GLI2-HRD1 axis facilitates 5-FU resistance in gastric cancer cells by regulating ubiquitination degradation of UCK2

Highlights

• •UCK2 identified as key gene for 5-FU resistance in gastric cancer cells.
• •GLI2 and HRD1 upregulation linked to decreased UCK2 and increased 5-FU resistance.
• •HRD1 promotes 5-FU resistance by ubiquitinating and degrading UCK2.
• •GLI2 overexpression activates HRD1, enhancing GC cell proliferation and resistance.
• •GLI2-HRD1-UCK2 axis identified as crucial pathway for 5-FU resistance in GC.

Abstract

5-Fluorouracil (5-FU) is a primary chemotherapeutic agent for treating gastric cancer (GC), yet resistance to 5-FU frequently limits its effectiveness and contributes to poor patient outcomes. This study investigated the molecular mechanisms by which uridine-cytidine kinase 2 (UCK2) influences 5-FU resistance in GC. Using a genome-wide CRISPR knockout (GeCKO v2) library, we identified UCK2 as a critical gene for 5-FU sensitivity in GC cells. In 5-FU-resistant GC cells, the transcription factor GLI2 and the E3 ubiquitin ligase HRD1 were both upregulated, while UCK2 expression was significantly reduced. Functional assays demonstrated that lowering UCK2 or increasing HRD1 expression enhanced GC cell proliferation and 5-FU resistance, with HRD1 mediating 5-FU resistance through the ubiquitination and degradation of UCK2. Furthermore, GLI2 overexpression promoted cell proliferation and resistance to 5-FU by transcriptionally activating HRD1. In vivo experiments confirmed that GLI2 knockdown effectively reduced tumor growth under 5-FU treatment, an effect that was reversed by HRD1 overexpression. These findings reveal the GLI2-HRD1-UCK2 axis as a crucial pathway for modulating 5-FU resistance in GC, suggesting new potential targets for overcoming chemoresistance in GC therapy.

Introduction

Gastric cancer (GC) ranks as the fifth most common malignant tumor globally and is the third leading cause of cancer-related deaths [1]. For advanced stages of GC, surgical interventions have limited efficacy [2]. Chemotherapy remains a primary treatment modality for GC, with 5-fluorouracil (5-FU) being a key chemotherapeutic agent used [3]. However, the recurrence of GC in many patients following multiple rounds of 5-FU chemotherapy due to drug resistance poses a significant challenge in clinical treatment [4]. Consequently, addressing 5-FU resistance is crucial for enhancing the survival of GC patients, and understanding the mechanisms regulating this resistance is key to achieving this goal.

Uridine‐cytidine kinase 2 (UCK2) is an enzyme involved in the synthesis of pyrimidine nucleotides, which are essential for DNA and RNA production [5]. By catalyzing the phosphorylation of uridine and cytidine to their monophosphate forms, UCK2 plays a crucial role in the anti-cancer effects of certain nucleoside analogs, such as 3′-ethynyl nucleosides, against GC [6]. High-throughput genetic screening methods have become valuable tools for exploring the molecular mechanisms that drive specific cellular behaviors, including drug resistance. The CRISPR/Cas9 system, known for its ease of use and high efficiency in gene editing, has been widely adopted for studying gene function across various biological systems. Recently, CRISPR/Cas9 library screens have been developed for genetic studies in both cell cultures and animal models, enabling the identification of genes that are crucial for cancer cell survival, proliferation, migration, and drug resistance [[7], [8], [9]]. By using genome-wide CRISPR/Cas9 library screening, we identify UCK2 as one of critical factors associated with 5-FU sensitivity in gastric cancer cells. Thus, a deeper investigation into UCK2’s role in 5-FU resistance could be instrumental in overcoming chemotherapy resistance in GC patients.

Glioma-associated oncogene family zinc finger 2 (GLI2), a transcription factor in the hedgehog signaling pathway, plays a crucial role in the development of various human malignancies, including GC [10]. Increased GLI2 expression is associated with poorer survival outcomes in GC patients [11]. Moreover, inhibiting GLI2 can reduce GC cell migration and proliferation [12]. Notably, GLI2 expression is heightened in GC cells that survive after 5-FU treatment, and silencing GLI2 increases their sensitivity [13]. However, the precise mechanisms through which GLI2 influences 5-FU resistance in GC remain unclear and warrant further investigation. 3-Hydroxy-3-methylglutaryl reductase degradation (HRD1), an E3 ubiquitin ligase, plays a role in endoplasmic reticulum-associated degradation and is implicated in cancer progression through its regulation of protein ubiquitination [14]. For instance, HRD1 has been shown to promote hepatocellular carcinoma development by mediating the ubiquitination and degradation of PTEN [15]. HRD1 expression is notably high in GC [16], yet its role in 5-FU resistance within GC remains to be fully elucidated.

In this study, we utilized the GeCKOv2 library [17] to perform genome-wide knockout in gastric cancer cells aimed to screen and identify 5-FU resistance-related genes, and discovered that knockout of UCK2 resulted in a significant decrease in the sensitivity of gastric cancer cells to 5-FU. We observed that HRD1 could mediate the ubiquitination and degradation of UCK2, and GLI2 could bind to the HRD1 promoter. This suggests that GLI2 may transcriptionally activate HRD1 expression in GC cells, leading to increased UCK2 ubiquitination degradation, and consequently, promoting 5-FU resistance in GC cells. Our findings provide a theoretical basis for reversing 5-FU resistance in gastric cancer.

Methods

Cell culture

The gastric mucosal cells (GES-1), gastric cancer cell lines MKN-45, SNU-16, HGC-27, NUGC-3 and AGS and a human embryonic kidney cell line 293T were obtained from the American Type Culture Collection (ATCC; VA, USA). These cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, MD, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco) in an incubator with 5 % CO₂ atmosphere at 37 °C. The 5-FU-resistant GC cell lines (HGC-27/5-FU and AGS/5-FU cells) were developed by progressively increasing exposure to 5-FU (Sigma-Aldrich, MO, USA) as previously reported [18].

Tissue specimens

Six pairs of gastric cancer (GC) tissues and their corresponding adjacent normal gastric tissues were obtained from the tumor biobank of Fujian Medical University. All patients included in this study underwent surgical resection without receiving preoperative radiotherapy or chemotherapy. Written informed consent was obtained from each patient before tissue collection. Immediately after resection, the tissues were rapidly frozen in liquid nitrogen and stored at −80 °C. All experimental procedures adhered to the ethical guidelines of the Declaration of Helsinki.

Genome-wide CRISPR/Cas9 knockout library screen

The Human CRISPR Knockout Pooled Library, GeCKOv2, was sourced from Addgene (http://www.addgene.org/crispr/libraries/geckov2/). This library was transfected into HEK293T cells, followed by lentivirus packaging and purification according to the GeCKO v2 protocol [[7], [8], [9]]. To identify the effective concentration of 5-FU required to inhibit the proliferation of AGS cells, a preliminary dose-response study was performed. AGS cells were subsequently infected with the lentivirus at a low multiplicity of infection (0.3), ensuring that the majority of cells received the viral construct. The concentrated GeCKOv2 library (30 µl) was added to each well containing 3 × 10⁶ cells, achieving a transduction efficiency of 30 %, which corresponds to approximately 270 cells per lentiCRISPR construct. Post-infection, the cells were selected with puromycin (1 µg/ml) for 72 h to enrich the population of transduced cells, followed by an additional 5 days of incubation in the presence of puromycin to enhance viral integration and gene expression. After selection, the transduced AGS cells were divided into two equal groups, each containing a minimum of 3 × 10⁶ cells. One group was cryopreserved for subsequent genomic DNA analysis, while the other group was split into two replicates: one cultured in DMEM supplemented with 62 ng/ml 5-FU (equivalent to IC₉₀), and the other in DMEM with an equivalent volume of DMSO as a control. The cells were passaged or provided with fresh media every 3 days. Fourteen days after the addition of 5-FU, cell pellets (3 × 10⁶ cells) were collected. The frozen cell pellets were thawed, and genomic DNA was extracted using the TIANamp Genomic DNA Kit, DP304 (Tiangen, China). The sgRNA sequences were then amplified using NEBNext® High-Fidelity 2 × PCR Master Mix and subjected to high-throughput amplicon sequencing, performed by Novogene Technology (Beijing, China). The analysis of sgRNA read counts and identification of significant hits were conducted using the MAGeCK v0.5.7 algorithm.

Plasmid construction

The overexpression vectors of pcDNA3.1-UCK2, pcDNA3.1-GLI2, and pcDNA3.1-HRD1 were constructed by inserting the cDNA of UCK2 (GenBank accession number: NM_001363568.2), GLI2 (GenBank accession number: 001371271.1) or HRD1 (GenBank accession number: NM_032431.3) chemically synthesized by General Biosystems into the pcDNA3.1/myc-His(-)A (Invitrogen, Cat#: V855-20). Short hairpin RNA (shRNA) sequences targeting UCK2 and GLI2 were cloned into the pLKO.1-puro vector, respectively. Lentivirus production was carried out in HEK293T cells using the second-generation packaging system, comprising pMD2.G (Cat#12259, Addgene) and psPAX2 (Cat#12260, Addgene). The plasmid pGL4.10-HRD1 Pro-Luc with the HRD1 promoter driving firefly luciferase was constructed by ligating the PCR-generated full-length HRD1 promoter (nucleotides -2000 nt to +100 nt, relative to the transcription start site) into the XhoI and HindIII (NEB, Ipswich, MA, USA) cleaved sites of the luciferase reporter plasmid pGL4.10-Basic (Promega, Madison, Wisconsin, USA). The pGL4.10-HRD1 Pro-Luc plasmid was utilized as a template to generate mutants of predicted GLI2 binding sites. Taq DNA Polymerase High Fidelity (Vazyme, Nanjing, China) was used for PCR amplification, and the amplified PCR products were confirmed by DNA sequencing. Transfection was performed using Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer’s instructions.

Cell counting kit-8 (CCK-8) assay

1 × 10³ cells per well were plated into a 96-well plate and allowed to grow for 12 h, facilitating their attachment and initial proliferation. Subsequently, the specific drug being studied was introduced into the culture medium and left for an additional 72 h. The viability of the cells was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo, Japan), adhering to the instructions provided by the manufacturer. The absorbance for each well was measured at a wavelength of 450 nm (A450) using a microplate reader (Bio-Tek, Winooski, VT, USA). To calculate cell viability, the average absorbance (A) of the experimental group was divided by the average absorbance of the control group, and this ratio was then multiplied by 100 %. The half-maximal inhibitory concentration (IC₅₀) was determined using nonlinear regression analysis in GraphPad Prism (version 8.0.2; GraphPad Software Inc, San Diego, USA).

Colony formation assay

The colony formation assay involved seeding 1 × 10³ cells into separate wells of a 6-well plate. These cells were treated with either a vehicle control or the drug for 48 h. Following this, they were incubated for a period ranging from 10 to 14 days. Post incubation, the cultures were fixed using 25 % (v/v) methanol and then stained with a solution of 1 % crystal violet (Sigma-Aldrich, MO, USA). The resultant colonies were then counted manually.

Western blot analysis

Cells were lysed using RIPA buffer (Beyotime Biotechnology, Shanghai, China), and protein concentrations were determined using a BCA Protein Assay Kit (Beyotime Biotechnology). A total of 20 μg of protein per sample was separated by 10 % SDS-PAGE and transferred onto Millipore PVDF membranes. Membranes were blocked and incubated with primary antibodies against GLI2 (ab277800), HRD1 (ab170901), UCK2 (ab308252), γ-H2AX (ab229914) and β-actin (ab8226) overnight, followed by incubation with a secondary antibody (Abcam, 1:5000, ab7090) for 60 minutes. Blots were visualized using a Bio-Rad GEL imaging system and analyzed with ImageJ software. All antibodies were sourced from Abcam (Cambridge, UK).

Coimmunoprecipitation (Co-IP)

Cell lysates were prepared using a lysis buffer containing protease inhibitors. Sepharose CL-4B beads (Sigma-Aldrich) were incubated with IgG (Abcam, 1:50, ab172730) and UCK2 antibodies (Abcam, 1:50, ab241281) for 4 h, followed by overnight incubation with cell lysates at 4 °C. Bound proteins were eluted for Western blot analysis.

UCK2 ubiquitination analysis

Following treatment, cells were incubated with 10 μM MG132 for 6 h, lysed in 1 % SDS buffer, and boiled for 10 minutes. Lysates were sonicated, diluted, and the supernatant was incubated with UCK2 Ub-IP antibody (Abcam, 1:50, ab241281), followed by protein A/G IP magnetic beads for 12 h at 4 °C. Ubiquitination of UCK2 was assessed by Western blot using an anti-Ub antibody (Abcam, 1:1000, ab140601).

Chromatin immunoprecipitation (ChIP) assay

In this assay, cells underwent fixation and quenching, followed by DNA fragmentation through sonication. Overnight incubation of cell lysates at 4 °C was done with either anti-GLI2 (Abcam, 1:100, ab226390) or anti-IgG (Abcam, 1:100, ab172730) antibodies. Protein A/G PLUS Agarose (Santa Cruz, TX, USA) was employed to isolate chromatin-antibody complexes. Subsequently, DNA was purified and subjected to analysis via quantitative PCR (qPCR).

Animal experiments

All animal experiments were performed in compliance with the ethical guidelines for animal research set by the Institution. Male BALB/c nude mice (8-week-old) were randomly divided into five groups: control, NC, shGLI2, HRD1, and shGLI2 + HRD1, with each group consisting of six mice. These BALB/C nude mice were subcutaneously injected with 0.2 mL of HGC-27 and AGS cells (2 × 10⁴ cells), which were stably transfected with oe-HRD1, shGLI2, shNC, or oe-NC. The drug was administered to the mice intraperitoneal injections of 5-FU (50 mg/kg) every 4 days for a duration of 20 days, starting from the time when tumors became palpable. Tumor volume was calculated using the formula: (length × width²/2). Following the treatment period, the mice were euthanized, and tumor tissues were harvested.

Immunohistochemistry (IHC)

The tumor sections underwent deparaffinization and antigen retrieval, followed by blocking and overnight incubation with primary antibodies against Ki67, GLI2, HRD1, and UCK2. Subsequently, they were incubated with an appropriate secondary antibody (Abcam, 1:500, ab150077). The sections were then stained with DAB, dried, counterstained with hematoxylin, and mounted. Stained tissue sections were analyzed under a light microscope at 40 × magnification. Five non-overlapping fields were randomly selected, and both positively and negatively stained cells in each field were recorded. The percentage of positive cells was calculated using the following formula: (Number of positive cells/total number of cells) × 100. The final percentage was obtained by averaging the values from all analyzed fields, providing a reliable measure of positive staining across the tissue section.

Statistical analysis

The results are presented as the mean ± standard deviation, based on a minimum of three independent experiments. All statistical calculations were performed using GraphPad Prism 8.0.2 software (GraphPad Software Inc). The comparison between two distinct groups was made using a two-tailed Student’s t-test. For situations involving comparisons among multiple groups, a one-way analysis of variance (ANOVA) was executed, with subsequent analysis using Tukey’s post hoc test. Statistical significance was established at a P-value threshold of less than 0.05.

Results

Identification of genes associated with 5-FU resistance via genome-wide CRISPR knockout screening

In this study, we conducted a genome-wide CRISPR/Cas9 knockout screening to identify key genes involved in resistance to 5-fluorouracil (5-FU) in the human gastric cancer cell line AGS. The Human GeCKO v2 CRISPR library (http://www.addgene.org/crispr/libraries/geckov2/), containing 123,411 unique sgRNAs targeting 19,050 human genes, was used to create a pool of mutant cells. First, a 5-FU concentration-survival curve was generated for the parental AGS cells (Fig. 1A) to determine the optimal drug concentration for treatment. The mutant cell pool was then treated with either vehicle or a concentration of 5-FU equivalent to the IC₉₀, the concentration required to inhibit 90 % of cell growth. Viable cells were collected to assess sgRNA enrichment. Our hypothesis was that the knockout of genes essential for 5-FU sensitivity would confer resistance to the drug, and the corresponding sgRNAs would be enriched in the surviving cell population. The sgRNA-coding regions were PCR-amplified and subjected to next-generation sequencing (NGS) to quantify the representation of sgRNAs and evaluate their enrichment. As shown in Fig. 1B, the sgRNA coverage in the 5-FU-treated group was significantly lower than in the vehicle-treated control group. However, the control group exhibited higher median read counts and a more uniform distribution with fewer outliers, indicating that the CRISPR screen successfully identified genes whose loss allowed cells to survive in a highly cytotoxic 5-FU environment. Among the genes identified, uridine-cytidine kinase 2 (UCK2), a key enzyme in the pyrimidine salvage pathway responsible for phosphorylating uridine and cytidine to form uridine monophosphate (UMP) and cytidine monophosphate (CMP), emerged as the most positively selected gene in the 5-FU-treated cells (Fig. 1C). This suggests that the loss of UCK2 might confer resistance to 5-FU in gastric cancer cells. Further analysis of the hits using Gene Ontology (GO) enrichment revealed that the most enriched biological process was “macromolecule biosynthetic process” (Fig. 1D). This finding underscores the role of key biosynthetic pathways in 5-FU resistance.

Fig. 1.

CRISPR library screening identified UCK2 as a key factor in 5-FU sensitivity. (A) Survival curve of AGS cells treated with increasing concentrations of 5-FU, assessed using the CCK-8 assay, showing a dose-dependent decrease in cell viability. (B) Box plot comparing the median read count of all sgRNAs between vehicle-treated and 5-FU-treated samples. The reduction in the median sgRNA read count and the presence of more outliers in the 5-FU group indicate the effectiveness of the CRISPR library screening. (C) Scatterplot showing the enrichment of specific sgRNAs after 5-FU treatment, with UCK2 emerging as the most positively selected gene. (D) Gene Ontology (GO) analysis revealed that the positively selected genes in the 5-FU-treated AGS cells were primarily involved in biological processes such as macromolecule biosynthesis, cellular macromolecule biosynthesis, and macromolecule metabolism.

Altered expression of GLI2, HRD1, and UCK2 in gastric cancer cells and the role of UCK2 in 5-FU resistance

To expand upon the insights gained from the genome-wide CRISPR knockout screen, we next investigated the expression patterns of key genes implicated in 5-FU resistance. Given their potential roles in drug sensitivity [[7], [8], [9]], we specifically focused on GLI2, HRD1, and UCK2. To this end, we first assessed the expression levels of these genes in both gastric mucosal cells and a range of gastric cancer (GC) cell lines. Comparative analysis revealed that in GC cells (MKN-45, SNU-16, HGC-27, NUGC-3, and AGS), GLI2 and HRD1 were expressed at considerably higher levels compared to gastric mucosal cells (GES-1), while UCK2 expression was notably lower (Fig. 2A). To further validate these findings, we examined the expression levels of GLI2, HRD1, and UCK2 in six paired primary gastric cancer tissues (T) and their corresponding adjacent non-cancerous tissues (N) by Western blot analysis (Fig. 2B). Consistent with the results observed in GC cell lines, GLI2 and HRD1 were significantly upregulated in tumor tissues compared to adjacent normal tissues, whereas UCK2 expression was markedly reduced in tumor samples. Quantification of protein expression levels further confirmed these differences, showing statistically significant increases in GLI2 and HRD1, while UCK2 levels were significantly lower in tumor tissues compared to adjacent normal tissues. These results support the hypothesis that the GLI2-HRD1 axis is upregulated, while UCK2 is downregulated in GC, reinforcing its potential role in GC progression and chemoresistance. Given the moderate expression of UCK2 in HGC-27 and AGS cells among all GC cell lines, these were chosen for further study. In the established 5-FU-resistant GC cell lines (HGC-27/5-FU and AGS/5-FU), GLI2 and HRD1 exhibited higher expression, whereas UCK2 expression was lower compared to parent cells (Fig. 2C). To explore UCK2’s role in 5-FU resistance in GC cells, a targeted knockdown of UCK2 was executed in two GC cell lines, HGC-27 and AGS. The efficacy of this knockdown was evident, as there was a notable reduction in UCK2 protein levels in these cell lines, confirmed by the Western blot analysis (Fig. 2D). There was a significant increase in cell proliferative ability (Fig. 2E) and clonogenic potential (Fig. 2F) in the GC cells where UCK2 had been knocked down. This suggests that UCK2 plays a role in regulating cell growth, with its downregulation leading to enhanced cellular proliferation and survival. Most notably, the knockdown of UCK2 led to a decrease in the sensitivity of GC cells to 5-FU (Fig. 2G). The findings from these experiments paint a clear picture of the role of UCK2 in GC. The decreased expression of UCK2 seems to be associated with an aggressive GC cell phenotype characterized by enhanced growth and a diminished response to 5-FU. Hence, targeting UCK2, either directly or indirectly, might be a potential strategy for overcoming 5-FU resistance in GC treatment.

Fig. 2.

Dysregulation of GLI2, HRD1, and UCK2 in gastric cancer cells and its impact on 5-FU resistance. (A) Protein levels of GLI2, HRD1, and UCK2 in gastric cancer (GC) cell lines (MKN-45, SNU-16, HGC-27, NUGC-3, AGS) and gastric mucosal cells (GES-1) were assessed by western blot. (B) Western blot analysis was performed to compare the protein expression levels of GLI2, HRD1, and UCK2 in six pairs of primary gastric cancer tissues (T) and their corresponding adjacent non-cancerous tissues (N). The accompanying bar graph quantifies the relative expression levels of GLI2, HRD1, and UCK2, normalized to β-actin, in all six paired tissue samples. Data are presented as mean ± SD. (C) Expression of GLI2, HRD1 and UCK2 in 5-FU-resistant GC cell lines (HGC-27/5-FU and AGS/5-FU) examined by Western blot analysis. The histograms indicate the levels of the protein determined from 3 independent experiments expressed as the mean ratio relative to that in the control after normalization to β-actin. (D) Confirmation of shRNAi knockdown of UCK2 by Western blot analysis. (E) Enhanced proliferation rate in UCK2 knockdown cells measured by CCK-8 assay. (F) Elevated clonogenic potential in UCK2 knockdown cells determined through colony formation assay. (G) UCK2 knockdown confers resistance to 5-FU as demonstrated by the CCK-8 assay. Data are presented as mean ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, **p < 0.001 (Student’s t-test).

HRD1 overexpression promotes GC cell proliferation and 5-FU resistance by targeting UCK2 for degradation

To elucidate the role of HRD1 in promoting 5-FU resistance in GC cells, a series of functional and mechanistic studies were conducted. Overexpression of HRD1 in HGC-27 and AGS GC cell lines significantly increased HRD1 protein levels, which was accompanied by a marked reduction in UCK2 protein levels (Fig. 3A). This reduction correlated with enhanced GC cell proliferation (Fig. 3B) and clonogenic potential (Fig. 3C) as well as increased resistanceresistance to 5-FU treatment (Fig. 3D), suggesting a functional link between HRD1 and chemoresistance.

Fig. 3.

Overexpression of HRD1 enhances gastric cancer cell proliferation and resistance to 5-FU by promoting the degradation of UCK2. (A) Western blot analysis showing that HRD1 overexpression leads to a reduction in UCK2 protein levels in HGC-27 and AGS cells. The histograms indicate the levels of the protein determined from 3 independent experiments expressed as the mean ratio relative to that in the control after normalization to β-actin. (B) CCK-8 assay demonstrating increased proliferation in HRD1-overexpressing GC cells compared to control cells. Data are presented as mean ± SD from three independent experiments.. (C) Representative images (left) and quantification (right) of colony formation assays showing enhanced clonogenicity in HRD1-overexpressed GC cells. (D) CCK-8 assay assessing the impact of HRD1 overexpression on 5-FU sensitivity. HRD1-overexpressing cells exhibited reduced sensitivity to 5-FU, as evidenced by higher cell viability compared to controls. (E) Co-IP analysis in 293T cells revealing the interaction between HRD1 and UCK2. HRD1 and UCK2 proteins were pulled down using an anti-Flag antibody, confirming their direct interaction. (F) Co-IP analysis in GC cells showing both endogenous and exogenous interactions between HRD1 and UCK2 following HRD1 overexpression. Immunoprecipitation was performed using anti-HRD1 and anti-UCK2 antibodies. (G) Western blot analysis assessing UCK2 protein levels in GC cells treated with MG132, a proteasome inhibitor, to determine whether HRD1-mediated UCK2 degradation occurs via the ubiquitin-proteasome pathway. (H) Western blot analysis of UCK2 protein levels in GC cells treated with cycloheximide (CHX), a protein synthesis inhibitor, to evaluate the stability and degradation rate of UCK2 in HRD1-overexpressing cells. (I) Ubiquitination assay showing that HRD1 overexpression increases UCK2 ubiquitination, as assessed by immunoprecipitation of UCK2 followed by immunoblotting with an anti-ubiquitin antibody. (J) Western blot analysis comparing HRD1 and UCK2 protein expression in GC cells overexpressing UCK2, HRD1 or both. (K) Colony formation assay showing the clonogenic potential of GC cells overexpressing UCK2, HRD1, or both. Representative images (left) and quantification (right) indicate that HRD1 overexpression enhances clonogenicity, while UCK2 overexpression has an inhibitory effect.. (L) CCK-8 assay measuring the proliferation rate of GC cells overexpressing UCK2, HRD1 or both. (M) CCK-8 assay measuring the sensitivity of GC cells overexpressing UCK2, HRD1 or both to 5-FU treatment. Data are presented as mean ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (One-way ANOVA).

To further explore the underlying mechanism, co-immunoprecipitation (Co-IP) assays confirmed a direct interaction between HRD1 and UCK2 (Fig. 3E and F). Additionally, HRD1 overexpression led to a significant decrease in UCK2 protein levels, an effect that was reversed by MG132 treatment, a proteasome inhibitor (Fig. 3G), indicating proteasomal degradation of UCK2. Cycloheximide (CHX) chase assays further demonstrated that HRD1 facilitated UCK2 degradation (Fig. 3H), and ubiquitination assays revealed a substantial increase in UCK2 ubiquitination in the presence of HRD1 overexpression (Fig. 3I). To assess the functional consequences of HRD1-mediated UCK2 degradation, rescue experiments were performed by co-expressing HRD1 and UCK2. While HRD1 overexpression reduced UCK2 protein levels, UCK2 overexpression restored its expression, counteracting the effect of HRD1 (Fig. 3J). Moreover, UCK2 overexpression inhibited GC cell clonogenicity (Fig. 3K), proliferation (Fig. 3L), and reversed HRD1-induced 5-FU resistance (Fig. 3M), further supporting the role of HRD1 in chemoresistance via UCK2 degradation. To delineate the mechanism by which HRD1 selectively targets UCK2 for ubiquitination, site-directed mutagenesis experiments were conducted to identify the key ubiquitination sites on UCK2 that mediate its degradation by HRD1. Based on bioinformatic predictions and structural analysis, two lysine residues (K78 and K202) were individually and combinatorially mutated (K78R, K202R, and K78R/K202R mutants) to assess their role in HRD1-mediated ubiquitination. As shown in Fig. S1, ubiquitination assays and Western blot analysis revealed that mutation of K78 (K78R) significantly reduced UCK2 ubiquitination and led to increased UCK2 protein stability. In contrast, the K202R mutation had no significant effect on either ubiquitination or UCK2 protein levels. Notably, the double mutation (K78R/K202R) recapitulated the effects of K78R alone, confirming that K78 is the critical ubiquitination site required for HRD1-mediated UCK2 degradation. These results collectively demonstrate that K78 is the key lysine residue required for UCK2 ubiquitination and degradation, providing mechanistic insight into HRD1-mediated regulation of UCK2 stability.

GLI2 transcriptionally upregulates HRD1 to promote GC cell proliferation and 5-FU resistance

By overexpressing GLI2 in GC cells, it was observed that not only did GLI2 levels increase, but there was also a concomitant rise in HRD1 protein levels, while the expression of UCK2 decreased (Fig. 4A). This change in protein expression had significant physiological implications. Specifically, the upregulation of GLI2 led to a notable increase in GC cell growth (Fig. 4B) and clonogenic potential (Fig. 4C). Moreover, this alteration was also associated with an increased resistance of these cells to 5-FU (Fig. 4D). This suggests that GLI2 plays a crucial role in modulating the biological behavior of GC cells, particularly in how they respond to 5-FU treatment.

Fig. 4.

GLI2 promotes GC cell proliferation and 5-FU resistance by transcriptionally upregulating HRD1. (A) Western blot analysis of GLI2, HRD1, and UCK2 protein levels in HGC-27 and AGS cells with GLI2 overexpression. β-actin served as a loading control. GLI2 overexpression led to an increase in HRD1 expression and a decrease in UCK2 levels. The histograms indicate the levels of the protein determined from 3 independent experiments expressed as the mean ratio relative to that in the control after normalization to β-actin. (B) Cell proliferation of GC cells transfected with control (NC) or GLI2 overexpression plasmids was assessed using the CCK-8 assay at different time points (0, 24, 48, and 72 h). GLI2 overexpression significantly enhanced cell proliferation in both HGC-27 and AGS cells. (C) Representative images of colony formation assays in GC cells with GLI2 overexpression, demonstrating increased clonogenic potential compared to control cells. The quantification of colony numbers is shown in the bar graph. (D) 5-FU sensitivity was evaluated in GC cells with GLI2 overexpression by CCK-8 assay. The dose-response curves show that GLI2-overexpressing cells exhibited significantly reduced sensitivity to 5-FU compared to control cells. (E) Westen blot analysis of GLI2, HRD1 and UCK2 expression in GC cells transfected with GLI2-specific shRNA (shGLI2) or control shRNA (shNC). Knockdown of GLI2 led to a decrease in HRD1 expression and an increase in UCK2 levels. The histograms indicate the levels of the protein determined from 3 independent experiments expressed as the mean ratio relative to that in the control after normalization to β-actin. (F) Bioinformatic prediction of GLI2 binding sites in the HRD1 promoter region using the JASPAR database. Two potential GLI2-binding motifs were identified within the HRD1 promoter at positions –1734 to –1722 bp and –738 to –726 bp upstream of the transcription start site. (G) Chromatin immunoprecipitation (ChIP) assay confirming GLI2 binding to the HRD1 promoter in GC cells overexpressing GLI2. The enrichment of GLI2 at the predicted HRD1 promoter regions suggests direct transcriptional regulation. (H) ChIP assay in GLI2-knockdown GC cells demonstrating reduced GLI2 binding to the HRD1 promoter, further confirming the direct regulatory interaction. (I) Dual-luciferase reporter assay assessing the activity of HRD1 promoter constructs in GLI2-knockdown GC cells. Knockdown of GLI2 significantly decreased the luciferase activity of wild-type HRD1 promoter constructs (HRD1-WT), whereas mutation of the predicted GLI2-binding sites (HRD1-MUT) abrogated this effect. (J) Western blot analysis of GLI2, HRD1, and UCK2 protein levels in GC cells with GLI2 knockdown, HRD1 overexpression, or their combination. Overexpression of HRD1 restored the downregulation of HRD1 and the upregulation of UCK2 caused by GLI2 knockdown. (K) Cell proliferation was measured using the CCK-8 assay in GC cells transfected with shGLI2, HRD1 overexpression plasmid, or both. GLI2 knockdown reduced proliferation, whereas HRD1 overexpression rescued this effect. (L) Representative images of colony formation assays in GC cells transfected with shGLI2, HRD1 overexpression plasmid, or both. The quantification of colony numbers indicates that HRD1 overexpression restored the reduced clonogenic potential caused by GLI2 knockdown. (M) 5-FU sensitivity was assessed in GC cells with GLI2 knockdown, HRD1 overexpression, or their combination using CCK-8 assay. GLI2 knockdown enhanced 5-FU sensitivity, whereas HRD1 overexpression rescued this effect, indicating that HRD1 mediates GLI2-induced chemoresistance. Results are presented as mean ± SD, based on data from at least three independent experiments. Statistical significance was determined one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).

GLI2, a key transcription factor, plays a crucial role in modulating tumor growth and progression through its regulatory impact on gene expression [19]. Intriguingly, we found that GLI2 knockdown led to a decrease in HRD1 protein levels while concurrently causing an increase in UCK2 expression (Fig. 4E). This suggests a critical regulatory relationship where GLI2 modulates these proteins, impacting GC cell behavior. Further investigations revealed potential GLI2 binding sites within the promoter region of HRD1 (Fig. 4F), hinting at a direct regulatory mechanism. This binding was empirically confirmed through ChIP assay (Fig. 4G), establishing a direct interaction between GLI2 and the HRD1 gene. Notably, the knockdown of GLI2 was found to weaken its binding to the HRD1 promoter, leading to reduced activity of the HRD1-WT luciferase reporter (Fig. 4H), whereas the mutant version (HRD1-MUT) remained unaffected (Fig. 4I). This implies that GLI2 directly influences HRD1 transcription. In a reversal of roles, overexpression of HRD1 was observed to counteract the effects induced by GLI2 knockdown; this overexpression led to a decrease in UCK2 levels, essentially nullifying the impact of reduced GLI2 (Fig. 4J). This regulatory interaction had significant cellular consequences, as GLI2 knockdown suppressed cell proliferation (Fig. 4K), clonogenicity (Fig. 4L), and 5-FU resistance (Fig. 4M), while HRD1 overexpression restored these properties, demonstrating that HRD1 acts downstream of GLI2 to regulate UCK2 and confer 5-FU resistance in GC cells. To explore whether GLI2 directly regulates UCK2, a dual-luciferase assay was performed to assess GLI2 binding to the UCK2 promoter. The results demonstrated that GLI2 did not enhance UCK2 transcription (Fig. S2A and B), indicating that UCK2 is not a direct transcriptional target of GLI2. Furthermore, UCK2 overexpression did not alter GLI2 mRNA levels (Fig. S2C), protein expression (Fig. S2D), or stability (Fig. S2E), suggesting that UCK2 does not regulate GLI2 in a feedback-dependent manner. These findings support a model in which GLI2 transcriptionally activates HRD1, which in turn promotes UCK2 ubiquitination and degradation, ultimately leading to 5-FU resistance in GC cells. To assess the therapeutic potential of targeting this pathway, HGC-27 and AGS cells were treated with the GLI2 inhibitor GANT61 and the HRD1 inhibitor LS102. GANT61 treatment led to a reduction in GLI2 and HRD1 expression while increasing UCK2 levels (Fig. S3A), confirming that GLI2 transcriptionally regulates HRD1, which mediates UCK2 degradation. Similarly, LS102 significantly reduced HRD1 expression without affecting GLI2 levels, leading to UCK2 upregulation (Fig. S3B), further reinforcing HRD1’s role in UCK2 ubiquitination and degradation. Both inhibitors significantly enhanced 5-FU sensitivity, as evidenced by reduced cell viability (Fig. S3C) and suppressed clonogenic potential (Fig. S3D). These results highlight the translational relevance of targeting the GLI2-HRD1-UCK2 axis to improve 5-FU efficacy in GC treatment.

Since 5-FU disrupts RNA and DNA metabolism by generating toxic metabolites [20], the effect of the GLI2-HRD1-UCK2 axis on DNA replication stress was further examined by assessing γ-H2AX levels. Cells with elevated GLI2 or HRD1 expression exhibited reduced γ-H2AX accumulation following 5-FU treatment (Fig. 5A), suggesting that these proteins mitigate DNA damage and alleviate replication stress. In contrast, GLI2 or HRD1 knockdown significantly increased γ-H2AX levels (Fig. 5B), indicating that their loss exacerbates DNA damage and replication stress, potentially sensitizing cells to 5-FU. Moreover, UCK2 knockdown reduced γ-H2AX accumulation, whereas UCK2 overexpression had the opposite effect (Fig. 5C), further supporting the role of UCK2 in modulating replication stress and DNA damage in response to 5-FU treatment. To determine whether this pathway influences cellular responses to other chemotherapeutic agents, GC cells were treated with DNA damaging agent cisplatin following GLI2 knockdown or overexpression. Consistent with prior findings, GLI2 knockdown decreased GLI2 and HRD1 expression while increasing UCK2 levels, whereas GLI2 overexpression upregulated GLI2 and HRD1 while reducing UCK2 expression (Fig. 5D). Functionally, GLI2 knockdown significantly enhanced cisplatin sensitivity, whereas GLI2 overexpression conferred resistance, as determined by CCK-8 assay (Fig. 5E) and colony formation assay (Fig. 5F). These findings suggest that the GLI2-HRD1-UCK2 axis contributes to chemoresistance not only to 5-FU but also to cisplatin, highlighting its potential as a therapeutic target for overcoming resistance to multiple chemotherapeutic agents.

Fig. 5.

GLI2-HRD1-UCK2 axis modulates DNA replication stress and chemoresistance in gastric cancer cells. (A) Western blot analysis showing the effects of GLI2 and HRD1 overexpression on γ-H2AX expression, a marker of DNA damage, in HGC-27 and AGS cells treated with 5-FU. Overexpression of GLI2 or HRD1 (OE-GLI2, OE-HRD1) resulted in reduced γ-H2AX levels, indicating that both proteins protect against 5-FU-induced DNA damage and replication stress. (B) Western blot analysis of γ-H2AX expression in HGC-27 and AGS cells following GLI2 or HRD1 knockdown (sh-GLI2, sh-HRD1) in response to 5-FU treatment. Knockdown of GLI2 or HRD1 significantly increased γ-H2AX levels, suggesting that loss of these proteins exacerbates DNA damage accumulation and replication stress under 5-FU exposure. (C) Western blot analysis showing the effect of UCK2 knockdown or overexpression on γ-H2AX levels in response to 5-FU treatment. UCK2 knockdown (sh-UCK2) led to a reduction in γ-H2AX expression, while UCK2 overexpression significantly increased γ-H2AX levels, suggesting that UCK2 promotes DNA damage accumulation under 5-FU treatment. (D) Western blot analysis of GLI2, HRD1, and UCK2 protein levels in HGC-27 and AGS cells following GLI2 knockdown (sh-GLI2) or overexpression (GLI2). GLI2 knockdown significantly reduces HRD1 expression while increasing UCK2 levels, whereas GLI2 overexpression enhances HRD1 expression and decreases UCK2 levels. β-actin was used as a loading control. (E) Cell viability assay (CCK-8) evaluating the effect of GLI2 knockdown or overexpression on cisplatin sensitivity in HGC-27 and AGS cells. GLI2 knockdown significantly reduced cell viability in response to increasing concentrations of cisplatin, whereas GLI2 overexpression conferred resistance to cisplatin, suggesting that GLI2 plays a key role in cisplatin resistance in gastric cancer cells. (F) Colony formation assay assessing the long-term effects of GLI2 modulation on cisplatin resistance. Representative images (left) and quantification of colony numbers (right) show that GLI2 knockdown significantly reduced colony formation upon cisplatin treatment, whereas GLI2 overexpression increased colony survival, further supporting the role of GLI2 in cisplatin resistance. *P < 0.05, **P < 0.01 (One-way ANOVA).

The GLI2/HRD1 axis enhances 5-FU resistance in vivo by regulating UCK2 expression

To assess if the influence of the GLI2/HRD1 axis on 5-FU resistance observed in GC cells in vitro could be reflected in actual tumor response in vivo, the parental HGC-27 or AGS cells and their genetically modified versions with either GLI2 knockdown, HRD1 overexpression, or both, were subcutaneously transplanted into BALB/c nu/nu mice. As shown in Fig. 6A, the knockdown of GLI2 led to a significant reduction in tumor growth, highlighting its role in promoting tumor development. Conversely, the overexpression of HRD1 exhibited an opposite effect, stimulating tumor growth. Notably, the tumor-suppressing effect of GLI2 knockdown was counteracted when HRD1 was overexpressed, suggesting a complex interplay between these two factors in tumor progression and drug resistance. The study further explored the molecular alterations associated with these manipulations. GLI2 knockdown resulted in a marked decrease in the levels of Ki67 (a marker indicative of cell proliferation), GLI2, and HRD1 proteins in the tumor tissues while increasing UCK2 expression (Fig. 6B). These molecular changes align with the observed reduction in tumor growth following GLI2 silencing. Conversely, HRD1 overexpression reversed these effects, enhancing Ki67 and HRD1 levels while suppressing UCK2 expression. These findings were corroborated by Western blot analysis of tumor tissues (Fig. 6C). This suggests that HRD1 can counteract the molecular consequences of GLI2 knockdown, thereby influencing key pathways involved in tumor growth and chemotherapy resistance. As pictorially modeled in Fig. 6D, the GLI2/HRD1 axis plays a pivotal role in gastric cancer (GC) growth and resistance to 5-fluorouracil (5-FU) treatment. Through the suppression of UCK2, a crucial regulator in this pathway, the interaction between GLI2 and HRD1 creates a cellular environment that favors 5-FU resistance. These findings highlight the importance of the GLI2-HRD1 axis in modulating GC cell survival and drug resistance.

Fig. 6.

The GLI2/HRD1 axis enhances 5-FU resistance in vivo by modulating UCK2 expression. (A) Gross appearance of subcutaneous xenograft tumors derived from GC cells with different genetic modifications at the endpoint of the experiment. Tumor growth curves were plotted by measuring tumor volume at different time points. Tumor volume was measured every 3 days, and growth curves indicate that overexpression of GLI2 or HRD1 enhances tumor growth and 5-FU resistance, whereas UCK2 overexpression inhibits tumor proliferation. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test). (B) Representative immunohistochemistry (IHC) staining images of Ki67 (a proliferation marker), GLI2, HRD1, and UCK2 in xenograft tumor tissues. Tumors with GLI2 or HRD1 overexpression showed increased Ki67 and HRD1 expression, while UCK2 expression was reduced, indicating enhanced tumor proliferation and drug resistance. Scale bar = 50 μm. The histograms showing the quantification of IHC staining intensity for Ki67, GLI2, HRD1, and UCK2. Data are presented as mean ± SD from multiple tumor sections. The statistical significance of differences among groups was assessed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Western blot analysis of GLI2, HRD1, and UCK2 protein levels in the tumor tissues from different experimental groups. Results confirm that GLI2 and HRD1 overexpression reduces UCK2 expression, supporting the proposed regulatory mechanism in vivo. The histograms indicate the levels of the protein determined from 3 independent experiments expressed as the mean ratio relative to that in the control after normalization to β-actin. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test). (D) A schematic model illustrating the mechanism by which the GLI2-HRD1 axis regulates 5-FU resistance in GC cells. GLI2 transcriptionally activates HRD1, which promotes the ubiquitination and proteasomal degradation of UCK2, leading to reduced sensitivity to 5-FU. Pharmacological inhibition of GLI2 or HRD1 may restore UCK2 expression and enhance 5-FU efficacy.

Discussion

GC ranks among the most common cancers globally, known for its complex carcinogenesis, progression, and tendency to metastasize [1]. 5-FU is widely recognized as a fundamental drug in chemotherapy regimens, primarily due to its potential to improve the prognosis of GC patients [21]. However, the 5-year survival rate for GC remains approximately 30 % [22], with chemotherapy resistance being a major factor contributing to the poor survival rates in GC patients [21]. This study aimed to delve into the molecular mechanisms underlying 5-FU resistance in GC and to develop strategies to counteract this resistance. A key finding of this research is the role of the GLI2-HRD1 axis in enhancing 5-FU resistance in GC cells, primarily through the ubiquitination and subsequent degradation of UCK2.

Through an unbiased genome-wide CRISPR/Cas9 knockout (KO) library screening combined with transcriptome sequencing, we identified UCK2 as a key gene responsible for 5-FU sensitivity in gastric cancer (GC) cells. Our results demonstrated that the knockout of UCK2 confers resistance to 5-FU in GC cells. CRISPR/Cas9 knockout library screening is a powerful forward genetic approach that allows the identification of genes contributing to specific phenotypes on a genome-wide scale. In this study, we utilized the GeCKO v2 library for the screening, following previously established methods [[7], [8], [9]]. To ensure the robustness of our screening, we performed a comprehensive quality check by evaluating read depth, the number of enriched sgRNAs, and sgRNA coverage across all experimental groups.

UCK2, a critical enzyme in the pyrimidine salvage synthesis pathway, is responsible for phosphorylating uridine and cytosine [23]. Increased nucleoside metabolism is a hallmark of cancer, and while UCK2 has been known to catalyze nucleotides necessary for tumor cell proliferation, recent studies suggest that UCK2 might also promote tumor growth through mechanisms independent of its catalytic function, potentially by activating oncogenic signaling pathways [23]. Clinically, the phosphorylation ability of UCKs, including UCK2, is utilized to activate various cytotoxic ribonucleoside analogs, such as 5-FU [24]. Prior research indicated a link between UCK2 alterations and 5-FU resistance in colorectal cancer cells [25]. However, the role of UCK2 in mediating 5-FU resistance in GC had not been previously reported. This study found that UCK2 expression was significantly lower in 5-FU-resistant GC cells compared to parental cells, with its downregulation notably enhancing GC resistance to 5-FU. Therefore, UCK2 downregulation is associated with 5-FU resistance in GC.

The study then examined the upstream regulatory mechanisms of UCK2 in the development of 5-FU resistance in GC. HRD1, an E3 ligase, is known for its role in the degradation of misfolded proteins during endoplasmic reticulum-associated degradation processes [26]. It plays a dual role in cancer progression by mediating the ubiquitination and degradation of various downstream targets. For example, HRD1 inhibited breast cancer cell growth and metastasis by facilitating IGF-1R degradation [27], whereas it was previously reported to enhance colon cancer cell migration and invasion by promoting MMP-2 and MMP-9 degradation [28]. Thus, HRD1’s role in cancer can be either pro-cancerous or anti-cancerous, depending on the tumor type. The expression pattern of HRD1 in GC was not well understood until this study, which revealed that HRD1 was overexpressed in 5-FU-resistant GC cells compared to parental cells, with its overexpression significantly promoting GC cell proliferation and 5-FU resistance. Notably, the study demonstrated that HRD1 overexpression mediated the ubiquitination and degradation of UCK2 in GC cells. Furthermore, the upregulation of UCK2 counteracted the effect of HRD1 overexpression on 5-FU resistance. Therefore, HRD1 contributes to reduced 5-FU sensitivity in GC cells by facilitating the ubiquitination degradation of UCK2.

GLI2, a component of the hedgehog signaling pathway crucial for embryonic development [29], has been implicated in the chemotherapy resistance of various cancers. For instance, GLI2 upregulation was linked to increased cisplatin resistance in ovarian cancer cells [19], and its activation in hypoxic tumor microenvironments was associated with chemoresistance in colorectal cancer [30]. Previous studies have also indicated that silencing GLI2 increased 5-FU sensitivity in GC cells [13]. Consistent with these findings, this study observed that GLI2 was more highly expressed in 5-FU-resistant GC cells than in parental cells, and its overexpression significantly promoted GC cell proliferation and 5-FU resistance. Acting as a transcription factor, GLI2 regulates the expression of various target genes [31]. This research uncovered that GLI2 transcriptionally activates HRD1 expression in GC cells, a novel finding. Moreover, it was observed that the inhibition of 5-FU resistance caused by GLI2 knockdown was reversed by HRD1 overexpression. In summary, GLI2 upregulation promotes 5-FU resistance in GC cells by transcriptionally activating HRD1 expression.

Conclusions

In summary, this study represents the first instance of using an unbiased, whole-genome CRISPR-library screening to identify UCK2 as the most critical gene and pathway associated with 5-FU sensitivity in gastric cancer cells. Our findings reveal that GLI2 plays a pivotal role in enhancing GC cell resistance to 5-FU by promoting the ubiquitination and subsequent degradation of UCK2 through the transcriptional activation of HRD1. The identification of this GLI2-HRD1-UCK2 axis sheds new light on the molecular mechanisms underlying 5-FU resistance in GC. These insights not only deepen our understanding of the pathways involved in chemotherapy resistance but also provide a strong theoretical foundation for developing potential therapeutic strategies to overcome 5-FU resistance. Targeting the GLI2-HRD1-UCK2 pathway might offer novel avenues for improving the efficacy of 5-FU-based chemotherapy in patients with gastric cancer, potentially leading to better clinical outcomes. This study thus sets the stage for future research aimed at validating these mechanisms and exploring targeted therapies that could restore 5-FU sensitivity in resistant GC cells.

Ethics approval and consent to participate

All animal experiments employed in our study were approved by the Institutional Animal Care and User Committee of Fujian Medical University (IACUC-FJMU 2024-Y-1505).

Consent for publication

Not applicable

Funding

This work was supported in part by a grant from the National Natural Science Foundation of China (81672967) and the Natural Science Foundation of Fujian Province (2020J01122870).

Supplementary figure legends

Fig. S1. Identification of key ubiquitination sites on UCK2 through site-directed mutagenesis. Ubiquitination levels of wild-type (WT) and mutant UCK2 (K78R, K202R, and K78R/K202R) were assessed by immunoprecipitation (IP) and Western blot analysis following MG132 treatment. Top panel (IP: UCK2, WB: Ub): UCK2 ubiquitination was detected using an anti-ubiquitin (Ub) antibody after immunoprecipitation of UCK2. WT UCK2 exhibited strong ubiquitination, whereas the K78R mutation significantly reduced ubiquitination, indicating that K78 is a major ubiquitination site. The K202R mutation did not affect UCK2 ubiquitination, and the double mutant (K78R/K202R) exhibited a ubiquitination pattern similar to K78R alone, confirming that K78 is the critical residue required for HRD1-mediated UCK2 ubiquitination. Middle panels (WCL: UCK2): Western blot analysis of total UCK2 expression in whole-cell lysates (WCL) confirmed comparable protein expression across conditions, ensuring that differences in ubiquitination were not due to variations in protein levels. β-actin was used as a loading control to verify equal protein loading.

Fig. S2. GLI2 does not directly regulate UCK2 transcription, and UCK2 does not modulate GLI2 expression or stability. (A) Schematic representation of the putative GLI2-binding site (BS) within the UCK2 promoter region (–1720 to –1706 bp) predicted by the JASPAR database. (B) Dual-luciferase reporter assay assessing the effect of GLI2 knockdown on UCK2 promoter activity in HGC-27 and AGS gastric cancer cells. Both the wild-type (UCK2 WT) and mutant (UCK2 MUT) promoter constructs showed no significant changes in luciferase activity upon GLI2 knockdown, indicating that GLI2 does not transcriptionally regulate UCK2 expression. (C) UCK2 overexpression does not affect GLI2 mRNA levels. RT-qPCR analysis of UCK2 and GLI2 expression in HGC-27 and AGS cells transfected with either control (NC) or UCK2-overexpressing plasmid. While UCK2 mRNA levels significantly increased upon transfection (**P < 0.01), GLI2 expression remained unchanged. (D) UCK2 overexpression does not alter GLI2 protein levels. Western blot analysis of UCK2 and GLI2 protein expression in HGC-27 and AGS cells with UCK2 overexpression. Quantification of band intensities shows a significant increase in UCK2 expression (**P < 0.01) but no change in GLI2 levels. (E) UCK2 overexpression does not affect GLI2 protein stability. HGC-27 and AGS cells transfected with NC or UCK2-overexpressing plasmid were treated with cycloheximide (CHX, 100 µg/mL) for 0, 1, 2, and 4 h, followed by Western blot analysis of GLI2 degradation. Quantification of GLI2 protein levels over time shows no significant differences between control and UCK2-overexpressing cells, indicating that UCK2 does not influence GLI2 stability. Data are presented as mean ± SEM from three independent experiments.

Fig. S3. Pharmacological inhibition of GLI2 (GANT61) or HRD1 (LS102) increases UCK2 expression and enhances 5-FU sensitivity in gastric cancer cells. (A) Western blot analysis of GLI2, HRD1, and UCK2 protein levels in HGC-27 and AGS cells treated with either GANT61 (GLI2 inhibitor) or LS102 (HRD1 inhibitor). GANT61 treatment reduces GLI2 and HRD1 levels while increasing UCK2 expression, whereas LS102 treatment decreases HRD1 levels without affecting GLI2 but leads to a significant increase in UCK2 expression. β-actin was used as a loading control. (B) Cell viability assay (CCK-8) showing that treatment with GANT61 or LS102 enhances 5-FU sensitivity in HGC-27 and AGS cells. Cells pretreated with GANT61 or LS102 exhibited significantly lower viability in response to increasing concentrations of 5-FU compared to control-treated cells. (C) Colony formation assay evaluating the long-term effects of GANT61 or LS102 in combination with 5-FU. Representative images (left) and quantification of colony numbers (right) demonstrate that cells pretreated with GANT61 or LS102 exhibited significantly fewer surviving colonies upon 5-FU treatment compared to the control group. (*P < 0.05, **P < 0.01). These results suggest that targeting GLI2 or HRD1 enhances 5-FU sensitivity in gastric cancer cells by increasing UCK2 expression, highlighting the potential therapeutic value of GLI2 and HRD1 inhibition in overcoming chemoresistance.

CRediT authorship contribution statement

Chaorong Xue: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Xuanzi Zhang: Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation. Wanling Yu: Methodology, Data curation. Hanbin Lin: Project administration, Formal analysis, Data curation. Junrong Zhang: Methodology, Data curation. Jiawen Liu: Validation, Methodology. Zongqi Weng: Methodology, Formal analysis. Manduo Ouyang: Methodology, Data curation. Xinjian Lin: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Xinjian Lin reports financial support was provided by National Natural Science Foundation of China. Reports a relationship with that includes:. Has patent pending to. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.