Introduction

Hepatocellular carcinoma (HCC) ranks as the sixth most prevalent malignancy globally and represents the third primary contributor to cancer-associated mortality (Sung 2021). The insidious nature of early-stage HCC and poor treatment outcomes contribute to its dismal prognosis (Forner et al. 2018). While molecularly targeted agents and immunotherapies have enhanced survival outcomes in select HCC cohorts, intrinsic and acquired resistance driven by tumor microenvironment (TME) complexity and heterogeneity remain substantial clinical challenges (Pu 2022; Deng 2022; Li 2021; Chan et al. 2011; Tang 2024; Su 2023). This underscores the critical need for elucidating the fundamental molecular underpinnings driving hepatocarcinogenesis and TME evolution. Such comprehensive understanding could facilitate the development of novel therapeutic strategies, potentially enabling the design of synergistic treatment regimens that either prevent oncogenic progression or resensitize therapy-refractory neoplasms to immunological interventions (Zhang 2023; Donne and Lujambio 2023; Su 2024).

The ubiquitin-proteasome system (UPS) serves as the primary mechanism governing intracellular protein degradation through ubiquitination (Farinati 2009). As a highly dynamic and tightly controlled post-translational modification (PTM), ubiquitination is executed via a multi-enzyme cascade) (Ferlay 2015). This ATP-dependent process involves sequential activation by E1 ubiquitin-activating enzymes, conjugation via E2 ubiquitin-conjugating enzymes, and substrate-specific ligation mediated by E3 ubiquitin ligases (Sun et al. 2020). As the primary determinants of substrate specificity, E3 ligases critically regulate proteostasis; their dysregulation disrupts signaling cascades, promotes aberrant protein accumulation, and drives oncogenesis (Wang 2007; Hoeller and Dikic 2009).

FBXW11, a prominent member of the E3 ubiquitin-protein ligase family, serves as a crucial regulator of multiple cellular mechanisms. Its oncogenic significance has been established through modulation of tumor-associated proteins that drive cancer cell proliferation and metastatic potential (Wang 2014). Functioning as a core component of the SCF (SKP1-CUL1-F-box protein) ubiquitin ligase complex through CUL1 interaction, FBXW11 exerts substantial influence on cell cycle regulation and oncogenesis (Shi 2020). Emerging evidence reveals an interaction between FBXW11 and CUL7, demonstrating its capacity to mediate AID degradation and regulate IgA class switching in murine B lymphocytes (Luo 2019). Notably, the potential oncogenic implications of the FBXW11-CUL7 interaction remain unexplored in cancer biology. Clinical observations indicate FBXW11 protein overexpression in lymphocytic leukemia patients, where it accelerates cell cycle progression and activates NF-κB and β-catenin/TCF signaling cascades without triggering apoptotic pathways (Wang 2018). Parallel findings in murine skin carcinogenesis models demonstrate FBXW11 upregulation promoting neoplastic growth and metastatic behavior through NF-κB pathway activation (Bhatia 2002; Zhang et al. 2019). Paradoxically, human non-small cell lung carcinoma (NSCLC) tissues exhibit significant FBXW11 downregulation compared to adjacent normal tissues, with functional studies confirming its tumor-suppressive role in inhibiting NSCLC and pancreatic cancer cell proliferation/invasion (Chang 2018; Savita and Karunagaran 2013; Wang 2016). These contradictory findings FBXW11 plays an important role in tumorigenesis, functioning as either an oncogene or tumor suppressor across different malignancies. Despite these advancements, the pathophysiological role of FBXW11 in hepatocellular carcinoma remains uncharacterized. This knowledge gap necessitates comprehensive investigation into functional mechanisms of FBXW11 during hepatic carcinogenesis and tumor progression.

Materials and methods

Cell lines and cell culture

Human hepatocellular carcinoma (HCC) cell lines HCC-LM3 HuH7 Hep3B and SNU-449 were acquired from the cell bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (CTCC China). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco USA) containing 10% fetal bovine serum (FBS; Gibco USA) and 1% penicillin-streptomycin (Gibco USA). All cellLines were maintained at 37 °C in a humidified 5% CO2 incubator. Profiling and authentication of HCC-LM3, HuH7, Hep3B, and SNU-449 cell lines was performed using the short tandem repeat identification criteria developed by the International Cell Line Authentication Committee (Almeida 2004).

Clinical specimens

HCC and adjacent non-tumorous tissues were collected from the Department of General Surgery at the Second Hospital of Dalian Medical University (China). Written informed consent was obtained from all participants, and ethical approval was secured from the Institutional Review Boards of The Second hospital of Dalian Medical University. All methods involving human participants were performed in accordance with the Declaration of Helsinki.

Antibodies and reagents

MG132, cycloheximide (CHX) and SC79 were sourced from Selleck Chemicals (USA). Antibodies included: FBXW11 (Proteintech, #13149-1-AP), Vinculin (Proteintech, #CL594-26520), YB1 (Cell Signaling Technology, #4202), Myc (Proteintech, #60003-2-Ig), Flag (Sigma, #F7425), HA (Santa Cruz Biotechnology, #sc-7392), ub (Cell Signaling Technology, #20326), Akt (Cell Signaling Technology, #9272), p-Akt (s473) (Cell Signaling Technology, #9271), p-Akt (t308) (Cell Signaling Technology, #9275), mTOR (Cell Signaling Technology, #2972), p-mTOR (Cell Signaling Technology, #2971).

Plasmids

Short hairpin RNAs (shRNAs) targeting FBXW11 (shFBXW11-1/2), YB1 (shYB1-1/2), and non-targeting control (pGIPZ-shControl) were designed by GenePharma (China). Full-length human FBXW11 and YB1 cDNA sequences were cloned into pDONOR vectors via Gateway Technology (Thermo Fisher Scientific) to generate entry clones. These were subsequently recombined into Gateway-compatible vectors for FLAG- or Myc-tagged protein expression.

Western blotting

Total protein lysates were obtained from transfected cells through homogenization in RIPA buffer, with protein quantification performed using the Thermo Scientific™ BCA Assay Kit (Thermo Fisher Scientific). Protein samples underwent thermal denaturation (95 °C, 5 min) in a boiling water bath before electrophoretic separation on 10% SDS-polyacrylamide gels. Subsequent electroblotting transferred proteins onto PVDF membranes (0.45 μm pore size). Membranes were initially blocked with 5% non-fat dry milk in TBST for 60 min at ambient temperature, then probed with primary antibodies during overnight incubation at 4 °C. Following three TBST washes (10 min each), membranes were exposed to HRP-conjugated secondary antibodies for 1 h at room temperature. After additional TBST washes, chemiluminescent detection was performed using Biyuntian ECL substrate (Shanghai Biyuntian Bio-Technique), with signal acquisition conducted on a LI-COR Odyssey imaging system.

Immunohistochemistry (IHC)

Tissue sections were processed using an IHC Kit (Proteintech, KIHC-5). Antigen retrieval was performed with EDTA or citrate buffer, as specified. FBXW11 and YB1 expression was quantified via H-score analysis based on staining intensity.

Cell proliferation assays

For crystal violet assays, 3,000 cells/well were seeded in 6-well plates. Cells were fixed and stained at days 3, 5, 7, 9, and 11. Absorbance at 590 nm was measured after dissolving stained cells in acetic acid. Triplicate biological replicates were analyzed.

Proteome analysis

Cellular specimens were subjected to three cycles of ice-cold ultrasonic disruption in lysis solution (8 M urea 1% protease inhibitor cocktail) using a high-power sonicator. Post-homogenization particulate matter was pelleted via centrifugation (12000 rpm 15 min 4 °C) after whichSupernatants were isolated for protein quantification employing a BCA assay per manufacturer protocols. Reduction of disulfide bonds was achieved through 30 -min incubation with 5 mM DTT at 56 °CSucceeded by 15 -min alkylation using 11 mM iodoacetamide under dark ambient conditions. Protein solutions wereSubsequently diluted with 100 mM TEAB to reduce urea concentration below 2 M prior to enzymatic digestion. Sequential tryptic digestion was performed: initial overnight incubation at 1:50 enzyme-to-substrate ratio followed by 4-hour secondary digestion at 1:100 ratio. For LC-MS/MS interrogation digested peptides were reconstituted in 0.1% formic acid (mobile phase A) and chromatographed through a custom-packed reverse-phase column (75 μm × 15 cm). Separation utilized a multi-step gradient: 6–23% mobile phase B (0.1% formic acid 98% ACN) over 26 min 23–35% in 8 min 35–80% in 3 minSustained at 80 % for 3 min (400 nL/min flow, EASY-nLC 1000). Ionization occurred via NSI source at 2.0 kV coupled to a Q ExactiveTM Plus mass spectrometer. Full MS scans (m/z 350–1800) were acquired at 70,000 resolution, with top-20 data-dependent MS/MS fragmentation (NCE 28%) at 17,500 resolution. Dynamic exclusion duration was set to 15 s with AGC target 5 × 10⁴ and 100 m/z initial fragment mass. Statistical significance thresholds for differential protein expression were established at 1.5-fold change (p < 0.05) through comparative proteomic analysis.

Co-immunoprecipitation (Co-IP) and affinity pull-down

Cells were lysed in NETN buffer. For Co-IP, lysates were incubated with anti-FBXW11/YB1 antibodies. For pull-down assays, HEK-293T cells expressing Flag- or Myc-tagged proteins were lysed, and supernatants were incubated with Flag/Myc magnetic beads. Precipitated proteins were analyzed by immunoblotting.

Ubiquitination assay

Cells were lysed in SDS-containing buffer, denatured at 95 °C, and diluted to reduce SDS concentration. Lysates were immunoprecipitated with HA-Ub antibody and analyzed by immunoblotting using HA, Myc, or Flag antibodies.

CCK-8 viability assay

Cells (1500/well) were seeded in 96-well plates. At designated intervals, CCK-8 reagent was added, and absorbance at 450 nm was measured using a microplate reader.

In vivo tumorigenesis

This study is reported in accordance with ARRIVE guidelines. All work performed with animals was approved by the Institutional Animal Care and Use Committee of Wuhan Servicebio Technology Co. Ltd. Pathogen-free female athymic nude mice (4–5 weeks old; 18–22 g) were purchased fromWuhan Servicebio Technology Co., Ltd. Nude mice were Subcutaneously injected with 1 × 10⁷ cells/mL (100 µL) into the left flank. Tumor volume and weight were monitored weekly.

LinkedOmics database

LinkedOmics (http://linkedomics.org) aPublicly accessible web portal, facilitates integrative multi-omics analyses across 32 cancer types from The Cancer Genome Atlas (TCGA) consortium (Vasaikar 2018). The platform’s LinkInterpreter module was employed to perform Kyoto Encyclopedia of Genes and Genomes (KEGG;https://www.kegg.jp/) pathway enrichment analysis. Statistical significance was determined by Pearson correlation tests with a P-value threshold of 0.05.

Statistical analysis

Student’s t-test was used to compare the differences between two groups. One-way ANOVA with Bonferroni’s multiple comparisons test was used to compare the means from more than two groups. The correlations between the FBXW11 expression and clinicopathological factors were performed by Pearson’s chi-square test. Overall survival (OS) was analyzed using a log-rank test and the Kaplan–Meier method. The correlation between FBXW11 expression and YB1 expression was determined by Pearson’s correlation coefficient. Statistical analysis was performed using the spss 18.0 software package. P < 0.05 was considered statistically significant.

Results

FBXW11 is markedly reduced and correlates with unfavorable clinical outcomes in hepatocarcinoma

To investigate the role of FBXW11 in HCC pathogenesis, we analyzed protein extracts from 10 paired HCC tumors and adjacent non-tumorous tissues via Western blot. FBXW11 expression was substantially decreased in tumor tissues compared to matched normal counterparts (Fig. 1A, B). Immunohistochemical (IHC) analysis of 99 HCC tissues and adjacent normal specimens further confirmed reduced FBXW11 levels in malignant tissues (Fig. 1C). Stratification of patients based on FBXW11 expression (low vs. high) revealed a significant association between low FBXW11 levels and advanced TNM stages (Table 1). Univariate and multivariate regression analysis confirmed FBXW11 as an independent prognostic marker, with low expression correlating with reduced overall survival (OS: HR = 3.058, 95% CI = 1.362–6.849, P = 0.042) after adjusting for TNM stage (P = 0.036) and other clinicopathological variables (Table 2). Survival curve analysis demonstrated significantly worse clinical outcomes in patients with FBXW11-low tumors (Fig. 1D). These collective findings establish FBXW11 as both a tumor progression biomarker and independent prognostic indicator in HCC pathogenesis.

Fig. 1
figure 1

FBXW11 is downregulated and associated with poor prognosis in HCC. A Western blot assays evaluating FBXW11 protein levels in 10 paired non-tumorous adjacent tissues (NT) and hepatocellular carcinoma (Ca). B Quantitative analysis of band intensities from matched HCC tissues using Image J software, normalized to Vinculin. Statistical significance was determined by paired Student’s t-test (*P < 0.05). (C) Immunohistochemical images illustrating FBXW11 expression patterns in HCC specimens. Scale bar: 50 μm. Statistical significance was determined by unpaired Student’s t-test (***P < 0.001). DKaplan-MeierSurvival curves comparing overall Survival in 99 HCC patients categorized by FBXW11 expression levels.

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Table 1 Correlation between FBXW11 expression and clinicopathological characteristics
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Table 2 Univariate and multivariate analyses indicating associations between overallSurvival and various risk factors in 99 HCC patients
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FBXW11 suppresses HCC cell proliferation

Functional studies were conducted using gain- and loss-of-function approaches in multiple HCC cell lines (Fig. 2A, D). Ectopic FBXW11 expression in HCC-LM3 and HuH7 cells significantly impaired cell viabilities (Fig. 2B-C). Conversely, FBXW11 knockdown via two distinct shRNAs enhanced proliferative activity in Hep3B and SNU-449 cells (Fig. 2E-F). Similar results were obtained in the CCK-8 assay; overexpressing FBXW11 decreased the growth of HCC-LM3 and HuH7 cells (Fig. S1A-B), and FBXW11 knockdown in Hep3B and SNU-449 cells promoted cell viability (Fig. S1C-D). These experiments establish FBXW11 as a tumor suppressor that constrains HCC cell growth.

Fig. 2
figure 2

FBXW11 suppresses hepatocellular carcinoma cell proliferation. A Immunoblot analysis of FBXW11 expression with Vinculin loading control in HCC-LM3 and HuH7 cells following FBXW11 overexpression. B, C Representative images and cell growth curves of HCC-LM3 and HuH7 cells with overexpression of FBXW11. D Western blot validation of FBXW11 knockdown efficiency using two distinct shRNA constructs in Hep3B and SNU-449 cells, with Vinculin as internal control. E, F Representative images and cell growth curves of Hep3B and SNU-449 cells with knockdown of FBXW11. Triplicate experimental data presented as mean ± SD; statistical significance determined by unpaired t-test. *P < 0.05, ** P < 0.01, *** P < 0.001.

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FBXW11 interacts with the cold shock domain (CSD) of YB1

To investigate direct substrates of FBXW11 Flag-tagged FBXW11 was expressed in Huh7 cells followed by immunoprecipitation using an anti-FBXW11 antibody. Co-purified proteins wereSubjected to mass spectrometry, revealing 126 candidate interactors. Subsequent proteomic profiling of FBXW11-overexpressing cells identified 93 proteins with ≥ 1.5-fold expression modulation. Comparative analysis of affinity-purified proteins from anti-Flag-FBXW11Pull-down assays in HCC models identified 126 potential binding partners. Intersection analysis highlighted three overlapping candidates-YB1, β-catenin, and IkB-β-displaying significant abundance alterations (Fig. 3A). Given YB1’s established oncogenic role in HCC (Liu 2021), these findings suggest its potential as a critical FBXW11 substrate in HCC pathogenesis.

To validate FBXW11-YB1 interaction, HEK-293T cells were co-transfected with Flag/Myc-tagged FBXW11 and YB1 constructs. Reciprocal co-immunoprecipitation confirmed their physical association (Fig. 3B-E). Endogenous interaction was further verified in HuH7 cells through co-IP assays (Fig. 3F). Domain mapping revealed YB1’s tripartite architecture (AP, CSD, CTD; Fig. 3G) (Wolffe 1992). Serial deletion experiments demonstrated that full-length YB1 (YB1-FL), the N-terminal AP-CSD region (YB1-APD), and the isolated CSD domain retained binding capacity to FBXW11. Strikingly, reciprocal domain analysis identified the CSD domain as the critical mediator of this interaction (Fig.3H-I).

Fig. 3
figure 3

FBXW11 associates with the CSD of YB1. A Overlap analysis comparing proteins showing ≥ 1.5-fold expression alterations following FBXW11 overexpression (blue) and potential FBXW11 interactors identified through co-immunoprecipitation coupled with mass spectrometry (yellow). B-E Reciprocal co-IP assays in HEK-293T cells transiently transfected with specified constructs. Lysates were immunoprecipitated using anti-Flag or anti-Myc antibodies followed by immunoblotting with designated antibodies. F Endogenous interaction validation in HuH7 cells using FBXW11-specific antibodies for immunoprecipitation, with subsequent detection of YB1 and FBXW11 by immunoblotting. G Domain architecture schematic of YB1 protein. H, I Domain mapping experiments in HEK-293T cells transfected with truncated YB1 constructs. Immunoprecipitations were performed with anti-Flag/Myc antibodies followed by reciprocal immunoblot detection

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FBXW11 enhances K48-linked ubiquitination of YB1 to accelerate its proteasomal degradation

As a well-characterized E3 ubiquitin ligase, FBXW11 contributes to tumor progression through substrate-specific ubiquitination processes (Wang 2014). We investigated whether FBXW11 enhances YB1 ubiquitination. Initial experiments demonstrated that FBXW11 knockdown markedly reduced ubiquitination levels in HuH7 cells (Fig.4A). Subsequent co-transfection experiments with Flag-YB1, Myc-FBXW11, and HA-ubiquitin constructs revealed that FBXW11 overexpression significantly augmented YB1 ubiquitination (Fig. 4B). Given the critical roles of K48- and K63-linked polyubiquitination in cellular regulation (Tekcham 2020), we characterized the specific ubiquitin linkage involved. Co-transfection systems incorporating K48-specific or K48R ubiquitin mutants demonstrated that FBXW11 specifically enhanced K48-linked polyubiquitination of YB1 through anti-Flag immunoprecipitation analysis (Fig.4C). Parallel experiments with K63 chain variants showed no significant alteration in ubiquitination patterns (Fig. 4D), confirming linkage specificity.

Since K48-linked ubiquitination typically targets substrates for proteasomal degradation (Liu 2024), we examined FBXW11-mediated YB1 regulation. FBXW11 overexpression reduced YB1 protein levels in HCCLM3 and HuH7 cells, an effect reversed by proteasomal inhibition with MG132 (Fig.4E, F). Cycloheximide (CHX) chase experiments confirmed accelerated YB1 degradation upon FBXW11 overexpression, with reduced protein half-life observed in HuH7 cells (Fig. 4G, H). To further confirm the role of FBXW11 in the regulation of YB1 in HCC, we detected the expression of FBXW11 and YB1 in HCC tissues by IHC (Fig. 4I). The results showed that FBXW11 protein was notably negatively related to YB1 protein (Fig. 4J). Collectively, these findings establish that FBXW11 selectively induces K48-linked polyubiquitination of YB1, thereby promoting its proteasome-dependent degradation and effectively modulating intracellular YB1 concentrations.

Fig. 4
figure 4

FBXW11 enhances K48-specific ubiquitination of YB1 and reduces its protein stability. A YB1 immunoprecipitates from lysates of FBXW11-depleted and control cells were analyzed by immunoblotting with antibodies against Ub, FBXW11, YB1, and Vinculin. B, D HEK-293T cells pretreated with 10 µM MG132 for 6 h were transfected with specified plasmids for 24 h, followed by Flag affinity pull-down or direct immunoblotting with indicated antibodies. E, F Immunoblot analysis of YB1 expression in FBXW11-overexpressing or EV-transfected HCC-LM3 and HuH7 cells, maintained with or without MG132 (10 µM, 6 h). G Time-dependent YB1 degradation assessed by Western blot in Flag-FBXW11- or EV-expressing HuH7 cells exposed to 50 µM CHX. Vinculin served as loading control. H Densitometric quantification of YB1 levels normalized to Vinculin (ImageJ analysis). Data represent mean ± SD from triplicate experiments. Statistical significance was determined by unpaired t-test. *P < 0.05, ** P < 0.01. I Representative IHC staining of FBXW11 and YB1 in HCC tissues. Scale bars, 50 μm. J The association between FBXW11 and YB1 protein expression in HCC patients was evaluated by Pearson’s correlation coefficient.

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FBXW11 suppresses hepatocellular carcinoma proliferation through YB1 regulation

To validate the effect of YB1 on FBXW11-mediated HCC cell proliferation, HCC-LM3 and HuH7 cells were transfected with FBXW11 plasmid with or without YB1 plasmid. The results showed that silencing of YB1 significantly inhibited HCC cell proliferation (Fig. 5A-C), and rescue of YB1 could significantly block FBXW11-mediated cell proliferation (Fig. 5D-F). KEGG pathway enrichment analysis of FBXW11 co-expressed genes primarily include negative regulators based on the LinkedOmics database revealed significant enrichment in signaling pathways such as PI3K/Akt (Fig. S2). Previous studies have reported that YB1 promotes tumor progression by activating the PI3K/Akt/mTOR pathway (Zheng 2019; Delicato 2021; Liu 2021; Ha 2015; Wu 2023). Western blot assays further confirmed that FBXW11 overexpression inhibited the phosphorylation levels of Akt and its downstream target mTOR, while YB1 rescue restored their phosphorylation activity (Fig.5D). To validate the role of Akt signaling in FBXW11-YB1 regulation, FBXW11-overexpressing cells were treated with the Akt activator SC79. CCK-8 assays showed that SC79 treatment reversed the inhibitory effect of FBXW11 on hepatocarcinoma cell proliferation (Fig. 5G-H). These results indicate that FBXW11 regulates hepatocarcinoma cell proliferation by inhibiting the Akt/mTOR signaling pathway through YB1.

Fig. 5
figure 5

FBXW11 suppresses hepatocellular carcinoma cell proliferation via YB1-mediated Akt activation. A YB1 knockdown efficiency validation by immunoblotting in HCC-LM3 and Hep3B cells transduced with two distinct YB1-targeting shRNAs. B, C Cell proliferation assessed by CCK-8 assay 48 h post-seeding in 96-well plates following YB1 silencing. D Following co-transfection of YB1 and FBXW11 plasmids into Huh7 and Hep3B cells, Western blotting was performed on cell lysates with antibodies specific to the designated proteins. E, H CCK-8 assay evaluating YB1 overexpression or SC79 treatment effects on FBXW11-modulated proliferation. Triplicate experimental data presented as mean ± SD; statistical significance determined by One-way ANOVA with Bonferroni’s multiple comparisons test. ** P < 0.01, *** P < 0.001.

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FBXW11 suppressed hepatocellular carcinoma progression through the YB1/Akt pathway in vivo

To validate the in vivo antitumor effect of FBXW11, this study analyzed a subcutaneous xenograft model. HuH7 cells from the empty vector control group (EV), FBXW11 overexpression group (FBXW11), and FBXW11 overexpression combined with YB1 rescue group (FBXW11 + YB1) were subcutaneously inoculated into immunodeficient mice. The results showed that FBXW11 overexpression significantly inhibited tumor growth, while YB1 rescue partially reversed the proliferation-inhibiting effect of FBXW11 (Fig. 6A-D). Western blot analysis revealed that YB1 protein levels were reduced in xenograft tissues of the FBXW11 overexpression group, accompanied by inhibited expression of phosphorylated Akt and phosphorylated mTOR, whose phosphorylation levels were restored by YB1 rescue (Fig. 6E). Immunohistochemical staining further showed that YB1 protein expression intensity was decreased in the FBXW11 overexpression group and negatively correlated with FBXW11 expression (Fig. 6F). These results confirm that FBXW11 inhibits the in vivo growth of hepatocarcinoma cells by regulating the YB1/Akt/mTOR signaling pathway.

Fig. 6
figure 6

FBXW11 suppresses hepatocellular carcinoma progression through YB1 regulation in vivo. A–D Subcutaneous tumor formation analysis using HuH7 cells overexpressing FBXW11, including Mice images, tumor images, growth curve and weight. E Western blot analysis of indicated proteins in xenograft tumors derived from HuH7 cells transduced with FBXW11-overexpressing lentivirus, with or without YB1 co-transfection. F Representative IHC images demonstrating FBXW11 and YB1 protein expression in xenograft tumors derived from HuH7 cells transduced with FBXW11-overexpressing lentivirus, with or without YB1 co-transfection. Scale bars: 50 μm. G A model of hepatocarcinogenesis triggered by the decreased FBXW11 expression through the YB1-Akt axis. Illustrations in the diagrams were provided by Fig draw. Quantitative data presented as mean ± SD (biological replicates n = 5). ** P < 0.01, *** P < 0.001.

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Discussion

As a constituent of the F-box protein family’s FBXW subfamily, FBXW11 has been implicated in oncogenesis through its dysregulated expression across multiple malignancies, including hematopoietic cancers, prostate adenocarcinoma, colorectal carcinoma, and mammary tumors (Yao 2021; Fuchs et al. 2004). Our investigation expands this understanding by demonstrating, for the first time, significant downregulation of FBXW11 in hepatocellular carcinoma (HCC) specimens. This reduced expression pattern correlates with adverse clinical outcomes, suggesting FBXW11 may function as a critical regulatory element in HCC pathogenesis and disease progression. The observed association between diminished FBXW11 levels and poor prognostic indicators underscores its potential importance in hepatocarcinogenesis mechanisms.

YB1 is a versatile oncogenic protein that serves key roles in transcriptional regulation and protein synthesis. This multifunctional factor has been documented to be upregulated in numerous malignancies and frequently correlates with unfavorable clinical outcomes across various cancers, including breast (Bargou 1997), ovarian (Kamura 1999), hepatocellular (Yasen 2005), lung (Shibahara 2001), colorectal (Shibao 1999), prostate (Giménez-Bonafé 2004), multiple myeloma (Chatterjee 2008), melanoma (Schittek 2007), osteosarcoma (Oda 1998), glioblastoma (Faury 2007), mesothelioma (Goswami and Nakshatri 2014), and bladder urothelial carcinoma (Chen 2019). Substantial evidence reveals YB1’s critical involvement in modulating multiple oncogenic processes encompassing cellular proliferation, cell cycle regulation, cancer stemness maintenance, metastatic potential, DNA repair mechanisms, autophagic processes, immune evasion, and multidrug resistance (Alkrekshi 2021). Positioned upstream of key signaling networks and growth-related genes – including drug resistance mediators, HER-2, EGFR, proliferating cell nuclear antigen, and cyclins A/B–YB1 emerges as a promising therapeutic target for cancer intervention (Wu 2007). Current strategies focus on either direct YB1 inhibition or modulation of its upstream regulators such as PDK and RSK. Our investigation highlights YB1 overexpression in hepatocellular carcinoma (HCC) specimens and demonstrates that YB1 knockdown markedly suppresses HCC proliferation. Furthermore, we identify FBXW11 as a novel negative regulator of YB1 through ubiquitination-mediated protein degradation, suggesting this pathway’s potential therapeutic relevance in HCC management. These findings collectively reinforce the rationale for targeting YB1 regulatory networks in liver cancer treatment strategies.

Numerous studies have demonstrated a close regulatory relationship between YB1 and the Akt signaling pathway in cancer. YB1 enhances Akt phosphorylation by suppressing PTEN expression (reducing its mRNA levels) to relieve inhibition of the PI3K/Akt pathway (Delicato 2021), and by transcriptionally activating downstream target genes (e.g., eEF1A1) or directly binding to key molecules in the Akt pathway (e.g., Akt1, PIK3R1) (Liu 2021; Wu 2023). Additionally, YB1 promotes tumor cell resistance to chemotherapeutic agents (e.g., gemcitabine, sorafenib, gefitinib) by maintaining Akt/mTOR signaling activity (Liu 2021; Guo 2024; Lou 2021), and accelerates tumor invasion and metastasis by activating the Akt-dependent epithelial-mesenchymal transition (EMT) process (e.g., TGF-β1-induced mesenchymal phenotype) (Ha 2015; Lou 2021). Clinical data indicate that co-expression of YB1 and p-Akt is significantly associated with advanced clinical stages and poor prognosis in various cancers, including non-small cell lung cancer and nasopharyngeal carcinoma (Zheng 2019; Zhan 2022). Collectively, as a multifunctional nucleic acid-binding protein, YB1 forms a regulatory network with Akt through transcriptional regulation, signaling pathway cross-talk, and epigenetic modifications to drive tumor proliferation, metastasis, and drug resistance.

Conclusion

In conclusion, this investigation revealed FBXW11 as a crucial regulatory factor influencing YB1-mediated hepatocarcinogenesis through the following mechanistic insights: (1) FBXW11 demonstrates significant downregulation in HCC tissues and correlates with unfavorable clinical outcomes. (2) Functional analyses confirm its tumor-suppressive role in hepatocellular carcinoma. (3) Direct molecular interaction occurs between FBXW11 and YB1 through CSD domain binding. (4) FBXW11 specifically induces K48-linked polyubiquitination of YB1, thereby accelerating its proteasomal degradation. (5) The anti-proliferative effects of FBXW11 in HCC cells are mechanistically dependent on YB1 regulation. (Fig. 6G)