LRP8 Promotes colorectal cancer progression by suppressing ferroptosis through the SLC3A2/GPX4 signalling axis

Abstract

Background

Colorectal cancer (CRC) persists as one of the most lethal malignancies worldwide, with therapeutic resistance representing a significant obstacle in clinical management. Ferroptosis, a form of programmed cell death triggered by iron accumulation and lipid peroxidation, has recently emerged as a promising target for cancer therapy. Although low-density lipoprotein receptor-related protein 8 (LRP8) has been implicated in oncogenic processes across cancer types, its involvement in CRC progression and ferroptosis regulation has not been fully elucidated.

Methods

This study utilized an integrative multi-omics approach, incorporating transcriptomic profiling across the colorectal carcinogenesis spectrum (normal mucosa, adenoma, carcinoma; n = 5 each) and proteomic analysis via 4D-DIA mass spectrometry. LRP8 expression patterns were examined in 40 paired CRC and adjacent normal tissues and a tissue microarray comprising 94 cases. Functional investigations were conducted in CRC cell lines following LRP8 knockdown or overexpression. Xenograft models were employed for in vivo validation. Mechanistic insights were gained through co-immunoprecipitation, redox assays, and transmission electron microscopy.

Results

Transcriptomic data revealed a stepwise increase in LRP8 expression during CRC development. Clinical analyses demonstrated that elevated LRP8 levels correlated significantly with advanced tumour stage, lymphatic metastasis, and poorer patient prognosis. Functional assays indicated that LRP8 enhances oncogenic behaviors by interacting with SLC3A2. Reintroducing SLC3A2 in LRP8-depleted cells restored glutathione peroxidase 4 (GPX4) expression and mitigated oxidative stress, thereby rescuing ferroptosis resistance. In vivo, silencing LRP8 inhibited tumour growth and induced ferroptosis-associated alterations, including disrupted iron homeostasis and increased lipid peroxidation.

Conclusion

LRP8 facilitates CRC progression by antagonizing ferroptosis via modulation of the SLC3A2/GPX4 signalling axis. These findings highlight LRP8 as a previously unrecognized regulator of ferroptotic vulnerability and a potential therapeutic target in CRC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40001-026-03964-2.

Introduction

Globally, colorectal carcinoma (CRC) maintains its position as the third most frequently diagnosed malignancy and the second deadliest cancer type [1]. Statistical models anticipate substantial growth in disease burden, estimating 3.2 million new cases and 1.6 million annual deaths by 2040—equivalent to a 63% incidence jump and 73.4% mortality surge [2]. Although current therapeutic strategies, including surgical resection, neoadjuvant chemoradiotherapy, and precision molecular interventions, have achieved incremental improvements in clinical outcomes, persistent issues such as locoregional recurrence, distant metastasis, and resistance to treatment continue to limit long-term survival [3, 4]. These challenges highlight the pressing need to identify novel molecular drivers of CRC and develop more effective, targeted treatment approaches.

Ferroptosis, an iron-dependent programmed cell death mechanism driven by lipid peroxidation and antioxidant system failure [5–7], has become a potential cancer treatment strategy [8]. This process critically depends on the glutathione-GPX4 pathway, the primary cellular defence against oxidative membrane damage [9]. In CRC, GPX4 is frequently overexpressed in tumour tissues relative to adjacent normal mucosa, underscoring its potential role in tumour survival [10]. Experimental models have shown that induction of ferroptosis, whether through iron modulation, enhancement of reactive oxygen species (ROS), or inhibition of GPX4, effectively suppresses CRC progression [11]. Conversely, resistance to ferroptosis has been implicated in tumour relapse and treatment failure, positioning ferroptosis as a targetable vulnerability in CRC [12].

Using a multi-omics approach, we identified low-density lipoprotein receptor-related protein 8 (LRP8, also known as ApoER2) as a novel contributor to CRC pathogenesis and ferroptosis regulation. LRP8 has been shown to influence ferroptosis sensitivity by preserving the cellular pool of selenocysteine tRNAs required for the efficient translation of GPX4 [13] and by preventing ribosomal stalling at the UGA codon that encodes its essential selenocysteine residue [14]. LRP8 modulates lipid metabolism via the Wnt/β-catenin–stearoyl-CoA desaturase 1 (SCD1) signalling axis, further shaping the cellular redox landscape [15]. Beyond its role in ferroptosis, LRP8 has been implicated in promoting epithelial–mesenchymal plasticity, metastatic dissemination, and resistance to therapy across multiple malignancies, including gastric [16], ovarian [17], and breast cancers [18]. In CRC specifically, the miR-378a-5p/LRP8 regulatory axis has been associated with modulation of radiation response, suggesting broader oncogenic functions [19]. Despite these findings, the involvement of LRP8 in CRC ferroptosis regulation remains incompletely characterized. This study aims to elucidate the role of LRP8 in modulating ferroptotic sensitivity in CRC and to identify new therapeutic strategies that exploit this regulatory pathway.

Materials and methods

Patient samples and tissue microarray (TMA)

Fresh CRC tissues and matched adjacent non-tumorous samples were collected from 40 patients undergoing curative resection at Gansu Provincial People's Hospital (Lanzhou, China) between April and August 2024. None of the patients received preoperative chemotherapy or radiotherapy. All patients provided written informed consent. This study received approval from the Ethics Committee of Gansu Provincial Hospital (Approval document No.2024-807) and was conducted in accordance with the ethical guidelines delineated in the Helsinki Declaration.

Additionally, a commercially available tissue microarray (TMA) slide (Chip ID: HColA180Su21), comprising 94 pairs of CRC and adjacent normal tissues, was purchased from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China). All samples were processed according to standard procedures for downstream analyses, including qPCR and IHC.

Clinicopathological features-including age, sex, tumour size, vascular/perineural invasion, lymph node status, TNM classification, PD-1/PD-L1 expression, and KRAS, NRAS, and BRAF mutation status were retrieved from medical records.

Immunohistochemistry (IHC)

Following optimized protocols, IHC staining was conducted on TMA and xenograft tumour sections using primary antibodies against LRP8(1:300, PA5-109269,Invitrogen),GPX4(1:50,30388-1-AP,Proteintech),SLC3A2(1:50,15193-1-AP,Proteintech) and Ki67(1:300,NBP2-22112,Novus). Two independent pathologists, blinded to clinical information, evaluated the staining results. Ten random high-power fields (× 400) were assessed per slide, with 100 tumour cells counted per field.

Staining was scored based on the percentage of positive cells and staining intensity. Proportion scores were assigned as follows: 0 (none), 1 (< 25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). Intensity scores ranged from 0 (negative) to 3 (strong). Final IHC scores were calculated by multiplying the proportion and intensity scores.

Cell culture and lentiviral transduction

CRC cell lines (SW480, SW620, HT-29, HCT-116, LOVO) and normal human colonic epithelial cells (NCM-460) were sourced from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). CRC cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C with 5% CO₂. NCM-460 cells were maintained in specialized medium as per the supplier's instructions. All the cell lines used in the experiments were certified by the institution before the experiments and there was no mycoplasma contamination during the handling process.

Short hairpin RNAs (shRNAs; sh1, sh2, sh3) targeting LRP8 or SLC3A2, a scrambled control (NC), and an overexpression vector (OE-LRP8) were cloned into lentiviral vectors (GeneChem, Shanghai, China) and transduced into CRC cells.

Quantitative real-time PCR (qRT-PCR)

Total RNA extraction was performed with the M5 HiPer Universal RNA Mini Kit (Polymed, China), and cDNA synthesis was used with PrimeScript™ RT reagent Kit (Takara, Japan). Quantitative PCR analysis was conducted with TB Green Premix Ex Taq™ (Takara, RR820A), and relative gene expression was calculated via the 2^ΔΔCt method.

Western blotting

Cell lysates were prepared using ice-cold lysis buffer (20 mM Tris–HCl pH 7.5, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA, 50 mM NaF, 1 mM DTT, 10% glycerol) supplemented with protease inhibitors. Following protein quantification (BCA assay, Thermo Fisher), equal protein amounts were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% non-fat milk, membranes were incubated with primary antibodies against LRP8(1:500, PA5-109269,Invitrogen),GPX4(1:1000,30388–1-AP,Proteintech),SLC3A2(1:5000,15193–1-AP,Proteintech) and GAPDH(1:10000,ab181602,Abcam), then with HRP-conjugated secondary antibodies. Protein signals were visualized using enhanced chemiluminescence (Thermo Fisher).

Colony formation assay

Cells (1000/well) were seeded in 6-well plates and incubated for 10–14 days. Colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Colonies with > 50 cells were manually counted in five random fields.

Flow cytometry analysis

Cells were stained with Annexin V-FITC/PI (BD Biosciences, USA) and analyzed by flow cytometry (BD FACSCanto^™ II). For cell cycle analysis, ethanol-fixed cells were stained with PI and RNase A, and DNA content was quantified by flow cytometry.

EdU incorporation assay

Cell proliferation was assessed using the EdU Apollo567 kit (RiboBio, China). Cells were treated with 50 μM EdU, then fixed, permeabilized, and stained. Nuclear counterstaining was performed using Hoechst 33,342. Fluorescence images were captured using an Olympus microscope (Olympus).

Migration and invasion assays

Migration and invasion were assessed in Transwell chambers (8 μm pores; Corning, USA). For migration, serum-free cell suspensions were seeded in the upper chamber, with 10% FBS medium in the lower well. The chamber membrane was pre-coated with Matrigel (BD Biosciences, USA) for invasion. After 48 h, non-migrated cells were removed, and migrated/invaded cells were fixed, stained, and counted under a microscope.

RNA sequencing and bioinformatics

Novogene Co., Ltd. (Beijing, China) conducted RNA-seq using total RNA from 15 samples (normal mucosa, adenoma, and carcinoma; n = 5 each). After RNA quality control, sequencing was performed on the Illumina platform. Differentially expressed genes (DEGs) were identified, and functional annotations were performed using GO and KEGG analyses.

Proteomics analysis

Proteomic profiling was performed using 4D-DIA mass spectrometry (Bruker timsTOF Pro). SW480 cells transfected with siLRP8 or siNC were analyzed by LC–MS/MS. Raw data were processed with Spectronaut (Biognosys, Sweden), and differential protein expression was analyzed with functional enrichment of affected pathways (GO, KEGG) [29].

Animal experiments

Fifteen male BALB/c nude mice (4 weeks old, n = 5/group) were subcutaneously injected with 1 × 10⁶SW480 cells (wild-type, NC, or LRP8 knockdown). Tumour dimensions and body weight were measured every 2 days from day 10. After a 21-day alimentation period,All animals were euthanized by barbiturate overdose (intravenous injection, 150 mg/kg pentobarbital sodium),death was confirmed by loss of heartbeat, breathing and pupil response, and the tumor tissues were extirpated and weighed.Tumor tissues were immobilized in 4% paraformaldehyde for subsequent experimentation. All animal experimental procedures were sanctioned by the Bestcell Model Biological Center (IACUC Issue No.2025–04-18A).

Transmission electron microscopy (TEM)

Cells were fixed in glutaraldehyde, dehydrated, embedded in resin, and sectioned into ultrathin slices. After staining with uranyl acetate and lead citrate, mitochondrial ultrastructure was visualized using TEM (Thermo Fisher Scientific, USA).

Biochemical assays

BCA assays were used for protein quantification. Glutathione (total, reduced, oxidised), total iron, ferrous iron (Fe²⁺), cysteine, and ROS levels were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute and Elabscience), following manufacturer protocols and analysed via spectrophotometry.

Mitochondrial membrane potential (MMP)

JC-1 staining (Cat. C2003S) assessed mitochondrial membrane potential. Cells were incubated with JC-1 dye, washed, and analyzed by flow cytometry (Thermo Fisher Scientific, USA). The red-to-green fluorescence ratio reflected MMP status.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.0 and SPSS 26.0. Data were expressed as mean ± SD. The students’ t-test was used for two-group comparisons, and ANOVA was applied for multiple-group analyses. Associations between LRP8 expression and clinical variables were assessed using the χ² test. A p-value < 0.05 was considered statistically significant.

Results

Transcriptomic and bioinformatic analyses identify lrp8 as a key gene associated with CRC progression

To investigate dynamic gene expression changes during colorectal tumorigenesis, we conducted transcriptome sequencing on tissue samples representing three distinct stages: normal mucosa (N, n = 5), adenomas (T, n = 5), and carcinomas (C, n = 5) (Fig. 1A). A total of 3685 DEGs were identified and the expression change of LRP8 is the most significant. Volcano plots illustrated substantial transcriptional shifts in both A vs. T and C vs. N comparisons (Fig. 1B), with the number of DEGs increasing progressively along the typical adenoma–carcinoma sequence (Fig. 1C). Unsupervised hierarchical clustering revealed clear segregation among the three sample types (Fig. 1D), and all sequencing datasets passed standard quality control assessments, confirming the robustness of the transcriptomic analysis (Fig. 1E). Venn diagrams demonstrated both shared and unique DEGs among the pairwise comparisons (Fig. 1F).Functional enrichment based on GO highlighted that the DEGs were predominantly involved in biological processes such as extracellular matrix organization, epithelial cell migration, and regulation of the Wnt signalling pathway (Fig. 1G–H). KEGG pathway analysis further revealed significant enrichment in pathways implicated in tumour biology, including IL-17 signalling, the p53 pathway, and focal adhesion (Fig. 1I).Complementary annotations via Disease Ontology, DisGeNET, and Reactome databases consistently associated the identified DEGs with colorectal cancer-related pathways and mechanisms (Fig. 1J–L). Among the DEGs, the LRP8 is the top one. Therefore, we chose it for next analysis.

Fig. 1.

Transcriptomic profiling reveals dynamic gene expression changes during colorectal tumorigenesis. A Colorectal tissue samples were obtained from normal mucosa (N, n = 5), adenoma (T, n = 5), and carcinoma (C, n = 5) for transcriptomic analysis. B Volcano plots depicting differentially expressed genes (DEGs) between T vs. N (left) and C vs. N (right) comparisons. C Bar chart summarising the number of upregulated and downregulated DEGs in each comparison group. D A hierarchical clustering heatmap shows global expression patterns of DEGs across the three groups (T, A, and C). E Principal component analysis (PCA) illustrating distinct clustering of samples based on transcriptomic profiles. F Venn diagram indicating the overlap of DEGs across A vs. T and C vs. N comparisons. G Gene Ontology (GO) enrichment analysis of overlapping DEGs, highlighting key biological processes and cellular components involved in tumorigenesis. H Directed acyclic graph (DAG) visualizing hierarchical relationships among enriched GO terms. I KEGG pathway enrichment analysis identifies tumour-related signalling pathways that are significantly enriched among DEGs. J–L Functional enrichment of DEGs using Disease Ontology J, DisGeNET K, and Reactome L databases, demonstrating strong associations with colorectal cancer-related disease terms and pathways

LRP8 Is upregulated in colorectal cancer and correlates with poor prognosis

Notably, LRP8 expression exhibited a stepwise upregulation from normal mucosa to adenoma and carcinoma tissues, suggesting its potential involvement in the early and progressive stages of colorectal carcinogenesis. This observation positioned LRP8 as a promising candidate gene for subsequent functional validation.

Subsequently, we analyzed LRP8 mRNA levels in both colon adenocarcinoma (COAD) (Fig. 2A) and rectal adenocarcinoma (READ) (Fig. 2B) from TCGA. To validate these findings, we examined LRP8 expression in 40 paired CRC tumours and adjacent standard tissue samples. Immunofluorescence staining revealed that LRP8 protein was primarily localized to the cytoplasm and plasma membrane of cancer cells (Fig. 2C). RT-PCR confirmed a significant increase in LRP8 mRNA expression in tumor tissues compared to normal tissues (P = 0.006; Fig. 2D). Consistently, immunohistochemical (IHC) analysis of the same cohort demonstrated a marked overexpression of LRP8 in tumor specimens (Fig. 2E). This observation was further supported by semi-quantitative scoring of a tissue microarray containing 86 CRC cases, which revealed significantly higher LRP8 expression in tumor tissues (P < 0.0001; Fig. 2F). To explore the clinical relevance of LRP8, we examined its association with key clinicopathological features. Elevated LRP8 levels were significantly correlated with advanced TNM stage, presence of lymph node metastasis, increased infiltration of CD8⁺ T cells, PD-1/PD-L1 positivity, and KRAS mutations (Table 1). No significant associations were observed with patient age, sex, tumour size, or NRAS/BRAF mutation status. Importantly, Kaplan–Meier analysis of the tissue microarray cohort indicated that high LRP8 expression was an unfavourable prognostic indicator, predicting reduced 5-year overall survival (P < 0.05; Fig. 2G).

Fig. 2.

LRP8 is upregulated in colorectal cancer and associated with poor clinical outcomes. A Analysis of LRP8 mRNA expression in colon adenocarcinoma (COAD) tissues compared to adjacent normal tissues using data from The Cancer Genome Atlas (TCGA). B LRP8 mRNA expression in rectal adenocarcinoma (READ) versus matched normal tissues based on the TCGA dataset. C Representative immunofluorescence staining images showing increased LRP8 expression (red) in CRC tissues compared to adjacent normal mucosa. Nuclei are counterstained with DAPI (blue). D RT-PCR analysis of LRP8 mRNA levels in 40 pairs of CRC and matched adjacent normal tissues. E Representative immunohistochemistry (IHC) images displaying LRP8 expression in CRC and corresponding normal tissues. F Quantitative analysis of IHC staining scores for LRP8 in 86 CRC samples obtained from a commercial tissue microarray. G Kaplan–Meier survival analysis based on IHC data indicates that elevated LRP8 protein expression correlates with reduced overall survival in CRC patients. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Table 1.

The correlation between the expression level of LRP8 and clinicopathological characteristics as well as prognosis in CRC patients

Characteristics	No.of cases	LRP8 expression			χ2	P-value
		Low		High
Gender
Male	46	14	32		1.139	0.286
Female	48	10	38
Age (years)
≤ 60	35	11	24		1.020	0.313
> 60	59	13	46
Tumor size(cm)
≤ 5	45	9	36		1.389	0.238
> 5	49	15	34
Remote relocation
Yes	5	1	4		0.085	0.621
No	89	23	66
Vascular invasion
Yes	31	11	20		2.409	0.121
No	63	13	50
Nerve invasion
Yes	17	6	11		1.040	0.308
No	77	18	59
T staging
T1−T2	9	4	5		1.872	0.171
T3−T4	85	20	65
Lymph node status
N0	59	21	38		8.437	0.004
N1−N2	35	3	32
pTNM staging
I/II	57	20	37		6.955	0.008
III/IV	37	4	33
CD8 positive rate
< 1%	29	3	26		5.088	0.024
≧1%	65	21	44
PD1 positive rate
< 1%	30	12	18		4.851	0.028
≧1%	64	12	52
PDL1 positive rate
< 1%	44	16	28		5.104	0.024
≧1%	50	8	42
KRAS mutation
Yes	32	4	28		4.334	0.037
No	62	20	42
NRAS mutation
Yes	4	0	4		1.432	0.231
No	90	24	66
BRAF mutation
Yes	2	1	1		0.643	0.442
No	92	23	69

LRP8 promotes malignant behaviors of CRC cells

To elucidate the oncogenic role of LRP8 in colorectal carcinogenesis, we systematically characterized its expression profile and functional significance across multiple CRC cell lines. Comparative analysis revealed significant upregulation of LRP8 at both transcriptional and translational levels in all examined CRC cell lines (SW480, SW620, HT-29, HCT116, and LOVO) relative to normal colonic epithelial cells (NCM460) (Supplementary Fig.  1 A). Therefore, we chose SW480, SW620 and HCT116 to silence or overexpression LRP8 (Supplementary Fig. 1B−D). Cell functional analysis showed that LRP8 depletion in SW480 and SW620 cells resulted in profound suppression of malignant phenotypes, including impaired proliferative capacity (Fig. 3A), reduced clonogenic potential, attenuated migratory and invasive properties (Fig. 3B−D), as well as increased apoptotic susceptibility and G1 phase cell cycle arrest (Fig. 3E, F). Conversely, ectopic LRP8 expression in HCT116 cells significantly enhanced these oncogenic behaviors, as evidenced by accelerated cell cycle progression and diminished apoptosis (Fig. 3).

Fig. 3.

LRP8 regulates CRC cell proliferation, migration, invasion, apoptosis, and cell cycle. A Wound healing assay to evaluate the impact of LRP8 on cell migration. B EdU incorporation assay assessing DNA synthesis in CRC cells upon LRP8 modulation. C Transwell assays to assess cell migration and invasion in response to LRP8 modulation. D Colony formation assay showing the effect of LRP8 on CRC cell proliferation. E Flow cytometric analysis of apoptosis in cells with LRP8 silencing or overexpression. F Cell cycle analysis via flow cytometry, showing G1-phase arrest in LRP8-knockdown cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

LRP8 sustains GPX4 expression to inhibit ferroptosis in CRC

To explore the downstream effects of LRP8 in CRC, we performed proteomic profiling using 4D-DIA sequencing in SW480 cells following LRP8 knockdown by siRNA. The resulting volcano plot demonstrated a broad spectrum of differentially expressed proteins (DEPs), among which GPX4 and SLC3A2, key regulators of oxidative stress and ferroptosis, were significantly downregulated (Fig. 4D).

Fig. 4.

LRP8 modulates ferroptosis-related proteins in CRC cells. A SDS-PAGE and Coomassie Brilliant Blue staining were performed to evaluate protein integrity in CRC cell lines. B Bar graph depicting the distribution of differentially expressed proteins (DEPs) across various comparison groups. C Hierarchical clustering of DEPs, revealing distinct expression patterns in CRC cells. D Volcano plot showing DEPs between LRP8 siRNA and control groups, highlighting key proteins such as GPX4. E Bar chart of Clusters of Orthologous Groups (COG) analysis for the DEPs. F Pie chart illustrating the subcellular localization of DEPs. G Gene Ontology (GO) enrichment analysis of DEPs, identifying key biological processes. H Bubble plot displaying Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEPs. I Protein–protein interaction (PPI) network for DEPs, identifying key protein interactions. J KEGG pathway map highlighting ferroptosis-related pathways, focusing on GPX4-associated signalling. K Protein interaction network within ferroptosis-related pathways involving GPX4. L RT-qPCR validation of GPX4 and SLC3A2 expression in LRP8 knockdown CRC cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

GO and KEGG pathway enrichment analyses indicated that LRP8 silencing predominantly affected pathways related to oxidative stress response, ferroptosis, and metabolic regulation (Fig. 4E). Subcellular localization predictions revealed that LRP8 is mainly distributed in the nucleus (32.7%) and cytoplasm (30.29%), suggesting its potential involvement in organelle-specific regulatory mechanisms associated with ferroptotic cell death (Fig. 4F).Further GO enrichment analysis emphasised strong associations with ferroptosis-related biological processes, including organelle organisation, intracellular transport, and protein-binding functions (Fig. 4G). KEGG analysis showed that cell cycle progression and metabolic pathways were notably disrupted in LRP8-deficient cells, indicating broader impacts on cellular homeostasis (Fig. 4H). PPI network analysis identified clusters of DEPs with dense interconnections, particularly centered on ferroptosis-related proteins (Fig. 4I). A focused PPI subnetwork built around GPX4 highlighted its interactions with core antioxidant and metabolic regulators (Fig. 4K), reinforcing its potential role of LRP8 in ferroptosis. Subsequent qRT-PCR validation confirmed that LRP8 knockdown decreased GPX4, SLC3A2, YTHDF2, and DNMT1 expression, while SLC31A1 were upregulated (Fig. 4L). These results were consistent with the proteomic data and further supported the regulatory relationship between LRP8 and ferroptosis-associated genes. These findings demonstrate that LRP8 influences ferroptosis in CRC cells by controlling the expression of GPX4 and SLC3A2.

To determine whether LRP8 suppression triggers ferroptosis in CRC cells, we generated stable LRP8-knockdown and LRP8-overexpressing cell lines (Fig. 5A, B). TEM of LRP8-silenced SW480 and SW620 cells revealed classic features of ferroptosis, including reduced mitochondrial volume, condensed membrane density, loss or disappearance of cristae, and outer membrane rupture. In contrast, LRP8-overexpressing cells exhibited intact mitochondrial structures without these abnormalities (Fig. 5C). JC-1 staining was used to assess mitochondrial membrane potential. LRP8 knockdown led to a pronounced reduction in JC-1 aggregates, indicating mitochondrial depolarisation and dysfunction. Conversely, LRP8 overexpression preserved membrane potential, as evidenced by stronger JC-1 aggregation (Fig. 5D). Silencing LRP8 significantly increased intracellular ferrous iron (Fe²⁺) levels (Fig. 5E), total iron content (Fig. 5G), and glutathione (GSH) levels (Fig. 5H), suggesting an accumulation of labile iron and heightened redox activity. Interestingly, LRP8 overexpression in HCT116 cells also elevated Fe²⁺, total iron, and GSH levels, indicating that LRP8 regulates iron metabolism and redox balance across multiple CRC cell types. To confirm that these changes were ferroptosis-specific, ferrostatin-1 (Fer-1), a selective ferroptosis inhibitor, was applied to LRP8-knockdown cells. Fer-1 treatment reversed mitochondrial damage, restoring organelle morphology (Fig. 5I) and normalizing JC-1 fluorescence patterns (Fig. 5J), indicating recovery of membrane potential. Fer-1 significantly reduced Fe²⁺ accumulation (Fig. 5K, L) in the LRP8-silenced cells, supporting the iron dependence of the observed phenotype. Treatment also restored GSH levels (Fig. 5M) and reduced oxidized glutathione (GSSG) levels (Fig. 5N), further alleviating oxidative stress. Together, these findings demonstrate that LRP8 influences ferroptosis sensitivity in CRC cells by regulating intracellular iron levels and redox status, and that ferroptotic responses induced by LRP8 loss are reversible through ferroptosis inhibition.

Fig. 5.

LRP8 suppression triggers ferroptosis in colorectal cancer cells. A, B qRT-PCR and western blot assessing GPX4 expression following LRP8 knockdown in three CRC cell lines. C Transmission electron microscopy (TEM) revealing characteristic mitochondrial abnormalities associated with ferroptosis following LRP8 silencing. D JC-1 staining and flow cytometry assessing mitochondrial membrane potential in control and LRP8-deficient cells. E, F Quantifying intracellular ferrous iron (Fe²⁺) and total iron (t-Fe) in LRP8-modulated cells. G, H Measurement of total glutathione (GSH), reduced GSH, and oxidized GSH (GSSG) levels. I, J TEM imaging and JC-1 staining of cells co-treated with the ferroptosis inhibitor Ferrostatin-1 (Fer-1), showing mitochondrial rescue. K, L Iron content (Fe²⁺ and t-Fe) in cells after Fer-1 administration. (M–N) GSH and GSSG levels in Fer-1-treated cells, confirming ferroptosis attenuation. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

LRP8 binds SLC3A2 to promote cysteine uptake and ferroptosis resistance

To investigate the molecular mechanisms by which LRP8 influences ferroptosis, we conducted mass spectrometry-based immunoprecipitation (MS-IP) in LRP8-overexpressing HCT116 cells to identify its interacting protein partners (Fig. 6A, B). The results showed that SLC3A2 was identified as a potential binding partner (Fig. 6C).

Fig. 6.

LRP8 interacts with SLC3A2 to modulate stress responses and ferroptosis in colorectal cancer cells. A Western blot confirming the expression of Flag-tagged LRP8 in HCT116 cells. B Co-immunoprecipitation (Co-IP) assay demonstrating the physical interaction between LRP8 and SLC3A2. C Volcano plot of mass spectrometry–identified proteins showing differentially expressed interactors. D Gene Ontology (GO) enrichment analysis of LRP8-interacting proteins, emphasizing roles in protein localization, DNA damage response, and cellular stress adaptation. E KEGG pathway analysis reveals enrichment in phagosome formation and endoplasmic reticulum (ER) protein processing. F Subcellular compartment analysis indicating the localization patterns of LRP8-associated proteins. G Western blot assessing SLC3A2 expression following LRP8 knockdown in SW480 and SW620 cells. H protein–protein interaction (PPI) network analysis. I ELISA analysis of Cyc content when knockdown of SLC3A2 in SW480 and SW620 cells. J ELISA analysis of ROS levels when knockdown of SLC3A2 in SW480 and SW620 cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Functional enrichment analysis of the LRP8-associated protein complexes revealed significant enrichment in pathways related to protein trafficking, oxidative stress response, and endoplasmic reticulum (ER) stress (Fig. 6D–F), suggesting a broader role for LRP8 in maintaining cellular stress adaptation and metabolic homeostasis.Western blot analysis confirmed that LRP8 knockdown in SW480 and SW620 cells led to a marked reduction in SLC3A2 expression (Fig. 6G), supporting a regulatory relationship between these two proteins. In addition, protein–protein interaction (PPI) network analysis further reinforced the existence of a direct or functionally relevant interaction between LRP8 and SLC3A2 (Fig. 6H). Subsequently,ELISA analysis confirmed that knockdown of SLC3A2 in SW480 and SW620 cells led to a significant decrease in Cyc content and a significant increase in ROS levels (Fig. 6I, J). These findings indicate that LRP8 may regulate cysteine uptake and redox homeostasis in CRC cells by interacting with SLC3A2, thereby contributing to ferroptosis susceptibility through modulation of system Xc⁻ activity.

Building on the identified interaction between LRP8 and SLC3A2, we explored whether LRP8 promotes CRC progression by inhibiting SLC3A2-mediated ferroptosis. Efficient knockdown of SLC3A2 in HCT116 cells was validated at both the mRNA and protein levels (Fig. 7A, B). Co-transfection of LRP8-overexpressing cells with SLC3A2-targeting shRNA significantly reversed the upregulation of GPX4 and SLC3A2, implicating SLC3A2 as a downstream effector of LRP8's anti-ferroptotic activity (Fig. 7C). Biochemical assays revealed that simultaneous LRP8 overexpression and SLC3A2 silencing resulted in significant reductions in intracellular GSH and Cys levels (Fig. 7I, J), accompanied by increased reactive oxygen species (ROS) accumulation (Fig. 7K), indicating a breakdown in redox homeostasis. Functionally, LRP8 overexpression enhanced proliferation, as evidenced by increased EdU and Ki67 positivity. However, this proliferative effect was markedly attenuated by SLC3A2 knockdown, indicating that SLC3A2 is necessary for LRP8-driven cell proliferation. Similarly, LRP8 overexpression promoted cell migration and invasion, as shown by transwell assays, whereas co-silencing SLC3A2 significantly suppressed these metastatic properties. Colony formation assays further demonstrated that LRP8-driven clonogenic growth was reduced upon SLC3A2 depletion. Additionally, cell cycle analysis showed that knockdown of SLC3A2 in LRP8-overexpressing cells caused a significant decrease in S and G2/M phase populations, suggesting impaired cell cycle progression. These findings indicate that SLC3A2 is a key mediator of LRP8’s oncogenic effects in CRC, facilitating ferroptosis resistance.

Fig. 7.

LRP8 inhibits ferroptosis and enhances CRC cell proliferation and metastasis via the SLC3A2/GPX4 axis. HCT116 cells were co-transfected with LRP8 overexpression (OE-LRP8) and SLC3A2 shRNA (sh-SLC3A2) constructs. Forty-eight hours post-transfection: A Western blot confirming knockdown efficiency of SLC3A2. B qPCR analysis validating reduced SLC3A2 mRNA levels. C Western blot showing GPX4 protein expression following co-transfection. D Colony formation assay demonstrating changes in clonogenic potential with SLC3A2 knockdown. E Transwell assays assessing migration and invasion following SLC3A2 silencing. F EdU incorporation assay evaluating proliferative capacity under SLC3A2 knockdown with LRP8 rescue. G Flow cytometric analysis of apoptosis in cells with SLC3A2 knockdown and LRP8 overexpression. H Cell cycle analysis by flow cytometry shows cell population distribution after SLC3A2 silencing. I, K Biochemical assays measuring intracellular levels of total glutathione (GSH) I, reactive oxygen species (ROS) J and cysteine K after dual modulation of LRP8 and SLC3A2. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

LRP8 silencing triggers ferroptosis in CRC xenograft models

To explore the in vivo function of LRP8 in colorectal cancer (CRC), we established a subcutaneous xenograft model using SW480 cells with stable LRP8 knockdown. Tumours from the sh-LRP8 group exhibited significantly reduced volumes and growth rates compared to those in the negative control group, indicating that LRP8 is essential for efficient tumour progression (Fig. 8A–C). Immunohistochemistry confirmed successful knockdown, showing markedly decreased LRP8 expression in tumour sections from the sh-LRP8 cohort (Fig. 8D). TUNEL staining revealed a substantial increase in apoptotic cells within the sh-LRP8 tumors, suggesting that LRP8 contributes to tumor cell survival (Fig. 8E). Western blot analysis further validated these findings, demonstrating reduced levels of LRP8 and GPX4, a key regulator of ferroptosis, in tumor lysates from the LRP8-silenced group (Fig. 8F). Biochemical assays provided additional insight into the redox imbalance induced by LRP8 depletion. Tumours lacking LRP8 exhibited decreased concentrations of ferrous iron (Fe²⁺; Fig. 8J), total iron (t-Fe; Fig. 8K), GSH (Fig. 8L), and Cys (Fig. 8H). In contrast, oxidized glutathione (GSSG) levels were significantly elevated (Fig. 8M), accompanied by a marked increase in reactive oxygen species (ROS) accumulation (Fig. 8I). These metabolic shifts are indicative of ferroptotic cell death. Together, these in vivo results demonstrate that LRP8 promotes tumour growth in CRC by suppressing ferroptosis.

Fig. 8.

LRP8 knockdown impairs tumour growth and promotes ferroptosis in a colorectal cancer xenograft model. A Representative images of subcutaneous tumours derived from control and LRP8-knockdown SW480 cells in nude mice (n = 5). B, C Tumour volume measurements and growth curves were recorded over time. D Immunohistochemical (IHC) staining of LRP8, GPX4, SLC3A2, and Ki-67 in xenograft tumour sections. E TUNEL staining indicating apoptotic cell death in tumour tissues. F Western blot analysis of LRP8, GPX4, and SLC3A2 protein levels in excised tumours. G Tumour weight measurements in tumour tissues. H Assessment of cysteine (Cys) content in tumour samples. I Detection of reactive oxygen species (ROS) levels in tumour tissues. J, K Quantifying ferrous iron (Fe²⁺) and total iron (t-Fe) content in tumour tissues. L, M Measurement of reduced glutathione (GSH) and oxidized glutathione (GSSG) levels. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Discussion

Our integrated multi-omics and functional analyses establish LRP8 as a critical oncogenic driver in colorectal cancer. We firstly demonstrate that LRP8 overexpression, clinically correlated with advanced disease and poor prognosis. It overexpression promotes malignant transformation by enhancing proliferative, migratory, and anti-apoptotic capacities while maintaining redox homeostasis. The mechanistic elucidation reveals that LRP8 physically interacts with SLC3A2 to preserve system Xc⁻ integrity, thereby sustaining intracellular cysteine/GSH pools and GPX4-mediated lipid peroxide clearance. The finding is underscored by in vivo evidence showing LRP8 ablation induces tumor-suppressive ferroptosis, characterized by iron dyshomeostasis, mitochondrial degeneration, and lethal lipid peroxidation. This work defines a novel LRP8-SLC3A2-GPX4 regulatory axis with therapeutic potential for CRC treatment.

While accumulating evidence highlights LRP8 as a pivotal oncoprotein in various malignancies [16, 17, 20, 21], its role in CRC progression remains incompletely characterised. Previous studies have demonstrated that LRP8 facilitates tumorigenesis in ovarian cancer by antagonising p53 signalling [21], promotes metastasis in lung cancer via Spon1-mediated pathways [22], and silencing LRP8 promotes a luminal-epithelial differentiation state in TNBC cells, consequently enhancing their chemosensitivity [23]. In the present study, we observed a progressive increase in LRP8 expression throughout the colorectal adenoma-carcinoma sequence, with a marked upregulation in carcinoma tissues compared to normal mucosa and adenoma. Clinically, elevated LRP8 levels were significantly associated with advanced TNM stage, lymph node metastasis, and poor patient survival, underscoring its potential as a prognostic biomarker in CRC. In vitro experiments further revealed that LRP8 knockdown notably inhibited CRC cell proliferation, colony formation, migration, and invasion, while its overexpression amplified these malignant characteristics. These in vitro findings were corroborated in vivo, where LRP8 depletion significantly reduced tumour growth in xenograft models.

In this study, SLC3A2 was identified as a critical downstream effector of LRP8, mediating ferroptosis resistance in CRC. As a chaperone protein, SLC3A2 plays a key role in the membrane localisation and stabilisation of light chains of amino acid transporters, such as SLC7A11 and SLC1A5, facilitating cystine uptake and maintaining intracellular redox balance [24]. The upregulation of SLC3A2 has been associated with ferroptosis suppression and enhanced glutathione synthesis in various tumour types, including laryngeal [25], breast [26], and treatment-resistant prostate cancers [27]. Proteomic analysis using immunoprecipitation followed by mass spectrometry (IP-MS) revealed a direct interaction between LRP8 and SLC3A2, which was further confirmed through co-immunoprecipitation assays. This interaction suggests that LRP8 may exert part of its anti-ferroptotic effects by modulation of SLC3A2. Rescue experiments further supported this hypothesis, showing that reintroducing SLC3A2 into LRP8-silenced cells alleviated ferroptosis-associated phenotypes, such as excessive ROS production, glutathione depletion, and mitochondrial damage. These findings establish SLC3A2 as a functional mediator of the LRP8-regulated ferroptosis pathway in CRC. Beyond its metabolic role, SLC3A2 also shapes the tumour immune microenvironment by regulating immune cell activation and antigen presentation [28]. This suggests that the LRP8–SLC3A2 axis may govern ferroptosis resistance and contribute to immune evasion in CRC. Targeting this axis could therefore represent a promising therapeutic strategy, disrupting tumour redox homeostasis while enhancing anti-tumour immune responses [29].

In summary, our study systematically elucidates the oncogenic role of LRP8 in colorectal cancer through comprehensive multi-omics analyses and functional validation. We demonstrate that LRP8 exhibits progressive upregulation during CRC development, correlating with advanced disease stage and poor prognosis. Mechanistically, LRP8 promotes tumour progression by interacting with SLC3A2 to maintain redox homeostasis and suppress ferroptosis, while simultaneously enhancing CRC cells' proliferative, migratory, and invasive capacities. Identifying the LRP8-SLC3A2-GPX4 axis provides novel insights into the molecular regulation of ferroptosis resistance in CRC, revealing a previously unrecognized vulnerability that could be exploited therapeutically.

However, several limitations should be considered when interpreting these findings. First, while our multi-omics approach identified key pathways, the precise structural basis of LRP8-SLC3A2 interaction requires further biochemical characterization. Second, the xenograft models using immunocompromised mice, though validated for tumorigenicity studies, may not fully recapitulate the immune microenvironment of human CRC.

Author contributions

All authors made important contributions to this study. Chengzhang Zhu, Zhengpeng Qian are the co-first authors. Chengzhang Zhu: Conceptualization, Methodology, Investigation, Formal Analysis, Writing—Original Draft, Writing—Review and Editing. Zhengpeng Qian: Methodology, Validation, Investigation, Data Curation, Writing—Original Draft. Shijie Yang: Investigation, Resources, Visualization, Data Curation. Yongfeng Wang: Investigation, Software, Formal Analysis, Visualization. Xiongfei Yang: Investigation, Resources, Data Curation, Visualization. Binbin Du and Hui Cai: Supervision, Funding Acquisition, Project Administration, Writing—Review and Editing.

Funding

The work is supported by National Natural Science Foundation of China (No. 82360498), Gansu Joint Scientific Research Fund Major Project under Grant (No.23JRRA1537), The 2025 Central-Guided Local Science and Technology Development Found(No.25ZYJA003), Alpha isotope mass production technology and targeted radiopharmaceuticals research(No.GSTWS250108), and department of Health Commission of Gansu Province(No.GSWSKY2024-12).

Data availability

RNA sequencing:The datasets generated and analysed during the current study are available in the National Library of Medicine (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1276701) repository. Project ID:PRJNA1276701. Proteomics data generated:The datasets generated and analysed during the current study are available in the Integrated Proteome Resources (https://www.iprox.cn/page/project.html?id=IPX0012425000) repository. Project ID:IPX0012425000. The designated point of contact for data requests:Dr Chengzhang Zhu;The First Clinical Medical College, Lanzhou Universit, Lanzhou, Gansu Province, 730000, China. Email:zcz873558920@163.com.

Declarations

Ethics approval and consent to participate

All patients provided written informed consent. This study received approval from the Ethics Committee of Gansu Provincial Hospital (Approval document No.2024-807) and was conducted in accordance with the ethical guidelines delineated in the Helsinki Declaration. All animal experimental procedures were sanctioned by the Bestcell Model Biological Center (IACUC Issue No.2025-04-18A).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.