ABSTRACT Gallbladder cancer (GBC) is the most common malignant tumor of the biliary tract and is often prone to early distant metastasis. However, the mechanisms underlying GBC’s invasive

metastasis remain unclear. This study identified that F-box only protein 33 (FBXO33) expression is significantly elevated in GBC and is negatively associated with patient prognosis. In vivo

and in vitro experiments demonstrated that knockdown of FBXO33 inhibits epithelial-mesenchymal transition (EMT) progression in GBC, while overexpression of FBXO33 promotes EMT progression.

Mechanistically, FBXO33 regulates EMT progression by modulating the polyubiquitination of p53 at K291 and K292. Moreover, the upregulation of FBXO33 in GBC is driven by transcriptional

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PKA-DEPENDENT SNAI2 DEGRADATION Article Open access 18 August 2023 DEUBIQUITINASE USP39 AND E3 LIGASE TRIM26 BALANCE THE LEVEL OF ZEB1 UBIQUITINATION AND THEREBY DETERMINE THE PROGRESSION OF

HEPATOCELLULAR CARCINOMA Article Open access 01 March 2021 INTRODUCTION GBC is the most common malignant tumor of the biliary tract, characterized by a high mortality rate and poor

prognosis [1, 2]. Despite extensive research, no significant improvement in GBC prognosis has been achieved [3]. Early metastasis is a hallmark of GBC, often making surgical treatment

unfeasible at diagnosis [4]. Thus, identifying effective early biomarkers and therapeutic targets for GBC is essential. Studies suggest that EMT plays a critical role in initiating distant

metastasis in malignant tumors [5]; however, the molecular mechanisms driving EMT in GBC remain unclear. This highlights an urgent need to identify novel EMT-related biomarkers to improve

patient outcomes. Protein ubiquitination involves the covalent attachment of a 76-amino-acid ubiquitin protein to target proteins, mediated by ubiquitin-activating enzymes (E1),

ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) [6, 7]. This process regulates the biological functions of target proteins and is integral to key signaling pathways,

influencing metastasis and treatment responses in malignant tumors [8, 9]. The F-box family of E3 ubiquitin ligases has been shown to play a pivotal role in EMT progression [10]. For

example, FBXO11 targets Snail for ubiquitination and degradation, thereby inhibiting EMT and metastasis in breast cancer models [11], and also regulates EMT via ubiquitination of SAMD1 [12].

Conversely, FBXO32 facilitates EMT by ubiquitinating and degrading substrates such as CTBP1 and PTEN [13, 14]. Recent studies have identified that FBXO33, a member of the F-box family, acts

as a tumor suppressor by promoting ubiquitination and degradation of MYC, thereby inhibiting metastasis in non-small cell lung cancer [15]. However, the role of FBXO33 in other tumor types,

including GBC, remains unclear. This study demonstrates that FBXO33, a novel E3 ubiquitin ligase, is significantly upregulated in GBC. The effects of FBXO33 on EMT in GBC and its underlying

mechanisms were evaluated. The findings indicate that FBXO33 functions as an oncogene in GBC. Overexpression of FBXO33 promotes EMT progression, while its downregulation inhibits EMT

progression. Mechanistically, ubiquitination at K291 and K292 of p53 is critical for FBXO33-mediated regulation of EMT in GBC. Furthermore, transcriptional activation of FBXO33 in GBC

depends on regulation by the transcription factor YY1. This activation is further reliant on the lactylation modification at K183 of the YY1 protein. MATERIALS AND METHODS PATIENT DATA

COLLECTION Clinical samples were collected in strict accordance with the approved protocol by the Ethics Committee of Fujian Medical University’s Medical College, China. Eighty-two

paraffin-embedded tumor specimens diagnosed as GBC by the Clinical Pathology Department of Fujian Medical University Union Hospital from January 2013 to January 2024, along with paired

benign tissues, were collected. Informed consent was obtained from all patients. None of the patients received any radiotherapy or chemotherapy prior to tumor resection. CELL LINES AND CELL

CULTURE Human GBC cell lines GBC-SD and SGC-996 were purchased from the Shanghai Institute of Life Sciences, China. The 293T cell line was provided by the Department of Oncology, Tongji

University School of Medicine, China. All cell lines underwent mycoplasma detection and STR cell identification to ensure their authenticity and purity. All cells were maintained in

high-glucose Dulbecco’s Modified Eagle Medium (DMEM, Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, USA) and cultured at 37 °C in a humidified atmosphere containing 5%

CO2. All cells were tested negative for mycoplasma contamination. NEXT-GENERATION SEQUENCING Three random 0.8 mm tumor tissue cores were selected to collect tumor DNA. For samples with low

tumor volume, 5-10 tissue slides were used to microdissect the selected tumor regions for DNA enrichment. Although DNA was isolated from the same tumor block, the p53 IHC slides were not

used to guide the microdissection areas. The samples were sequenced using the AmpliSeq Cancer Hotspot Panel version 5. All p53 mutation calls were cross-referenced against publicly available

databases (ClinVar, available at https://www.ncbi.nlm.nih.gov/clinvar/ and COSMIC, available at https://cancer.sanger.ac.uk/cosmic) [16, 17]. The criteria for mutation reporting were a

minimum read depth of 500, an allele ratio ≥5%, a base quality score ≥30, a probability score for single nucleotide changes ≥0.90, and a quality score for insertions/deletions ≥1000.

REAL-TIME QUANTITATIVE POLYMERASE CHAIN REACTION (RT-QPCR) Total RNA was extracted from GBC tissues or cells using TRIzol (15596-026, Invitrogen, USA). mRNA was reverse transcribed using the

All-In-One 5×RT MasterMix (Abm, Canada). Subsequently, PCR was performed using Fast Start Universal SYBR Green Master Mix (Roche, Basel, Switzerland) and fluorescence quantification was

conducted according to the operation manual of the ABI 7500 real-time system. GAPDH was used as an internal control for normalization. The relative expression levels of mRNA were evaluated

using the 2 − ΔΔCt method. Detailed information on primer sequences is provided in Table 1. WESTERN BLOT ANALYSIS (WB) AND ANTIBODIES Total protein was extracted from GBC tissues or cells

using pre-cooled RIPA (Radioimmunoprecipitation assay) buffer (Beyotime, Shanghai, China). Subsequently, the protein lysates were quantified using the BCA (Bicinchoninic acid) protein assay

kit (Thermo Fisher Scientific). After separating the proteins by SDS-PAGE electrophoresis, they were transferred onto PVDF membranes. The membranes were then blocked with 5% non-fat milk for

2 h at room temperature and incubated with primary antibodies overnight at 4 °C. After incubation with secondary antibodies at room temperature for 1 h, the antibody-antigen complexes were

detected using the ECL assay kit (Advansta, USA). The following primary antibodies were purchased: rabbit anti-YY1 (ab227269), rabbit anti-YY1 (ab109237), rabbit anti-p53 (ab32049), mouse

anti-p53 (ab308609), rabbit anti-Flag (ab205606), mouse anti-Flag (ab125243), rabbit anti-His (ab213204), mouse anti-His (ab18184), rabbit anti-HA (ab9110), rabbit anti p65(ab32536), rabbit

anti Myc-tag(ab9106), mouse anti Myc-tag(ab32), rabbit anti-Ubiquitin (ab134953) from Abcam; lactylation pan-antibody (PTM-1401RM) from PTM BIO (Hangzhou, China); rabbit anti-FBXO33

(PA5-61275) from Thermo Fisher Scientific; rabbit anti-E-Cadherin (#3195), rabbit anti-Vimentin (#5741), rabbit secondary antibody (#7074), and mouse secondary antibody (#7076) from Cell

Signaling Technology. Rabbit anti-GAPDH (#5174) was used as an internal control for normalization. CHEMICAL INHIBITORS The following exogenous drugs used in this study were purchased from

MCE (MedChemExpress, USA): MG132 (HY-13259), Cycloheximide (CHX, HY-12320), and Lactate (HY-B2227). IMMUNOHISTOCHEMICAL ANALYSIS (IHC) Paraffin-embedded sections were deparaffinized in

xylene, dehydrated in graded ethanol, and subjected to antigen retrieval. The sections were washed with TBS-T and quenched in a 3% hydrogen peroxide TBS solution for 10 minutes to block

endogenous peroxidase/biotin activity. Non-specific binding was blocked with a serum-free blocking reagent, followed by overnight incubation with primary antibodies. Primary antibodies

specific for FBXO33 (1:500, Thermo Fisher Scientific), p53 (1:400, Abcam), E-Cadherin (1:200, #3195), and Vimentin (1:300, #5741) were incubated overnight at 4 °C. The sections were then

incubated with secondary antibodies for 20 min at room temperature in a humid chamber. Visualization was achieved using DAB (3,3’-Diaminobenzidine) solution, and the sections were

counterstained with hematoxylin. Digital pathology slide scanner (Olympus VS200, Japan) was used to scan the images, and Image-Pro Plus 6.0 (Media Cybernetics, USA) was employed for

analysis. The quantification of immunohistochemical evaluation was based on the product of staining intensity and the percentage of positively stained cells. The percentage of positively

stained cells was scored on a scale from 0 to 4 (0 = 0–10%; 1 = 11–25%; 2 = 26–50%; 3 = 51–75%; 4 = 76–100%), while staining intensity was rated on a scale from 0 to 3 (0 = no staining; 1 = 

weak; 2 = moderate; 3 = strong). The overall protein expression in each sample was expressed as histoscore, which was multiplication product of the staining intensity score(0–3) and

percentage of positively stained cells score(0–4) and is between 0 and 12. Low expression is scored 0–4. High expression is scored 6–12. The staining score was evaluated by two independent

pathologists. LENTIVIRUS CONSTRUCTION AND TRANSFECTION Lentiviral particles carrying Flag-tagged FBXO33-GFP (Flag-FBXO33) and its control vector lentivirus (Vector 1), as well as lentiviral

particles containing the YY1 gene coding region for overexpression (OE-YY1) and its control vector lentivirus (Vector 2), were obtained from GENE (Shanghai, China). GBC cells were infected

with lentivirus for 24 hours. Stable clones were selected with puromycin (5 μg/ml; Sigma). Lentivector-mediated short-hairpin FBXO33 (sh-FBXO33) and non-targeting plasmids (sh-Scr 1) and

lentivector-mediated short-hairpin YY1 (sh-YY1) and non-targeting plasmids (sh-Scr 2)were designed and synthesized by GENE (Shanghai, China). Stable transfer GBC cells with FBXO33 and YY1

knockdown were generated in the same way. PLASMID TRANSFECTION All plasmids used for cell transfection were obtained from GENE (Shanghai, China). Cells were seeded in 6-well plates or 10 cm2

culture dishes. When cells reached 70–80% confluence, transfection was performed using Lipofectamine 3000 reagent (Invitrogen Thermo Fisher Scientific, USA) according to the manufacturer’s

instructions. Cells were collected 48 h post-transfection for subsequent experiments. GENERATION OF P53-KNOCKOUT (P53-KO) GBC CELL LINES p53-knockout GBC cell lines were constructed using

the CRISPR–Cas9 gene-editing system. Lentiviruses containing Cas9-guide RNA targeting sequences were designed and synthesized by GENE (Shanghai, China). Lentivirus infection was performed on

GBC cells at 80% confluency. The cells were selected after culture for one week in a medium containing 5 μg/ml puromycin. Monocolonies were picked, and the knockout efficiency was

determined by western blotting. The sequence of single guide RNAs (sgRNAs) targeting human p53: 5ʹ-CCATTGTTCAATATCGTCCG-3ʹ and 5ʹ-CGGACGATATTGAACAATGG-3ʹ. The non-specific control sgRNA in

the same vector were defined as Vector-KO cells. DUAL-LUCIFERASE REPORTER ASSAY Bioinformatics analysis based on the JASPAR database indicated binding sites for YY1 in the FBXO33 promoter

region. Plasmids containing the wild-type (WT) and mutant (MUT) FBXO33 promoter regions were obtained from GENE (Shanghai, China) based on the sequences of these binding sites. The WT and

MUT FBXO33 promoter region plasmids were separately transfected into different experimental and corresponding control group cells. After 48 h, firefly luciferase (F-luc) activity was

measured using the Dual-Luciferase Reporter Assay System (Promega E2920, USA) according to the manufacturer’s instructions, with Renilla luciferase (R-luc) used as an internal control for

normalization. XENOGRAFT TUMOR MODEL Female athymic BALB/c nude mice (4–6 weeks old) were purchased from SIPEIFU (Beijing, China). All mice were housed under specific pathogen-free

conditions, strictly in accordance with the approved protocol of the Ethical Committee of Fujian Medical University’s Medical College (Approval No: IACUC FJMU 2023-0197). Mice were randomly

divided into experimental groups. GBC SGC-996 cells (sh-FBXO33 group and corresponding sh-Scr group (4–6 weeks old, _n_ = 6) or Flag-FBXO33 group and corresponding Vector group (4–6 weeks

old, _n_ = 5), 1 × 107 cells per group) were suspended in 100 μl of sterile PBS. The cells were subcutaneously implanted into the right hind limb of the mice using a syringe to establish the

xenograft tumor model. Tumor growth was monitored weekly, and tumor volume was calculated using the formula: (length × width2)/2. After 4 weeks of tumor formation, the mice were euthanized,

and xenograft specimens were collected by surgical excision and weighed. PROTEIN STABILITY CHX (final concentration 50 μg/ml) was added to the cells. After specified incubation times,

protein levels of p53 were assessed using western blotting. CHROMATIN IMMUNOPRECIPITATION (CHIP-QPCR) ANALYSIS The ChIP assay was conducted using a SimpleChIP Plus Enzymatic Chromatin IP Kit

(CST, 9004S). Briefly, GBC cells (1 × 107 cells per group) were prepared according to the kit instructions and resuspended in ChIP buffer. Chromatin DNA fragments were enzymatically

sheared, and antibody-magnetic bead complexes were used to pull down DNA fragments at 4 °C. Target DNA fragments were then detected using RT-qPCR. CO-IMMUNOPRECIPITATION (CO-IP) When cell

confluency reached 80–90% in 10 cm2 dishes, Co-IP was performed according to the instructions of the protein immunoprecipitation kit (Geneseed, Guangzhou, China). In brief, cell lysates were

prepared and protein concentrations were quantified using BCA protein assay. Subsequently, 1 mg of total protein was incubated overnight at 4 °C with antibody-magnetic bead complexes. The

following day, antibody-antigen complexes were eluted, and subsequent protein blotting was performed for relevant detection. Anti-rabbit IgG for IP (RA1009-01) was purchased from Vazyme

(Nanjing, China) to prevent detection of IP antibody heavy chains (55 kDa). STATISTICAL ANALYSIS In this study, all statistical analyses were performed using SPSS 19.0 software package (SPSS

Inc., Chicago, USA) and GraphPad Prism 8 software (GraphPad, USA). Clinical pathological data analysis employed chi-square analysis and Pearson correlation analysis. For two-group

comparisons, we employed the t-test to evaluate the significance of differences. When analyzing differences among multiple groups, we utilized analysis of variance (One-way ANOVA). All data

are presented as mean ± standard error of the mean (SEM) of independent replicates (_n_ ≥ 3). Differences with _p_ < 0.05 were considered statistically significant. RESULTS HIGH

EXPRESSION OF FBXO33 IN GBC IS NEGATIVELY CORRELATED WITH OVERALL SURVIVAL IN GBC To investigate the role of FBXO33 in GBC, mRNA expression levels of FBXO33 were measured in 20 paired GBC

tumor and adjacent gallbladder tissues using RT-qPCR. The results revealed that FBXO33 expression was significantly higher in GBC tissues compared to adjacent tissues (Fig. 1A, B).

Additionally, IHC staining was performed to assess protein expression levels of FBXO33 in 82 paired GBC tumor and adjacent tissues. The findings confirmed that FBXO33 expression was elevated

in cancer tissues compared to adjacent tissues (Fig. 1C, D). FBXO33 expression was positively associated with distant metastasis and TNM staging but showed no significant correlation with

gender, age, or the presence of gallstones (Table 2). Moreover, FBXO33 expression progressively increased from T1 to T4 stages of GBC (Fig. 1E). Kaplan-Meier survival analysis demonstrated

that GBC patients with low FBXO33 expression had higher overall survival rates compared to those with high FBXO33 expression (Fig. 1F). These results indicate that FBXO33 acts as an oncogene

and is highly expressed in GBC. FBXO33 REGULATES THE PROGRESSION OF EMT IN GBC IN VIVO AND IN VITRO To clarify the role of FBXO33 in GBC invasion and metastasis, GBC cells with FBXO33

knockdown (sh-FBXO33) or overexpression (Flag-FBXO33) were constructed using lentiviral vectors (Fig. 2A, B). Scratch and Transwell assays revealed that cell migration was significantly

reduced in the sh-FBXO33 group compared to the sh-Scr control group (Fig. 2C, D), while migration was significantly enhanced in the Flag-FBXO33 group compared to the Vector control group

(Fig. 2E, F). Western blot analysis showed that in the sh-FBXO33 group, E-Cadherin expression was significantly upregulated, and Vimentin expression was significantly downregulated compared

to the sh-Scr group (Fig. 2G and Supplementary Fig. S1A). Conversely, in the Flag-FBXO33 group, E-Cadherin expression was significantly downregulated, and Vimentin expression was

significantly upregulated compared to the Vector group (Fig. 2H and Supplementary Fig. S1B). To further validate these findings, a subcutaneous xenograft model of GBC was established using

SGC-996 cells with either FBXO33 knockdown or overexpression and their respective control cells. Tumors in the sh-FBXO33 group were smaller and lighter compared to those in the sh-Scr group,

indicating that FBXO33 knockdown inhibited GBC tumor growth (Fig. 2I–K). In contrast, tumors in the Flag-FBXO33 group were larger and heavier, suggesting that FBXO33 overexpression promoted

tumor growth (Supplementary Fig. S1C–E). IHC analysis of subcutaneous xenografts confirmed that E-Cadherin expression was significantly increased and Vimentin expression significantly

decreased in the sh-FBXO33 group compared to the sh-Scr group (Fig. 2L). Conversely, E-Cadherin expression was significantly reduced and Vimentin expression significantly elevated in the

Flag-FBXO33 group compared to the Vector group (Supplementary Fig. S1F). In summary, these findings demonstrate that FBXO33 regulates the progression of EMT in GBC both in vivo and in vitro.

FBXO33 INTERACTS WITH P53 E3 ubiquitin ligases perform their biological functions by recognizing and regulating substrates [18], and FBXO33 is a member of this family. To understand the

mechanism by which FBXO33 functions in GBC, the online tool Ubibrowser was used to predict potential substrates of FBXO33 [19]. Among the predicted substrates, RELA (p65) had the highest

confidence score, followed by p53 (Fig. 3A and Supplementary Fig. S2A). Substrate ubiquitination is essential for understanding the function of E3 ubiquitin ligases, as well as their

substrate recognition and regulatory roles [20]. To investigate the mechanism of FBXO33, Co-IP experiments were performed to assess the effect of Flag-FBXO33 on the ubiquitination of p65 and

p53. The results showed that Flag-FBXO33 significantly increased the ubiquitination level of p53 compared to the Vector group, while it had minimal effect on p65 (Supplementary Fig. S2B,

C). Thus, p53 was selected as the substrate of FBXO33 for further investigation. Co-IP experiments confirmed that FBXO33 binds to p53 (Fig. 3B, C). Given that many E3 ubiquitin ligases can

target both wild-type p53 (wtp53) and mutant p53 (mutp53) [21], we investigated whether FBXO33 exhibits the same behavior. The p53 gene in GBC cell lines remained unmutated [22], indicating

that the p53 interacting with FBXO33 in GBC-SD and SGC-996 cells is wtp53. Using CRISPR/Cas9, the endogenous p53 gene was knocked out in GBC-SD and SGC-996 cells (Supplementary Fig. S2D, E),

followed by transfection with plasmids encoding Myc-tagged wtp53 and mutp53 variants. Co-IP experiments showed that Flag-FBXO33 significantly increased the ubiquitination level of wtp53 but

had minimal effect on common mutp53 variants (p53R175H, p53R248W, and p53R273H) [23] (Supplementary Fig. S2F, G). Therefore, wtp53 was selected as the substrate of FBXO33 for further

analysis. To identify the interaction domains of FBXO33 and p53, truncated forms of p53 (WT-p53, ▲1-100-p53, ▲100–293-p53, ▲293-393-p53) (Fig. 3D) and FBXO33 (WT-FBXO33, ▲1-111-FBXO33,

▲111-555-FBXO33) were constructed (Fig. 3E). Co-IP results demonstrated that His-WT-p53, His-▲1-100-p53, and His-▲293-393-p53 could bind to Flag-FBXO33, while His-▲100–293-p53 lost this

ability, indicating that the 100–293 domain of p53 is crucial for binding to FBXO33 (Fig. 3F, G). Similarly, Flag-WT-FBXO33 and Flag-▲1-111-FBXO33 could bind to His-p53, while

Flag-▲111-555-FBXO33 lost this ability, showing that the 111-555 domain of FBXO33 is essential for binding to p53 (Fig. 3H, I). These findings confirm that FBXO33 interacts with p53. FBXO33

REGULATES THE STABILITY OF THE P53 PROTEIN Western blot analysis revealed that sh-FBXO33 significantly increased p53 protein expression (Fig. 4A and Supplementary Fig. S3A), while

Flag-FBXO33 significantly decreased it (Fig. 4B and Supplementary Fig. S3B). RT-qPCR results indicated that neither sh-FBXO33 nor Flag-FBXO33 altered p53 mRNA expression (Fig. 4C, D),

suggesting that FBXO33 regulates p53 protein expression at the post-translational level. Proteasome inhibitor MG132 alleviated the inhibitory effect of Flag-FBXO33 on p53, indicating that

FBXO33 affects p53 protein expression through the ubiquitin-proteasome pathway (Fig. 4E and Supplementary Fig. S3C). To further evaluate the impact of FBXO33 on p53 protein stability,

cycloheximide (CHX) was used to block protein synthesis. The CHX assay showed that p53 protein had a longer half-life in sh-FBXO33 GBC cells (Fig. 4F, G) and a shorter half-life in

Flag-FBXO33 GBC cells (Fig. 4H, I). These results demonstrate that FBXO33 regulates p53 protein stability via the ubiquitin-proteasome pathway. REGULATION OF P53 UBIQUITINATION BY FBXO33

This study examined how FBXO33 regulates the stability of p53 protein. Co-IP assays were performed to assess endogenous p53 polyubiquitination levels. The results showed that sh-FBXO33

significantly reduced p53 polyubiquitination after MG132 treatment (Fig. 5A, B), whereas Flag-FBXO33 markedly increased p53 polyubiquitination (Supplementary Fig. S2B, C). Furthermore,

deletion of the 111-555 domain of FBXO33 abolished its ability to inhibit p53 (Fig. 5C, D), indicating that this domain is essential for promoting p53 polyubiquitination. Key ubiquitination

sites within the 100–293 domain of p53 protein, lysine-291 (K291) and lysine-292 (K292), were identified [24]. To evaluate the roles of these sites in FBXO33-mediated p53 polyubiquitination,

lysine-to-arginine mutations were introduced, resulting in His-K291R-p53 and His-K292R-p53 mutant plasmids. His-WT-p53, His-K291R-p53, and His-K292R-p53 were transfected into Flag-FBXO33

and Vector control cells. Western blot analysis revealed that K291R and K292R mutations reduced the inhibitory effect of Flag-FBXO33 on p53 protein (Fig. 5E). Co-IP experiments confirmed

that these mutations weakened the promotion of p53 polyubiquitination by Flag-FBXO33 (Fig. 5E). These findings demonstrate that K291 and K292 are critical for FBXO33-mediated regulation of

p53 protein. P53 REGULATES THE FUNCTION OF FBXO33 IN GBC AND IS NEGATIVELY CORRELATED WITH FBXO33 EXPRESSION To assess whether p53 contributes to FBXO33-mediated regulation of GBC cell

migration and EMT, His-WT-p53, His-K291R-p53, and His-K292R-p53 were transfected into Flag-FBXO33 group cells. Scratch assays revealed that cell migration was significantly reduced in the

Flag-FBXO33+His-WT-p53 group compared to the Flag-FBXO33 group. Further reductions in migration were observed in the Flag-FBXO33+His-K291R-p53 and Flag-FBXO33+His-K292R-p53 groups compared

to the Flag-FBXO33+His-WT-p53 group (Fig. 6A, B). Similar trends were observed in Transwell assays (Fig. 6C, D). Western blot analysis showed increased E-Cadherin expression and decreased

Vimentin expression in the Flag-FBXO33+His-WT-p53 group compared to the Flag-FBXO33 group. These changes were further amplified in the Flag-FBXO33+His-K291R-p53 and Flag-FBXO33+His-K292R-p53

groups compared to the Flag-FBXO33+His-WT-p53 group (Fig. 6E). These findings suggest that His-WT-p53 reduces the promotive effects of Flag-FBXO33 on GBC cell migration and EMT. Mutations

at K291 and K292 (His-K291R-p53 and His-K292R-p53) further attenuated the inhibitory effects of Flag-FBXO33 on p53, resulting in significantly enhanced anti-cancer activity of p53 in these

mutant groups compared to His-WT-p53. In conclusion, FBXO33 regulates GBC cell migration and EMT through p53, with K291 and K292 ubiquitination sites of p53 playing critical roles in this

process. The experimental results confirm that FBXO33 regulates GBC cell migration and EMT through p53. To further validate this conclusion, Western blot was used to analyze the protein

expression levels of FBXO33 and p53 in 15 GBC patients, regardless of the mutational status of p53. In 11 cases, FBXO33 was highly expressed in cancer tissues compared to adjacent tissues,

while p53 was more highly expressed in adjacent tissues than in cancer tissues. Conversely, in 3 cases, FBXO33 was more highly expressed in adjacent tissues, while p53 was more highly

expressed in cancer tissues (Fig. 6F). IHC analysis of 30 consecutive GBC pathological sections revealed that high expression of FBXO33 in GBC tissues corresponded to low p53 expression, and

vice versa (Fig. 6G). Correlation analysis indicated a negative relationship between the expression of FBXO33 and p53 (Fig. 6H). When examining the mutational status of p53 in 82 GBC

patients (Fig. 1C), Kaplan-Meier curves showed that patients with wtp53 had higher overall survival rates compared to those with mutp53 (Supplementary Fig. S4A). These findings align with

the results reported by Sunwang Xu et al. [22]. Correlation analysis revealed a negative association between FBXO33 and wtp53 expression (Supplementary Fig. S4B), but no correlation between

FBXO33 and mutp53 expression (Supplementary Fig. S4C). Moreover, high FBXO33 expression was significantly associated with poor prognosis in patients with wtp53 but showed no association with

prognosis in patients with mutp53 (Supplementary Fig. S4D, E). These results suggest that wtp53 regulates the function of FBXO33 in GBC and is negatively correlated with its expression.

TRANSCRIPTION FACTOR YY1 PROMOTES FBXO33 EXPRESSION IN GBC The results demonstrate that FBXO33 is significantly upregulated in GBC tissues compared to adjacent tissues and regulates EMT

progression, indicating its potential as a therapeutic target for GBC. To explore the regulatory mechanism of FBXO33 expression, the promoter sequence of the human FBXO33 gene (2000 bp

upstream of the transcription start site) was obtained from the NCBI database. Potential transcription factors for FBXO33 were predicted using the PROMO, Harmonizome, and GTRD databases.

When the PROMO database was set to 0% Maximum matrix dissimilarity rate, YY1 was identified as the only intersecting transcription factor across all three databases (Fig. 7A). To evaluate

whether YY1 regulates FBXO33 expression in GBC, GBC cells with YY1 knockdown (sh-YY1) and overexpression (OE-YY1) were constructed (Fig. 7B, C). RT-qPCR analysis revealed that sh-YY1

significantly reduced FBXO33 mRNA expression (Fig. 7D), while OE-YY1 significantly increased it (Fig. 7E). Western blot results confirmed similar effects at the protein level (Fig. 7F, G).

To further investigate, a wild-type plasmid (WT-PRO) containing the potential YY1 binding sequence in the FBXO33 promoter and a mutant plasmid (MUT-PRO) with a mutated binding sequence were

constructed (Fig. 7H). Dual-luciferase reporter assays demonstrated that fluorescence intensity in the WT-PRO group was higher than in the MUT-PRO group (Fig. 7I, J). Furthermore, sh-YY1

reduced fluorescence intensity in the WT-PRO group but had no significant effect on the MUT-PRO group (Fig. 7I). Conversely, OE-YY1 increased fluorescence intensity in the WT-PRO group but

had no significant effect on the MUT-PRO group (Fig. 7J). ChIP-qPCR analysis showed that YY1 was enriched in the FBXO33 promoter region. Knockdown of YY1 significantly reduced enrichment,

while overexpression of YY1 significantly increased it (Fig. 7K, L). These results confirm that in GBC, YY1 promotes the transcriptional activation of FBXO33. TRANSCRIPTIONAL ACTIVATION OF

FBXO33 BY YY1 IN GBC DEPENDS ON YY1 LACTYLATION MODIFICATION Protein lactylation modification (Kla) is a recently identified post-translational modification that plays a vital role in

regulating gene expression [25]. Notably, YY1 is one of the proteins susceptible to lactylation modification [26], with the lactylation site K183 playing a critical role in its

transcriptional function [27]. Based on this, it was hypothesized that the transcriptional activation of FBXO33 by YY1 is also regulated by lactylation modification. To test this hypothesis,

experiments were conducted. RT-qPCR and Western blot analysis showed that exogenous lactate did not alter the protein expression of YY1 (Fig. 8A) but significantly increased the mRNA and

protein expression of FBXO33 (Fig. 8B, C). Co-IP experiments demonstrated that lactate enhanced the lactylation level of YY1 (Fig. 8D, E). Since YY1 has only one lactylation site (K183)

[27], lysine K183 was mutated to arginine, creating a wild-type YY1 plasmid (Flag-WT-YY1) and a K183 mutant plasmid (Flag-K183R-YY1). Co-IP results indicated that mutation of the K183

lactylation site reduced the lactylation level of YY1 (Fig. 8F, G). Further, Western blot analysis revealed that in the Flag-K183R-YY1 group, exogenous lactate failed to increase FBXO33 mRNA

and protein expression, unlike in the Flag-WT-YY1 group (Fig. 8H, I). Dual-luciferase reporter assays confirmed that lactate significantly enhanced fluorescence intensity in the WT-PRO

group but had no significant effect on the MUT-PRO group (Fig. 8J). After K183 mutation, lactate could no longer increase fluorescence intensity in the WT-PRO group (Fig. 8J). ChIP-qPCR

analysis showed that lactate significantly elevated the enrichment level of YY1 at the FBXO33 promoter region (Fig. 8K), but this effect was lost after K183 mutation (Fig. 8K). In

conclusion, the K183 lactylation modification of YY1 enhances its ability to bind to the FBXO33 promoter region, thereby regulating the transcriptional activation of FBXO33. DISCUSSION In

recent years, studies have increasingly highlighted the critical role of protein ubiquitination in tumor development and progression [28, 29]. Targeting ubiquitin signaling pathways presents

significant potential for cancer therapy [20, 30]. A detailed understanding of ubiquitination targets and their specific mechanisms in GBC could serve as a foundation for developing novel

therapeutic strategies. Among E3 ubiquitin ligases, the F-box family has emerged as a key regulator of EMT progression and holds promise for cancer treatment [31, 32]. For instance, FBXO43,

which is highly expressed in various malignancies, promotes oncogenic activity by stabilizing SKP2, a cell cycle regulator [33]. Similarly, FBXO45 facilitates pancreatic cancer progression

by regulating the stability of the tumor suppressor USP49 [34]. FBXO33, an E3 ubiquitin ligase associated with Cullin 1 [35], is a member of the F-box protein family, characterized by

uncharacterized domains [31, 36]. Experimental results in this study confirmed that FBXO33 is upregulated in GBC and positively correlates with distant metastasis and TNM staging. Knockdown

of FBXO33 inhibits EMT progression in GBC, while its overexpression promotes EMT. These findings demonstrate that FBXO33 functions as an oncogene in GBC. E3 ubiquitin ligases link target

proteins with specific E2 ubiquitin-conjugating enzymes [8, 37], modulating the biological functions of their substrates [38]. However, limited studies have explored the target proteins of

FBXO33, with only EIF3F [39],YBX1 [40], and MYC [15] confirmed as its ubiquitination targets. p53, one of the first identified tumor suppressors [41], has been extensively studied in cancer

research [42, 43]. When mutated, p53 loses its tumor-suppressive functions, and mutp53 gains oncogenic properties, promoting cancer cell survival, proliferation, and metastasis [44].

Mutations in p53 are common in GBC [45] and are associated with poorer patient prognoses [22]. The ubiquitination of p53 has been extensively studied in recent years. MDM2, a well-known

ubiquitin ligase, promotes p53 ubiquitination and represents a promising target for cancer therapy through the MDM2/p53 axis [46, 47]. Additionally, p53 has been identified as a substrate

for FBXL8 [48], VPRBP [49], TRIM31 [50], and SCML2 [51]-mediated ubiquitination. Interestingly, both wtp53 and mutp53 can undergo ubiquitination [52], with some E3 ubiquitin ligases

targeting both forms [53, 54], while others exhibit specificity for either wtp53 or mutp53 [55,56,57]. In this study, p53 was predicted to be a ubiquitination target of FBXO33 through the

Ubibrowser platform and subsequently confirmed by Co-IP experiments. Further analysis revealed that FBXO33’s effect on wtp53 is significantly stronger than on mutp53. Mechanistic studies

identified the DNA binding domain (DBD) of p53 as the critical region for FBXO33-mediated ubiquitination, with K291 and K292 in the DBD playing essential roles in regulating GBC EMT

progression. Analysis of tissue samples from GBC patients further confirmed the correlation between FBXO33 and wtp53, but not mutp53. It was also observed that high expression of FBXO33 is

negatively correlated with prognosis in patients with wtp53, whereas no such correlation was observed in patients with mutp53. The transcription factor YY1 regulates the expression of

various genes in malignant tumors by mediating enhancer-promoter interactions [58, 59]. Using predictions from multiple databases, potential binding sequences of YY1 were identified in the

promoter region of FBXO33. Experimental verification confirmed that YY1 acts as a transcription factor for the FBXO33 gene, partially explaining the high expression of FBXO33 in GBC. In the

context of protein post-translational modification, protein lactylation induced by tumor metabolite lactate plays a critical role in regulating protein function [60]. The K183 site on YY1 is

a key lactylation site [26, 27]. Dual-luciferase reporter assays and ChIP-qPCR analysis demonstrated that lactate enhances the enrichment of YY1 in the promoter region of FBXO33. Further

experiments confirmed that the binding ability of YY1 to the FBXO33 promoter region is strengthened following lactylation modification at K183, revealing a novel mechanism for the

transcriptional activation of FBXO33 in GBC. In conclusion, this study highlights the positive correlation between FBXO33 expression and the prognosis of GBC. As an oncogene, FBXO33 promotes

EMT progression by enhancing p53 ubiquitination at K291 and K292, leading to decreased p53 expression. Furthermore, YY1 activates FBXO33 transcription in GBC through K183 lactylation

modification, sustaining high FBXO33 expression in GBC. These findings elucidate the mechanism of action of FBXO33 in GBC and provide potential strategies for targeted ubiquitin signaling

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2024;187:294–311.e21. Article  CAS  PubMed  Google Scholar  Download references FUNDING This study was funded by the Natural Science Foundation of Fujian Province (Grant No. 2022J011300).

AUTHOR INFORMATION Author notes * These authors contributed equally: Zhenheng Wu, You Peng, Wen Chen. AUTHORS AND AFFILIATIONS * Department of Hepatobiliary Surgery and Fujian Institute of

Hepatobiliary Surgery, Fujian Medical University Union Hospital, Fuzhou, 350001, Fujian, China Zhenheng Wu * The Third Affiliated Hospital of Sun Yat-sen University, Zhaoqing Hospital,

Health Management Center, Zhaoqing, 526070, Guangdong, China You Peng * Department of Hepatobiliary Surgery, Fuzhou First Hospital Affiliated with Fujian Medical University, Fuzhou, 350009,

Fujian, China Wen Chen * Department of Hepatic Surgery Center, Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China Feng Xia * The Basic

Medical School, Hubei University of Science and Technology, Xianning, 437100, Hubei, China Tieshan Song & Qiming Ke * Department of General Surgery, Tianjin Medical University General

Hospital, Tianjin, 300052, China Qiming Ke Authors * Zhenheng Wu View author publications You can also search for this author inPubMed Google Scholar * You Peng View author publications You

can also search for this author inPubMed Google Scholar * Wen Chen View author publications You can also search for this author inPubMed Google Scholar * Feng Xia View author publications

You can also search for this author inPubMed Google Scholar * Tieshan Song View author publications You can also search for this author inPubMed Google Scholar * Qiming Ke View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Study design (Qiming Ke, Zhenheng Wu), immunohistochemistry analysis (Zhenheng Wu, Wen Chen, Feng Xia),

cell and animal experiments (Zhenheng Wu, Qiming Ke, Wen Chen), qPCR and WB experiments (Qiming Ke, You Peng, Tieshan Song), data analysis (Qiming Ke, Zhenheng Wu, You Peng, Wen Chen),

drafting of the manuscript (Qiming Ke), approval of the final version of the manuscript (all authors). CORRESPONDING AUTHOR Correspondence to Qiming Ke. ETHICS DECLARATIONS COMPETING

INTERESTS The authors declare no competing interests. ETHICS APPROVAL Research involving human subjects is reviewed and approved by the Ethics Committee of Fujian Medical University Union

Hospital. Patients/participants provide written informed consent to participate in this study. The animal study has been approved by the Ethics Committee of the Medical Faculty of the Fujian

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CITE THIS ARTICLE Wu, Z., Peng, Y., Chen, W. _et al._ Lactylation-driven transcriptional activation of FBXO33 promotes gallbladder cancer metastasis by regulating p53 polyubiquitination.

_Cell Death Dis_ 16, 144 (2025). https://doi.org/10.1038/s41419-025-07372-y Download citation * Received: 01 June 2024 * Revised: 20 December 2024 * Accepted: 21 January 2025 * Published: 28

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