Introduction

CRC stands as a common malignant tumor of the digestive system, characterized by high incidence and mortality rates1. Oxaliplatin-based chemotherapy, exemplified by FOLFOX regimens, is a primary neoadjuvant treatment for advanced CRC patients. However, resistance to Oxaliplatin in tumor cells can substantially compromise clinical efficacy and patient prognosis2. Thus, elucidating the resistance mechanisms in CRC is vital for enhancing the outcomes for patients with drug resistance.

The intricacies of tumor drug resistance stem from a multitude of contributing factors. A burgeoning area of focus within this domain is ferroptosis, an emerging form of regulated cell death that is tightly interwoven with the progression and resistance mechanisms of cancer3. Characterized by its reliance on iron accumulation and the propagation of lipid peroxidation, ferroptosis represents a distinct pathway of cell demise4. The disruption of cellular iron equilibrium by excessive intracellular iron is recognized as a pivotal trigger for ferroptosis5. Upholding a balanced iron metabolism is paramount for tumor cells to sidestep the onset of ferroptosis. Utilizing bioinformatics, our study scrutinized the variances in gene expression between FOLFOX chemotherapy-resistant and -sensitive colorectal cancer, uncovering an overabundance of iron-responsive element-binding protein 2 (IREB2) in instances of resistance. IREB2, a key modulator of iron homeostasis, exerts its influence by regulating the expression of Transferrin receptor (TFRC) mRNA and Ferritin Heavy Chain 1 (FTH1) protein, thereby significantly impacting the modulation of ferroptosis6. It is subject to regulation by the upstream E3 ubiquitin ligase, F-box and leucine-rich repeat protein 5 (FBXL5)7. Despite its potential significance, the role of the FBXL5/IREB2/TFRC axis in the interplay between ferroptosis and drug resistance in CRC has yet to be thoroughly elucidated.

Our investigation employs a multifaceted approach, integrating bioinformatics, clinical research, and cellular experimentation to evaluate the expression profiles of FBXL5 alongside critical iron metabolism factors IREB2, TFRC, and FTH1. We have concurrently examined the fluctuating expressions of Fe2+, MDA, GSH, and ROS—indicators of ferroptosis—to delineate their interactions and the underlying molecular mechanisms that dictate drug resistance. The capacity to manipulate the FBXL5/IREB2/TFRC axis to perturb iron metabolic equilibrium and consequently incite ferroptosis, offering a novel avenue to abrogate drug resistance in CRC, is currently under rigorous exploration.

Materials and methods

Materials

Clinical data

This study collected 119 cases of T3 and T4 stage colorectal cancer specimens archived in the Department of Pathology at Binzhou Medical University Hospital and Liaocheng People’s Hospital between December 2018 and December 2023. Fresh tissue samples were immediately frozen in liquid nitrogen, while paraffin-embedded specimens were fixed in 10% buffered neutral formalin. Normal colorectal mucosal tissues, located ≥ 5 cm from the tumor margin, were selected as the control group.

The inclusion criteria were as follows: (1) All cases were re-evaluated and confirmed as colorectal cancer by two senior pathologists through double-blind review according to the WHO Classification of Digestive System Tumors (2019); (2) Patients with T3 or T4 stage colorectal cancer (any N stage) who underwent radical surgery after neoadjuvant chemotherapy were selected based on the Chinese Guidelines for Diagnosis and Treatment of Colorectal Cancer (2020 edition); (3) All patients received preoperative neoadjuvant chemotherapy with FOLFOX regimen; (4) Complete pathological evaluation, including tumor regression grade, was available for all cases postoperatively. The exclusion criteria were as follows: (1) patients lost to follow-up, (2) those who died from causes unrelated to colorectal cancer, (3) cases with T1 or T2 stage disease, (4) patients who did not receive FOLFOX regimen treatment, and (5) cases with TRG 4 (complete pathological response with no residual tumor).

All procedures performed in the study involving human participants were in accordance with the ethical standards of the institutional and/or national research councils and the 1964 Declaration of Helsinki and its subsequent amendments or similar ethical standards. The study design was approved by the Ethics Committee of the Affiliated Hospital of Binzhou Medical College (approval number: KYLL−112). All patients provided informed consent. Informed consent was obtained from all participants. Detailed clinical case information is provided in Supplemental Table S1.

Cell lines

The human colon cancer cell lines COLO205, SW480, SW620, HCT−116, and HCT−8 were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The oxaliplatin-resistant colon cancer cell line HCT−116/OXA was derived from HCT−116 cells through induction by Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Reagents

The specific lentiviral sequences targeting the FBXL5 gene, the nonsense sequence lentivirus, and transfection enhancement reagents were designed and provided by GeneChem Co., Ltd. (Shanghai, China). Antibodies against FBXL5 (ab140175), IREB2 (ab2329994), TFRC (ab214039), and FTH1 (ab65080) were purchased from Abcam (USA). Goat anti-rabbit secondary antibody was obtained from Proteintech, and the immunohistochemistry kit was purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). The Cell Counting Kit−8 (CCK−8) was acquired from Dojindo Laboratories (Japan). Oxaliplatin (OXA), FerroOrange fluorescent probe, Erastin, and Fer−1 were purchased from GlpBio. MDA and GSH assay kits were obtained from Beyotime Biotechnology (Shanghai, China), the iron ion assay kit was from Solarbio, the ROS detection kit was from Meilun Biotechnology (Jiangsu, China), and the PrimeScript RT-PCR Kit was purchased from Takara (Japan).

Methods

Immunohistochemistry

Primary antibodies included IREB2 (1:100), TFRC (1:400), and FTH1 (1:200). Positive controls were selected as follows: FBXL5 from human placental tissue, IREB2 from human kidney tissue, TFRC from human esophageal cancer tissue, and FTH1 from human liver tissue. PBS was used as a negative control by replacing the primary antibody.Evaluation criteria: FBXL5, IREB2, and FTH1 were primarily expressed in the cytoplasm, while TFRC was mainly localized to the cell membrane and cytoplasm. Quantification was performed using Image Pro Plus software. The integrated optical density (IOD) of positive cells in 10 high-power fields was measured, and the average IOD value was calculated. Samples with IOD values above the average were considered positive, while those with values equal to or below the average were deemed negative. The positive expression rate was calculated based on the number of positively stained cells.

Tumor regression grading criteria

According to the tumor regression grading (TRG) criteria for post-neoadjuvant chemotherapy, all cases in this study were pathologically evaluated using the Dowrak/Rödel five-tier grading system. The Dowrak/Rödel criteria are defined as follows: TRG0 indicates no response, TRG1 represents fibrosis in < 25% of the tumor area, TRG2 indicates fibrosis in 25%−50% of the tumor area, TRG3 represents fibrosis in > 50% of the tumor area, and TRG4 denotes complete remission. In this study, TRG4 cases with no residual tumor were excluded. Cases classified as TRG0 or TRG1 were categorized as the resistant group, while those classified as TRG2 or TRG3 were designated as the sensitive group.

Cell culture

The human colorectal cancer cell lines HCT−8, COLO205, SW480, SW620, and HCT−116, as well as the drug-resistant cell line HCT−116/OXA, were cultured in medium supplemented with 10% fetal bovine serum. To maintain the drug-resistant phenotype, HCT−116/OXA cells were cultured in medium containing 2 µg/mL OXA. OXA treatment was discontinued one week prior to experiments. Mycoplasma was regularly checked, with no detection in any of the cell lines during the experiment.

Cell transfection

Two specific sequences targeting the FBXL5 gene were designed and synthesized: one for FBXL5 overexpression (OE-FBXL5) and the other for FBXL5 knockdown (sh-FBXL5). A nonsense interference sequence (Vector) was also designed as a control. Transfection was performed according to the RNAi-mate product instructions. Seventy-two hours post-transfection, puromycin was added to the culture medium for two weeks to select transfected cells. Transfection efficiency was subsequently evaluated using Western blotting and RT-qPCR.

CCK−8 assay

Prepare a cell suspension at an appropriate density for the experiment. Seed the cells at a density of 1,000 cells per well in a 96-well plate. Add culture medium containing varying concentrations of oxaliplatin (OXA) and incubate for 24 h. After removing the culture medium, add 10 µL of CCK−8 reagent to each well and incubate at 37 °C. Measure the optical density (OD) at 450 nm using a spectrophotometer, with each condition performed in triplicate. Calculate cell viability and the half-maximal inhibitory concentration (IC50). Generate a dose-response curve based on cell viability and drug concentration.

Transwell assay

Cells were suspended in serum-free medium and adjusted to a concentration of 1 × 10⁵ cells/mL. A total of 100 µL of the cell suspension was added to the upper chamber, while 600 µL of medium supplemented with 20% fetal bovine serum (FBS) was added to the lower chamber. Each experimental group was performed in triplicate, and the cells were incubated for 24 h. Following incubation, the cells were fixed with methanol and stained with Giemsa solution. Non-invading cells and Matrigel on the upper side of the membrane were gently removed using a cotton swab. Three random fields were selected under a microscope, and the number of invading cells was counted and averaged.

Colony formation assay

Cells were seeded in 6-well plates at a density of 1 × 10³ cells per well and cultured in 2 mL of complete medium for 14 days until visible colonies formed. The colonies were fixed with methanol and stained with Giemsa solution. Images were captured, and the number of colonies in each group was counted and recorded.

Database analysis

The GSE28702 dataset from the GEO database was downloaded, which includes triplicate technical replicates for both sensitive and resistant samples. Differential gene expression analysis was performed using the limma package to identify genes associated with Oxaliplatin resistance, with a significance threshold of p < 0.05 and an absolute log-fold change (|logFC|) > 0.4. The WP_FERROPTOSIS gene set was retrieved from the MSigDB database, resulting in the inclusion of 64 relevant genes. A Venn diagram was utilized to identify overlapping genes between those related to ferroptosis and Oxaliplatin resistance. Additionally, the Kaplan-Meier Plotter database was employed to conduct survival analysis of the target genes within the colorectal cancer microarray cohort.

Statistical analysis

All statistical analyses were conducted using the SPSS26.0 software. GraphPad 9. 0 and Image J were used for image and data processing. Count data were expressed as n (%), and χ2 test was used for group comparison. The correlation between expressions was analyzed using Spearman’s correlation or linear regression analysis. All results were considered to be statistically significant at P < 0.05.

Results

Elevated iron metabolism characterizes oxaliplatin-resistant CRC

Resistant CRC tissues exhibited significantly higher Fe²⁺ levels versus sensitive controls (P < 0.05, Fig. 1A), while MDA, ROS, and GSH showed no difference (Fig. 1B-D). This indicates dysregulated iron homeostasis under resistance.

Fig. 1
figure 1

Elevated iron metabolism characterizes oxaliplatin-resistant CRC. (A)Levels of Fe2+; (B) Levels of MDA; (C) Levels of ROS; (D) Levels of GSH.

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IREB2 links chemoresistance to ferroptosis and predicts poor prognosis

Bioinformatics analysis of GSE28702 identified 2,606 oxaliplatin resistance-associated genes. Intersection with ferroptosis gene set (WP_FERROPTOSIS) revealed five key regulators, including iron homeostasis master gene IREB2 (Fig. 2A). IREB2 expression was elevated in resistant clinical specimens (Fig. 2B) and correlated with reduced survival (Fig. 2C). Protein interaction analysis demonstrated IREB2 binding to ferroptosis effectors (TFRC/FTH1) and upstream regulator FBXL5 (Fig. 2D).

Fig. 2
figure 2

IREB2 links chemoresistance to ferroptosis and predicts poor prognosis. (A) Venn diagram showing the intersection between iron homeostasis-related genes and oxaliplatin resistance-related genes. (B) Analysis of the GSE28702 dataset from the GEO database revealed significant upregulation of IREB2 in oxaliplatin-resistant colorectal cancer cases. (C) Survival analysis indicated that high IREB2 expression is associated with poor prognosis. (D) Protein-protein interaction network constructed using the STRING database demonstrated interactions between IREB2 and FBXL5, TFRC, and FTH1.

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FBXL5-IREB2-TFRC axis correlates with iron overload and chemotherapy response

Immunohistochemistry of 119 CRC samples confirmed IREB2/TFRC overexpression and FBXL5 downregulation in resistant tumors (Fig. 3A). Western blot and RT-qPCR analysis of drug-resistant and sensitive colorectal cancer tissues, as well as adjacent normal mucosa, confirmed these findings, showing consistent trends with the immunohistochemistry results(Fig. 3B, Supplemental Fig. S1).

Tumor regression was analyzed in relation to the expression of various factors in CRC tissues. Tumor regression rates positively correlated with FBXL5 (P < 0.05, Fig. 3C), but inversely with IREB2/TFRC and Fe2+(P < 0.05, Fig. 3D, E,G). No associations were observed with FTH1, GSH, MDA, or ROS (Fig. 3F, H,I, J).

Spearman analysis revealed: Fe²⁺ levels were positively correlated with the expression of IREB2 and TFRC, but negatively correlated with the expression of FBXL5 and FTH1 (Table 1). These findings suggest that iron ion metabolism in colorectal cancer is closely associated with the expression of FBXL5, IREB2, TFRC, and FTH1.

Fig. 3
figure 3

FBXL5-IREB2-TFRC axis correlates with iron overload and chemotherapy response. (A) Immunohistochemical staining showing the expression of FBXL5, IREB2, TFRC, and FTH1 in normal intestinal mucosa and oxaliplatin-sensitive and oxaliplatin-resistant colorectal cancer tissues. (B) Expression levels of FBXL5, IREB2, TFRC, and FTH1 in colorectal cancer tissues. Membranes were sectioned prior to antibody hybridization. β-Actin served as the loading control. Data are presented as mean ± standard deviation (SD) from three independent experiments. Full-length blots/gels are presented in Supplementary Fig. 1.(C)Correlation between tumor regression and FBXL5 expression.(D)Correlation between tumor regression and IREB2 expression. (E) Correlation between tumor regression and TFRC expression. (F)Correlation between tumor regression and FTH1 expression. (G)Correlation between tumor regression and Fe2+ levels. (H)Correlation between tumor regression and GSH levels.(I)Correlation between tumor regression and MDA levels. (J) Correlation between tumor regression and ROS levels.

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Table 1 Correlation of Fe2+ with the expression of FBXL5, IREB2, TFRC, and FTH1 in colorectal cancer.
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Activation of ferroptosis enhances the sensitivity of tumor cells to oxaliplatin

To further validate the findings from clinical samples and elucidate the underlying regulatory mechanisms, we examined the expression of FBXL5 in human colorectal cancer cell lines, including COLO205, SW480, HCT−116, HCT−8, and SW620 (Fig. 4A). In vitro studies used HCT−116/OXA cells (IC₅₀=146.7 µg/mL vs. parental 14.5 µg/mL, P < 0.05, Fig. 4B). We then evaluated the expression of ferroptosis-related factors in both sensitive and resistant colorectal cancer cell lines. The results were consistent with the trends observed in colorectal cancer tissue samples. Resistant cells showed elevated Fe²⁺ (Fig. 4C, F) but unchanged ROS/GSH/MDA (Fig. 4D, E,G).

To further investigate whether increasing intracellular iron levels and disrupting iron homeostasis could activate ferroptosis and enhance the sensitivity of resistant cells to oxaliplatin, we examined the effect of ferroptosis activation on oxaliplatin sensitivity in colorectal cancer cells. Erastin (ferroptosis inducer) synergized with oxaliplatin, reducing cell viability (Fig. 4H-I).

Fig. 4
figure 4

Activation of Ferroptosis Enhances the Sensitivity of Tumor Cells to Oxaliplatin. (A) Expression of FBXL5 in different colorectal cancer cell lines. Membranes were sectioned prior to antibody hybridization. β-Actin served as the loading control. Data are presented as mean ± standard deviation (SD) from three independent experiments. (B) Sensitivity of HCT−116 and HCT−116/OXA cells to oxaliplatin (dose-response curves). (C-E) Detection of Fe2+, GSH, and MDA levels in the two cell groups using commercial assay kits. (F) Fluorescence images of FerroOrange staining in the two cell groups. (G) Detection of ROS levels in the two cell groups using the DCFH-DA fluorescent probe. (H-I) Enhanced cell viability and drug sensitivity in HCT−116/OXA cells treated with oxaliplatin combined with Erastin. Full-length blots/gels are presented in Supplementary Fig. 2.

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Modulating FBXL5 triggers ferroptosis and reverses oxaliplatin resistance

To further investigate the role of the FBXL5-IREB2-TFRC axis in ferroptosis and colorectal cancer drug resistance, HCT−116/OXA cells were transfected with lentiviruses carrying either OE-FBXL5 (overexpression) or sh-FBXL5 (knockdown) sequences. Transfection efficiency was confirmed by Western blot and qRT-PCR. Western blot analysis revealed that the sh-FBXL5 group showed significantly upregulation of IREB2/TFRC protein (Fig. 5A-D). qRT-PCR results indicated no significant differences in IREB2 mRNA expression among the groups, whereas TFRC mRNA expression was significantly reduced in the OE-FBXL5 group and increased in the sh-FBXL5 group (Fig. 5E-G). Furthermore, FBXL5 knockdown elevated intracellular Fe2+ and MDA levels(Fig. 5H-I, K). No significant changes were observed in GSH levels(Fig. 5J).

Fig. 5
figure 5

Modulating FBXL5 triggers ferroptosis. (A-D)Protein expression of FBXL5, IREB2, and TFRC in cells transfected with OE-FBXL5 or sh-FBXL5. Membranes were sectioned prior to antibody hybridization. β-Actin served as the loading control. Data are presented as mean ± standard deviation (SD) from three independent experiments. (E-G) mRNA expression of FBXL5, IREB2, and TFRC in transfected cells. (H-J) Detection of Fe2+, MDA, and GSH levels in cells after FBXL5 modulation. (K) Fluorescence images of FerroOrange staining in cells after FBXL5 modulation. Full-length blots/gels are presented in Supplementary Fig. 3.

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The subsequent evaluation of the growth-inhibitory effects of FBXL5 knockdown in HCT−116/OXA revealed significantly suppressed proliferation and migration capabilities(Fig. 6A-C). To determine the impact of FBXL5 modulation on oxaliplatin sensitivity, CCK−8 assays revealed that FBXL5 knockdown significantly enhanced cellular sensitivity to oxaliplatin(Fig. 6D).

Fig. 6
figure 6

FBXL5 knockdown suppresses proliferation and migration while enhancing oxaliplatin sensitivity in HCT−116/OXA cells. (A)CCK−8 assay to determine the proliferation levels of HCT−116/OXA cells after FBXL5 modulation. (B) Transwell migration assay to evaluate the effect of FBXL5 modulation on the migratory ability of HCT−116/OXA cells. (C) Colony formation assay to assess the effect of FBXL5 modulation on the proliferative ability of HCT−116/OXA cells. (D) Dose-response curves showing the chemosensitivity of HCT−116/OXA cells to oxaliplatin after FBXL5 modulation.

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Ferroptosis Inhibition rescues FBXL5-knockdown effects

To determine whether ferroptosis mediated the observed resistance reversal, we applied ferroptosis inhibitor Ferrostatin−1 (Fer−1). Figure 7A, B showed that oxaliplatin treatment significantly reduced cell viability in sh-FBXL5 cells compared to the control group, while co-treatment with Fer−1 partially restored cell viability. Colony formation and Transwell assays performed at the IC₅₀ concentration of oxaliplatin (146.7 µg/mL) demonstrated that the proliferation and migration capabilities of oxaliplatin-treated sh-FBXL5 cells were decreased (P < 0.05, Fig. 7C-D). These findings suggest that FBXL5 knockdown induces ferroptosis, significantly enhances the sensitivity of oxaliplatin-resistant colorectal cancer cells to oxaliplatin, and ultimately reverses drug resistance.

Fig. 7
figure 7

Ferroptosis inhibition rescues FBXL5-knockdown effects. (A-B)(A-B) Cell viability (A) and chemosensitivity to oxaliplatin (OXA) (B) in HCT−116/OXA cells following FBXL5 knockdown and combined treatment with Fer−1. (C) Transwell migration assay to evaluate the effect of FBXL5 knockdown and combined treatment with Fer−1 and OXA on the migratory ability of HCT−116/OXA cells. (D) Colony formation assay to assess the effect of FBXL5 knockdown and combined treatment with Fer−1 and OXA on the proliferative ability of HCT−116/OXA cells.

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Discussion

Ferroptosis, a regulated form of cell death driven by iron-dependent lipid peroxidation, involves complex mechanisms, including endogenous enzymatic antioxidant pathways and exogenous iron transporter-dependent processes. Dysregulation of iron transport, leading to intracellular iron accumulation, is a key initiator of ferroptosis8. Excess iron ions catalyze Fenton reactions, exacerbating lipid peroxidation and ultimately causing cell death9. Iron, an essential micronutrient, plays a dual role in cancer: it supports tumor growth and proliferation while also inducing ferroptosis under iron overload conditions10. Tumor cells, characterized by high metabolic activity and iron demand, exhibit elevated iron metabolism, which enhances their growth and resistance to therapy11. Chen et al. demonstrated that tumor cells in liver, lung, and breast cancers exhibit high iron metabolism, promoting tumor progression12. Conversely, iron overload can trigger ferroptosis by increasing reactive oxygen species (ROS), depleting glutathione (GSH), and elevating malondialdehyde (MDA) levels, leading to membrane damage6. Thus, inducing ferroptosis in iron-rich tumor cells has emerged as a novel therapeutic strategy. For instance, Wang et al. showed that iron overload sensitizes non-small cell lung cancer cells to PD-L1 therapy13, while Yu et al. found that increasing iron content in doxorubicin-resistant breast cancer cells induces ferroptosis and reverses drug resistance14. These findings highlight the intricate relationship between ferroptosis, tumor progression, and drug resistance, with iron homeostasis playing a pivotal role in tumor cell survival.

In this study, we observed higher iron levels in oxaliplatin-resistant colorectal cancer (CRC) tissues and cell lines compared to sensitive groups, while ferroptosis-related markers (ROS, GSH, MDA) showed no significant changes, suggesting that resistant cells maintain iron homeostasis but remain susceptible to ferroptosis. Using the ferroptosis inducer Erastin, we demonstrated that activating ferroptosis significantly enhances oxaliplatin sensitivity in resistant cells. These results indicate that iron-rich CRC cells are prone to drug resistance but can be sensitized to ferroptosis, offering a potential therapeutic avenue.

To explore the molecular mechanisms underlying iron homeostasis in resistant CRC, we performed bioinformatics analysis and identified IREB2, a key regulator of iron metabolism, as a co-expressed gene associated with oxaliplatin resistance and ferroptosis. IREB2, encoding the iron regulatory protein IRP2, modulates iron homeostasis by regulating downstream targets such as TFRC (transferrin receptor) and FTH1 (ferritin heavy chain)6,15. TFRC facilitates iron uptake, while FTH1 sequesters free iron, maintaining iron pool stability and preventing ferroptosis16,17. IREB2 achieves this balance by inhibiting TFRC mRNA degradation and suppressing FTH1 protein translation18. Studies have shown that targeting IREB2 inhibits ferroptosis and suppresses CRC cell proliferation, migration, and invasion19, while activating IREB2 induces ferroptosis in lung epithelial cells20. In our clinical samples, IREB2 and TFRC expression, along with Fe2+ levels, were higher in resistant groups and positively correlated with each other but negatively correlated with tumor regression. Cellular experiments confirmed these findings, suggesting that the IREB2-TFRC axis plays a critical role in maintaining iron-rich, drug-resistant tumor cells.

To further explore the regulatory mechanisms of IREB2, we utilized the STRING database to construct a protein-protein interaction network and identified FBXL5 as the most closely associated upstream gene of IREB2. FBXL5, a member of the F-Box protein family, is a key component of the E3 ubiquitin ligase complex. Its C-terminal leucine-rich repeat (LRR) domains enable substrate recognition21. The role of FBXL5 as an E3 ubiquitin ligase in tumor biology has gained increasing attention. For instance, Vinas et al. demonstrated that FBXL5 inhibits gastric cancer cell invasion and metastasis by ubiquitinating and degrading Snail122. In contrast, Yao et al. reported that FBXL5 promotes colorectal cancer progression by regulating the PI3K/AKT signaling pathway23, highlighting its context-dependent functions in different cancers. Recent studies have revealed that FBXL5 senses intracellular iron levels and acts as a negative regulator of IREB2, promoting its ubiquitination and proteasomal degradation24. While FBXL5 has been implicated in tumor progression, its role in ferroptosis and drug resistance remains underexplored. In our study, FBXL5 expression was higher in CRC tissues than in normal mucosa but lower in resistant versus sensitive tumors. FBXL5 expression negatively correlated with IREB2, TFRC, and iron levels but positively correlated with tumor regression.

We selected the HCT−116 colorectal cancer cell line, which exhibited moderate expression of FBXL5 and sensitivity to Oxaliplatin, and induced it to develop resistance, creating the HCT−116/OXA cell line for subsequent experiments. Consistent with clinical sample studies, drug-resistant colorectal cancer cells were in a state of high iron compared to sensitive cell lines. In cellular transfection experiments, compared to the overexpression of FBXL5, the knockdown of FBXL5 further increased the expression of IREB2 and TFRC, significantly elevated Fe2+ and MDA levels, and enhanced the sensitivity of colorectal cancer cells to Oxaliplatin, while also markedly diminishing their proliferative and migratory capabilities. To confirm that the knockdown of FBXL5 and the upregulation of IREB2 and TFRC expression are related to the reversal of Oxaliplatin resistance through the activation of Ferroptosis, we conducted rescue experiments with the Ferroptosis inhibitor Fer−1. The results showed that Fer−1 could rescue the cytotoxic effects of Oxaliplatin on colorectal cancer drug-resistant cells post-FBXL5 knockdown, enhancing their sensitivity to Oxaliplatin and improving their proliferative and migratory abilities. Our study demonstrates that targeting FBXL5 to affect the FBXL5-IREB2-TFRC axis, inducing Fe2+ overload, and thereby activating Ferroptosis in colorectal cancer cells, could be a novel approach to enhance their sensitivity to Oxaliplatin and reverse drug resistance.

Oxaliplatin, a first-line chemotherapy for advanced CRC, induces DNA crosslinking and oxidative stress to trigger apoptosis25,26. However, drug resistance remains a significant challenge. Emerging evidence highlights ferroptosis as a potential mechanism to reverse resistance27. For example, Tao et al. demonstrated that downregulating USP3 increases iron levels and ferroptosis, enhancing cisplatin sensitivity in lung cancer28, while Du et al. reported that dihydroartemisinin induces ferroptosis in pancreatic ductal adenocarcinoma, overcoming cisplatin resistance29. Our study reveals that modulating the FBXL5-IREB2-TFRC axis increases Fe2+ levels, activates ferroptosis, and enhances oxaliplatin sensitivity in CRC cells. Conversely, inhibiting ferroptosis restores resistance, suggesting that iron-induced ferroptosis synergizes with oxaliplatin to overcome drug resistance.

In conclusion, our study demonstrates that the FBXL5-IREB2-TFRC axis plays a critical role in oxaliplatin resistance and prognosis in CRC. Targeting FBXL5 to upregulate IREB2 and TFRC, increase intracellular iron levels, and induce ferroptosis represents a novel therapeutic approach to reverse drug resistance. While FTH1 expression did not differ between sensitive and resistant groups, its role in CRC iron metabolism warrants further investigation30. Overall, our findings provide new insights into the molecular mechanisms of CRC resistance and highlight the FBXL5-IREB2-TFRC-ferroptosis axis as a potential target for developing small-molecule therapies to improve patient outcomes.