2.1 Patient samples

This study encompassed rectal cancer patients who underwent nCRT at the First Affiliated Hospital of Fujian Medical University between January 2020 and January 2022. Detailed inclusion and exclusion criteria for patient selection are provided in Supplementary Material 1. The study ultimately included a total of 17 tumor specimens collected before radiotherapy, along with the corresponding clinical data. All participants received standard long- course neoadjuvant radiotherapy prior to surgery. TME was consistently performed by the same experienced colorectal surgeon 6 to 8 weeks following the completion of radiotherapy. The study adhered to the Helsinki Declaration and was approved by the Ethics Committee of First Affiliated Hospital of Fujian Medical University (NO. [2021]274). All patients provided written informed consent.

2.2 Radiotherapy sensitivity evaluation

This study focused on the short-term effects of preoperative radiotherapy in rectal cancer, using pathological response as an indicator of radiosensitivity, rather than local recurrence or survival rates. Experienced pathologists assessed all available tumor samples to confirm histological type and evaluated them using the tumor regression grade (TRG) criteria from the 7th edition of the AJCC manual. TRG0 represented complete response with no viable tumor cells; TRG1 represented moderate response with rare tumor cells; TRG2 represented minimal response with residual tumor outgrowing fibrosis; and TRG3 represented poor response with extensive residual tumor. Tumors with TRG0-1 were categorized as radiosensitive, whereas those with TRG2-3 were categorized as radioresistant.

2.3 Immunohistochemical (IHC) staining

Immunohistochemistry analysis was performed as previously described [12]. Sections were incubated overnight at 4 °C with anti-NUAK1 (1:200 dilution, 22723-1-AP, Proteintech) and anti-4-HNE (1:400 dilution, ab46545, Abcam) antibodies, with PBS as the negative control. Finally, DAB development was performed using a DAB kit (Servicebio, China). Images of five random fields per section were captured using an Olympus MVX10 microscope (Olympus, Japan). The staining results were assessed by two independent, experienced pathologists. The immunohistochemical score was quantified by calculating the product of the staining intensity and the percentage of positive cells. The percentage of positively stained cells was scored on a scale from 0 to 5 (0 = 0–5%; 1 = 6–25%; 2 = 26–50%; 3 = 51–75%; 4 = 76–100%), and the staining intensity was rated from 0 to 3 (0 = negative staining; 1 = weak; 2 = moderate; 3 = strong).

2.4 Cell culture and lentiviral transfection

Colorectal cancer cell lines (HCT116, SW620, SW837, and DLD-1) were purchased from GeneChem (Shanghai, China). All cell lines were authenticated by short tandem repeat (STR) profiling and confirmed to be free of mycoplasma contamination. Cells were maintained at 37 °C in a 5% CO2 incubator using RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Lentiviral vectors for NUAK1 and GPX4 overexpression and NUAK1 knockdown were purchased from Zolgene Biotechnology (Fuzhou, China). Cells were infected with the viral supernatant and then selected with puromycin (2 µg/mL). Empty vector was used as a negative control. The primer sequences used for lentiviral transfection in this study were listed in Table S1 (Supplementary Material 2). Transfection efficiency was verified by Western blot and Quantitative real-time polymerase chain reaction (qPCR).

2.5 Cell irradiation

During the logarithmic growth phase, cells were irradiated with X-rays at a dose rate of 3 Gy/min using a medical electron linear accelerator (Varian, USA). Following ionizing radiation (IR), the external surfaces of the culture flasks were wiped with 75% ethanol and returned to the incubator for continued cultivation.

2.6 CCK-8 assays

Cell viability was assessed using the Cell Counting Kit-8 (APExBIO, USA) according to the manufacturer’s instructions. Cells were seeded at 3000 per well in 96-well plates. In specified experiments, cells were pre-treated with cell death inhibitors for 24 h before exposure to X-ray irradiation. Optical density (OD) at 450 nm was measured using an enzyme reader (Bio-Rad, USA). A blank containing only RPMI 1640 medium was used to correct the OD values.

2.7 Colony formation assay

The radiosensitivity of CRC cells was determined using a colony formation assay. Cells (200–2000/well) were seeded in 6-well plates and treated with different doses of IR (2, 4, 6, 8 Gy) after attachment. In specified experiments, cells were pre-treated with Ferrostatin-1 (2 µM, HY-100579, MedChemExpress) or ML385 (10 µM, HY-100523, MedChemExpress) for 24 h before exposure to X-ray irradiation. Following irradiation, the medium was replaced with fresh medium containing the same concentration of the respective inhibitor. The inhibitors were replenished with every medium change (every 2 day) and maintained throughout the subsequent colony formation period. After 14 days, colonies were fixed with 4% paraformaldehyde for 15 min and stained with 0.2% crystal violet for 30 min. Colonies containing > 50 cells were counted to determine the survival fraction, and the survival curves were fitted using the single-hit multi-target model.

2.8 ROS and lipid peroxidation assay

In experiments involving Nrf2 inhibition, cells were pre-treated with ML385 (10 µM) or vehicle control (DMSO) for 24 h before exposure to irradiation. At 24 h post-irradiation, cells were harvested for assessment. Total ROS levels were measured using 10 µM DCFH-DA (Beyotime, China), while lipid peroxidation was assessed with 5 µM BODIPY 581/591 C11 dye (Invitrogen, USA). After a 30-minute dark incubation, cells were trypsinized, washed with PBS to remove unbound dye, and resuspended in 0.5 ml PBS for analysis using a flow cytometer (Bio-Rad, USA).

2.9 Measurement of intracellular MDA and Fe2+ levels

Cells were exposed to 6 Gy X-rays and then cultured for an additional 48 h. The cells were then rinsed with PBS, and the intracellular MDA or Fe2+ levels were measured using the MDA Assay kit (Nanjing Jiancheng Institute, China) and Ferrous Iron Colorimetric Assay Kit (Elabscience, China) according to the manufacturer’s instructions.

2.10 Transmission electron microscopy (TEM)

Mitochondrial morphology was examined using a transmission electron microscope. Briefly, cells cultured in 6-well plates were fixed with buffer containing 2.5% glutaraldehyde and 0.1 M phosphate. The samples were then washed, dehydrated, and embedded in resin according to standard procedures. The resin blocks were sectioned into ultrathin slices, which were then observed using a transmission electron microscope (Hitachi, Japan).

2.11 RNA extraction and qPCR assay

Total RNA was extracted from CRC cells using the RNAeasy™ Kit (Beyotime, China) and cDNA was synthesized with the RevertAid Kit (ThermoFisher, USA). Qpcr was performed using PowerUp™ SYBR™ Green Master Mix (ThermoFisher, USA) on an ABI QuantStudio 5 system (Applied Biosystems, USA), with β-Actin as an internal control. Gene expression levels were calculated using the 2 − ΔΔCt method. The primer sequences used for qPCR in this study were listed in Table S2 (Supplementary Material 2).

2.12 Western blot assay

Cells were lysed in buffer containing protease inhibitor and phosphatase inhibitor cocktails. BCA Protein Assay Kits (Beyotime, China) were used to determine protein concentrations. Total proteins were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were then incubated overnight at 4 °C with primary antibodies against NUAK1 (22723-1-AP, Proteintech), GPX4 (ab125066, Abcam), β-actin (bs-0061R, Bioss), Nrf2 (16396-1-AP, Proteintech), Lamin B1 (12987-1-AP, Proteintech), MYPT1 (22117-1-AP, Proteintech), Phospho-MYPT1 (Ser445) (CABT-BL6360, Creative Diagnostics), and NQO1 (11451-1-AP, Proteintech). The membranes were washed three times and then incubated with a secondary antibody for 1 h at room temperature. After incubation, the membranes were washed three times again. Finally, the antibody-antigen complex signal was detected using the ECL chemiluminescence kit (Beyotime, China), according to the manufacturer’s instructions.

2.13 Co-immunoprecipitation (Co-IP) assay

Co-IP was performed to assess protein interactions. Cell lysates prepared in RIPA buffer with protease inhibitors were incubated overnight at 4 °C with antibodies against NUAK1 (22723-1-AP, Proteintech), Nrf2 (16396-1-AP, Proteintech), or control IgG, followed by capture with Protein A/G magnetic beads. Beads were washed and bound proteins were eluted in SDS loading buffer. Co-precipitated proteins and input lysates were analyzed by Western blotting as described above, using antibodies against NUAK1, Nrf2, and MYPT1 (22117-1-AP, Proteintech).

2.14 Dual-luciferase reporter assay

The dual-luciferase reporter assay was performed to investigate the impact of NUAK1 depletion on the activity of both the GPX4 promoter and the ARE. A reporter plasmid containing the GPX4 promoter was synthesized and constructed by Zolgene Biotechnology (Fuzhou, China). The ARE-Luc reporter plasmid was purchased from Yeasen Biotechnology (Shanghai, China). Cells were co-transfected with the respective reporter plasmids and the Renilla luciferase plasmid (pRL-TK) as an internal control. At 48 h post-transfection, cells were harvested, and the firefly and Renilla luciferase activities were sequentially measured using the Dual Luciferase Reporter Assay Kit (Vazyme Biotech, China) following the manufacturer’s instructions. The relative luciferase activity was calculated by normalizing the firefly luminescence to the Renilla luminescence for each sample.

2.15 Xenograft tumor mouse model

Male BALB/c nude mice (4–6 weeks old, n = 18) were purchased from Shanghai Slake Laboratory Animal Co., LTD. Nude mice were randomly assigned to six groups of more than three mice each. Cells were irradiated with 4 Gy, and within 2 hours, 3 × 10⁶ irradiated cells were resuspended and injected subcutaneously into mice to establish xenograft models. A caliper was used to measure the length and width of each tumor every other day. After 2 weeks of injection, mice were euthanized, and xenograft specimens were surgically excised. The tumor volume was calculated using the following formula: Tumor volume (mm3) = (length×width2) / 2. All animal procedures were approved by the Animal Ethics Committee of Fujian Medical University (NO. 2023-Y-0548).

2.16 Bioinformatic analyses

The GSE123924 dataset from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) was used to explore the potential mechanism underlying NUAK1 knockdown. This dataset comprises three samples of control cells and three samples of NUAK1-knockdown cells. The ‘limma’ R package was used to identify differentially expressed genes (DEGs) between them, with a threshold adjusted P value < 0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the DEGs were performed using the ‘clusterProfiler’ R package. Separately, to evaluate the clinical predictive value of key pathway genes, pre-treatment expression data from LARC patient cohorts (GSE35452, GSE45404, and GSE119409) were used for analyzed. For each dataset, receiver operating characteristic (ROC) curves were constructed based on the expression levels of NUAK1, GPX4, and Nrf2 to assess their ability to distinguish between patients with poor versus good response to neoadjuvant chemoradiotherapy.

2.17 Statistical analysis

Statistical analyses were performed using GraphPad Prism 10 software (GraphPad, USA). Bioinformatics analyses were performed using R software (Version 4.3.0). Two-group comparisons were analyzed using the Student’s t-test (two-tailed). One-way analysis of variance (ANOVA) was used for multiple group comparisons. The Chi-square test was used to compare categorical variables between different groups. When the assumptions of the Chi-square test were not met, Fisher’s exact test was used for the comparison. The gene expression correlations were computed using the Pearson correlation test. Quantitative data are presented as mean ± standard deviation (SD). The number of biologically independent replicates (n) for each experiment is specified in the corresponding figure legends. A P value of ≤ 0.05 was considered statistically significant.