TRIM23 promotes 5-Fluorouracil resistance in colorectal cancer by upregulating GALNT4 expression
Colorectal cancer (CRC) is the third most common cancer worldwide and ranks second for cancer-related mortality . As a key component of CRC chemotherapy, 5-Fluorouracil (5-FU) exerts anti-tumor effects and prolongs the survival of patients primarily by inhibiting the biological activity of thymidylate synthase, which in turn disrupts the intracellular pool of deoxyribonucleotides essential for DNA replication [ – ]. However, the development of chemotherapy resistance leads to treatment failure and poor survival . Despite extensive research into 5-FU resistance mechanisms, such as enzyme abnormalities, dysregulated apoptosis and autophagy, epithelial-mesenchymal transition, metabolic reprogramming, cell cycle disturbances, multidrug resistance, tumor microenvironment regulation, and epigenetic changes [ – ], the mechanisms are still not fully understood and further efforts are needed. Tripartite motif containing 23 (TRIM23), a member of the tripartite motif (TRIM) family, encodes a protein with dual activities as an E3 ubiquitin ligase and GTPase . Accumulating evidence has indicated that TRIM family proteins are extensively involved in tumor progression and treatment resistance [ – ]. It has been shown that TRIM3, which mediates breast cancer resistance to tamoxifen through the TRIM3/UBC9/ESR1 axis, correlates with lower survival rates during therapy . TRIM22 promotes glioblastoma growth and progression by activating the nuclear factor-κB (NF-κB) signaling pathway . TRIM28 enhances the recruitment of chemokine-driven myeloid-derived suppressor cells by stimulating the NF-κB signaling pathway, which in turn inhibits infiltrating activated CD8 + T cells, leading to resistance to programmed cell death protein 1 inhibitor in non-small cell lung cancer . TRIM23 has been found to exhibit abnormal expression in diverse tumors, including lung adenocarcinoma (LUAD), hepatocellular carcinoma (HCC), gastric cancer (GC), head and neck squamous cell carcinoma (HNSCC), and malignant peripheral nerve sheath tumor (MPNST) [ – ]. It contributes to tumor proliferation, invasion, metastasis, and chemoresistance by mediating ubiquitination, activating NF-κB, participating in glycolysis, and regulating cell cycle [ – ]. However, whether and how TRIM23 acts in CRC chemotherapy resistance is unclear. Here, we investigated the expression of TRIM23 in CRC and its relationship with patient prognosis and chemotherapeutic efficacy. Our results indicated that TRIM23 increased resistance to 5-FU in CRC and preliminarily elucidated the molecular mechanisms. It may provide new insights for screening 5-FU-sensitive patients and reversing chemoresistance. 

Tissue samples The tissue array with 101 colon cancer samples and 79 adjacent normal samples was obtained from ShGnghGi Outdo Biotech CompGny (Shanghai, China). Additionally, tumor tissues from 99 RAS/BRAF wild-type, microsatellite-stable (MSS) metastatic colorectal cancer (mCRC) patients treated with cetuximab and 5-FU-based combination chemotherapy at the Third Affiliated Hospital of Soochow University from 2017 to 2022 were collected. Progression-free survival (PFS) was defined as the time from the first administration of 5-FU to disease progression or death. All the samples were confirmed by pathological analysis. All experiments complied with the principles of the Declaration of Helsinki and were approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University (Approval No. (2023) Education 038). Immunohistochemistry (IHC) Tissue sections were deparaffinized, rehydrated using graded alcohol, and subjected to antigen retrieval with heated citrate buffer. After blocking with 3% bovine serum albumin, sections were incubated overnight at 4°C with a TRIM23 antibody (1:200), followed by a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP). The sections were then stained with 3,3’-diaminobenzidine and counterstained with hematoxylin. Images were observed under a microscope. All antibodies used are listed in Table . Cell culture Human colon cancer cell lines HT-29 and HCT-116 were sourced from the Cell Bank, Type Culture Collection, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (TransGen Biotech, Beijing, China) with 10% fetal bovine serum (FBS), maintained at 37 °C with 5% CO 2 . 5-FU-resistant cell lines HT-29-R and HCT-116-R were established through stepwise dosage increases. Cell counting kit-8 (CCK-8) proliferation assay For drug sensitivity testing, 1 × 10³ cells were seeded into 96-well plates and treated with various concentrations of 5-FU. After 48 h, 10µL of CCK-8 solution was added to each well, and the plates were incubated at 37 °C for 2 h. Absorbance at 450 nm was measured with a microplate reader (Molecular Devices, Sunnyvale, CA, USA) to calculate cell viability and half maximal inhibitory concentration (IC 50 ). Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted with Trizol reagent (Thermo Fisher, Waltham, MA, USA), and cDNA was synthesized using the PrimeScript RT reagent kit (Beyotime, Shanghai, China). qRT-PCR was performed using the SYBR Green qRT-PCR kit (Beyotime), with GAPDH as the internal control. All primer sequences were designed and synthesized by General Biosystems (Chuzhou, Anhui, China), and the primer sequences are listed in Table . Western blot Cells and tissues were collected, and total protein was extracted using RIPA lysis buffer (Beyotime). Protein concentration was measured with the BCA Protein Assay Kit (Beyotime). Protein samples were separated by SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk at 37 °C for 1 h, incubated with primary antibodies (TRIM23 1:1000, GALNT4 1:1500) overnight at 4 °C, and then with HRP-conjugated goat anti-rabbit secondary antibody (1:5000). Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) kit (Pierce, Waltham, MA, USA) and analyzed using ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). All antibodies used are listed in Table . Immunofluorescence (IF) HT-29 and HCT-116 cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. After blocking with 10% goat serum, the cells were incubated with TRIM23 primary antibody (1:200) overnight at 4 °C, followed by Alexa Fluor 594-labeled secondary antibody (1:1000). For tumor tissue sections, Ki-67 polyclonal antibody (1:400) was incubated overnight at 4 °C, followed by incubation with Alexa Fluor 594-conjugated secondary antibody (1:1000). Images were observed and captured using a fluorescence microscope (Olympus, Tokyo, Japan). All antibodies used are listed in Table . Colony formation assay Cells were seeded in 6-well plates (500 cells per well) and cultured in DMEM with 10% FBS for 2 weeks. Colonies were then fixed with 4% paraformaldehyde and stained with 1% crystal violet for counting. Cell transfection TRIM23 overexpressing plasmids, short hairpin RNA (shRNA) targeting TRIM23, GALNT4 overexpression plasmids, and negative control plasmids were obtained from General Biosystems (Chuzhou, Anhui, China). The shTRIM23 sequence was: 5’-GGCAGCAACAAGAAGATATTTCAAGAGAATATCTTCTTGTTGCTGCCTTTTTT-3’. Transfection was conducted using Lipofectamine 3000 (Thermo Fisher) following the manufacturer’s instructions, and transfection efficiency was verified by qRT-PCR and Western Blot. Animal experiments Five-week-old female BALB/c mice weighing approximately 15–17 g were randomly divided into four groups (five mice per group, with no significant difference in weight between each group) and inoculated with HT-29-R cells transfected with Lv-shNC, Lv-shTRIM23, Lv-oe-NC, and Lv-oe-TRIM23. The mice received 5-FU injections (25 mg/kg) every two days, and tumor volume was measured every five days to generate the tumor growth curve. On day 30, the mice were sacrificed, and tumor volume and weight were measured. Tumor tissues were analyzed for TRIM23 and GALNT4 expression using Western Blot, while tumor sections underwent IHC, IF, and TUNEL staining. For mechanism validation, twenty mice with similar characteristics were divided into four groups and inoculated with HT-29-R cells transfected with Lv-shNC, Lv-shTRIM23, Lv-shNC + Lv-oe-GALNT4, and Lv-shTRIM23 + Lv-oe-GALNT4. The same 5-FU treatment regimen and measurement schedule were followed. The mice were executed on day 40, and the tumor volume and weight were measured. Tumor tissues were analyzed for TRIM23 and GALNT4 expression via Western Blot. All experiments were approved by the Animal Experiment Ethics Committee of the Third Affiliated Hospital of Soochow University. TUNEL staining Subcutaneous tumors from mice were sectioned and stained using the TUNEL apoptosis detection kit (Beyotime) according to the manufacturer’s instructions. Statistical analysis The statistical significance of differences was assessed using one-way ANOVA, two-way ANOVA or Student’s t test. χ 2 test and Spearman’s correlation coefficient evaluated the relationship between TRIM23 expression and clinicopathological parameters. Kaplan-Meier curves were used for survival analysis, assessed with the log-rank test. Analyses were conducted using GraphPad Prism 9.5.1 or SPSS 26.0. A P -value < 0.05 indicates statistical significance. 

The tissue array with 101 colon cancer samples and 79 adjacent normal samples was obtained from ShGnghGi Outdo Biotech CompGny (Shanghai, China). Additionally, tumor tissues from 99 RAS/BRAF wild-type, microsatellite-stable (MSS) metastatic colorectal cancer (mCRC) patients treated with cetuximab and 5-FU-based combination chemotherapy at the Third Affiliated Hospital of Soochow University from 2017 to 2022 were collected. Progression-free survival (PFS) was defined as the time from the first administration of 5-FU to disease progression or death. All the samples were confirmed by pathological analysis. All experiments complied with the principles of the Declaration of Helsinki and were approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University (Approval No. (2023) Education 038). 

Tissue sections were deparaffinized, rehydrated using graded alcohol, and subjected to antigen retrieval with heated citrate buffer. After blocking with 3% bovine serum albumin, sections were incubated overnight at 4°C with a TRIM23 antibody (1:200), followed by a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP). The sections were then stained with 3,3’-diaminobenzidine and counterstained with hematoxylin. Images were observed under a microscope. All antibodies used are listed in Table . 

Human colon cancer cell lines HT-29 and HCT-116 were sourced from the Cell Bank, Type Culture Collection, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (TransGen Biotech, Beijing, China) with 10% fetal bovine serum (FBS), maintained at 37 °C with 5% CO 2 . 5-FU-resistant cell lines HT-29-R and HCT-116-R were established through stepwise dosage increases. 

For drug sensitivity testing, 1 × 10³ cells were seeded into 96-well plates and treated with various concentrations of 5-FU. After 48 h, 10µL of CCK-8 solution was added to each well, and the plates were incubated at 37 °C for 2 h. Absorbance at 450 nm was measured with a microplate reader (Molecular Devices, Sunnyvale, CA, USA) to calculate cell viability and half maximal inhibitory concentration (IC 50 ). 

Total RNA was extracted with Trizol reagent (Thermo Fisher, Waltham, MA, USA), and cDNA was synthesized using the PrimeScript RT reagent kit (Beyotime, Shanghai, China). qRT-PCR was performed using the SYBR Green qRT-PCR kit (Beyotime), with GAPDH as the internal control. All primer sequences were designed and synthesized by General Biosystems (Chuzhou, Anhui, China), and the primer sequences are listed in Table . 

Cells and tissues were collected, and total protein was extracted using RIPA lysis buffer (Beyotime). Protein concentration was measured with the BCA Protein Assay Kit (Beyotime). Protein samples were separated by SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk at 37 °C for 1 h, incubated with primary antibodies (TRIM23 1:1000, GALNT4 1:1500) overnight at 4 °C, and then with HRP-conjugated goat anti-rabbit secondary antibody (1:5000). Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) kit (Pierce, Waltham, MA, USA) and analyzed using ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). All antibodies used are listed in Table . 

HT-29 and HCT-116 cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. After blocking with 10% goat serum, the cells were incubated with TRIM23 primary antibody (1:200) overnight at 4 °C, followed by Alexa Fluor 594-labeled secondary antibody (1:1000). For tumor tissue sections, Ki-67 polyclonal antibody (1:400) was incubated overnight at 4 °C, followed by incubation with Alexa Fluor 594-conjugated secondary antibody (1:1000). Images were observed and captured using a fluorescence microscope (Olympus, Tokyo, Japan). All antibodies used are listed in Table . 

Cells were seeded in 6-well plates (500 cells per well) and cultured in DMEM with 10% FBS for 2 weeks. Colonies were then fixed with 4% paraformaldehyde and stained with 1% crystal violet for counting. 

TRIM23 overexpressing plasmids, short hairpin RNA (shRNA) targeting TRIM23, GALNT4 overexpression plasmids, and negative control plasmids were obtained from General Biosystems (Chuzhou, Anhui, China). The shTRIM23 sequence was: 5’-GGCAGCAACAAGAAGATATTTCAAGAGAATATCTTCTTGTTGCTGCCTTTTTT-3’. Transfection was conducted using Lipofectamine 3000 (Thermo Fisher) following the manufacturer’s instructions, and transfection efficiency was verified by qRT-PCR and Western Blot. 

Five-week-old female BALB/c mice weighing approximately 15–17 g were randomly divided into four groups (five mice per group, with no significant difference in weight between each group) and inoculated with HT-29-R cells transfected with Lv-shNC, Lv-shTRIM23, Lv-oe-NC, and Lv-oe-TRIM23. The mice received 5-FU injections (25 mg/kg) every two days, and tumor volume was measured every five days to generate the tumor growth curve. On day 30, the mice were sacrificed, and tumor volume and weight were measured. Tumor tissues were analyzed for TRIM23 and GALNT4 expression using Western Blot, while tumor sections underwent IHC, IF, and TUNEL staining. For mechanism validation, twenty mice with similar characteristics were divided into four groups and inoculated with HT-29-R cells transfected with Lv-shNC, Lv-shTRIM23, Lv-shNC + Lv-oe-GALNT4, and Lv-shTRIM23 + Lv-oe-GALNT4. The same 5-FU treatment regimen and measurement schedule were followed. The mice were executed on day 40, and the tumor volume and weight were measured. Tumor tissues were analyzed for TRIM23 and GALNT4 expression via Western Blot. All experiments were approved by the Animal Experiment Ethics Committee of the Third Affiliated Hospital of Soochow University. 

Subcutaneous tumors from mice were sectioned and stained using the TUNEL apoptosis detection kit (Beyotime) according to the manufacturer’s instructions. 

The statistical significance of differences was assessed using one-way ANOVA, two-way ANOVA or Student’s t test. χ 2 test and Spearman’s correlation coefficient evaluated the relationship between TRIM23 expression and clinicopathological parameters. Kaplan-Meier curves were used for survival analysis, assessed with the log-rank test. Analyses were conducted using GraphPad Prism 9.5.1 or SPSS 26.0. A P -value < 0.05 indicates statistical significance. 

Upregulated TRIM23 expression associated with poor prognosis and reduced chemotherapy efficacy in CRC IHC assay of the tissue array showed that TRIM23 protein expression was significantly upregulated in colon cancer tissues compared to adjacent normal tissues (Fig. a-b). There was no significant correlation between TRIM23 protein expression levels and gender, age, tumor size, American Joint Committee on Cancer (AJCC) 8th edition stage, T stage, N stage, or M stage (Table ). Tumor specimens and clinical data from 99 patients with mCRC were retrospectively collected. IHC analysis revealed high TRIM23 expression in 84 cases (84.8%) and low expression in 15 cases (15.2%). Representative tissue samples with positive and negative TRIM23 protein expression were shown in Fig. c. Compared to the low-expression group, the high-expression group showed a lower disease control rate (DCR) (73.3% vs. 91.7%, P = 0.037) and shorter median progression-free survival (mPFS) (6.7 months vs. 11.3 months, P = 0.003), indicating a poorer response to 5-FU-based combination therapy (Fig. d). Additionally, TRIM23 protein expression showed no significant correlation with clinicopathological factors such as gender, age, tumor site, gross type, histological grade, disease status, presence of liver metastasis, and treatment regimen (Table ). These data demonstrated that TRIM23 may function as an oncogene in CRC, reducing the sensitivity of patients to 5-FU. TRIM23 is upregulated in 5-FU-resistant CRC cells The sensitivity of HT-29 and HCT-116 cells to 5-FU was determined using the CCK-8 assay (Fig. a). Compared to the control group, treatment with 10µM 5-FU significantly increased TRIM23 mRNA and protein expression in HT-29 and HCT-116 cells but did not alter the intracellular localization of TRIM23 protein (Fig. b-d). The qRT-PCR and Western Blot analyses demonstrated that TRIM23 mRNA and protein levels were higher in HT-29-R and HCT-116-R cells than in HT-29 and HCT-116 cells (Fig. a-b). Compared with HT-29 and HCT-116 cells, HT-29-R and HCT-116-R cells with elevated TRIM23 expression exhibited increased proliferation ability and resistance to 5-FU (Fig. c-d), which appeared that TRIM23 may be associated with the growth and 5-FU resistance of CRC cells. TRIM23 promotes CRC cell proliferation and 5-FU resistance To further investigate the relationship of TRIM23 expression with CRC cell proliferation and 5-FU resistance, HT-29-R and HCT-116-R cells were treated in four groups: (1) Lv-shNC, (2) Lv-shTRIM23, (3) Lv-oe-NC, and (4) Lv-oe-TRIM23. The expression levels of TRIM23 mRNA and protein were detected in these groups (Fig. a-b). Under 5-FU treatment, colon cancer cells with TRIM23 knockdown showed reduced proliferation ability and resistance to 5-FU compared to the control group, whereas overexpressing TRIM23 did not significantly alter these properties (Fig. c-d), suggesting that TRIM23 enhanced both proliferative ability and 5-FU resistance in colon cancer cells, and its downregulation decreased cell proliferation while restoring sensitivity to 5-FU. GALNT4 is a regulatory target of TRIM23 RNA sequencing (RNA-seq) and bioinformatics methods were applied to search for candidate targets of TRIM23. The sequencing data have been deposited in the National Genomics Data Center ( https://ngdc.cncb.ac.cn/gsa-human ), accession number: HRA007713. Differentially expressed genes (DEGs) between TRIM23 knockdown cells (Lv-shTRIM23-HT-29-R, Lv-shTRIM23-HCT-116-R) and control cells (Lv-shNC-HT-29-R, Lv-shNC-HCT-116-R) were identified via RNA-seq (Fold change > 2, P < 0.05) (Fig. a). Significant functions and pathways enriched by these DEGs were revealed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses (Supplementary Material 1: Fig. S1, S2). Specifically, 705 DEGs were found between Lv-shTRIM23-HT-29-R cells and Lv-shNC-HT-29-R cells, and 287 DEGs were identified between Lv-shTRIM23-HCT-116-R cells and Lv-shNC-HCT-116-R cells, with 33 genes commonly differentially expressed in both comparisons, of which 12 were protein-coding genes (Fig. b). The qRT-PCR results indicated that knocking down or overexpressing TRIM23 in HT-29-R and HCT-116-R cells affected the mRNA expression of ANKRD36B, GALNT4, and MBL2 (Fig. c). Validation in the GSE14095 database showed that TRIM23 correlated with ANKRD36B, GALNT4, and PLCXD2 expression. ANKRD36B was negatively correlated with TRIM23 expression, while GALNT4 was positively correlated (Fig. d), consistent with the cell experiment results. Consequently, GALNT4 was selected for further study. Then, Lv-shNC, Lv-shTRIM23, Lv-oe-NC, and Lv-oe-TRIM23 were transfected into HT-29-R and HCT-116-R cells, respectively. Western Blot results showed that TRIM23 knockdown decreased GALNT4 expression, while TRIM23 overexpression increased GALNT4 protein levels (Fig. e), indicating that TRIM23 positively regulated GALNT4 expression at the protein level. TRIM23 promotes 5-FU resistance in CRC cells by regulating GALNT4 expression HT-29-R and HCT-116-R cells were divided into four treatment groups: (1) Lv-shNC, (2) Lv-shTRIM23, (3) Lv-shNC + Lv-oe-GALNT4, and (4) Lv-shTRIM23 + Lv-oe-GALNT4. The mRNA and protein expression levels of TRIM23 and GALNT4 were measured in each group (Fig. a-b). Under 5-FU treatment, the overexpression of GALNT4 combined with the knockdown of TRIM23 increased proliferation ability and resistance to 5-FU in HT-29-R and HCT-116-R cells compared to knockdown of TRIM23 alone (Fig. c-d), suggesting that TRIM23 mediated 5-FU resistance in colon cancer cells by upregulating GALNT4, and GALNT4 overexpression counteracted the 5-FU sensitization caused by TRIM23 downregulation. TRIM23 promotes CRC resistance to 5-FU in vivo by modulating GALNT4 expression A mouse xenograft tumor model was established with HT-29-R cells. In the TRIM23 knockdown group, tumor volume and weight were reduced compared to the control group, while no significant changes were observed in the TRIM23 overexpression group (Fig. a-c). Western Blot demonstrated that TRIM23 positively regulated GALNT4 expression in the mouse xenograft tumor model, consistent with in vitro findings (Fig. d). IHC and IF staining showed decreased proliferative activity and increased apoptosis in the TRIM23 knockdown group compared to the control group, with opposite effects in the TRIM23 overexpression group (Fig. e). These findings indicated that TRIM23 reduced CRC sensitivity to 5-FU and enhanced GALNT4 expression in vivo. To further investigate, HT-29-R cells transfected with Lv-shNC, Lv-shTRIM23, Lv-shNC + Lv-oe-GALNT4, and Lv-shTRIM23 + Lv-oe-GALNT4 were injected into mice. Tumor growth curves, tumor weight, and expression levels of TRIM23 and GALNT4 were analyzed. The tumor volume and weight in the TRIM23 knockdown group were significantly reduced compared to the control group, while the GALNT4 overexpression group showed the opposite results (Fig. f-i). However, simultaneous overexpression of GALNT4 and TRIM23 knockdown resulted in increased tumor volume and weight compared to TRIM23 knockdown alone (Fig. f-i), suggesting reduced sensitivity to 5-FU. These findings indicated that TRIM23 regulated CRC 5-FU resistance through modulation of GALNT4 expression in vivo. 

IHC assay of the tissue array showed that TRIM23 protein expression was significantly upregulated in colon cancer tissues compared to adjacent normal tissues (Fig. a-b). There was no significant correlation between TRIM23 protein expression levels and gender, age, tumor size, American Joint Committee on Cancer (AJCC) 8th edition stage, T stage, N stage, or M stage (Table ). Tumor specimens and clinical data from 99 patients with mCRC were retrospectively collected. IHC analysis revealed high TRIM23 expression in 84 cases (84.8%) and low expression in 15 cases (15.2%). Representative tissue samples with positive and negative TRIM23 protein expression were shown in Fig. c. Compared to the low-expression group, the high-expression group showed a lower disease control rate (DCR) (73.3% vs. 91.7%, P = 0.037) and shorter median progression-free survival (mPFS) (6.7 months vs. 11.3 months, P = 0.003), indicating a poorer response to 5-FU-based combination therapy (Fig. d). Additionally, TRIM23 protein expression showed no significant correlation with clinicopathological factors such as gender, age, tumor site, gross type, histological grade, disease status, presence of liver metastasis, and treatment regimen (Table ). These data demonstrated that TRIM23 may function as an oncogene in CRC, reducing the sensitivity of patients to 5-FU. 

The sensitivity of HT-29 and HCT-116 cells to 5-FU was determined using the CCK-8 assay (Fig. a). Compared to the control group, treatment with 10µM 5-FU significantly increased TRIM23 mRNA and protein expression in HT-29 and HCT-116 cells but did not alter the intracellular localization of TRIM23 protein (Fig. b-d). The qRT-PCR and Western Blot analyses demonstrated that TRIM23 mRNA and protein levels were higher in HT-29-R and HCT-116-R cells than in HT-29 and HCT-116 cells (Fig. a-b). Compared with HT-29 and HCT-116 cells, HT-29-R and HCT-116-R cells with elevated TRIM23 expression exhibited increased proliferation ability and resistance to 5-FU (Fig. c-d), which appeared that TRIM23 may be associated with the growth and 5-FU resistance of CRC cells. 

To further investigate the relationship of TRIM23 expression with CRC cell proliferation and 5-FU resistance, HT-29-R and HCT-116-R cells were treated in four groups: (1) Lv-shNC, (2) Lv-shTRIM23, (3) Lv-oe-NC, and (4) Lv-oe-TRIM23. The expression levels of TRIM23 mRNA and protein were detected in these groups (Fig. a-b). Under 5-FU treatment, colon cancer cells with TRIM23 knockdown showed reduced proliferation ability and resistance to 5-FU compared to the control group, whereas overexpressing TRIM23 did not significantly alter these properties (Fig. c-d), suggesting that TRIM23 enhanced both proliferative ability and 5-FU resistance in colon cancer cells, and its downregulation decreased cell proliferation while restoring sensitivity to 5-FU. 

RNA sequencing (RNA-seq) and bioinformatics methods were applied to search for candidate targets of TRIM23. The sequencing data have been deposited in the National Genomics Data Center ( https://ngdc.cncb.ac.cn/gsa-human ), accession number: HRA007713. Differentially expressed genes (DEGs) between TRIM23 knockdown cells (Lv-shTRIM23-HT-29-R, Lv-shTRIM23-HCT-116-R) and control cells (Lv-shNC-HT-29-R, Lv-shNC-HCT-116-R) were identified via RNA-seq (Fold change > 2, P < 0.05) (Fig. a). Significant functions and pathways enriched by these DEGs were revealed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses (Supplementary Material 1: Fig. S1, S2). Specifically, 705 DEGs were found between Lv-shTRIM23-HT-29-R cells and Lv-shNC-HT-29-R cells, and 287 DEGs were identified between Lv-shTRIM23-HCT-116-R cells and Lv-shNC-HCT-116-R cells, with 33 genes commonly differentially expressed in both comparisons, of which 12 were protein-coding genes (Fig. b). The qRT-PCR results indicated that knocking down or overexpressing TRIM23 in HT-29-R and HCT-116-R cells affected the mRNA expression of ANKRD36B, GALNT4, and MBL2 (Fig. c). Validation in the GSE14095 database showed that TRIM23 correlated with ANKRD36B, GALNT4, and PLCXD2 expression. ANKRD36B was negatively correlated with TRIM23 expression, while GALNT4 was positively correlated (Fig. d), consistent with the cell experiment results. Consequently, GALNT4 was selected for further study. Then, Lv-shNC, Lv-shTRIM23, Lv-oe-NC, and Lv-oe-TRIM23 were transfected into HT-29-R and HCT-116-R cells, respectively. Western Blot results showed that TRIM23 knockdown decreased GALNT4 expression, while TRIM23 overexpression increased GALNT4 protein levels (Fig. e), indicating that TRIM23 positively regulated GALNT4 expression at the protein level. 

HT-29-R and HCT-116-R cells were divided into four treatment groups: (1) Lv-shNC, (2) Lv-shTRIM23, (3) Lv-shNC + Lv-oe-GALNT4, and (4) Lv-shTRIM23 + Lv-oe-GALNT4. The mRNA and protein expression levels of TRIM23 and GALNT4 were measured in each group (Fig. a-b). Under 5-FU treatment, the overexpression of GALNT4 combined with the knockdown of TRIM23 increased proliferation ability and resistance to 5-FU in HT-29-R and HCT-116-R cells compared to knockdown of TRIM23 alone (Fig. c-d), suggesting that TRIM23 mediated 5-FU resistance in colon cancer cells by upregulating GALNT4, and GALNT4 overexpression counteracted the 5-FU sensitization caused by TRIM23 downregulation. 

A mouse xenograft tumor model was established with HT-29-R cells. In the TRIM23 knockdown group, tumor volume and weight were reduced compared to the control group, while no significant changes were observed in the TRIM23 overexpression group (Fig. a-c). Western Blot demonstrated that TRIM23 positively regulated GALNT4 expression in the mouse xenograft tumor model, consistent with in vitro findings (Fig. d). IHC and IF staining showed decreased proliferative activity and increased apoptosis in the TRIM23 knockdown group compared to the control group, with opposite effects in the TRIM23 overexpression group (Fig. e). These findings indicated that TRIM23 reduced CRC sensitivity to 5-FU and enhanced GALNT4 expression in vivo. To further investigate, HT-29-R cells transfected with Lv-shNC, Lv-shTRIM23, Lv-shNC + Lv-oe-GALNT4, and Lv-shTRIM23 + Lv-oe-GALNT4 were injected into mice. Tumor growth curves, tumor weight, and expression levels of TRIM23 and GALNT4 were analyzed. The tumor volume and weight in the TRIM23 knockdown group were significantly reduced compared to the control group, while the GALNT4 overexpression group showed the opposite results (Fig. f-i). However, simultaneous overexpression of GALNT4 and TRIM23 knockdown resulted in increased tumor volume and weight compared to TRIM23 knockdown alone (Fig. f-i), suggesting reduced sensitivity to 5-FU. These findings indicated that TRIM23 regulated CRC 5-FU resistance through modulation of GALNT4 expression in vivo. 

CRC is a prevalent malignancy globally, with rising incidence and mortality rates . The combination of 5-FU with oxaliplatin or irinotecan is a first-line treatment for advanced CRC, extending survival by about two years . However, at present, chemotherapy resistance poses a major challenge to the treatment of CRC, highlighting the necessity of research into the resistance mechanisms and overcoming strategies. Previous studies have established that TRIM23 promotes cancer progression in a variety of tumors, including CRC . TRIM23 expression is upregulated in cisplatin-resistant LUAD cells and mediates cisplatin resistance by regulating glucose metabolism through the TRIM23/NF-κB/GLUT1/3 axis. Knockdown of TRIM23 restores cisplatin sensitivity in LUAD cells . Through the circPVT1/miR-377/TRIM23 network, TRIM23 is involved in regulating HCC cell proliferation, apoptosis, glycolysis, and tumor progression . High TRIM23 expression in GC patients is associated with poor prognosis . In addition, TRIM23 may influence HNSCC progression and is a potential therapeutic target for MPNST . TRIM23 is an independent risk factor for poor prognosis in CRC, which promotes cell proliferation and tumor growth by physically interacting with P53 and enhancing its ubiquitination . TRIM23 is involved in the SNHG17/TRIM23/PES1 axis by mediating the ubiquitination and degradation of PES1, promoting the growth and metastasis of CRC . This study confirmed that TRIM23 expression was upregulated in CRC and was associated with poorer chemotherapy efficacy and prognosis in patients undergoing 5-FU-based therapy. Treatment with 5-FU elevated TRIM23 mRNA and protein levels in CRC cells and higher TRIM23 expression was observed in 5-FU-resistant CRC cells compared to parental cells. Mechanistically, TRIM23 was found to contribute to CRC resistance to 5-FU in vitro and in vivo by upregulating GALNT4 expression, as shown in Fig. . Knockdown of TRIM23 in 5-FU-resistant colon cancer cells restored their sensitivity to 5-FU, while overexpression of GALNT4 in TRIM23 knockdown colon cancer cells counteracted this effect. The TRIM23/GALNT4 axis appears to play a crucial role in CRC 5-FU resistance, suggesting that targeting suppression of TRIM23 and GALNT4 may enhance CRC sensitivity to 5-FU. GALNT4, an enzyme responsible for initiating mucin-type O-glycosylation, is dysregulated in expression in diverse tumors and associated with tumor proliferation, invasion, differentiation, and prognosis . In clear cell renal cell carcinoma, GALNT4 is markedly downregulated and correlates with tumor size, necrosis level, and TNM stage, and is an independent risk factor for overall survival (OS) and relapse-free survival (RFS). A GALNT4-based nomogram predicts patient prognosis better than the TNM staging system . In HCC, GALNT4 acts as a tumor suppressor . Low GALNT4 expression is an independent adverse prognostic factor, associated with poorer differentiation, higher rates of vascular invasion, higher Barcelona Clinic Liver Cancer stage and TNM stage, and shorter RFS and OS. GALNT4 inhibits migration, invasion, and stemness of HCC cells in vitro as well as tumor growth in vivo . In addition, GALNT4 is overexpressed in estrogen receptor-positive breast epithelial cells compared to basal-like subtypes, regulating the estrogen pathway through FOXA1 glycosylation. Silencing GALNT4 reduces the expression of estrogen receptor α, FOXA1, and cyclin D1 in breast cancer cells, leading to G0/G1 phase arrest . MiR-4262 has been reported to regulate apoptosis and proliferation in colon cancer cells by targeting the 3’ untranslated region of GALNT4, resulting in its downregulation . It has also been confirmed that miR-4262 inhibits colon cancer cell proliferation by inducing GALNT4-mediated G1/S phase arrest . Our study demonstrated that GALNT4 increased CRC resistance to 5-FU. Nevertheless, the precise mechanism remains unclear and may relate to GALNT4-mediated O-glycosylation modifications, which require further investigation. There are some limitations to this study. The development of tumor chemotherapy resistance is a complex process that implicates multiple pathways and targets. In this study, we found that the TRIM23/GALNT4 axis may significantly contribute to CRC resistance to 5-FU. Nonetheless, further research is required to elucidate how TRIM23 and GALNT4 interact, identify other targets of TRIM23, and determine whether the TRIM23/GALNT4 axis interacts with other signaling pathways involved in CRC 5-FU resistance. The results of RNA-seq and subsequent cellular experiments indicated that ANKRD36B and MBL2 were potential targets of TRIM23. ANKRD36B expression varies with racial differences in CRC . Mannan-binding lectin (MBL), encoded by the MBL2 gene and produced by the liver, is a C-type serum lectin . Low preoperative levels of MBL are predictive of postoperative pneumonia in CRC patients, and those who develop pneumonia have higher recurrence rates and poorer survival . Furthermore, the single-nucleotide polymorphism at codon 54 of MBL2 may affect CpG island hypermethylation in the rectal mucosa of patients with ulcerative colitis, contributing to ulcerative colitis related carcinogenesis . Whether ANKRD36B and MBL2 are targets of TRIM23 and whether they are associated with 5-FU resistance in CRC requires further investigation. TRIM23 is highly expressed in CRC and appears to be a biomarker for identifying 5-FU-sensitive patients and predicting prognosis. By upregulating GALNT4 expression, TRIM23 contributes to CRC resistance to 5-FU. Thus, targeting the TRIM23/GALNT4 axis may be a strategy to reverse 5-FU resistance in CRC. 

Below is the link to the electronic supplementary material. Supplementary Material 1 

