H3K18 La-METTL5-CCT2 signaling axis promotes colorectal cancer progression by inhibiting pyroptosis

Abstract

BACKGROUND

Histone lactylation, a novel epigenetic mark, has been revealed to be involved in tumorigenesis. However, its role in colorectal cancer (CRC) remains unclear. In this study, the functional roles and mechanisms of histone lactylation in CRC progression were explored.

AIM

To investigate the functional roles and molecular mechanisms of histone H3K18 lactylation (H3K18 La) in CRC progression, focusing on its regulation of pyroptosis and the downstream METTL5/CCT2 axis.

METHODS

Using 80 paired CRC/normal tissues, we assessed histone H3K18 La levels via immunohistochemistry and western blotting. Lactate levels were modulated pharmacologically. Cleavage under targets and tagmentation, transcriptome sequencing, and RNA immunoprecipitation assays were used to investigate transcriptional regulation and mRNA stability. The functional roles of METTL5 and CCT2 were tested in vitro via proliferation, migration, and invasion assays and in vivo. Pyroptosis-related protein levels were analyzed by flow cytometry and western blotting.

RESULTS

Significantly elevated H3K18 La levels were observed in CRC tissues and correlated with advanced stage and poor prognosis. Lactate depletion reduced global lactylation and H3K18 La levels, thus suppressing CRC malignancy. Mechanistically, H3K18 La enrichment at the METTL5 promoter drove its transcription. METTL5, in turn, stabilized CCT2 mRNA via m6A modification, upregulating CCT2 expression. METTL5 and CCT2 promoted CRC proliferation and metastasis in vitro and in vivo, whereas CCT2 overexpression reversed the effects of METTL5 knockdown. This H3K18 La-METTL5-CCT2 axis suppressed pyroptosis by downregulating GSDMD-N, NLRP3, and cleaved caspase-1. CCT2 can regulate inflammasome activation by directly binding to NLRP3, thereby inhibiting the progression of pyroptosis in CRC cells. Furthermore, the AKT inhibitor AZD6482 synergized with oxaliplatin to increase cytotoxicity in CRC cells.

CONCLUSION

This study systematically reveals for the first time that the H3K18 La-METTL5-CCT2 axis promotes the malignant progression of CRC through the inhibition of pyroptosis. This mechanism integrates the crosstalk among metabolic reprogramming, histone lactylation, and m6A RNA methylation, providing a novel perspective for understanding the multilevel regulatory network underlying CRC progression. On the basis of these findings, the therapeutic strategy of combining AZD6482 with oxaliplatin has promising translational potential in chemosensitization. This study provides a new theoretical basis and potential intervention targets for the prognostic evaluation and targeted therapy of CRC.

Key Words: Colorectal cancer; Histone lactylation; H3K18 La; METTL5; CCT2; Pyroptosis; M6A methylation; Lactate

Core Tip: Lactate-induced histone H3K18 lactylation drives colorectal cancer progression by activating METTL5 transcription. METTL5 stabilizes CCT2 mRNA via m6A modification, upregulating CCT2 expression and inhibiting pyroptosis. This H3K18 lactylation-METTL5-CCT2 signaling axis links metabolic reprogramming, epigenetics, and RNA methylation to suppress cell death and promote malignancy. Targeting this pathway with the PI3Kβ inhibitor AZD6482 increases oxaliplatin cytotoxicity in high-lactate microenvironments, offering a novel therapeutic strategy for colorectal cancer.

INTRODUCTION

Overview of lactylation and its role in cancer

Histone lactylation, an emerging epigenetic modification mechanism, plays a crucial role in tumor initiation, progression, chemoresistance, and metastasis. Lactate accumulation in the tumor microenvironment can induce the lactylation of histone and nonhistone proteins, thereby regulating gene expression and cellular functions. Both lactate and histone lactylation levels are markedly higher in colorectal cancer (CRC) tissues than in normal tissues[1]. Compared with those in differentiated cancer cells, histone lactylation levels are considerably elevated in CRC stem cells. The levels of histone H4 lysine 12 lactylation are substantially increased in CRC stem cells, which results in the activation of the transcription of the glutamate-cysteine ligase catalytic subunit, the inhibition of ferroptosis, and the promotion chemotherapy resistance[2]. The levels of H3K18 La are also markedly increased in CRC; this modification increases the expression of the autophagy-related protein RUBCNL and mediates resistance to bevacizumab treatment[3]. In KRAS-mutant CRC, the lactylation level is further elevated, particularly the level of histone H3K9 La, which is closely associated with poor prognosis. KRAS mutation promotes lactate production, increases H3K9 La levels, and upregulates the cholesterol transporter GRAMD1A, thereby driving CRC growth and metastasis[4].

Recently, the mechanisms underlying the role of lactylation in CRC have been increasingly investigated. Lactylation alters chromatin accessibility by modifying histone-DNA interactions, thereby increasing gene transcriptional activity. For instance, H3K18 La increases AURKB transcription; AURKB interacts with heterogeneous nuclear ribonucleoprotein M to stabilize PSAT1 mRNA, promoting serine biosynthesis and accelerating CRC progression[5]. The long noncoding RNA STEAP3-AS1 promotes CRC liver metastasis by recruiting the BRG1/ERG/P300 complex to activate H3K18 La and induce MMP9 expression[6]. Lactylation also promotes tumor angiogenesis by upregulating pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and provides nutrients and oxygen to support cancer cell proliferation[1].

Nonhistone lactylation and therapeutic challenges in CRC

Nonhistone lactylation also contributes to tumor initiation, progression, and drug resistance[7]. For example, alanine-tRNA synthetase 1 functions as a lactyltransferase. It senses intracellular lactate levels, translocates to the nucleus, and directly lactylates YAP and TEAD1, thereby activating downstream target genes that promote tumor proliferation[8]. Alanine-tRNA synthetase 1 can also mediate global lysine lactylation, including the lactylation of K120 and K139 within the p53 DNA-binding domain. This modification impairs p53 liquid-liquid phase separation, reduces its DNA-binding capacity, and inhibits its transcriptional activation function, ultimately promoting tumorigenesis[9]. SIRT3 inhibits hepatocellular carcinoma progression by deacetylating cyclin E2 at lysine 348[10]. Lactylation of pyruvate kinase M2 at K62 inhibits the conversion of its tetramer to a dimer, reduces its nuclear localization, and increases its kinase activity. These changes inhibit glycolysis and induce macrophage polarization toward a reparative phenotype[11]. Lactylation of the noncanonical methyltransferase METTL16 at K229 can promote its m6A modification of FDX1 mRNA, upregulate FDX1 expression, and induce cuproptosis[12]. In summary, lactate affects multiple processes, including metabolic reprogramming, epigenetic regulation, protein function, and gene transcription, through the lactylation of histones and nonhistones, thereby driving tumor progression[13].

CRC is the third most common cancer worldwide and the second leading cause of cancer-related death[14]. Currently employed treatment strategies include surgery, chemotherapy (5-fluorouracil, oxaliplatin, and irinotecan), targeted therapy (anti-VEGF/VEGFR drugs bevacizumab and apatinib and anti-EGFR drug cetuximab), and immunotherapy (programmed death-1 inhibitors pembrolizumab and nivolumab). However, lactylation facilitates CRC resistance to chemotherapy, targeted therapy, and immunotherapy by regulating gene expression and remodeling the tumor microenvironment. For example, lactylation of the NBS1 protein at K388 promotes the formation of the MRE11-RAD50-NBS1 complex and the recruitment of homologous recombination repair proteins to DNA double-strand breaks. This activity improves the DNA damage repair capacity of tumor cells and ultimately leads to chemotherapy failure[15]. Lactylation also upregulates M2 macrophage-related genes, such as Arg1 and VEGF, driving macrophage polarization toward the M2 phenotype. M2 macrophages promote the formation of the immunosuppressive tumor microenvironment by secreting cytokines, including interleukin-10 and transforming growth factor-beta[16]. Accordingly, histone lactylation constitutes a critical bridge between high-rate glycolytic (metabolic) reprogramming and epigenetic regulation in CRC, providing a novel metabolic-epigenetic perspective on tumor initiation, progression, and treatment resistance. Further studies on the role and mechanisms of lactylation in CRC may yield new diagnostic and therapeutic targets and strategies.

Summary of the present study

In this study, we discovered that lactate and lactylation have multiple regulatory effects on CRC, including influencing malignant phenotypes such as cell proliferation, cell cycle progression, and metastasis. These findings suggest that lactate levels and lactylation levels may serve as prognostic biomarkers in CRC. Mechanistically, lactate can increase the transcriptional activity of METTL5 by promoting H3K18 La lactylation at the promoter region of the METTL5 gene. Upregulated METTL5 subsequently binds to CCT2 mRNA, catalyzes its m6A modification, and increases its stability. This H3K18 La-METTL5-CCT2 signaling axis ultimately promotes the malignant progression of CRC by inhibiting pyroptosis. Additionally, we found that AZD6482 enhanced the cytotoxic effect of oxaliplatin on CRC cells by inhibiting the downstream signaling pathway of lactate, providing experimental evidence for the efficacy of combination therapy targeting the downstream signaling pathway of lactate.

MATERIALS AND METHODS

Sample collection

A total of 80 patients diagnosed with CRC at Anyang Tumor Hospital between January and December 2018 were enrolled in this study. None of the patients received medication before sample collection. Resected CRC tissues and matched adjacent non-tumor tissues were immediately snap-frozen in liquid nitrogen after ex vivo removal and stored at -80 °C for subsequent experiments. The study protocol was approved by the Medical Research Ethics Committee of Anyang Tumor Hospital, No. 2017WZ11K02, and all patients signed informed consent forms before surgery. All procedures involving human participants were performed in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Cells and lentiviruses

Human normal colonic epithelial cells (FHC, Cat. No. FH1283) and seven CRC cell lines (SW620, Caco2, HT-29, HCT-8, HCT-15, HCT-116, and SW480) were obtained from Shanghai Fuheng Biotechnology Co., Ltd., with catalog numbers FH0021, FH0029, FH0020, FH0042, FH0026, FH0031, and FH0022, respectively. All cells were cultured in Dulbecco’s modified Eagle medium (Gibco, 12800082, CA, United States) supplemented with 10% fetal bovine serum at 37 °C in a 5% CO₂ incubator. The medium was replaced every 24 hours.

The METTL5 knockdown construct, METTL5-overexpressing plasmid, CCT2-overexpressing plasmid, and their corresponding negative control vectors were synthesized by GeneBiogist Co., Ltd. Transfections were performed using Lipofectamine™ 2000 (Invitrogen, 11668019, United States) following the manufacturer’s instructions. For lentivirus production, pLKO.1-based short hairpin RNA vectors were co-transfected with packaging plasmids pRev, pGag, and pVSVG (2:2:2:1 ratio), and overexpression vectors (pSIN/pCDH) were co-transfected with psPAX2 and pMD2.G (2:2:1 ratio) into HEK293T cells. After 48 hours, viral supernatants were harvested, filtered through a 0.45 μm filter, and supplemented with 8 μg/mL polybrene (Sigma, TR-1003, MO, United States) to enhance infection efficiency before being co-incubated with target cells. Transfected cells were selected with 2 mg/mL puromycin to establish stable cell lines. The inhibitors oxamate and 2-DG were sourced from Macklin (Cat. No.: S818460-25 g; Shanghai, China) and APExBIO (Cat. No.: B1027-1C; TX, United States), respectively.

Immunohistochemistry and hematoxylin-eosin staining

Paraffin-embedded sections of tumor tissues from patients with CRC, matched adjacent non-tumor tissues, and mouse tumor tissues were sequentially dewaxed with xylene and hydrated with gradient ethanol. Subsequently, the sections were treated with peroxidase blocking solution for 10 minutes to inactivate endogenous peroxidase activity, followed by three washes with phosphate-buffered saline (PBS).

Immunohistochemistry staining: After blocking with freshly prepared 3% goat serum, sections were incubated overnight at 4 °C with primary antibodies. Subsequently, sections were incubated with the corresponding secondary antibody at 25 °C for 30 minutes, and the chromogenic reaction was performed using DAB chromogenic solution. Cell nuclei were counterstained with hematoxylin, and sections were mounted with neutral balsam for image acquisition. Blind scoring was independently performed by two experienced pathologists, and the mean score was used for analysis.

Hematoxylin-eosin staining: Sections were stained with hematoxylin solution for 30 seconds to visualize cell nuclei, differentiated with hydrochloric acid-ethanol for 2-5 seconds, and blued under running water. The sections were stained with eosin solution for 1 minutes to highlight cytoplasmic structures. After dehydration with gradient ethanol and clearing with xylene, sections were mounted with neutral balsam, cured in a fume hood, and stored at room temperature for subsequent analysis.

Cell counting kit-8 assay

Cell proliferation assay: Cells in the logarithmic growth phase were harvested, counted, and seeded in 6-well plates at the experimentally defined density. Starting from the second day after seeding, cell proliferation was assessed daily for 5 days. Briefly, the original medium in the wells was aspirated and discarded, and 500 μL of fresh medium containing 50 μL of cell counting kit-8 (CCK-8) reagent (DOJINDO, Cat. No. CK04; Japan) was added to each well. After gentle mixing, the plates were incubated at 37 °C for 2 hours in the dark. Subsequently, the supernatant was collected, mixed, and 100 μL of it was transferred to a 96-well plate. Absorbance was measured at 450 nm (OD450) using a microplate reader. Optical density values at different time points were used to plot cell growth curves.

Drug toxicity assay: Cells in the logarithmic growth phase were collected, counted, and seeded in 96-well plates at the predetermined density. After cell attachment to the plate, cells were treated with different drug combinations for 48 hours. Cell viability was assessed following the CCK-8 protocol as above, and dose-response curves were plotted to evaluate drug effects on cell viability.

Colony formation assay

Cells from different treatment groups were seeded in 6-well plates at a density of 5 × 10² cells/well, with three replicate wells set for each group. The cells were cultured continuously in RPMI-1640 medium for 14 days until visible cell colonies formed. Colonies were fixed with 75% ethanol at room temperature for 30 minutes and stained with 0.5% crystal violet solution for 20 minutes. Residual staining solution was removed by gentle rinsing with PBS. A cell cluster containing more than 50 cells was defined as a colony. After drying at 37 °C, colony images were captured using a Nikon D5600 camera (Nikon Corporation, Japan), and the number of colonies in each well was counted.

EdU assay

Cells from different treatment groups were seeded into 96-well plates at a density of 5 × 10³ cells/well. A total of 100 μL of medium containing 20 μM EdU was added to each well, and the cells were incubated at 37 °C in a 5% CO₂ environment for 2 hours. Subsequently, the cells were fixed with 4% paraformaldehyde for 30 minutes and permeabilized with PBS containing 0.5% Triton X-100 for 20 minutes. Nuclei were stained with Hoechst 33342 (Invitrogen, Cat. No.: 34580; United States), and the cell proliferation rate was calculated according to the instructions of the EdU Detection Kit (Acmec, Cat. No. AC10869-100T; Shanghai, China). Images from five randomly selected fields of view per well were captured using a fluorescence microscope, followed by quantitative analysis.

Transwell assay

Cell migration assay: CRC cells from different treatment groups were seeded into the upper chamber of a Transwell insert (Corning, Cat. No. 3450; NY, United States) at a density of 2 × 10⁴ cells/chamber. The lower chamber was filled with a medium containing 20% fetal bovine serum as a chemoattractant. After incubation at 37 °C in a 5% CO₂ environment for 24 hours, the cells were stained with 0.1% crystal violet (Macklin, Cat. No. C805210-25 g; Shanghai, China). The number of transmembrane cells was observed and counted under an inverted microscope.

Cell invasion assay: The upper surface of the Transwell membrane was pre-coated with 30 μL Matrigel (Corning, Cat. No. 354230; NY, United States) and solidified at 37 °C in a 5% CO₂ environment for 1 hour. Subsequent steps for seeding, incubation, and staining were performed as described for the cell migration assay.

Immunofluorescence

The pretreatment of paraffin-embedded sections of CRC tissues and matched adjacent non-tumor tissues was performed as described for the immunohistochemistry (IHC) assay. After blocking with 5% bovine serum albumin, sections were incubated overnight at 4 °C with primary antibodies. Following three washes with PBS, sections were incubated at room temperature for 1 hour in the dark with AF555-labeled goat anti-rabbit secondary antibody (Bioss, Cat. No. Bs-0294P-AF555; Beijing, China), followed by three washes with PBS. Nuclei were counterstained with Hoechst, and sections were mounted with anti-fluorescence quenching mounting medium. Images were captured using a fluorescence microscope, and quantitative analysis was performed using Image Pro Plus software by counting H3K18 La-positive cells and total cells in each field of view to calculate the proportion of positive cells.

Extracellular lactate detection

The detection was performed following the manufacturer’s protocol (Abcam, Cat. No. Ab65331; United Kingdom). Standards were first diluted to a series of concentrations, sample volumes were adjusted, and all reagents and samples were equilibrated to room temperature. Duplicate wells were prepared for standards (50 μL standard), samples (2-50 μL sample + buffer to 50 μL), and sample background controls (2-50 μL sample + enzyme-free background reaction mixture to 50 μL). Reaction mixtures were prepared as follows: Enzyme-containing reaction mixture (46 μL assay buffer + 2 μL chromogen/substrate mixture + 2 μL enzyme mixture) and enzyme-free background reaction mixture (48 μL assay buffer + 2 μL chromogen/substrate mixture), each at a volume of “total number of assay wells + 1”. After adding the appropriate mixture to each well, the plates were incubated at room temperature for 30 minutes. Absorbance was measured at 450 nm using a microplate reader. Lactate concentration in samples was calculated from the standard curve (standard concentration vs absorbance) after subtracting the background control signal. The original lactate content in samples was obtained by multiplying the calculated concentration by the dilution factor. This method exhibited stable chromogenicity (within 4 hours) and enabled accurate quantification of lactate in cell or tissue extracts.

Western blotting

CRC cells from different treatment groups were washed with pre-cooled PBS and lysed on ice for 30 minutes by adding RIPA lysis buffer (Macklin, Cat. No. R874810-50 mL; Shanghai, China) containing 1% protease inhibitor and 1% phosphatase inhibitor. Lysate was centrifuged at 12000 rpm for 10 minutes at 4 °C, and the supernatant was collected as the total protein extract. Protein concentration was determined using the BCA assay (Solarbio, Cat. No. PC0020; Beijing, China). Equal amounts of protein (20 μg per sample) were separated by sodium-dodecyl sulfate gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Biosharp, Cat. No. BS-PVDF-22-S; Beijing, China) using the wet transfer method. Membranes were blocked with 5% non-fat milk at room temperature for 1 hour and incubated overnight at 4 °C with primary antibodies. Subsequently, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour and treated with enhanced chemiluminescence substrate (Thermo Fisher Scientific, Cat. No. 34580; United States). Protein band images were captured using the ChemiScope 6200 chemiluminescent imaging system (Clinx) for grayscale analysis. Antibodies are listed in Supplementary Table 1.

Real-time quantitative polymerase chain reaction assays

Total RNA was extracted from CRC cells under different treatment conditions using the Trizol method. Subsequently, RNA was reverse-transcribed into complementary DNA using the Hifair^® II First Strand cDNA Synthesis Kit (Yeasen Biotechnology, Cat. No. 11121ES60; Shanghai, China). Amplification was performed on the Applied Biosystems™ 7500 Real-Time Fluorescence Quantitative PCR System (Invitrogen, United States) using HotStart™ 2 × FAST Green qPCR Master Mix (APExBIO, TX, United States). Glyceraldehyde-3-phosphate dehydrogenase was used as the internal reference gene, and the relative expression level of the target gene was calculated using the 2^-ΔΔCt method. The sequences of the primers used are provided in Supplementary Table 2.

Cleavage under targets and tagmentation

Cleavage under targets and tagmentation (CUT&Tag) library construction was performed using the Vazyme kit (Cat. No.: TD903-01)[17] according to the manufacturer’s instructions. Library quality control and sequencing were conducted by Frasergen Bioinformatics Co., Ltd. Data processing involved the following steps: (1) Adapter trimming and removal of low-quality bases from raw paired-end reads using Trim_Galore software; (2) Alignment of clean reads to the hg38 reference genome using Bowtie2 software; (3) Removal of polymerase chain reaction (PCR) duplicates using Picard; (4) Generation of genome browser tracks in bigwig format with samtools software after merging biological replicates; (5) Genome-wide peak calling using MACS2 software (parameters: “-f BAMPE -broad -nomodel”); and (6) Visualization of bigwig signals using IGV software (Broad Institute) and generation of peak plots using Deeptools.

Chromatin immunoprecipitation

A total of 1 × 10⁷ CRC cells were cross-linked with 1% formaldehyde at room temperature for 10 minutes, and the reaction was terminated with glycine for 5 minutes. Cells were washed with pre-cooled PBS and lysed using the lysis buffer provided in the Magna ChIP™ Kit (Cell Signaling Technology, Cat. No. 9005; MA; United States) to isolate chromatin fragments. The resulting protein-DNA complexes were incubated overnight at 4 °C with anti-H3K18 La antibody (PTM BIO, Cat. No. PTM-1427RM; Zhejiang, China) and A/G agarose magnetic beads. The following day, non-specifically bound complexes were removed with sequential washes, and specific complexes were eluted at 65 °C, followed by cross-link reversal. DNA was recovered using a purification kit. The enrichment level of the METTL5 promoter region was quantified by real-time fluorescence quantitative PCR (qPCR). Primer sequences used for chromatin immunoprecipitation (ChIP)-qPCR are listed in Supplementary Table 3.

RNA sequencing

Total cellular RNA was extracted using the phenol/chloroform method. RNA sequencing was performed on both the BGISEQ platform (BGI Genomics, Shenzhen, China) and the Illumina HiSeq 2500 platform. High-quality clean reads were aligned to the hg38 reference genome using Hisat2 software (default parameters). Gene expression quantification was performed using HT-seq software under default settings. Differential expression analysis was conducted using the DESeq2 R package, which applies statistical tests to digital gene expression data based on a negative binomial distribution model; genes with an adjusted P < 0.05 were considered differentially expressed. Gene set enrichment analysis was conducted using the online tool provided by the Broad Institute.

RNA immunoprecipitation assay

The experiment was performed following the standard protocol provided by the manufacturer (Sigma-Aldrich, Cat. No. RIP-12RXN; United States)[18]. Cells cultured to 80% confluence were collected and lysed on ice for 15 minutes using lysis buffer containing protease and RNase inhibitors. After centrifugation, the supernatant was collected, and 10 μL was retained as the 5% input sample. Protein A magnetic beads were washed, aliquoted, and incubated with at room temperature for 30 minutes to form antibody-bead complexes. The complexes were resuspended in immunoprecipitation buffer, mixed with diluted cell lysate, and incubated at 4 °C overnight with rotation. Subsequent washing was performed under mild conditions (five washes with wash buffer) or stringent conditions (gradient washing with wash buffer, strict wash buffer containing Igepal and NaCl, followed by standard wash buffer). Finally, the beads were resuspended in 0.2 mL wash buffer, and the volume of the input sample was adjusted to 0.2 mL. Tri reagent and chloroform were added to each sample, and after centrifugation, the upper aqueous phase was collected and transferred to a centrifuge tube containing linear acrylamide, sodium acetate, and isopropanol, followed by precipitation at -80 °C for at least 1 hour. RNA pellets were collected by centrifugation, washed with 70% ethanol, air-dried, and dissolved in 20 μL RNase-free water. The purified RNA was used for subsequent analysis.

Flow cytometric analysis of cell pyroptosis

The FAM-FLICA Caspase-1 Detection Kit (Acmec, Cat. No. 10604-100T; Shanghai, China) was used to quantitatively assess Caspase-1 enzymatic activity in CRC cells via flow cytometry, following the manufacturer’s protocols. Briefly, treated cells were washed with PBS, collected by centrifugation, and resuspended in medium to obtain a single-cell suspension. The FLICA probe was added, and cells were incubated at 37 °C in the dark for 60 minutes to label active Caspase-1. Propidium iodide staining solution was added to evaluate cell membrane integrity. After washing with buffer and resuspension, cells were analyzed by flow cytometry to detect FAM (FLICA) and propidium iodide fluorescence, allowing the quantification of Caspase-1 activation and determination of the proportion of pyroptotic cells.

Nude mouse subcutaneous xenograft model

This study was conducted and reported in accordance with the ARRIVE guidelines. Six-week-old male BALB/c nude mice were sourced from Henan Scopus Biotechnology Co., Ltd. (China). All animal studies were conducted in compliance with institutional guidelines and were approved by the Medical Research Ethics Committee of Anyang Tumor Hospital, No. 2025WZ04K03. Sample size was determined based on power analysis using data from preliminary experiments, with a minimum of six mice per group to ensure adequate statistical power. SW480 cells (2 × 10⁶) from different treatment groups were resuspended in 50 μL PBS, mixed with an equal volume of Matrigel, and slowly inoculated subcutaneously into the right abdominal region of nude mice using a 1 mL syringe. When xenograft tumors reached approximately 100 mm³, mice were randomly assigned to experimental groups using a computer-generated random number table to ensure unbiased allocation. Mice in the experimental groups received corresponding drug treatments via intraperitoneal injection, while the control group received an equal volume of PBS. All investigators involved in tumor measurement and data analysis were blinded to the group assignments. Tumor volumes were measured by an independent researcher who was not informed of the treatment allocation. Mice were euthanized 27 days after inoculation, and tumor tissues were collected for measurement of weight and volume. Portions of the tumor tissues were subjected to IHC or hematoxylin-eosin staining. Tumor volume was calculated using the formula V = (length × width²)/2. All animal experimental procedures were performed in accordance with the ethical guidelines approved by the Institutional Animal Care and Use Committee. All animal experimental procedures were performed in accordance with the ethical guidelines approved by the Institutional Animal Care and Use Committee. For euthanasia, mice were anaesthetized via intraperitoneal injection of sodium pentobarbital (150 mg/kg) and then subjected to cervical dislocation, in compliance with the American Veterinary Medical Association Guidelines for the Euthanasia of Animals.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software (version 8.0). All quantitative data were obtained from at least three independent experiments and are presented as the mean ± SD. All the statistical tests were two-sided unless otherwise specified. Differences between two groups were assessed using an unpaired Student’s t test. Comparisons among three or more groups were analyzed using one-way or two-way ANOVA, followed by appropriate post hoc multiple-comparison tests (e.g., Tukey’s multiple-comparisons test for all pairwise comparisons or Bonferroni correction for selected comparisons), as applicable. A P value > 0.05 was considered statistically nonsignificant, whereas P < 0.05 was considered statistically significant.

RESULTS

Lactylation levels are closely associated with the prognosis of patients with CRC

CRC progression is a complex, multistep process. To investigate the role of lactylation in this process, we collected primary tumor and adjacent nontumor tissues from 80 patients with CRC (Supplementary Table 4) and followed up with all the patients for 5 years to assess overall survival. The expression level of H3K18 La, a marker of lactylation-mediated transcriptional activation, was significantly lower in nonmetastatic (stage T1/2) tumors than in metastatic (stage T3/4) tumors (Figure 1A). These findings suggest that lactation levels gradually increase with CRC progression and are associated with patient prognosis. To test this hypothesis, the 80 patients were divided into low- and high-H3K18 La groups. Survival analysis revealed that the 5-year survival rate was significantly greater in the low-expression group than in the high-expression group (Figure 1B), indicating that elevated lactylation promotes CRC progression. Moreover, lactylation levels were abnormally higher in CRC tissues than in adjacent nontumor tissues (Supplementary Figure 1A and B). This finding is consistent with those of previous reports[1]. To systematically analyze the value of clinical characteristics and histone H3K18 lactylation (H3K18 La) in the prognosis of patients with colon cancer, Cox proportional hazards regression analysis was performed on 80 patients with CRC. Univariate Cox regression results revealed that both the lymph node metastasis stage (N stage) and the H3K18 La expression level were significantly associated with patient prognosis (P < 0.05). After these significant variables, together with potential confounding factors, including sex and age, were incorporated into the multivariate Cox regression analysis, N stage and H3K18 La expression level were confirmed to be independent prognostic factors for colon cancer (P < 0.05). These results suggest that the combination of N stage and H3K18 La expression level can provide a reliable reference for the prognostic evaluation and individualized treatment of patients with colon cancer (Supplementary Tables 5-7).

Figure 1 Lactylation is closely associated with the prognosis of patients with colorectal cancer. A: Immunohistochemistry detection of H3K18 La in 80 colorectal cancer (CRC) tissue samples, including 18 cases at stage T1/2 and 62 cases at stage T3/4 (left panel, scale bar: 100 μm). H3K18 La expression was quantified using the Allred scoring system (right). Stage T1/2: In situ CRC without lymph node or distant metastasis; stage T3/4: Advanced CRC with lymph node or distant metastasis; B: Survival analysis of the 80 patients. Patients were divided into low-expression (Allred score ≤ 2, n = 24) and high-expression (score ≥ 3, n = 56) based on H3K18 La levels; C: SW480 cells (1 × 10⁴ cells/well) cultured with different concentrations of 2-DG or oxamate for 5 days. Cell viability was detected using the cell counting kit-8 for 5 consecutive days, and cell growth curves were plotted; D: SW480 cells (5 × 10² cells/well) cultured with different concentrations of 2-DG or oxamate for 14 days. Subsequently, crystal violet staining was performed to visualize the formation of cell colonies, and the number of monoclonal cell clusters was counted and subjected to quantitative analysis; E: SW480 cells (5 × 10³ cells/well) cultured with different concentrations of 2-DG or oxamate for 24 hours and incubated with EdU for an additional 2 hours. The number of EdU-positive cells was detected by fluorescence microscopy and subjected to quantitative comparison; F: SW480 cells (1.5 × 10⁴ cells/chamber) seeded in Transwell insert or Matrigel-coated Transwell insert with medium containing 2-DG or oxamate. After 48 hours of culture, cells were stained with crystal violet (scale bar: 100 μm). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001.

To investigate the mechanism underlying the role of lactylation in CRC progression, we inhibited lactate production in CRC cell lines. Both the glycolysis inhibitor 2-DG and the lactate dehydrogenase inhibitor oxamate dose-dependently reduced lactate production in SW480 and HCT116 cells (Supplementary Figure 1C and D). Treatment with either inhibitor significantly decreased global lactylation and H3K18 La levels in tumor cells (Supplementary Figure 1E and F). These results confirm a direct link between lactate production and lactylation in CRC cells. We further assessed the effects of lactate and lactylation on CRC cell behavior. The results of the CCK-8 assay revealed that treatment with 2-DG and oxamate significantly inhibited the proliferative activity of tumor cells, suggesting that lactate and lactylation are crucial for maintaining the proliferative capacity of CRC cells (Figure 1C). The results of the colony formation assay indicated that both inhibitors reduced the sustained proliferative potential of tumor cells, suggesting that lactate and lactylation may be associated with the “stem cell-like” properties or malignancy of cells (Figure 1D). EdU incorporation assays demonstrated that both inhibitors significantly suppressed DNA replication, suggesting that lactate and lactylation are involved in cell cycle regulation (Figure 1E and F). Because metastasis risk is directly related to metastatic potential, we evaluated the effects of lactate and lactylation on CRC cell migration and invasion using Transwell assays. Treatment with 2-DG and oxamate significantly suppressed both processes, suggesting that lactate and lactylation can increase CRC cell motility and the ability of CRC cells to degrade and penetrate the extracellular matrix. Collectively, these results indicate that lactate and lactylation regulate key malignant traits of CRC cells, including proliferation, cell cycle progression, and metastasis, and may serve as prognostic biomarkers for patients with CRC.

Lactate and H3K18 La promote METTL5 transcription

To identify key genes regulated by lactylation during CRC progression, we analyzed three gene datasets from CRC tissues and two gene datasets with high LDHA and LDHB expression. Seven overlapping genes were identified: CCDC59, ENO1, PDHX, HOMER1, METTL5, INTS12, and PARP2 (Figure 2A). To verify the effect of lactate on the mRNA expression of these genes, we simulated low- and high-lactate microenvironments in cell models. The glycolysis inhibitor 2-DG inhibited the mRNA expression of the seven genes, with the strongest effect on METTL5 (Figure 2B). Conversely, sodium lactate (Nala) increased the mRNA expression of HOMER1, METTL5, INTS12, and PARP2, with METTL5 exhibiting the greatest increase (Figure 2B). At the protein level, 2-DG inhibited METTL5 expression, whereas Nala enhanced it (Figure 2C). These findings suggest that METTL5 is a key gene through which lactylation regulates CRC progression. To validate the above hypothesis, we examined METTL5 and LDHA expression in CRC and adjacent nontumor tissues. Both METTL5 and LDHA were highly expressed in CRC tissues, and the proportion of METTL5-positive cells was positively correlated with that of LDHA-positive cells (Figure 2D). Given that LDHA is highly expressed in most tumors and is directly associated with lactate production[19], these findings indicate that METTL5 expression may be regulated by lactate and lactylation. Consistent with these findings, METTL5 mRNA and protein levels were significantly increased in multiple CRC cell lines compared with normal colonic epithelial cells (Supplementary Figure 2A and B), suggesting that METTL5 may be involved in the malignant progression of CRC.

Figure 2 Lactate and H3K18 La promote METTL5 transcription. A: Venn diagram illustrating overlapping genes among gene sets highly expressed in colorectal cancer (CRC) tissues (GSE21510, GSE26692, and GSE180948) and gene sets positively correlated with LDHA or LDHB expression (LDHA_cor_positive, LDHB_cor_positive); B: SW480 cells treated with 5 mmol/L 2-DG or 5 mmol/L sodium lactate (Nala) for 24 hours. Quantitative polymerase chain reaction (qPCR) to detect the mRNA expression levels of CCDC59, ENO1, PDHX, HOMER1, METTL5, INTS12, and PARP2; C: SW480 cells treated with 5 mmol/L 2-DG or 5 mmol/L Nala for 24 hours. Western blotting to detect METTL5 protein expression; β-actin served as the internal reference; D: Immunohistochemistry (IHC) of METTL5 and LDHA protein expression in 15 randomly selected CRC tissues (T) and adjacent non-tumor tissues (N) (left panel). Positive cell proportion and correlation between METTL5 and LDHA expression were analyzed; E: SW480 cells treated with 5 mmol/L Nala for 24 hours, followed by the cleavage under targets and tagmentation assay using H3K18 La antibody and IgG control antibody. The upper panel depicts the genome-wide enrichment of H3K18 La near transcription start sites; the lower panel displays the sequencing signal peaks of H3K18 La and IgG in the chromosomal region of METTL5; F: Chromatin immunoprecipitation in SW480 and HCT116 cells using H3K18 La antibody and IgG, respectively. qPCR to detect the enrichment level of H3K18 La in the region near the METTL5 promoter; G: SW480 cells transfected with LDHA-overexpressing plasmid (left) or treated with 10 mmol/L oxamate for 24 hours (right). A subsequent chromatin immunoprecipitation assay was conducted, and qPCR was used to analyze the enrichment of H3K18 La in the METTL5 promoter region; H: IHC quantification of METTL5 expression in 80 CRC tissue samples (left panel, scale bar: 100 μm). The Allred scoring system to quantify METTL5 expression level (right panel); I: Survival analysis of the 80 patients. Based on the Allred score of METTL5, patients were divided into low-expression (score ≤ 2, n = 23) and high-expression (score ≥ 3, n = 57) groups. Differences in overall survival between the two groups were compared; J: SW480 cells stably transfected with empty vector plasmid or METTL5-overexpressing plasmid (1 × 10⁶ cells/mouse) were subcutaneously inoculated into BALB/c nude mice to establish a cell-derived xenograft model. Tumor volume was measured every 3 days starting from day 3 post-inoculation. On day 6 post-inoculation, mice were administered phosphate-buffered saline (control group) or oxamate (750 mg/kg, intraperitoneal injection daily). Mice were euthanized on day 27 post-inoculation; tumors were dissected and photographed (left), weighed for statistical analysis (middle, n = 6), and tumor growth curves were plotted (right); K: IHC to detect Ki67 and METTL5 expression in xenograft tumors from different treatment groups (as in J), followed by quantitative analysis. PBS: Phosphate-buffered saline. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001.

Although METTL5 has a protumor effect on some cancers, its role in CRC progression remains unclear[20,21]. In this study, we investigated the role of METTL5 using CRC cell models. First, we established SW480 cell lines with stable METTL5 knockdown or overexpression (Supplementary Figure 2C-E). The results of the CCK-8 and colony formation assays revealed that METTL5 knockdown significantly reduced the proliferative capacity of SW480 cells, whereas METTL5 overexpression significantly enhanced this ability (Supplementary Figure 2F and G). EdU assays indicated that METTL5 knockdown reduced the DNA replication ability of SW480 cells, whereas METTL5 overexpression enhanced this ability (Supplementary Figure 2H). Transwell assays revealed that METTL5 knockdown inhibited the migration and invasion abilities of SW480 cells, whereas METTL5 overexpression enhanced these abilities (Supplementary Figure 2I). These results confirm that METTL5 plays a crucial role in CRC progression.

Protein lactylation is involved in various biological processes, with histone lactylation often associated with transcriptional activation[22,23]. Because lactate increased METTL5 mRNA expression (Figure 2C), we hypothesized that lactate regulates METTL5 transcription. To test this hypothesis, we used a CUT&Tag assay to analyze the genome-wide distribution of H3K18 La and its distribution on METTL5. The results revealed that H3K18 La enrichment signals were widely present across the genome, with significant H3K18 La enrichment in the promoter region of METTL5, suggesting that lactate and H3K18 La may regulate METTL5 transcription (Figure 2E). ChIP confirmed that H3K18 La was specifically enriched at the METTL5 promoter region (Figure 2F). Previous studies have demonstrated that the transcription of RUBCNL can be directly regulated by H3K18 La modification[3]. Our results revealed that H3K18 La was significantly enriched in the promoter region of the RUBCNL gene (Figure 2F), further validating the reliability of the experimental methods and findings in this study. Moreover, LDHA overexpression increased H3K18 La levels at this locus, whereas oxamate treatment reduced H3K18 levels, indicating a direct association between lactate and H3K18 La at the METTL5 promoter region (Figure 2G). In clinical samples, METTL5 expression was significantly higher in stage T3/4 CRC tissues than in stage T1/2 CRC tissues (Figure 2H). Moreover, patients in the METTL5 low-expression group had a significantly longer 5-year survival period than those in the high-expression group did (Figure 2I), suggesting that high METTL5 expression is closely associated with malignant progression and poor prognosis in CRC patients. These results confirm that METTL5 undergoes H3K18 La and is directly regulated by lactate, indicating that METTL5 is a key gene through which lactate and H3K18 La promote the malignant progression of CRC.

To determine whether lactate and H3K18 La promote CRC progression through METTL5, we conducted a series of rescue experiments. The oxamate-induced downregulation of METTL5 mRNA expression was completely reversed by exogenous METTL5 overexpression (Supplementary Figure 2J). Moreover, the reduced proliferative capacity of SW480 cells caused by oxamate was reversed by exogenous METTL5 overexpression, indicating that lactate enhances the proliferative capacity of CRC cells by increasing METTL5 expression (Supplementary Figure 2K and L). Similarly, the inhibition of the migration and invasion abilities of SW480 cells induced by oxamate was reversed by exogenous METTL5 overexpression, suggesting that lactate enhances the metastatic potential of CRC cells by upregulating METTL5 expression (Supplementary Figure 2M). We subsequently used a cell line-derived xenograft (CDX) model to evaluate the role of METTL5 in lactate- and H3K18 La-mediated tumor growth. Oxamate significantly inhibited the growth of SW480-derived subcutaneous xenografts, whereas exogenous METTL5 overexpression reversed this effect (Figure 2J). Immunohistochemical analysis revealed that oxamate treatment reduced METTL5 and Ki67 expression in xenografts, whereas exogenous METTL5 overexpression restored Ki67 expression. These findings further confirm that METTL5 plays a key role in lactate-mediated CRC cell proliferation (Figure 2K). Collectively, these in vitro and in vivo results confirm that METTL5 is a key target gene through which lactate and H3K18 La promote CRC progression. Lactate increases METTL5 transcription by promoting H3K18 La at the METTL5 promoter region, thereby driving the malignant progression of CRC cells. Collectively, these results indicate that lactate can increase the transcriptional activity of the METTL5 gene by promoting H3K18 La modification in the METTL5 gene promoter region, thereby driving the malignant progression of CRC cells.

METTL5 promotes CCT2 expression in an m6A-dependent manner

The canonical function of METTL5 is to catalyze the m6A modification of 18S ribosomal RNA (rRNA), which is crucial for ribosome biogenesis and for ensuring the accuracy and efficiency of protein translation[24]. However, whether METTL5 directly regulates mRNA function remains unclear. To address this, we performed transcriptome sequencing in SW480 cells after METTL5 knockdown. A large number of genes exhibited decreased mRNA expression, suggesting that METTL5 may regulate mRNA expression (Figure 3A). We further validated the top five most significantly downregulated genes (RANBP1, ANXA3, PSAT1, CCT2, and EMC1). METTL5 knockdown did not significantly affect PSAT1 or EMC1 mRNA expression but did downregulate RANBP1, ANXA3, and CCT2 expression, with the strongest effect observed for CCT2 (Figure 3A and B). These findings suggest that CCT2 is a key target gene regulated by METTL5. Additionally, CCT2 mRNA and protein levels were significantly greater in multiple CRC cell lines than in normal colonic epithelial cells, suggesting that CCT2 expression is associated with CRC progression (Supplementary Figure 3A and B). These findings indicate that METTL5 may perform a noncanonical rRNA catalytic function to directly regulate CCT2 mRNA expression. Given that METTL5 typically functions by binding to RNA, we examined the interaction between METTL5 and CCT2 mRNA using an RNA immunoprecipitation (RIP) assay. The results revealed that the anti-METTL5 antibody significantly enriched the CCT2 mRNA fragments, suggesting direct binding between METTL5 and CCT2 mRNA (Figure 3C). We further performed an m6A methylated RIP assay using an m6A-specific antibody. The m6A antibody significantly enriched CCT2 mRNA fragments, and Nala treatment further enhanced this enrichment, indicating that lactate promotes m6A modification of CCT2 mRNA (Figure 3D). Given the regulatory effect of METTL5 on CCT2 mRNA expression (Figure 3A and B), we hypothesized that METTL5 may affect the stability of CCT2 mRNA. METTL5 knockdown significantly shortened the half-life of CCT2 mRNA, confirming that METTL5 increases the stability of CCT2 mRNA (Figure 3E). Together, these results indicate that METTL5 can directly bind to CCT2 mRNA and increase its stability, serving as a key mediator of the lactate- and lactylation-mediated m6A modification of CCT2 mRNA.

Figure 3 METTL5 promotes CCT2 expression in an m6A-dependent manner. A: Total RNA from SW480 cells before and after METTL5 knockdown subjected to transcriptome sequencing. Volcano plot to visualize the global changes in mRNA expression; genes with |logFC| > 2 and adj.P.Val < 0.01 are labeled; B: SW480 cells with stable METTL5 knockdown were analyzed via quantitative polymerase chain reaction for mRNA expression of RANBP1, ANXA3, PSAT1, CCT2, and EMC1; C: RNA immunoprecipitation in SW480 cells using a METTL5 antibody, with an IgG antibody as the negative control. The enrichment of METTL5 protein on CCT2 mRNA (left panel). The enrichment difference (right panel); D: SW480 cells cultured in normal medium (con) or medium containing 5 mmol/L sodium lactate for 24 hours. MeRIP using an m6A antibody, with an IgG antibody as the negative control. Quantitative polymerase chain reaction to analyze the level of m6A modification on CCT2 mRNA; E: METTL5-knockdown and control SK-Hep1 cells treated with cycloheximide at a final concentration of 100 μM for 0, 1, 2, and 4 hours. Western blotting was used to detect METTL5 protein expression, and grayscale value quantitative analysis was performed; F: SW480 cells with non-targeted knockdown (sh-NC) or METTL5 knockdown (sh-METTL5) were stably transfected with oe-NC or CCT2-overexpressing plasmid (oe-CCT2), respectively. These cells (1 × 10⁶ cells/mouse) were subcutaneously inoculated into BALB/c nude mice to establish a cell line-derived xenograft model. Tumor photograph (left panel), tumor weight (middle panel, n = 6), and tumor volume plot (right panel); G: Immunohistochemistry to detect the expression levels of Ki67, METTL5, and CCT2 in xenograft tumors from different treatment groups (as in F), followed by quantitative analysis. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001.

To determine whether CCT2 is a key downstream effector of METTL5 in CRC progression, we conducted a series of rescue experiments. First, we established an SW480 cell model with stable METTL5 knockdown and exogenous CCT2 overexpression. The results revealed that the reduction in CCT2 mRNA expression caused by METTL5 knockdown was completely restored by exogenous CCT2 overexpression (Supplementary Figure 3C). The results of functional experiments indicated that the reduced proliferative capacity of SW480 cells induced by METTL5 knockdown was reversed by exogenous CCT2 overexpression, indicating that METTL5 promotes CRC cell proliferation by increasing CCT2 expression (Supplementary Figure 3D and E). Similarly, the reduced migration and invasion abilities of SW480 cells caused by METTL5 knockdown were restored by exogenous CCT2 overexpression, indicating that METTL5 enhances the metastatic potential of CRC cells by upregulating CCT2 expression (Supplementary Figure 3F). We used a CDX model to evaluate the role of CCT2 in facilitating METTL5-mediated tumor growth. METTL5 knockdown significantly inhibited the growth of SW480 cell-derived subcutaneous xenografts, whereas exogenous CCT2 overexpression reversed this effect (Figure 3F). Immunohistochemical analysis revealed that METTL5 knockdown reduced CCT2 and Ki67 expression in xenografts, whereas exogenous CCT2 overexpression restored their expression. These findings further confirm that CCT2 is crucial for METTL5-mediated CRC cell proliferation (Figure 3G). Collectively, these in vitro and in vivo findings demonstrate that CCT2 is a key target gene through which METTL5 promotes CRC progression.

H3K18 La-METTL5-CCT2 signaling axis promotes tumor progression by inhibiting pyroptosis in CRC cells

Lactate and H3K18 La promote METTL5 transcription, and METTL5 stabilizes CCT2 mRNA by promoting its m6A modification, thereby driving the malignant progression of CRC cells. However, the specific biological functions regulated by this signaling axis remain unclear. To investigate this, we analyzed the transcriptome data of CRC samples from public databases and divided the samples into CCT2 high- and low-expression groups. Functional enrichment analysis indicated that the CCT2-associated genes were involved mainly in biological processes such as pyroptosis, osteoclast differentiation, platelet activation, and natural killer cell-mediated cytotoxicity (Figure 4A). Differential gene expression analysis revealed that core molecules of the pyroptosis signaling pathway were significantly enriched and potentially activated in the CCT2 low-expression group but inhibited in the CCT2 high-expression group (Figure 4B). Moreover, differential gene expression analysis before and after METTL5 knockdown revealed that core molecules of the pyroptosis signaling pathway were significantly upregulated after METTL5 knockdown (Figure 4C). These results suggest that pyroptosis is a key downstream target of the H3K18 La-METTL5-CCT2 signaling axis in CRC progression.

Figure 4 H3K18 La-METTL5-CCT2 signaling axis promotes tumor progression by inhibiting pyroptosis in colorectal cancer cells. A and B: Colorectal cancer samples were divided into high- and low-expression groups based on CCT2 expression levels, and differentially expressed genes between the two groups were identified. KEGG pathway enrichment analysis (A). Gene set enrichment analysis for the pyroptosis signaling pathway (B). Bubbles size represents GeneRatio, and bubble color indicates -log10(P value); C: Gene set enrichment analysis of transcriptome sequencing data of METTL5-knockdown SW480 cells for pyroptosis-related genes; D: SW480 cells in the non-targeted short hairpin RNA control (sh-NC) and METTL5-knockdown (sh-METTL5) groups were treated with medium containing phosphate-buffered saline or 5 mmol/L sodium lactate for 24 hours. Pyroptosis was assessed by flow cytometry. Propidium iodide staining was used to indicate cell membrane permeability, and Caspase-1 activity was used to reflect the activation status of inflammatory caspases; E: SW480 cells in the sh-NC and sh-METTL5 groups were stably transfected with oe-NC or oe-CCT2. Pyroptosis was assessed via flow cytometry and quantified for comparison. PBS: Phosphate-buffered saline; Nala: Sodium lactate. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001.

To clarify the effect of the H3K18 La-METTL5-CCT2 signaling axis on pyroptosis in CRC cells, we conducted a series of experiments. Western blotting indicated that the glycolysis inhibitor 2-DG upregulated the expression of pyroptosis-related proteins (GSDMD-N, NLRP3, and cleaved Caspase-1), whereas Nala inhibited their expression (Supplementary Figure 4A and B). Flow cytometry confirmed that 2-DG significantly increased the proportion of pyroptotic cells, whereas Nala reduced it (Supplementary Figure 4C and D). Collectively, these results indicate that lactate inhibits pyroptosis in CRC cells. Further investigations on the roles of METTL5 and CCT2 in this process revealed that METTL5 knockdown reversed the Nala-induced reduction in pyroptotic cells (Figure 4D), whereas CCT2 overexpression reversed the increase in pyroptotic cells caused by METTL5 knockdown (Figure 4E). These findings suggest that METTL5 and CCT2 are key downstream effectors through which lactate inhibits pyroptosis in CRC cells, confirming that the H3K18 La-METTL5-CCT2 signaling axis inhibits pyroptosis in CRC cells.

We subsequently attempted to elucidate the key molecular mechanism underlying lactate metabolic signaling in regulating pyroptosis in CRC cells at the protein-protein interaction level. In a high-lactate microenvironment, we screened and identified five proteins that potentially interact with CCT2 and are involved in the regulation of cell pyroptosis, namely, NLRP3, GSDMD, HMGB1, NEK7, and P2X7 (Supplementary Figure 4E). Subsequent coimmunoprecipitation assays confirmed that CCT2 specifically binds to NLRP3, whereas no significant interaction was detected between CCT2 and other candidate proteins (Supplementary Figure 4F). Immunofluorescence colocalization assays further validated the interaction between CCT2 and NLRP3 (Supplementary Figure 4G). Collectively, these results demonstrate that CCT2 regulates inflammasome activation by directly binding to NLRP3, thereby suppressing the initiation and progression of pyroptosis in CRC cells. Collectively, these results indicate that the H3K18 La-METTL5-CCT2 signaling axis promotes CRC progression by inhibiting pyroptosis.

AZD6482 enhances the cytotoxic effect of oxaliplatin on CRC cells in a high-lactate microenvironment

On the basis of predictions from our previous data analysis[25], three small-molecule inhibitors, AZD6482 (targeting PI3Kβ)[26], SB216763 (targeting GSK-3α and GSK-3β)[27], and tozasertib (targeting Aurora A/B/C kinases)[28], were identified as potentially effective against CRC cells in a high-lactate microenvironment. This part of the study aimed to verify these predictions and evaluate the regulatory specificity of the H3K18 La-METTL5-CCT2 signaling axis in CRC progression. A CCK-8 assay was used to evaluate the effects of the three inhibitors on lactate-induced chemoresistance. The results revealed that treatment with Nala significantly increased the half-maximal inhibitory concentration (IC₅₀) of oxaliplatin for SW480 cell cytotoxicity (Figure 5A-C). In the absence of Nala, AZD6482, SB216763, and tozasertib reduced the IC₅₀ of oxaliplatin by 1.87-, 2.07-, and 1.81-fold, respectively. Under Nala treatment, these inhibitors decreased the IC₅₀ of oxaliplatin by 3.27-, 2.27-, and 2.06-fold, respectively (Figure 5A-C). Comparative analysis revealed that compared with the other inhibitors, only AZD6482 significantly increased the sensitivity of SW480 cells to oxaliplatin under Nala treatment, with a 1.74-fold stronger effect. As a selective PI3Kβ inhibitor, AZD6482 significantly suppressed the phosphorylation and activation of downstream AKT proteins, confirming its efficacy and specificity (Supplementary Figure 5A).

Figure 5 AZD6482 enhances the cytotoxic sensitivity of oxaliplatin to colorectal cancer cells in a high-lactate microenvironment. A: SW480 cells (5 × 10³ cells/well) cultured in medium containing different concentrations of oxaliplatin (0, 1, 2, 5, 10, 25, 50, and 100 μM) with the following treatments added simultaneously: Phosphate-buffered saline (PBS), 5 mmol/L sodium lactate (Nala), PBS + 20 μM AZD6482, or 5 mmol/L Nala + 20 μM AZD6482. After 48 hours of further culture, the cell counting kit-8 (CCK-8) kit was used to detect cell viability, and cell survival curves were plotted; B: SW480 cells were seeded as in (A), cultured in medium with gradient concentrations of oxaliplatin (0-100 μM), and treated with PBS, 5 mmol/L Nala, PBS + 5 μM SB216763, or 5 mmol/L Nala + 5 μM SB216763, respectively. After 48 hours of culture, the CCK-8 method was used to detect cell viability, and survival curves were plotted; C: Cells were seeded as in (B), cultured under conditions with different concentrations of oxaliplatin, and supplemented with PBS, 5 mmol/L Nala, PBS + 5 μM Tozasertib, or 5 mmol/L Nala + 5 μM Tozasertib. After 48 hours, cell viability was detected using the CCK-8 assay, and dose-response curves were generated; D: SW480 cells (1 × 10⁶ cells/mouse) were subcutaneously inoculated into BALB/c nude mice to establish a cell line-derived xenograft model. Tumor volume was measured every 3 days starting from day 6 post-inoculation. From day 7 post-inoculation, mice received the following treatments via daily gavage: PBS (control), 200 mg/kg Nala, or 200 mg/kg Nala + 50 mg/kg AZD6482. Mice were euthanized 28 days later. Tumor photographs (left panel), tumor weight (middle panel, n = 6), and tumor growth curves (right panel); E: A portion of the xenograft tumor tissues obtained in (D) was subjected to hematoxylin-eosin staining, and another portion was used for immunohistochemistry detection of the expression levels of Ki67 and CCT2, followed by quantitative analysis; F: SW480 cells stably overexpressing oe-NC (control) or oe-CCT2 were treated with 20 μM AZD6482 for 24 hours. Western blotting was performed to detect the protein expression levels of METTL5, CCT2, GSDMD-N, NLRP3, and cleaved Caspase-1 (C-caspase1), and grayscale quantitative analysis of the bands was conducted. PBS: Phosphate-buffered saline; Nala: Sodium lactate; HE: Hematoxylin-eosin. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001.

To further assess the role of AZD6482 in a high-lactate microenvironment, additional experiments were performed. The results indicated that AZD6482 reversed the Nala-induced increase in SW480 cell proliferation, suggesting that it can inhibit the proliferative activity of CRC cells in a high-lactate microenvironment (Supplementary Figure 5B and C). Similarly, AZD6482 reversed the Nala-mediated increase in the DNA replication capacity of SW480 cells, indicating that it can suppress DNA replication in tumor cells under high-lactate conditions (Supplementary Figure 5D). To directly verify the regulatory effects of the lactate metabolic pathway and AZD6482 on pyroptosis in SW480 cells, electron microscopy was used to observe the ultrastructural changes in the cells in each group, with a focus on the core characteristic of pyroptosis, plasma membrane pore formation. Compared with the control treatment, treatment with the lactate donor Nala significantly reduced the number and size of plasma membrane pores in SW480 cells, confirming that the lactate metabolic pathway negatively regulates pyroptosis in CRC cells. In contrast, combined treatment with Nala and AZD6482 effectively reversed the above changes in membrane pores, restoring their number and size to levels close to those of the control group, indicating that AZD6482 significantly antagonizes the inhibitory effect of pyroptosis on the lactate metabolic pathway and promotes pyroptosis in CRC cells in a high-lactate microenvironment. These findings further validate the regulatory role of AZD6482 at the ultrastructural level. (Supplementary Figure 5E). A CDX model was used to assess the effects of Nala and AZD6482 on the growth of SW480 cell-derived subcutaneous xenografts. The results demonstrated that Nala treatment significantly accelerated xenograft growth; however, this effect was reversed by AZD6482 (Figure 5D). Hematoxylin-eosin staining and IHC further confirmed that AZD6482 reversed the Nala-induced acceleration of cancer cell proliferation and simultaneously suppressed the Nala-mediated increase in CCT2 expression (Figure 5E). Furthermore, our experimental results revealed that exogenous addition of the lactate donor Nala markedly elevated the phosphorylation level of AKT and the level of H3K18 La in SW480 cells compared with those in the control group. Conversely, simultaneous treatment with AZD6482 effectively reversed the Nala-induced upregulation of these indicators, restoring both AKT phosphorylation and H3K18 La levels to nearly those of the control group. These findings suggest that AZD6482 can intervene in signaling downstream of lactate metabolism by targeting the PI3Kβ-AKT pathway (Supplementary Figure 5F). Mechanistically, AZD6482 inhibited CCT2 expression, and its upregulation of pyroptosis-activated proteins (GSDMD-N, NLRP3, interleukin-1β, and cleaved Caspase-1) was reversed by exogenous CCT2 overexpression (Figure 5F). Collectively, these in vitro and in vivo experiments confirm that AZD6482 specifically inhibits the activity of lactate and its downstream signaling pathways. Collectively, these results indicate that AZD6482 can enhance the cytotoxic effect of oxaliplatin on CRC cells by inhibiting the activity of lactate and its downstream signaling pathways.

DISCUSSION

This study reveals the molecular mechanism through which lactylation influences the biological behavior of CRC cells through crosstalk between epigenetic regulation and RNA methylation. For the first time, this study demonstrated that the H3K18 La-METTL5-CCT2 signaling axis promotes tumor progression by inhibiting pyroptosis and verified the potential of the PI3Kβ inhibitor AZD6482 in increasing chemotherapy sensitivity in a high-lactate microenvironment. These findings provide a multidimensional theoretical basis for prognostic evaluation, mechanistic research, and targeted therapy in CRC. Schematic representation of the role of the lactic acid-driven H3K18 La-METTL5-CCT2 axis in the malignant progression of CRC (Supplementary Figure 5G).

Crosstalk between tumor metabolic reprogramming and epigenetic modification has become a central focus in cancer research. In this study, we found that H3K18 La levels were significantly elevated in CRC tissues, positively correlated with clinical stage, and negatively correlated with patient survival, supporting its role as an independent prognostic biomarker. These results are consistent with previous reports that “histone lactylation is activated under enhanced tumor metabolic stress”[29-31]; however, our findings clarify the specific clinical significance of H3K18 La in CRC. Experiments using glycolysis and lactate dehydrogenase inhibitors revealed a dose-dependent regulatory relationship between lactate and H3K18 La and demonstrated that this modification can drive malignant phenotypes, including proliferation and metastasis. These findings overturn the traditional perception of lactate as a mere metabolic byproduct, highlighting its role as an epigenetic regulator and providing direct experimental evidence that the Warburg effect promotes tumor progression through epigenetic mechanisms.

Through integrated analysis of multiple datasets, we identified METTL5 as a core target gene regulated by lactylation. While METTL5 can catalyze m6A modification of 18S rRNA to maintain ribosome function[32,33], we found that METTL5 is specifically activated by H3K18 La in CRC. CUT&Tag and ChIP experiments confirmed that H3K18 La enrichment at the METTL5 promoter increases METTL5 transcription; this process is directly regulated by lactate. This mechanism links histone lactylation to the transcriptional regulation of RNA methyltransferases, expanding the downstream effect network of epigenetic modification. Functionally, high METTL5 expression is correlated with CRC progression and poor prognosis and directly promotes malignant tumor phenotypes by regulating cell proliferation, DNA replication, and metastatic capacity. In vitro and in vivo rescue experiments further demonstrated that METTL5 is an essential molecule for the lactate-H3K18 La pathway to drive CRC progression. These results fill an important gap in METTL5 research in CRC and suggest that METTL5 enables tumor cells to adapt to the microenvironment under metabolic stress.

We also discovered that METTL5 directly binds to CCT2 mRNA through a noncanonical mechanism to promote its m6A modification and increase its stability, overturning the traditional view that “METTL5 regulates only rRNA”. High expression of CCT2, a subunit of the chaperonin complex, is closely associated with tumor stress adaptation and drug resistance[34,35]. This study is the first to confirm that CCT2 expression is regulated by m6A modification and that this process depends on the RNA-binding ability of METTL5. Notably, while METTL5 has been reported to promote tumor progression in hepatocellular carcinoma and intrahepatic cholangiocarcinoma primarily through regulating 18S rRNA m6A modification and translation, our study reveals a distinct noncanonical mechanism in CRC, in which METTL5 directly binds to and stabilizes CCT2 mRNA via m6A modification. This context-dependent function further highlights the novelty of METTL5 in CRC pathogenesis. Experiments demonstrated that METTL5 deficiency significantly shortened the half-life of CCT2 mRNA, whereas exogenous CCT2 overexpression completely reversed the tumor-suppressive effects of METTL5 knockdown. These findings confirm that CCT2 is a key downstream effector of METTL5. This pathway connects epigenetic regulation (H3K18 La) with RNA modification (m6A), forming a cascade regulatory network of “metabolite-histone modification-methyltransferase-mRNA stability”. Together, these findings provide a new paradigm for understanding how tumor cells maintain malignant phenotypes through multilevel regulation.

As an inflammatory programmed cell death pathway, pyroptosis plays a crucial role in tumor immune surveillance[36]. In this study, transcriptome analysis revealed that the pyroptosis pathway was significantly suppressed in the CCT2 high-expression group, whereas interference with METTL5 or CCT2 activated the expression of key pyroptosis proteins (GSDMD-N, NLRP3, and cleaved Caspase-1). Functional experiments further confirmed that the H3K18 La-METTL5-CCT2 signaling axis reduces tumor cell death by inhibiting pyroptosis, representing the core mechanism through which it drives CRC progression. This finding links the metabolic-epigenetic regulatory network to pyroptosis for the first time, revealing an adaptive tumor strategy summarized as “metabolic reprogramming-epigenetic modification-pyroptosis evasion”. Unlike previous studies suggesting that pyroptosis is regulated mainly by inflammatory signals, this study demonstrates that metabolism-driven epigenetic modification can directly inhibit pyroptosis, offering a novel perspective on the nonimmunomodulatory mechanisms of tumor immune escape. Lactate metabolism is closely related to the immune microenvironment and epigenetic modifications. As a metabolic intermediate, lactate serves not only as an energy substrate but also as a signaling molecule that modulates the epigenetic status of immune cells. Accumulating evidence has demonstrated that lactate influences histone acetylation and methylation via histone lactylation, thereby shaping macrophage polarization, T-cell function, and the tumor immune microenvironment. This metabolic-epigenetic-immune axis plays a pivotal role in diverse diseases, including sepsis and cancer, providing novel strategies for targeted therapy[37,38]. Nevertheless, we acknowledge that other histone lactylation marks, such as H3K9 La, may also contribute to CRC progression. Whether these additional lactylation sites converge on the METTL5-CCT2 axis remains an important question that warrants further investigation.

Chemoresistance induced by a high-lactate microenvironment is a major challenge in CRC treatment[39]. This study revealed that the PI3Kβ inhibitor AZD6482 specifically enhanced the cytotoxic effect of oxaliplatin on CRC cells in a high-lactate microenvironment, and its efficacy was significantly superior to that of other candidate inhibitors. We propose that AZD6482 acts by inhibiting the PI3K-AKT signaling pathway, thereby downregulating METTL5 and CCT2 expression and activating pyroptosis signaling; however, this requires further experimental validation. These findings provide a novel strategy to overcome chemotherapy insensitivity in high-lactate microenvironments and establish an experimental foundation for the precise application of PI3Kβ inhibitors in CRC.

This study has several limitations. First, the specific transcription factor coregulation mechanisms underlying H3K18 La of the METTL5 promoter remain to be elucidated. Second, the downstream molecular mechanism through which CCT2 inhibits pyroptosis requires further elucidation through protein interaction studies. Third, the translational potential of AZD6482 needs to be validated in patient-derived xenograft models with larger sample sizes. Fourth, we investigated the role of AZD6482 as a PI3Kβ inhibitor in counteracting lactate-induced signaling downstream of lactate metabolism, specifically through the PI3Kβ-AKT pathway. Whether AZD6482 directly affects lactate production or transport is an interesting question that warrants further investigation. Future research should focus on three directions: (1) Exploring the crosstalk between lactylation and other epigenetic modifications (acetylation and methylation); (2) Examining the association between the H3K18 La-METTL5-CCT2 signaling axis and immune checkpoint molecules to assess the potential for combined immunotherapy; and (3) Optimizing the combination regimens of AZD6482 and chemotherapeutic drugs to facilitate translation from preclinical studies to clinical trials.

Beyond the specific context of CRC, the H3K18 La-METTL5-CCT2 signaling axis exemplifies a broader paradigm in which metabolic reprogramming interfaces with epigenetic and posttranscriptional regulatory layers to orchestrate tumor progression. Lactate, long considered a metabolic waste product, has emerged as a central signaling molecule that links cellular metabolism to gene regulation through histone lactylation. This metabolic-epigenetic crosstalk operates within a complex regulatory network: Lactate accumulation not only drives histone lactylation but also modulates the expression and activity of RNA-modifying enzymes, which in turn regulate the stability of transcripts involved in metabolic adaptation and cell death[40]. Recent studies have revealed reciprocal crosstalk between lactylation and RNA modifications, resulting in the formation of positive feedback loops that amplify oncogenic signaling; for example, histone lactylation can upregulate glycolytic genes, further increasing lactate production and sustaining lactylation in a self-reinforcing circuit[40,41]. As an inflammatory form of programmed cell death, pyroptosis is increasingly recognized as a critical determinant of tumor immune surveillance, and its regulation by metabolic-epigenetic mechanisms represents an emerging topic in cancer biology[42,43]. The interplay between lactate metabolism, histone lactylation, and pyroptosis described in this study highlights how tumor cells exploit multilevel regulatory networks to evade immune destruction while maintaining metabolic fitness. Collectively, these insights position the “metabolite-histone modification-RNA methyltransferase-mRNA stability-cell death” axis as a recurring regulatory network across diverse cancer types, offering a framework for developing combination therapies that simultaneously target metabolic, epigenetic, and cell death pathways[29,44].

CONCLUSION

In conclusion, this study is the first to reveal that the H3K18 La-METTL5-CCT2 signaling axis promotes CRC progression by inhibiting pyroptosis. This study is also the first to verify the therapeutic value of targeting this pathway. This study provides important insights for basic research and clinical practice in CRC.