5 μM, 5 μM) elevated total P53 protein, and P21 expression was significantly upregulated in a concentration-dependent manner (Fig 7K) . These findings indicate that the anticancer mechanism of YHB-5 involves binding to GLG1, suppressing its expression, and subsequently activating the P53/P21 signaling pathway. Discussion Abnormal gene expression, including the upregulation of oncogenes and the silencing of tumor suppressor genes, has been identified as a critical driver of tumorigenesis. In this study, we first employed a comprehensive approach by leveraging multiple databases to procure transcriptomic and proteomic data from normal liver tissue, adjacent non-cancerous tissue, and HCC tissue. Our findings substantiated that GLG1 mRNA and protein levels exhibited a marked increase in HCC tissue compared to normal liver tissue and adjacent non-cancerous tissue. This finding is consistent with the previously reported high expression patterns of GLG1 in colorectal cancer (Hao et al., 2020) and prostate cancer (Guo et al., 2023). Furthermore, our findings indicate that elevated GLG1 expression is associated with unfavorable clinical pathological characteristics, including high grade and advanced stage, TP53 mutations, reduced survival, and immune infiltration in HCC. This suggests that GLG1 may function as a driver of HCC progression, with alterations in its expression levels reflecting the malignancy of the tumor. GLG1 expression was found to be notably higher in male patients, which is consistent with the epidemiological finding that the incidence of HCC is significantly higher in men than in women (Llovet et al., 2021). The high recurrence rate of HCC following surgical intervention has historically posed a significant clinical challenge. The present study found that high GLG1 expression is an independent prognostic risk factor for shorter overall survival in HCC patients. Consequently, by monitoring changes in GLG1 expression, it may be possible to identify patients with poorer prognoses at an early stage. This, in turn, could enable the development of more aggressive and personalized treatment plans for these patients. Abnormal cell proliferation and apoptosis, as well as enhanced invasion and migration, are common phenotypes of tumor cells. PCNA performs several functions in cells, including DNA replication, repair, and cell cycle regulation, and it serves as a marker indicating cell proliferation status (Strzalka & Ziemienowicz, 2011). The role of MMP7 in HCC is primarily its ability to degrade the extracellular matrix (ECM), thereby promoting tumor cell migration and invasion (Lin et al., 2017). EMT is another common indicator for evaluating cell invasion capacity, primarily manifested by the loss of epithelial cell markers such as E-cadherin (Debnath et al., 2021; Liu et al., 2023). In this experiment, it was found that after GLG1 was knocked down, the proliferation capacity of HCC cells (as measured by CCK-8, clonogenic assay, and subcutaneous tumor formation in BALB/c-nu) and migration/invasion capacity (as measured by wound healing assay and Transwell assay) were significantly inhibited. This inhibition was accompanied by a decrease in proliferation markers PCNA and invasion-related protein MMP7, while the EMT marker E-cadherin was upregulated. This finding indicates that GLG1 may promote the malignant potential of HCC by modulating cell proliferation and EMT-related pathways, which is in accordance with the outcomes of GO enrichment analysis. In order to further identify the downstream signaling pathways regulated by GLG1, we first performed GESA enrichment analysis and validated the results by Western blot. In summary, it was determined that GLG1 functions as an inhibitor of the P53/P21 pathway, thereby promoting the progression of HCC. This function is characterized by a reduction in P53 ser15 phosphorylation and a suppression of P53 stability. However, while this study confirmed the existence of the GLG1/P53/P21 axis, the specific mechanism of action between GLG1 and P53 remains uncertain. Determining this mechanism will be a key focus of our future research. The development of targeted therapy for HCC has been hindered by tumor heterogeneity and drug resistance, underscoring the need for the development of highly effective, low-toxicity small-molecule inhibitors that target new mechanisms. In previous studies, we demonstrated the potential of GLG1 as a therapeutic target. Consequently, the development of novel small-molecule inhibitors that specifically target GLG1 and restore P53 pathway activity may represent a promising direction for improving HCC treatment. In recent years, structure-based drug design (SBDD) and computer-aided screening technologies have significantly accelerated the discovery of anticancer small molecules (Cortes-Garcia et al., 2017; Druker et al., 2006). The Drug Gene Interaction Database (DGIdb) was screened to identify TTK21, a compound with potential binding activity to GLG1. Preliminary research suggests a correlation between TTK21 and increased acetyl transferase activity, axonal regeneration, neural development, and repair (Singh et al., 2022; Müller et al., 2022; Singh et al., 2024). However, the anticancer potential of TTK21 remains to be elucidated. The CCK-8 experiments yielded findings that indicated TTK21 exhibited moderate anticancer activity exclusively in Huh-7 and HCCLM3 cells, while it proved ineffective in MHCC97H and HLF cells. Such discrepancies in drug response may be attributable to tumor heterogeneity. Consequently, to enhance the anticancer activity and universality of the drug, we structurally modified and optimized TTK21, and screened the optimized compounds to obtain YHB-5, which exhibits the best anticancer activity against HCC. Through the implementation of molecular simulation docking techniques, we observed a certain degree of binding activity between GLG1 and YHB-5. Furthermore, preliminary validation was conducted to assess the potential of YHB-5 in the inhibition of HCC occurrence and development through the GLG1/P53/P21 axis. It is noteworthy that while YHB-5 displays promising anticancer properties in in vitro models, further evaluation of its pharmacokinetics and toxicity in cell and animal models is necessary to fully ascertain its potential therapeutic benefits. In summary, GLG1 regulates the development of HCC through the P53/P21 pathway, thereby deepening our understanding of the molecular mechanisms by which GLG1 promotes HCC. The identification of YHB-5 signifies a novel paradigm for the targeted development of anticancer drugs. Conclusion GLG1, a novel driver of HCC, has been shown to disrupt p21-mediated tumor suppression by inhibiting p53 stability and phosphorylation at Ser15. The targeted compound YHB-5 has been shown to effectively block this pathway, providing new insights for the development of potential small-molecule compounds for the treatment of HCC. Ethics approval and informed consent All animal experiments in this study were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised edition, 1978). All animal experiments used female mice and were reviewed by the Institutional Animal Care and Use Committee of Shenzhen Rongwan Biotechnology Co., Ltd., in compliance with animal protection, animal ethics, and moral principles. Availability of data and materials None. Funding This work was supported by the National Natural Science Foundation of China (81700056 to SS, 22201184 to ZR), Research Founding of post-doctor who came to Shenzhen (20211063010052 to SS), Natural Science Foundation of Top Talent of SZTU (GDRC202105 to ZR). Authors’ contributions Jiayao Lu and Qiuju Bu conducted most of the experiments, analyzed the data, and wrote the manuscript. Haibo Yue synthesized compounds for bioassays. Mingming Gao and Danyan Huang measured conducted cell culture and transfection experiments. Yongqi Lv and Ziwe Zhang made contributions to animal experiments. Xiaohui Zhu, Zhi Ren and Shasha Song provided assistance with research design and conducted a critical review of the manual. Acknowledgments We wish to thank the publicly available databases, including The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO)] and so on, for making their data accessible, which was instrumental to our analysis. Declaration of competing interest The authors have no conflicts of interest to declare. References Barrett, T., Wilhite, S. E., Ledoux, P., Evangelista, C., Kim, I. F., Tomashevsky, M., Marshall, K. A., Phillippy, K. H., Sherman, P. M., Holko, M., Yefanov, A., Lee, H., Zhang, N., Robertson, C. L., Serova, N., Davis, S., & Soboleva, A. (2013). NCBI GEO: archive for functional genomics data sets–update. Nucleic acids research, 41, D991–D995. https://doi.org/10.1093/nar/gks1193 Bray, F., Laversanne, M., Sung, H., Ferlay, J., Siegel, R. L., Soerjomataram, I., & Jemal, A. (2024). Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians, 74(3), 229–263. https://doi.org/10.3322/caac.21834 Bunz, F., Dutriaux, A., Lengauer, C., Waldman, T., Zhou, S., Brown, J. P., Sedivy, J. M., Kinzler, K. W., & Vogelstein, B. (1998). Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science, 282(5393), 1497–1501. https://doi.org/10.1126/science.282.5393.1497 Chen, X., Wang, Y., Tao, J., Shi, Y., Gai, X., Huang, F., Ma, Q., Zhou, Z., Chen, H., Zhang, H., Liu, Z., Sun, Q., Peng, H., Chen, R., Jing, Y., Yang, H., Mao, Y., & Zhang, H. (2015). mTORC1 Up-Regulates GP73 to Promote Proliferation and Migration of Hepatocellular Carcinoma Cells and Growth of Xenograft Tumors in Mice. Gastroenterology, 149(3), 741–52.e14. https://doi.org/10.1053/j.gastro.2015.05.005 Cheng, A. L., Kang, Y. K., Chen, Z., Tsao, C. J., Qin, S., Kim, J. S., Luo, R., Feng, J., Ye, S., Yang, T. S., Xu, J., Sun, Y., Liang, H., Liu, J., Wang, J., Tak, W. Y., Pan, H., Burock, K., Zou, J., Voliotis, D., … Guan, Z. (2009). Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. The Lancet. Oncology, 10(1), 25–34. https://doi.org/10.1016/S1470-2045(08)70285-7 Cortes-Garcia, C. J., Suárez-Castro, A., Gamez-Montaño, R., & Chacón-García, L. (2017). Docking studies of 1,5-disubtituted tetrazoles analogs of the anticancer drug imatinib as probable inhibitors of the ABL kinase and the T315I mutant kinase. In Proceedings of the 21st International Electronic Conference on Synthetic Organic Chemistry (pp. 1-30). MDPI. https://doi.org/10.3390/ecsoc-21-04734. Cuijuan, Q., Zhurong, X., Luyan, C., Yao, S., & Jun, Y. (2020). lncRNA MAFG-AS1 promotes migration, invasion and aerobic glycolysis of gastric cancer AGS cells by regulating miR-11181-3p/GLG1 axis. Translational Cancer Research, 12(9), 2185–2197. https://doi.org/10.21037/tcr-23-581 Debnath, P., Huirem, R.S., Dutta, P., and Palchaudhuri, S. (2021). Epithelial–mesenchymal transition and its transcription factors. Bioscience Reports. 42(1), BSR20211754. https://doi.org/10.1042/BSR20211754. Druker, B. J., Guilhot, F., O’Brien, S. G., Gathmann, I., Kantarjian, H., Gattermann, N., Deininger, M. W., Silver, R. T., Goldman, J. M., Stone, R. M., Cervantes, F., Hochhaus, A., Powell, B. L., Gabrilove, J. L., Rousselot, P., Reiffers, J., Cornelissen, J. J., Hughes, T., Agis, H., Fischer, T., … IRIS Investigators (2006). Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. The New England journal of medicine, 355(23), 2408–2417. https://doi.org/10.1056/NEJMoa062867 El-Khoueiry, A. B., Sangro, B., Yau, T., Crocenzi, T. S., Kudo, M., Hsu, C., Kim, T. Y., Choo, S. P., Trojan, J., Welling, T. H., Rd, Meyer, T., Kang, Y. K., Yeo, W., Chopra, A., Anderson, J., Dela Cruz, C., Lang, L., Neely, J., Tang, H., Dastani, H. B., … Melero, I. (2017). Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet, 389(10088), 2492–2502. https://doi.org/10.1016/S0140-6736(17)31046-2 Esposito, M., Mondal, N., Greco, T. M., Wei, Y., Spadazzi, C., Lin, S. C., Zheng, H., Cheung, C., Magnani, J. L., Lin, S. H., Cristea, I. M., Sackstein, R., & Kang, Y. (2019). Bone vascular niche E-selectin induces mesenchymal-epithelial transition and Wnt activation in cancer cells to promote bone metastasis. Nature cell biology, 21(5), 627–639. https://doi.org/10.1038/s41556-019-0309-2 GTEx Consortium (2013). The Genotype-Tissue Expression (GTEx) project. Nature genetics, 45(6), 580–585. https://doi.org/10.1038/ng.2653 European Association for the Study of the Liver (2018). EASL Clinical Practice Guidelines: Management of hepatocellular carcinoma. Journal of hepatology, 69(1), 182–236. https://doi.org/10.1016/j.jhep.2018.03.019 Guo, J., Wang, F., Hu, Y., Luo, Y., Wei, Y., Xu, K., Zhang, H., Liu, H., Bo, L., Lv, S., Sheng, S., Zhuang, X., Zhang, T., Xu, C., Chen, X., & Su, J. (2023). Exosome-based bone-targeting drug delivery alleviates impaired osteoblastic bone formation and bone loss in inflammatory bowel diseases. Cell reports. Medicine, 4(1), 100881. https://doi.org/10.1016/j.xcrm.2022.100881 Hao, S., Qu, R., Hu, C., Wang, M., & Li, Y. (2020). A Circular RNA Derived from Golgi Glycoprotein 1 mRNA Regulates KRAS Expression and Promotes Colorectal Cancer Progression by Targeting microRNA-622. OncoTargets and therapy, 13, 12637–12648. https://doi.org/10.2147/OTT.S284032 Hobor, S., Al Bakir, M., Hiley, C. T., Skrzypski, M., Frankell, A. M., Bakker, B., Watkins, T. B. K., Markovets, A., Dry, J. R., Brown, A. P., van der Aart, J., van den Bos, H., Spierings, D., Oukrif, D., Novelli, M., Chakrabarti, T., Rabinowitz, A. H., Ait Hassou, L., Litière, S., Kerr, D. L., … Swanton, C. (2024). Mixed responses to targeted therapy driven by chromosomal instability through p53 dysfunction and genome doubling. Nature communications, 15(1), 4871. https://doi.org/10.1038/s41467-024-47606-9 Hoffman, D., & Mehta, N. (2021). Recurrence of hepatocellular carcinoma following liver transplantation. Expert review of gastroenterology & hepatology, 15(1), 91–102. https://doi.org/10.1080/17474124.2021.1823213 Joerger, A. C., & Fersht, A. R. (2016). The p53 Pathway: Origins, Inactivation in Cancer, and Emerging Therapeutic Approaches. Annual review of biochemistry, 85, 375–404. https://doi.org/10.1146/annurev-biochem-060815-014710 Kudo, M., Finn, R. S., Qin, S., Han, K. H., Ikeda, K., Piscaglia, F., Baron, A., Park, J. W., Han, G., Jassem, J., Blanc, J. F., Vogel, A., Komov, D., Evans, T. R. J., Lopez, C., Dutcus, C., Guo, M., Saito, K., Kraljevic, S., Tamai, T., … Cheng, A. L. (2018). Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial. Lancet, 391(10126), 1163–1173. https://doi.org/10.1016/S0140-6736(18)30207-1 Kulesza, M., Dansonka-Mieszkowska, A., & Pieńkowska-Grela, B. (2019). Repair or perish—the role of p53 protein in a cell’s life.Nowotwory. Journal of Oncology, 69(4), 168–178. https://doi.org/10.5603/NJO.2019.0031 Levine, A. J., & Oren, M. (2009). The first 30 years of p53: growing ever more complex. Nature reviews. Cancer, 9(10), 749–758. https://doi.org/10.1038/nrc2723 Lin, Y., Liu, J., Huang, Y., Liu, D., Zhang, G., & Kan, H. (2017). microRNA-489 Plays an Anti-Metastatic Role in Human Hepatocellular Carcinoma by Targeting Matrix Metalloproteinase-7. Translational oncology, 10(2), 211–220. https://doi.org/10.1016/j.tranon.2017.01.010 Liu, S., Li, L., & Ren, D. (2023). Anti-Cancer Potential of Phytochemicals: The Regulation of the Epithelial-Mesenchymal Transition. Molecules (Basel, Switzerland), 28(13), 5069. https://doi.org/10.3390/molecules28135069 Llovet, J. M., Kelley, R. K., Villanueva, A., Singal, A. G., Pikarsky, E., Roayaie, S., Lencioni, R., Koike, K., Zucman-Rossi, J., & Finn, R. S. (2021). Hepatocellular carcinoma. Nature Reviews Disease Primers, 7, 6. https://doi.org/10.1038/s41572-020-00240-3 Mao, Y., Yang, H., Xu, H., Lu, X., Sang, X., Du, S., Zhao, H., Chen, W., Xu, Y., Chi, T., Yang, Z., Cai, J., Li, H., Chen, J., Zhong, S., Mohanti, S. R., Lopez-Soler, R., Millis, J. M., Huang, J., & Zhang, H. (2010). Golgi protein 73 (GOLPH2) is a valuable serum marker for hepatocellular carcinoma. Gut, 59(12), 1687–1693. https://doi.org/10.1136/gut.2010.214916 Miyachi, M., Kakazu, N., Yagyu, S., Katsumi, Y., Tsubai-Shimizu, S., Kikuchi, K., Tsuchiya, K., Iehara, T., & Hosoi, H. (2009). Restoration of p53 pathway by nutlin-3 induces cell cycle arrest and apoptosis in human rhabdomyosarcoma cells. Clinical cancer research : an official journal of the American Association for Cancer Research, 15(12), 4077–4084. https://doi.org/10.1158/1078-0432.CCR-08-2955 Mourelatos, Z., Gonatas, J. O., Nycum, L. M., Gonatas, N. K., & Biegel, J. A. (1995). Assignment of the GLG1 gene for MGF-160, a fibroblast growth factor and E-selectin binding membrane sialoglycoprotein of the Golgi apparatus, to chromosome 16q22-q23 by fluorescence in situ hybridization. Genomics, 28(2), 354–355. https://doi.org/10.1006/geno.1995.1156 Müller, F., De Virgiliis, F., Kong, G., Zhou, L., Serger, E., Chadwick, J., Sanchez-Vassopoulos, A., Singh, A. K., Eswaramoorthy, M., Kundu, T. K., & Di Giovanni, S. (2022). CBP/p300 activation promotes axon growth, sprouting, and synaptic plasticity in chronic experimental spinal cord injury with severe disability. PLoS biology, 20(9), e3001310. https://doi.org/10.1371/journal.pbio.3001310 Orth, M. F., Hölting, T. L. B., Dallmayer, M., Wehweck, F. S., Paul, T., Musa, J., Baldauf, M. C., Surdez, D., Delattre, O., Knott, M. M. L., Romero-Pérez, L., Kasan, M., Cidre-Aranaz, F., Gerke, J. S., Ohmura, S., Li, J., Marchetto, A., Henssen, A. G., Özen, Ö., Sugita, S., … Grünewald, T. G. P. (2020). High Specificity of BCL11B and GLG1 for EWSR1-FLI1 and EWSR1-ERG Positive Ewing Sarcoma. Cancers, 12(3), 644. https://doi.org/10.3390/cancers12030644 Sangro, B., Park, J., Finn, R., Cheng, A., Mathurin, P., Edeline, J., Kudo, M., Han, K., Harding, J., Merle, P., Rosmorduc, O., Rizzo, A., Neely, J., Yan, L., & Breder, V. (2020). LBA-3 CheckMate 459: Long-term (minimum follow-up 33.6 months) survival outcomes with nivolumab versus sorafenib as first-line treatment in patients with advanced hepatocellular carcinoma. Annals of Oncology, 31(Suppl 4), S241–S242. https://doi.org/10.1016/j.annonc.2020.04.078 Sidali, S., Trépo, E., Sutter, O., & Nault, J. C. (2022). New concepts in the treatment of hepatocellular carcinoma. United European gastroenterology journal, 10(7), 765–774. https://doi.org/10.1002/ueg2.12286 Singh, A. K., Neo, S. H., Liwang, C., Pang, K. K. L., Leng, J. C. K., Sinha, S. H., Shetty, M. S., Vasudevan, M., Rao, V. J., Joshi, I., Eswaramoorthy, M., Pavon, M. V., Sheila, A. R., Navakkode, S., Kundu, T. K., & Sajikumar, S. (2022). Glucose derived carbon nanosphere (CSP) conjugated TTK21, an activator of the histone acetyltransferases CBP/p300, ameliorates amyloid-beta 1-42 induced deficits in plasticity and associativity in hippocampal CA1 pyramidal neurons. Aging cell, 21(9), e13675. https://doi.org/10.1111/acel.13675 Singh, A. K., Rai, A., Joshi, I., Reddy, D. N., Guha, R., Alka, K., Shukla, S., Rath, S. K., Nazir, A., Clement, J. P., & Kundu, T. K. (2024). Oral Administration of a Specific p300/CBP Lysine Acetyltransferase Activator Induces Synaptic Plasticity and Repairs Spinal Cord Injury. ACS chemical neuroscience, 15(15), 2741–2755. https://doi.org/10.1021/acschemneuro.4c00124 Song, Q., He, X., Xiong, Y., Wang, J., Zhang, L., Leung, E. L., & Li, G. (2021). The functional landscape of Golgi membrane protein 1 (GOLM1) phosphoproteome reveal GOLM1 regulating P53 that promotes malignancy. Cell death discovery, 7(1), 42. https://doi.org/10.1038/s41420-021-00422-2 Spano, D., & Colanzi, A. (2022). Golgi Complex: A Signaling Hub in Cancer. Cells, 11(13), 1990. https://doi.org/10.3390/cells11131990 Strzalka, W., & Ziemienowicz, A. (2011). Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation. Annals of botany, 107(7), 1127–1140. https://doi.org/10.1093/aob/mcq243 Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., & Bray, F. (2021). Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: a cancer journal for clinicians, 71(3), 209–249. https://doi.org/10.3322/caac.21660 Swisher, S. G., Roth, J. A., Nemunaitis, J., Lawrence, D. D., Kemp, B. L., Carrasco, C. H., Connors, D. G., El-Naggar, A. K., Fossella, F., Glisson, B. S., Hong, W. K., Khuri, F. R., Kurie, J. M., Lee, J. J., Lee, J. S., Mack, M., Merritt, J. A., Nguyen, D. M., … Schrump, D. S. (1999). Adenovirus-mediated p53 gene transfer in advanced non-small-cell lung cancer. JNCI: Journal of the National Cancer Institute, 91(9), 763–771. https://doi.org/10.1093/jnci/91.9.763 Vogel, A., Meyer, T., Sapisochin, G., Salem, R., & Saborowski, A. (2022). Hepatocellular carcinoma. The Lancet, 400(10356), 1345–1362. https://doi.org/10.1016/S0140-6736(22)01200-4 Wang, C., Tan, J. Y. M., Chitkara, N., & Bhatt, S. (2024). TP53 Mutation-Mediated Immune Evasion in Cancer: Mechanisms and Therapeutic Implications. Cancers, 16(17), 3069. https://doi.org/10.3390/cancers16173069 Wang, C. H., Wey, K. C., Mo, L. R., Chang, K. K., Lin, R. C., & Kuo, J. J. (2015). Current trends and recent advances in diagnosis, therapy, and prevention of hepatocellular carcinoma. Asian Pacific journal of cancer prevention : APJCP, 16(9), 3595–3604. https://doi.org/10.7314/apjcp.2015.16.9.3595 Wiman K. G. (2006). Strategies for therapeutic targeting of the p53 pathway in cancer. Cell death and differentiation, 13(6), 921–926. https://doi.org/10.1038/sj.cdd.4401921 Yamaguchi, F., Hayakawa, S., Kawashima, S., Asakura, T., & Oishi, Y. (2022). Antitumor effect of memantine is related to the formation of the splicing isoform of GLG1, a decoy FGF‑binding protein. International journal of oncology, 61(1), 80. https://doi.org/10.3892/ijo.2022.5370 Yang, T., Mendoza-Londono, R., Lu, H., Tao, J., Li, K., Keller, B., Jiang, M. M., Shah, R., Chen, Y., Bertin, T. K., Engin, F., Dabovic, B., Rifkin, D. B., Hicks, J., Jamrich, M., Beaudet, A. L., & Lee, B. (2010). E-selectin ligand-1 regulates growth plate homeostasis in mice by inhibiting the intracellular processing and secretion of mature TGF-beta. The Journal of clinical investigation, 120(7), 2474–2485. https://doi.org/10.1172/JCI42150 Yasmin-Karim, S., King, M. R., Messing, E. M., & Lee, Y. F. (2014). E-selectin ligand-1 controls circulating prostate cancer cell rolling/adhesion and metastasis. Oncotarget, 5(23), 12097–12110. https://doi.org/10.18632/oncotarget.2503 Ye, Q. H., Zhu, W. W., Zhang, J. B., Qin, Y., Lu, M., Lin, G. L., Guo, L., Zhang, B., Lin, Z. H., Roessler, S., Forgues, M., Jia, H. L., Lu, L., Zhang, X. F., Lian, B. F., Xie, L., Dong, Q. Z., Tang, Z. Y., Wang, X. W., & Qin, L. X. (2016). GOLM1 Modulates EGFR/RTK Cell-Surface Recycling to Drive Hepatocellular Carcinoma Metastasis. Cancer cell, 30(3), 444–458. https://doi.org/10.1016/j.ccell.2016.07.017 Zhang, X., Wu, L. N., Li, X. Q., Luo, X., Liu, S. W., Zhang, L., Nawaz, S., Ma, L. N., & Ding, X. C. (2023). Whether the Golgi protein 73 could be a diagnostic serological marker in hepatocellular carcinoma: a meta analysis. BMC gastroenterology, 23(1), 85. https://doi.org/10.1186/s12876-023-02685-8 Zuber, M. E., Zhou, Z., Burrus, L. W., & Olwin, B. B. (1997). Cysteine-rich FGF receptor regulates intracellular FGF-1 and FGF-2 levels. Journal of cellular physiology, 170(3), 217–227. https://doi.org/10.1002/(SICI)1097-4652(199703)170:33.0.CO;2-R Information & Authors Information Version history V1 Version 1 15 January 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Jiayao Lu Shenzhen Technology University College of Pharmacy View all articles by this author Qiuju Bu Shenzhen Technology University College of Pharmacy View all articles by this author Haibo Yue Shenzhen Technology University College of Pharmacy View all articles by this author Mingming Gao Shenzhen Technology University College of Pharmacy View all articles by this author Danyan Huang Shenzhen Technology University View all articles by this author Yongqi Lv Jiangsu University School of Pharmacy View all articles by this author Ziwe Zhang Shenzhen Second People's Hospital Department of Pharmacy View all articles by this author Xiaohui Zhu Shenzhen Technology University College of Pharmacy View all articles by this author Zhi Ren Shenzhen Technology University College of Pharmacy View all articles by this author Shasha Song [email protected] Shenzhen Technology University College of Pharmacy View all articles by this author Metrics & Citations Metrics Article Usage 188 views 75 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jiayao Lu, Qiuju Bu, Haibo Yue, et al. 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