Lactylation affects p53 Nuclear Translocation to Promote Colorectal Cancer Progression

Lactylation affects p53 Nuclear Translocation to Promote Colorectal Cancer Progression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Lactylation affects p53 Nuclear Translocation to Promote Colorectal Cancer Progression Jie Ma, Yao Dai, Wenxin Da, bo shen, Yan Zhang, Pengtao Bao, Wei Zhu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5586218/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Lysine lactylation is a post-translational modification that connects lactate metabolism with protein function. Our study identifies lysine lactylation of p53 in colorectal cancer tissues and cells. This modification results in increased cytoplasmic accumulation and reduced nuclear accumulation of p53, along with enhanced protein degradation via the proteasome pathway. These changes collectively promote the proliferation, migration, and invasion of colorectal cancer cells. Specifically, we observe enrichment of lactate groups at lysine 291 within the p53 DNA-binding domain and lysine 370 in its C-terminal regulatory domain. Mutating these lysine residues to arginine decreased cytoplasmic accumulation and increased nuclear localization of p53, thereby inhibiting colorectal cancer cells proliferation and migration. Our findings suggest that p53 lactylation contributes to tumorigenesis by modulating its nuclear translocation. Biological sciences/Cancer/Cancer metabolism Biological sciences/Diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Colorectal cancer is the third most common cancer worldwide and the second leading cause of cancer death 1 . The development of such as diet, disease, genetics, and age, but the exact causes and mechanisms of its development are not fully understood 2 . Metabolic reprogramming, which refers to alterations in metabolism or nutrient supply, is a hallmark of cancer 3 . Lactate is one of the most consistently upregulated metabolites, while glucose is consistently downregulated in various cancers 4 , 5 , 6 , a phenomenon known as the "Warburg effect" 7 . Growing evidence suggests that lactate plays a crucial role in metabolic activities such as cell fate, embryonic development, inflammation and immune responses, and tumorigenesis, and it regulates gene expression by modifying histone lysine residues 8 , 9 , 10 , 11 . However, the relationship between lactylation and non-histone proteins remains unclear in the pathogenesis of colorectal cancer. The p53 protein is a tumor suppressor protein that plays a key role in many cellular processes, including cell division, maintaining genomic stability, apoptosis, autophagy, and immune responses 12 , 13 , 14 . The multi-modular structure of p53 facilitates various covalent modifications, including phosphorylation, ubiquitination, acetylation, methylation, sumoylation, neddylation, glycosylation, hydroxylation, and β-hydroxybutyrylation 15 , 16 , 17 . These covalent modifications can alter the activity and function of p53 to a certain extent 18 , 19 , so understanding the mechanisms of p53 regulation may offer new avenues for cancer treatment strategies to enhance the tumor suppressor function of p53 in cancer cells. In this study, we found that lactylation levels are closely related to the biological functions of colorectal cancer cells, and we demonstrated the impact of lactylation on the nuclear translocation and stability of the p53 protein. We then discovered that p53 protein lactylation exists in both colorectal cancer tissues and cells. We performed LC-MS/MS analysis and identified K291 and K370 as the most abundant sites of p53 protein lactylation modification in HCT116 cells. By mutating lysine (K) at positions 291 and 370 to arginine (R), we found that lactylation at these two sites promotes proliferation and migration of colorectal cancer cells and affects the nuclear translocation of the p53 protein. Taken together, our findings suggest that p53 protein lactylation can promote proliferation and metastasis of colorectal cancer cells, and targeting p53 protein lactylation may be an effective strategy for preventing the development of colorectal cancer. Results Lactate and lactylation levels are elevated in colorectal cancer tissues and cell lines Lactate and lactylation play important roles in regulating tumorigenesis 20 , 21 , 22 , but their specific roles in the development of colorectal cancer remain unclear. We collected colorectal cancer tissue and performed HE staining. Compared to adjacent tissues, colorectal cancer tissues showed signs of carcinogenesis, including disordered glandular structure and nuclear translocation of cancer cells (Figure S1 ). To verify the characteristics of aerobic glycolysis in tumors, we used lactate kit to measure the lactate level in colorectal cancer tissues. The results showed that the lactate level in colorectal cancer tissues was significantly higher than that in adjacent tissues (Fig. 1 A), indicating strong glycolysis in colorectal cancer cells. To simulate the characteristics of aerobic glycolysis of colorectal cancer cells in vitro, HCT116 cells were divided into four groups according to different treatments: low glucose group (5 mM glucose), high glucose group (25 mM glucose), LA group (20 mM lactate), and Nala group (20 mM sodium lactate). The results showed that the intracellular lactate level was significantly increased in the high glucose group, LA group, and Nala group (Fig. 1 B). Subsequently, HCT116 cells were treated with glycolysis inhibitor 2-deoxy-D-glucose (2-DG) or LDHA inhibitor oxamate. The results showed that the intracellular lactate level was significantly reduced (Fig. 1 C). These results suggest that colorectal cancer cells have active aerobic glycolysis metabolism and high lactate levels. To investigate the association between protein lactylation and colorectal cancer, we observed that high glucose treatment significantly elevated pan-lactylation levels in HCT116 cells (Fig. 1 D). Conversely, treatment with the glycolysis inhibitors 2-DG or oxamate resulted in a decrease in pan-lactylation levels (Fig. 1 E). Furthermore, treatment with LA and Nala, which promote lactate, led to an increase in pan-lactylation in HCT116 cells (Fig. 1 F). Importantly, pan-lactylation levels were significantly higher in colorectal cancer tissues compared to adjacent tissues (Figs. 1 G and 1 H). These findings strongly suggest that elevated lactylation is a characteristic of both colorectal cancer cells and tissues, and it is likely associated with glycolytic metabolism. Hyperlactylation of colorectal cancer cells in vitro promotes cell proliferation and metastasis Recent studies have consistently reported a positive correlation between elevated lactylation and disease progression in cancer 23 , 24 . Yu et al. found that elevated histone lactylation (H3K18la) levels were observed in human ocular melanoma tissues and cell lines, proving to be an adverse prognostic factor for patients with ocular melanoma 22 . In non-small cell lung cancer (NSCLC), histone lactylation is enriched on promoters of metabolic-related genes, thereby promoting NSCLC proliferation and migration 25 . However, the impact of protein hyperlactylation on the biological functions of colorectal cancer cells remains unclear. Here, we found that high glucose treatment increased the proliferative capacity of HCT116 cells, while low glucose treatment or oxamate treatment decreased the proliferative capacity of HCT116 cells (Fig. 2 A). However, both LA treatment and Nala treatment did not enhance the proliferative capacity of HCT116 cells (Fig. 2 B). High glucose treatment increased the wound healing rate of HCT116 cells at 24 h, while oxamate treatment decreased the wound healing rate of HCT116 cells at 24 h (Fig. 2 C). In cell migration and invasion experiments, we found that high glucose treatment increased the number of migrating cells in HCT116 cells at 14 h, while oxamate treatment or 2-DG treatment showed a contrast outcome (Fig. 2 D). High glucose treatment increased the number of invading cells in HCT116 cells at 18 h, while oxamate treatment or 2-DG treatment showed a contrast outcome (Fig. 2 F). Regrettably, the number of migrating cells and invading cells in the LA and Nala treatment groups did not change significantly (Figs. 2 E and 2 G). This suggests that under conditions of high lactylation, the proliferation, migration, and invasion abilities of colorectal cancer cells are significantly enhanced. Lactylation affects the nuclear translocation and stability of p53 protein Recently, Zong, Z. et al. evaluated the TCGA breast cancer dataset and found that patients carrying wild-type p53 with high serum lactate levels showed lower p53 signaling pathway scores, suggesting that lactylation in tumors may inhibit p53 function. Colorectal cancer HCT116 cells highly express wild-type p53 and have high lactylation levels 26 , so we hypothesized whether lactylation might affect the function of the p53 protein in colorectal cancer cells. The p53 protein acts as a transcription factor in tumor cells and plays a powerful role in the nucleus 27 . In human colorectal cancer tissue, we observed significantly higher p53 levels compared to adjacent tissue, consistent with previous reports (Fig. 1 H). We investigated its subcellular localization in cells. In cells treated with low glucose, p53 was primarily nuclear. Conversely, p53 predominantly localized to the cytoplasm in cells treated with high glucose (Fig. 3 A). This pattern was also observed in cells treated with oxamate or 2-DG (Figs. 3 B and 3 C). However, this phenomenon was not observed in cells treated with LA and Nala (Fig. 3 D). This suggests that the mechanism by which lactylation promotes proliferation and metastasis of colorectal cancer cells may be to reduce the expression of the p53 protein in the nucleus, thereby reducing its function as a transcription factor, and ultimately promoting the development and metastasis of colorectal cancer. Next, we further investigated the effect of lactylation on the stability of the p53 protein. Cycloheximide (CHX) is a protein synthesis inhibitor that works by binding to the 80S ribosome and blocking the translocation of tRNA, thereby inhibiting protein synthesis 28 . Therefore, we used CHX to treat HCT116 cells to ensure that no new proteins were produced, so that we could observe the degradation of the p53 protein. The results showed that the p53 protein degradation rate was slower in cells treated with low glucose or oxamate, while the p53 protein degradation rate was faster in cells treated with high glucose (Fig. 3 E). Since lactylation can affect the degradation rate of the p53 protein, we further treated HCT116 cells with MG-132 to observe whether lactylation-mediated degradation of the p53 protein occurs through the proteasome pathway. MG-132 is a proteasome inhibitor that effectively blocks the proteolytic activity of the 26S proteasome complex 29 . The results showed that after 8 h of treatment with MG-132, the p53 protein content was higher in cells treated with high glucose, while the p53 protein content was lower in cells treated with low glucose or oxamte (Fig. 3 F). However, this phenomenon was not observed in cells treated with LA or Nala (Figs. 3 G and 3 H). These results suggest that lactylation may reduce the stability of the p53 protein through the proteasome pathway, thereby reducing its function as a tumor suppressor protein. p53 lactylation levels are elevated in colorectal cancer tissues and cell lines Given the possibility of various post-translational modifications occurring on lysine residues of p53 30 and the previously observed high levels of lactylation in colorectal cancer tissues and cells, we predicted that p53 protein is subject to lactylation. We first observed co-localization of p53 protein and lactylation in both colorectal cancer tissues and cells (Figs. 4 B and 4 C). Subsequently, we used co-immunoprecipitation experiments to confirm the presence of p53 lactylation. Our results indicated the presence of p53 lactylation in a portion of colorectal cancer tissues (Fig. 4 B). We then observed the same phenomenon in colorectal cancer cells. Results showed that the level of p53 protein lactylation was significantly increased in HCT116 cells treated with high glucose (Fig. 4 D), while the opposite result was observed in the Oxamate and 2-DG treatment groups (Fig. 4 E). Lactate and sodium lactate also increased the level of p53 protein lactylation in HCT116 cells (Fig. 4 F). Lactylation is a new form of acylation modification that may share similar regulatory enzymes with other lysine acylation modifications 23 . p300 is currently the most common "writer" in lactylation research 31 , 32 , 33 , 34 . We detected the level of p300 in HCT116 cells and found that the level of p300 protein was slightly increased in cells treated with high glucose, but the level of p300 protein was significantly reduced in the Oxamate and 2-DG treatment groups (Figs. 4 G and 4 H). To further validate whether p300 mediates p53 lactylation, we treated cells with the p300 inhibitor (C646) and found that p53 lactylation was significantly inhibited (Fig. 4 I). We therefore deduce that p53 lactylation is mediated by p300. p53 lactylation promotes colorectal cancer cell proliferation and migration p53 is a well-characterized tumor suppressor protein with six major domains: two intrinsically disordered N-terminal transactivation domains (TADs), a proline-rich domain (PRD), a central DNA-binding domain (DBD), a tetramerization domain (TD), and an intrinsically disordered C-terminal regulatory domain (CTD) 35 . To identify the specific sites of p53 lactylation, we performed LC-MS/MS analysis, revealing abundant lactylation modifications on multiple lysine residues within the p53 protein in HCT116 cells (Figure S2A). Notably, K291 and K370 emerged as the most prominent sites of lactylation (Fig. 5 A). To investigate the functional significance of p53 K291 and K370 lactylation in colorectal cancer cells, we overexpressed Flag-tagged wild-type p53 (WT) and Flag-tagged mutant p53 (K291R/K370R) in HCT116 and H1299 cells (Fig. 5 B). The mutant, with lysine (K) residues at positions 291 and 370 replaced by arginine (R), mimicked the de-lactylated state. H1299 cells, deficient in p53, served as a control to eliminate potential variations in endogenous p53 levels. Immunoprecipitation experiments confirmed a significant reduction in p53 lactylation in the mutant group (K291R/K370R) compared to the wild-type group (Figs. 5 C and 5 D), aligning with the LC-MS/MS findings. Further functional analysis revealed that p53 K291 and K370 lactylation significantly promoted tumor cell proliferation (Figs. 5 E and 5 F) and migration (Fig. 5 G and 5 F), but had no effect on cell invasion (Figures S2B and S2C). These observations suggest that lactylation at K291 and K370 may play a critical role in regulating the proliferative and migratory capabilities of tumor cells. p53 lactylation affects nuclear translocation of p53 protein Our previous findings indicated that lactylation influences the nuclear translocation and stability of the p53 protein. We aimed to further investigate whether lactylation at K291/K370 sites affects p53 protein function. Immunofluorescence results showed that lactylated proteins were widely distributed in both the nucleus and cytoplasm. However, in the WT group, Flag-p53 expression was higher in the cytoplasm than in the nucleus. In contrast, Flag-p53 expression was significantly increased in the nucleus of the mutant group (K291R/K370R) cells (Figs. 6 A and 6 B). To clarify this phenomenon, we performed nuclear-cytoplasmic separation on transfected and pretreated cells. The results revealed that, compared to the WT group, Flag-p53 expression was significantly increased in the nucleus and decreased in the cytoplasm of the mutant group (K291R/K370R) cells (Figs. 6 C and 6 D). Next, to investigate the impact of K291/K370 lactylation on p53 protein stability, we treated transfected cells with CHX. The results showed that K291/K370 lactylation did not affect the degradation rate of p53 protein (Figs. 6 E and 6 F). Similarly, after treating transfected cells with MG-132, we observed that despite the inhibition of the proteasome pathway, Flag-p53 expression was not reduced in the mutant group (K291R/K370R) cells (Figs. 6 G and 6 H). This indicates that lactylation at K291 and K370 does not affect p53 protein stability. K291 is located at the end of the DBD region of the p53 protein, which is the region responsible for p53 binding to DNA 36 . K370 is located at the CTD region of the p53 protein, which is a regulatory domain containing nuclear export signals and nuclear localization signals 37 . This domain is crucial for the function of p53 as a transcription factor in the nucleus and for exporting p53 to the cytoplasm for degradation 38 . Our results show that lactylation at K291 and K370 promotes the accumulation of p53 in the nucleus of tumor cells while reducing its expression in the cytoplasm (Figs. 6 I). This suggests that lactylation at K291 and K370 may inhibit the tumor suppressor activity of p53 as a transcription factor by this mechanism, thereby promoting tumor cell proliferation and migration. Discussion Since the discovery of the “Warburg effect”, researchers have been interested in the following questions: (1) why cancer cells prefer aerobic glycolysis over oxidative phosphorylation; (2) how cancer cells utilize lactate metabolism; (3) the potential of targeting lactate metabolic pathways for cancer treatment 39 . Zhang et al. used mass spectrometry analysis to identify a 72.021 Da mass shift on lysine residues in human breast cancer cells, confirming the existence of histone lactylation modification 11 . This discovery opened up new insights into lactate metabolism. With the advancement of mass spectrometry technology, Wan et al. mined publicly available human MeltomeAtlas datasets and found that lactylation modifications are also widespread on human non-histone proteins, particularly prevalent on glycolytic enzymes 40 . This discovery ushered in a new era of research on protein lactylation modifications. While histone lactylation primarily plays a significant role at the transcriptional level, the function of non-histone lactylation remains largely unknown. P53, known as the "guardian of the genome", plays a crucial role in maintaining genomic stability and preventing tumor development 41 . Dysregulation of p53 can lead to disruptions in cell division, genomic stability, apoptosis, autophagy, and immune responses, ultimately increasing the risk of cancer 42 . Precise regulation of p53 is therefore essential for safeguarding genomic integrity and preventing tumorigenesis. Mechanisms regulating p53 function are multifaceted, with post-translational modifications (PTMs) of p53 being the most widespread and effective 17 . p53 can act as an upstream regulator of aerobic glycolysis 43, 44 . Researchers have combined the expression levels of six proteins (PTEN, p53, GLUT1, PKM2, LDHA, and MCT4) into a scoring system and categorized colorectal cancer patients into three Warburg subtypes (low/medium/high) based on their scores. They found that colorectal cancer patients with Warburg-high subtype tumors had poorer overall survival 44 . These findings suggest a complex regulatory relationship between glycolysis, lactate metabolism, and p53 in colorectal cancer. In this study, we observed high expression of lactylation and p53 in colorectal cancer tissues and cells. These lactate-mediated proteins undergo lactylation modifications, which subsequently promote proliferation and metastasis of colorectal cancer cells. We further discovered that p53 is lactylated by p300 and demonstrated that p53 is specifically lactylated at its DBD K291 and CTD K370 sites. Interestingly, our findings indicate that lactylation of p53 at K291 and K370 promotes proliferation and migration of colorectal cancer cells by affecting p53 nuclear translocation. The DBD region is crucial for the transcriptional activity of p53, while the CTD region regulates subcellular localization of p53 45 . Based on this, we hypothesize that lactylation may affect transcriptional activity of p53, thereby promoting tumorigenesis. Recent studies have shown that AARS1 can catalyze lactylation of K120 and K139 in the DBD of p53, leading to impaired DNA binding, weakened liquid-liquid phase separation, and reduced transcriptional activity 26 . This research conclusion validates our hypothesis and is highly consistent with our findings. Moreover, we discovered that lactylation can increase p53 degradation through the proteasome pathway, affecting p53 stability. Unfortunately, this phenomenon was not observed in subsequent studies on p53 lactylation sites. We speculate that this discrepancy might be attributed to the existence of other abundant PTMs in the DBD and CTD regions, which, together with lactylation modifications, collectively regulate p53 stability. Overall, our study reveals a significant role of p53 lactylation in colorectal cancer progression. While lactylation of p53 at K291 and K370 does not affect p53 stability, it strongly influences p53 nuclear translocation. The specific mechanism involves reducing p53 accumulation in the nucleus, thereby lowering its transcriptional activity. Our research sheds light on a novel regulatory mechanism linking lactate to p53 function, potentially paving the way for new strategies in colorectal cancer prevention and treatment. Methods Cell culture and treatment H1299 and the colorectal cancer cell lines HCT116 were purchased from Procell. Unless otherwise stated, cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 ºC in 5% (v/v) CO 2 . In follow-up experiments, cells were usually treated with glucose at a concentration of 5 or 25 mM for 24 h, Lactic acid (Sigma, 50-21-5) at a concentration of 25 mM for 24 h, Sodium lactate(Sigma, 867-56-1) at a concentration of 25 mM for 24 h, oxamate (Macklin, S818460-5g) at a concentration of 20 mM for 24 h, 2-Deoxy-D-glucose (MCE, 154-17-6) at a concentration of 20 mM for 24 h, Cycloheximide (MKBio, MS0035) at a concentration of 10 μM, MG-132 (MCE, HY-13259) at a concentration of 10 μM. Human tissue sample Human tissue samples of colorectal cancer were obtained from the Fourth Affiliated Hospital of Jiangsu University (all confirmed by the Department of Pathology). The patients had not received any prior treatment and complete clinical data was available. This study was approved by the Ethics Committee of the Fourth Affiliated Hospital of Jiangsu University and informed consent was obtained from all patients. Quantification of lactate levels The LA Assay Kit (Jiancheng, A019-2-1) was used to determine the content of lactate in tissues and cell lysates. According to the manufacturer's instructions, the sample, chromogenic reagent, and enzyme reaction solution were incubated at 37°C for 10 min, followed by the addition of the stop solution. Lactate content was determined by absorbance measurement at 530 nm. Each experiment was performed in triplicate. CCK-8 assay Cells were digested, resuspended, and seeded at a density of 2 x 10 3 cells per well in a 96-well plate. After incubation at 37°C for 24 hours, the supernatant was removed, and the cells were washed once with PBS. Different culture media were then added, and the cells were incubated for varying durations. At 0, 24, 48, and 72 hours, the culture medium was removed, and the cells were washed once with PBS. 10% CCK-8 solution in culture medium was then added to each well, and the plates were incubated at 37°C for 30 minutes. Finally, the optical density (OD) values were measured at 450 nm using a microplate reader. Wound healing assay Cells were digested, resuspended, and seeded at a density of 6 x 10 4 cells per well in a 6-well plate. Once the cells reached 90% confluence, three vertical lines were uniformly drawn on the cells using a sterile 200 μL pipette tip. The culture medium was then removed, and the cells were washed twice with PBS. Different culture media containing 1% FBS were added, and the cells were incubated at 37°C. Microscopic images were captured at 0 and 24 h, and the cell migration was quantified using ImageJ software. Transwell assay Transwell assays were performed using a 24-well Transwell system (Corning). Cells were pretreated, digested, resuspended in serum-free media, and seeded at a density of 4 x 10 4 cells per well in the upper chamber. 600 μL of culture medium containing 10% FBS was added to the lower chamber. After incubation for 14 hours, cells that migrated to the lower chamber were stained with crystal violet. The migrated cells were then imaged and counted. For invasion assays, a diluted Matrigel solution was pre-coated in the upper chamber before seeding the cells. Coimmunoprecipitation (Co‑IP) The tissue or cell lysates were incubated with p53 antibody (Santa Cruz, DO-1) for 2 h at 4 ºC. Forty microliters of protein A/G beads (Santa Cruz, sc-2003) was prewashed and then incubated with beads for another 2 h. After full washing, After full washing with IP lysate (Servicebio, G2038), 5x protein buffer (Biosharp, BL502A) was added and denatured at 99ºC for 5 min. Proteins were processed by Western blot using the corresponding antibodies. Western blot Tissue and cell lysates were prepared with RIPA lysis buffer (Beyotime, P0013B), and the protein concentrations were determined using a BCA assay kit (Beyotime, P0012S). The same amount of protein (30 μg) was separated by 10% SDS-PAGE and transferredonto PVDF membranes (Millipore, USA). The membranes were blocked with sealing fluid, followed by incubation with primary antibodies overnight at 4 ºC and secondary antibodies for 1 h at 37 ºC. The signals were detected using an ECL kit (Biosharp, BL520B) and quantified with ImageJ software. The following primary antibodies were used: Pan-Kla (PTM-1401, diluted 1:1000); p53 (Santa Cruz, DO-1, diluted 1:2000); p53 (Proteintech, 60283-2-Ig, diluted 1:1000) ; p300 (CST, D1M7C, diluted 1:500); β Actin (Proteintech, 20,536–1-AP, diluted 1:2000); DYKDDDDK tag Monoclonal antibody (Proteintech, 66008-4-Ig, diluted 1:100). Immunofluorescence Cells were fixed overnight with 4% paraformaldehyde. The cells were treated with 0.3% TritonX-100 at room temperature for 10 min. Then cells were blocked with 5% goat serum for 1 h, followed by incubation with primary antibodies at 4 ºC overnight. Then the cells were washed carefully and incubated with secondary antibody combinations for 1 h. Images were taken by fluorescence microscope (Zeiss, Germany). The following primary antibodies were used: p53 (Santa Cruz, DO-1, diluted 1:200); Pan-Kla (PTM-1401, diluted 1:100); DYKDDDDK tag Monoclonal antibody (Proteintech, 66008-4-Ig, diluted 1:100). The following secondary antibodies were used: DyLight 488 Conjugated AffiniPure Goat Anti-rabbit IgG (H+L) (Boster, BA1127); CY3 Conjugated AffiniPure Goat Anti-mouse IgG (H+L) (Boster, BA1031). All secondary antibodies were diluted 1:500. Plasmid transfection H1299 and HCT116 cells were seeded in 12-well plates at over 60% confluence and used for plasmid transfection. Mutants of p53 K291R and K370R with flag p53 WT or flag were constructed and cloned, which were purchased from Shanghai Genechem Co, Ltd. Transfected into H1299 and HCT116 cells with Lipofectamine™3000 (Invitrogen, L3000015). Pan antibody‑based PTM enrichment HCT116 cells were treated with either 25 mM glucose hypoxia or 25 mM lactate for 24 hours. Cells were collected and lysated with IP lysate, lysates were incubated with p53 antibody (Santa Cruz, DO-1) for 2 h at 4 ºC. Then pre-washed protein A/G beads were incubated at 4 ºC for 2 h. After full washing with IP lysate (Servicebio, G2038), 5x protein buffer (Biosharp, BL502A) was added and denatured at 99ºC for 5 min. LC-MS /MS analysis was performed. LC‒MS/MS analysis and database search LC-MS/MS analysis was performed with support from BiotechPack. Samples were subjected to SDS-PAGE followed by staining with Coomassie Brilliant Blue and destaining until complete. After enzymatic digestion, the peptides were desalted using self-packed desalting columns and the solvent was evaporated using a vacuum concentrator at 45°C. LC-MS/MS analysis was then performed, and the resulting raw spectra were searched against a target protein database using Byonic. Statistical analysis Data analysis and graph generation were performed using GraphPad Prism 8.0. Quantitative data are presented as mean ± standard deviation. For comparisons between multiple groups, one-way ANOVA was employed, followed by LSD-t test for pairwise comparisons. For comparisons between groups within a randomized block design, two-way ANOVA was used. The differences at p < 0.05 were considered statistically significant. Declarations Ethical Approval The methods were performed in accordance with relevant guidelines and regulations and approved by the Institutional Animal Care and Use Committee of Jiangsu University (UJS-IACUC-AP-20222030702). Conflicts of interest The authors declare no competing interests. Funding This work was supported by National Natural Science Foundation of China (32270964). Jiangsu Social Development Project (BE2022779). Author Contributions Jie Ma and Shengjun Wang designed the experiments. Yao Dai and Wenxin Da performed the experiments and data analysis. Yao Dai wrote the manuscript. Yan Zhang and Pengtao Bao provided technical support. Bo Shen and Deqiang Wang provided material support. Wei Zhu provided advice and comments. Jie Ma organized and supervised the study. All the authors critically reviewed the manuscript and approved the submitted version. Data availability statement Data are available on reasonable request. References Morgan E, Arnold M, Gini A, Lorenzoni V, Cabasag CJ, Laversanne M, et al. Global burden of colorectal cancer in 2020 and 2040: incidence and mortality estimates from GLOBOCAN. Gut 2023, 72(2): 338–344. Sedlak JC, Yilmaz Ö H, Roper J. Metabolism and Colorectal Cancer. Annu Rev Pathol 2023, 18: 467–492. Xia L, Oyang L, Lin J, Tan S, Han Y, Wu N, et al. The cancer metabolic reprogramming and immune response. Mol Cancer 2021, 20(1): 28. Reznik E, Luna A, Aksoy BA, Liu EM, La K, Ostrovnaya I, et al. A Landscape of Metabolic Variation across Tumor Types. Cell Syst 2018, 6(3): 301–313.e303. Li X, Yang Y, Zhang B, Lin X, Fu X, An Y, et al. Lactate metabolism in human health and disease. Signal Transduct Target Ther 2022, 7(1): 305. Wang T, Ye Z, Li Z, Jing DS, Fan GX, Liu MQ, et al. Lactate-induced protein lactylation: A bridge between epigenetics and metabolic reprogramming in cancer. Cell Prolif 2023, 56(10): e13478. Racker E, Spector M. Warburg effect revisited: merger of biochemistry and molecular biology. Science 1981, 213(4505): 303–307. Liu X, Zhang Y, Li W, Zhou X. Lactylation, an emerging hallmark of metabolic reprogramming: Current progress and open challenges. Front Cell Dev Biol 2022, 10: 972020. Dou X, Fu Q, Long Q, Liu S, Zou Y, Fu D, et al. PDK4-dependent hypercatabolism and lactate production of senescent cells promotes cancer malignancy. Nat Metab 2023, 5(11): 1887–1910. Wang C, Xue L, Zhu W, Liu L, Zhang S, Luo D. Lactate from glycolysis regulates inflammatory macrophage polarization in breast cancer. Cancer Immunol Immunother 2023, 72(6): 1917–1932. Zhang D, Tang Z, Huang H, Zhou G, Cui C, Weng Y, et al. Metabolic regulation of gene expression by histone lactylation. Nature 2019, 574(7779): 575–580. Timofeev O. Editorial: Mutant p53 in Cancer Progression and Personalized Therapeutic Treatments. Front Oncol 2021, 11: 740578. Hassin O, Oren M. Drugging p53 in cancer: one protein, many targets. Nat Rev Drug Discov 2023, 22(2): 127–144. D'Orazi G. p53 Function and Dysfunction in Human Health and Diseases. Biomolecules 2023, 13(3). Zhang L, Hou N, Chen B, Kan C, Han F, Zhang J, et al. Post-Translational Modifications of p53 in Ferroptosis: Novel Pharmacological Targets for Cancer Therapy. Front Pharmacol 2022, 13: 908772. Marques MA, de Andrade GC, Silva JL, de Oliveira GAP. Protein of a thousand faces: The tumor-suppressive and oncogenic responses of p53. Front Mol Biosci 2022, 9: 944955. Wen J, Wang D. Deciphering the PTM codes of the tumor suppressor p53. J Mol Cell Biol 2022, 13(11): 774–785. Liu Y, Su Z, Tavana O, Gu W. Understanding the complexity of p53 in a new era of tumor suppression. Cancer Cell 2024, 42(6): 946–967. Marx C, Sonnemann J, Beyer M, Maddocks ODK, Lilla S, Hauzenberger I, et al. Mechanistic insights into p53-regulated cytotoxicity of combined entinostat and irinotecan against colorectal cancer cells. Mol Oncol 2021, 15(12): 3404–3429. Yang H, Zou X, Yang S, Zhang A, Li N, Ma Z. Identification of lactylation related model to predict prognostic, tumor infiltrating immunocytes and response of immunotherapy in gastric cancer. Front Immunol 2023, 14: 1149989. Yu X, Yang J, Xu J, Pan H, Wang W, Yu X, et al. Histone lactylation: from tumor lactate metabolism to epigenetic regulation. Int J Biol Sci 2024, 20(5): 1833–1854. Yu J, Chai P, Xie M, Ge S, Ruan J, Fan X, et al. Histone lactylation drives oncogenesis by facilitating m(6)A reader protein YTHDF2 expression in ocular melanoma. Genome Biol 2021, 22(1): 85. Lv X, Lv Y, Dai X. Lactate, histone lactylation and cancer hallmarks. Expert Rev Mol Med 2023, 25: e7. Yang Z, Yan C, Ma J, Peng P, Ren X, Cai S, et al. Lactylome analysis suggests lactylation-dependent mechanisms of metabolic adaptation in hepatocellular carcinoma. Nat Metab 2023, 5(1): 61–79. Jiang J, Huang D, Jiang Y, Hou J, Tian M, Li J, et al. Lactate Modulates Cellular Metabolism Through Histone Lactylation-Mediated Gene Expression in Non-Small Cell Lung Cancer. Front Oncol 2021, 11: 647559. Zong Z, Xie F, Wang S, Wu X, Zhang Z, Yang B, et al. Alanyl-tRNA synthetase, AARS1, is a lactate sensor and lactyltransferase that lactylates p53 and contributes to tumorigenesis. Cell 2024, 187(10): 2375–2392.e2333. Engeland K. Cell cycle regulation: p53-p21-RB signaling. Cell Death Differ 2022, 29(5): 946–960. Li J, Cai Z, Vaites LP, Shen N, Mitchell DC, Huttlin EL, et al. Proteome-wide mapping of short-lived proteins in human cells. Mol Cell 2021, 81(22): 4722–4735.e4725. Kisselev AF. Site-Specific Proteasome Inhibitors. Biomolecules 2021, 12(1). Kon N, Churchill M, Li H, Mukherjee S, Manfredi JJ, Gu W. Robust p53 Stabilization Is Dispensable for Its Activation and Tumor Suppressor Function. Cancer Res 2021, 81(4): 935–944. Wang X, Fan W, Li N, Ma Y, Yao M, Wang G, et al. YY1 lactylation in microglia promotes angiogenesis through transcription activation-mediated upregulation of FGF2. Genome Biol 2023, 24(1): 87. Yang K, Fan M, Wang X, Xu J, Wang Y, Tu F, et al. Lactate promotes macrophage HMGB1 lactylation, acetylation, and exosomal release in polymicrobial sepsis. Cell Death Differ 2022, 29(1): 133–146. Fan M, Yang K, Wang X, Chen L, Gill PS, Ha T, et al. Lactate promotes endothelial-to-mesenchymal transition via Snail1 lactylation after myocardial infarction. Sci Adv 2023, 9(5): eadc9465. Li F, Si W, Xia L, Yin D, Wei T, Tao M, et al. Positive feedback regulation between glycolysis and histone lactylation drives oncogenesis in pancreatic ductal adenocarcinoma. Mol Cancer 2024, 23(1): 90. Ho TLF, Lee MY, Goh HC, Ng GYN, Lee JJH, Kannan S, et al. Domain-specific p53 mutants activate EGFR by distinct mechanisms exposing tissue-independent therapeutic vulnerabilities. Nat Commun 2023, 14(1): 1726. Han CW, Lee HN, Jeong MS, Park SY, Jang SB. Structural basis of the p53 DNA binding domain and PUMA complex. Biochem Biophys Res Commun 2021, 548: 39–46. Kumar A, Kumar P, Kumari S, Uversky VN, Giri R. Folding and structural polymorphism of p53 C-terminal domain: One peptide with many conformations. Arch Biochem Biophys 2020, 684: 108342. Laptenko O, Tong DR, Manfredi J, Prives C. The Tail That Wags the Dog: How the Disordered C-Terminal Domain Controls the Transcriptional Activities of the p53 Tumor-Suppressor Protein. Trends Biochem Sci 2016, 41(12): 1022–1034. Vaupel P, Multhoff G. Revisiting the Warburg effect: historical dogma versus current understanding. J Physiol 2021, 599(6): 1745–1757. Wan N, Wang N, Yu S, Zhang H, Tang S, Wang D, et al. Cyclic immonium ion of lactyllysine reveals widespread lactylation in the human proteome. Nat Methods 2022, 19(7): 854–864. Wang H, Guo M, Wei H, Chen Y. Targeting p53 pathways: mechanisms, structures, and advances in therapy. Signal Transduct Target Ther 2023, 8(1): 92. Lee SC, Lin KH, Balogh A, Norman DD, Bavaria M, Kuo B, et al. Dysregulation of lysophospholipid signaling by p53 in malignant cells and the tumor microenvironment. Cell Signal 2021, 78: 109850. Molinari F, Frattini M. Functions and Regulation of the PTEN Gene in Colorectal Cancer. Front Oncol 2013, 3: 326. Zhang M, Zhang Z, Lou Q, Zhang X, Yin F, Yin Y, et al. SIRT1/P53 pathway is involved in the Arsenic induced aerobic glycolysis in hepatocytes L-02 cells. Environ Sci Pollut Res Int 2023, 30(29): 73799–73811. Levine AJ. p53: 800 million years of evolution and 40 years of discovery. Nat Rev Cancer 2020, 20(8): 471–480. Additional Declarations (Not answered) Supplementary Files sfig1.tif Fig. S1 HE staining of colorectal cancer tissues and adjacent tissues (scale bar, 20 mm). s2.tif Fig. S2 p53 lactylation fails to influence colorectal cancer cell invasion. A Multiple lysine residues of p53 undergo lactylation modification in HCT116 cells treated with glucose (25 mM) and LA (25 mM). B Invasion ability of HCT116/H1299 cells overexpressing WT or K291R in 14 h were analyzed by transwell assay (scale bar, 100 mm; n = 3 per group). CInvasion ability of HCT116/H1299 cells overexpressing WT or K370R in 14 h were analyzed by transwell assay (scale bar, 100 mm; n = 3 per group). uneditedwb.pdf unedited blot Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5586218","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":395870726,"identity":"575414d3-8105-4adb-a18a-74faa83bf8a7","order_by":0,"name":"Jie Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBACxgYGAyDFxsPADmEBBYjWwnOASC1AYAChJBJgZhAAzP2Ht0ndqOGT4Zd8Y1D4g8FGdsMB5mcP8DpsRlqxcc4xNh7J2TkGxjwMacYbDrCZG+DXwmP4OIeNjcfgNlALA8PhxA0HeNgk8GrpP2NwOOcfG4/9zTMGhj8Y/hOhpSHH8HFuG9AWCR4DAx6GA0RoAfklt4+NR+JMWoExj0Gy8czDbGZ4tRgCQ0w659sxe/72w9sMf1TYyfYdb36GX0sDmDoGItgMwHHEjE89EMhDqBoQwfyAgOJRMApGwSgYoQAAFqlDqEvth04AAAAASUVORK5CYII=","orcid":"","institution":"The Affiliated Hospital of Jiangsu University","correspondingAuthor":true,"prefix":"","firstName":"Jie","middleName":"","lastName":"Ma","suffix":""},{"id":395870727,"identity":"ee95f27f-1d4f-4d95-a8a4-2872d1fc791b","order_by":1,"name":"Yao Dai","email":"","orcid":"","institution":"The Affiliated Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Dai","suffix":""},{"id":395870728,"identity":"624abe1c-6029-47f4-bdc7-8ca305e71107","order_by":2,"name":"Wenxin Da","email":"","orcid":"","institution":"Jurong Hospital Affiliated to Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Wenxin","middleName":"","lastName":"Da","suffix":""},{"id":395870729,"identity":"50a45917-583e-49c6-a712-1a0589fac6d8","order_by":3,"name":"bo shen","email":"","orcid":"https://orcid.org/0000-0001-5709-5213","institution":"Jiangsu Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"bo","middleName":"","lastName":"shen","suffix":""},{"id":395870730,"identity":"b7315bc2-a738-4ef5-b357-7df7740a42fa","order_by":4,"name":"Yan Zhang","email":"","orcid":"","institution":"The Affiliated Cancer Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Zhang","suffix":""},{"id":395870731,"identity":"9ab03198-d87c-44ea-93fd-81f1f240fdb4","order_by":5,"name":"Pengtao Bao","email":"","orcid":"","institution":"College of Pulmonary \u0026 Critical Care Medicine","correspondingAuthor":false,"prefix":"","firstName":"Pengtao","middleName":"","lastName":"Bao","suffix":""},{"id":395870732,"identity":"db7289ba-8b8d-46e2-956e-ec32c1cf10c0","order_by":6,"name":"Wei Zhu","email":"","orcid":"","institution":"Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zhu","suffix":""},{"id":395870733,"identity":"a78390be-cec4-42fc-96be-109e134d501d","order_by":7,"name":"Shengjun Wang","email":"","orcid":"https://orcid.org/0000-0001-6584-1183","institution":"Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Shengjun","middleName":"","lastName":"Wang","suffix":""},{"id":395870734,"identity":"40b9cabb-488f-4a03-9b58-951e8c25dd56","order_by":8,"name":"Deqiang Wang","email":"","orcid":"","institution":"The Affiliated Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Deqiang","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-12-05 10:46:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5586218/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5586218/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72732141,"identity":"792e6ffb-778a-4c57-a293-4224a252f39c","added_by":"auto","created_at":"2025-01-01 06:54:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10691449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactate and lactylation levels are elevated in colorectal cancer tissues and cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Lactate level of colorectal cancer tissuesand adjacent tissues (n = 10). \u003cstrong\u003eB\u003c/strong\u003e Lactate level of HCT116 cells treated with glucose (25 mM), oxamate (20 mM) and 2-DG (20 mM) for 24 h (n = 3 per group). \u003cstrong\u003eC\u003c/strong\u003e Lactate level of HCT116 cells treated with glucose (5 or 25 mM), LA (25 mM) and Nala (25 mM) for 24 h (n = 3 per group). \u003cstrong\u003eD\u003c/strong\u003e Level of Pan-kla in the HCT116 cells treated with glucose (5 or 25 mM) for 24 h were analyzed by Western blot. \u003cstrong\u003eE\u003c/strong\u003e Pan-kla level in the HCT116 cells treated with glucose (25 mM), oxamate (20 mM) and 2-DG (20 mM) for 24 h were analyzed by Western blot. \u003cstrong\u003eF\u003c/strong\u003e Pan-kla level in the HCT116 cells treated with glucose (25 mM), LA (25 mM) and Nala (25 mM) for 24 h were analyzed by Western blot. \u003cstrong\u003eG\u003c/strong\u003e Pan-kla level in the colorectal cancer tissues and adjacent tissues were analyzed by Western blot (1,2,3 represent different patients; N= adjacent tissues; T=cancer tissues). \u003cstrong\u003eH\u003c/strong\u003e Pan-kla and p53 levels of colorectal cancer tissues and adjacent tissues were analyzed by immunohistochemistry (1,2 represent different patients; scale bar, 20 mm).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5586218/v1/64804836e8f9aefc09dc4d6d.png"},{"id":72732333,"identity":"b3716d20-00c2-4775-b929-2c03845e0495","added_by":"auto","created_at":"2025-01-01 07:02:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30678478,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHyperlactylation of colorectal cancer cells in vitro promotes cell proliferation and metastasis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Proliferative ability of HCT116 cells treated with glucose (5 or 25 mM) and oxamate (20 mM) for 0, 24, 48, 72 h were analyzed by CCK-8 (n = 3 per group). \u003cstrong\u003eB\u003c/strong\u003e Proliferative ability of HCT116 cells treated with glucose (25 mM), LA (25 mM) and Nala (25 mM) for 0, 24, 48, 72 h were analyzed by CCK-8 (n = 3 per group). \u003cstrong\u003eC\u003c/strong\u003e Wound healing ability of HCT116 cells treated with glucose (5 or 25 mM) and oxamate (20 mM) for 24 h were analyzed by wound-healing assay (scale bar, 100 mm; n = 3 per group). \u003cstrong\u003eD\u003c/strong\u003e Migration ability of HCT116 cells treated with glucose (5 or 25 mM), oxamate (20 mM) and 2-DG (20 mM) for 14 h were analyzed by transwell assay (scale bar, 100 mm; n = 3 per group). \u003cstrong\u003eE\u003c/strong\u003e Migration ability of HCT116 cells treated with glucose (25 mM), LA (25 mM) and Nala (25 mM) for 14 h were analyzed by transwell assay (scale bar, 100 mm; n = 3 per group). \u003cstrong\u003eF\u003c/strong\u003e Invasion ability of HCT116 cells treated with glucose (5 or 25 mM), oxamate (20 mM) and 2-DG (20 mM) for 18 h were analyzed by transwell assay (scale bar, 100 mm; n = 3 per group). \u003cstrong\u003eG\u003c/strong\u003e Migration ability of HCT116 cells treated with glucose (25 mM), LA (25 mM) and Nala (25 mM) for 18 h were analyzed by transwell assay (scale bar, 100 mm; n = 3 per group).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5586218/v1/1583870cf2b8b3b9cd7b6f4d.png"},{"id":72732327,"identity":"4b1868c6-228c-4153-947f-fcc58b41260d","added_by":"auto","created_at":"2025-01-01 07:02:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4864290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactylation affects the nuclear translocation and stability of p53 protein.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e p53 level in the cytoplasm and nucleus of HCT116 cells treated with glucose (5 or 25 mM) in 24 h were analyzed by Western blot (n = 3 per group). \u003cstrong\u003eB\u003c/strong\u003e p53 level in the cytoplasm and nucleus of HCT116 cells treated with glucose (25 mM) and oxamate (20 mM) for 24 h were analyzed by Western blot (n = 3 per group). \u003cstrong\u003eC\u003c/strong\u003e p53 level in the cytoplasm and nucleus of HCT116 cells treated with glucose (25 mM) and 2-DG (20 mM) for 24 h were analyzed by Western blot (n = 3 per group). \u003cstrong\u003eD\u003c/strong\u003e p53 level in the cytoplasm and nucleus of HCT116 cells treated with glucose (25 mM), LA (25 mM) and Nala (25 mM) for 24 h were analyzed by Western blot (n = 3 per group). \u003cstrong\u003eE\u003c/strong\u003e p53 level in the HCT116 cells that were pretreated with glucose (5 or 25 mM) and oxamate (20 mM) for 24 h and then treated with CHX (10 mM) for 0, 1, 2, 4 h were analyzed by Western blot (n = 3 per group). \u003cstrong\u003eF\u003c/strong\u003e p53 level in the HCT116 cells that were pretreated with glucose (5 or 25 mM) and oxamate (20 mM) for 24 h and then treated with MG-132 (10 mM) for 0, 2, 4, 8 h were analyzed by Western blot (n = 3 per group). \u003cstrong\u003eG\u003c/strong\u003e p53 level in the HCT116 cells that were pretreated with glucose (25 mM), LA (25 mM) and Nala (25 mM) for 24 h and then treated with CHX (10 mM) for 0, 1, 2, 4 h were analyzed by Western blot (n = 3 per group). \u003cstrong\u003eH\u003c/strong\u003e p53 level in the HCT116 cells that were pretreated with glucose (25 mM), LA (25 mM) and Nala (25 mM) for 24 h and then treated with MG-132 (10 mM) for 0, 2, 4, 8 h were analyzed by Western blot (n = 3 per group).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5586218/v1/a118a66dc81b47fd4035cbb3.png"},{"id":72732167,"identity":"b8b9c1a5-e363-4ecb-a9e3-ac81392d0a89","added_by":"auto","created_at":"2025-01-01 06:54:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10495603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ep53 lactylation levels are elevated in colorectal cancer tissues and cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eRepresentative images of Pan-kla co-stained with p53 in colorectal cancer tissues and adjacent tissues (1, 2 represent different patients; scale bar, 50 mm). \u003cstrong\u003eB\u003c/strong\u003e IP analysis of p53 lactylation in colorectal cancer tissues and adjacent tissues (1, 2, 3, 4, 5 represent different patients; N= adjacent tissues; T= cancer tissues). \u003cstrong\u003eC\u003c/strong\u003e Representative images of Pan-kla co-stained with p53 in HCT116 cells treated with glucose (5 or 25 mM), oxamate (20 mM), LA (25 mM) and Nala (25 mM) for 24 h (scale bar, 20 mm). \u003cstrong\u003eD\u003c/strong\u003e IP analysis of p53 lactylation in HCT116 cells treated with glucose (5 or 25 mM) for 24 h. \u003cstrong\u003eE\u003c/strong\u003e IP analysis of p53 lactylation in HCT116 cells treated with glucose (25 mM), oxamate (20 mM) and 2-DG (20 mM) for 24 h. \u003cstrong\u003eF\u003c/strong\u003e IP analysis of p53 lactylation in HCT116 cells treated with glucose (25 mM), LA (25 mM) and Nala (25 mM) for 24 h. \u003cstrong\u003eG\u003c/strong\u003e p300 level in the HCT116 cells t treated with glucose (5 or 25 mM) by Western blot. \u003cstrong\u003eH\u003c/strong\u003e p300 level in the HCT116 cells that were pretreated with glucose (25 mM), oxamate (20 mM) and 2-DG (20 mM) for 24 h were analyzed by Western blot. \u003cstrong\u003eI\u003c/strong\u003e IP analysis of p53 lactylation in HCT116 cells treated with glucose (25 mM), DMSO and C646 (10 mM) for 24 h were analyzed by Western blot.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5586218/v1/d72191793bbb1e59426efa83.png"},{"id":72732155,"identity":"82374152-6a28-4f1e-9e24-c4e8369f0e5e","added_by":"auto","created_at":"2025-01-01 06:54:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":17623331,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ep53 lactylation promotes colorectal cancer cell proliferation and migration.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eThe MS/MS spectrum of modified “R.TEEENLRK(kla)K.G” (glucose:25 mM) and\u003c/p\u003e\n\u003cp\u003e“R.AHSSHLK(kla)SK.K” (LA:25 mM) were shown. \u003cstrong\u003eB\u003c/strong\u003e HCT116/H1299 cells overexpressing WT or K291R/ K370R Flag-p53 were analyzed by Western blot. \u003cstrong\u003eC\u003c/strong\u003e IP analysis of Flag-p53 lactylation in HCT116/ H1299 cells overexpressing WT or K291R. \u003cstrong\u003eD\u003c/strong\u003e IP analysis of p53 lactylation in HCT116 cells Flag-p53 lactylation in HCT116/H1299 cells overexpressing WT or K370R. \u003cstrong\u003eE\u003c/strong\u003e Proliferative ability of HCT116/ H1299 cells overexpressing WT or K291R in 0 and 72 h were analyzed by CCK-8 (n = 3 per group). \u003cstrong\u003eF\u003c/strong\u003e Proliferative ability of HCT116/H1299 cells overexpressing WT or K370R in 0 and 72 h were analyzed by CCK-8 (n = 3 per group). \u003cstrong\u003eG\u003c/strong\u003e Migration ability of HCT116/H1299 cells overexpressing WT or K291R in 14 h were analyzed by transwell assay (scale bar, 100 mm; n = 3 per group). \u003cstrong\u003eH\u003c/strong\u003e Migration ability of HCT116/H1299 cells overexpressing WT or K370R in 14 h were analyzed by transwell assay (scale bar, 100 mm; n = 3 per group).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5586218/v1/898ab6a4bf0b722e4a80be1e.png"},{"id":72732330,"identity":"21f9be40-68a5-4e1c-819b-01bb507b883d","added_by":"auto","created_at":"2025-01-01 07:02:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6152744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ep53 lactylation affects nuclear translocation of p53 protein.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Representative images of Pan-kla co-stained with Flag-p53 in HCT116/H1299 cells overexpressing WT or K291R (scale bar, 20 mm). \u003cstrong\u003eB\u003c/strong\u003e Representative images of Pan-kla co-stained with Flag-p53 in HCT116/H1299 cells overexpressing WT or K370R (scale bar, 20 mm). \u003cstrong\u003eC\u003c/strong\u003e Flag-p53 level in the cytoplasm and nucleus of HCT116/H1299 cells overexpressing WT or K291R were analyzed by Western blot (n = 3 per group). \u003cstrong\u003eD\u003c/strong\u003e Flag-p53 level in the cytoplasm and nucleus of HCT116/H1299 cells overexpressing WT or K370R were analyzed by Western blot (n = 3 per group). \u003cstrong\u003eE\u003c/strong\u003e Flag-p53 level in the HCT116/H1299 cells overexpressing WT or K291R and then treated with CHX (10 mM) for 0, 4 h were analyzed by Western blot (n = 3 per group). \u003cstrong\u003eF\u003c/strong\u003e Flag-p53 level in the HCT116/H1299 cells overexpressing WT or K370R and then treated with CHX (10 mM) for 0, 4 h were analyzed by Western blot (n = 3 per group). \u003cstrong\u003eG\u003c/strong\u003e Flag-p53 level in the HCT116/H1299 cells overexpressing WT or K291R and then treated with MG-132 (10 mM) for 0, 4 h were analyzed by Western blot (n = 3 per group). \u003cstrong\u003eH\u003c/strong\u003e level Flag-p53 in the HCT116/H1299 cells overexpressing WT or K370R and then treated with MG-132 (10 mM) for 0, 4 h were analyzed by Western blot (n = 3 per group). \u003cstrong\u003eI\u003c/strong\u003e Schematic diagram illustrates the molecular mechanism how p53 lactylation promotes colorectal cancer cells proliferation and migration.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5586218/v1/801aeadf100c907af0931849.png"},{"id":90034630,"identity":"8737f1b7-0c2b-46d1-9692-d305c890fbe4","added_by":"auto","created_at":"2025-08-27 15:40:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":72295215,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5586218/v1/938d859d-4f74-4508-8375-1951bd665abc.pdf"},{"id":72732566,"identity":"a1e10b85-7025-49f2-bf2d-d7bda0e2e5da","added_by":"auto","created_at":"2025-01-01 07:10:15","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2070752,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1 HE staining of colorectal cancer tissues and adjacent tissues \u003c/strong\u003e(scale bar, 20 mm).\u003c/p\u003e","description":"","filename":"sfig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5586218/v1/da6cbf434c5589da2cce12c9.tif"},{"id":72732567,"identity":"e1624b41-cbd5-425f-a93b-7ac8a0039750","added_by":"auto","created_at":"2025-01-01 07:10:16","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5342520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2 p53 lactylation fails to influence colorectal cancer cell invasion.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Multiple lysine residues of p53 undergo lactylation modification in HCT116 cells treated with glucose (25 mM) and LA (25 mM). \u003cstrong\u003eB\u003c/strong\u003e Invasion ability of HCT116/H1299 cells overexpressing WT or K291R in 14 h were analyzed by transwell assay (scale bar, 100 mm; n = 3 per group). \u003cstrong\u003eC\u003c/strong\u003eInvasion ability of HCT116/H1299 cells overexpressing WT or K370R in 14 h were analyzed by transwell assay (scale bar, 100 mm; n = 3 per group).\u003c/p\u003e","description":"","filename":"s2.tif","url":"https://assets-eu.researchsquare.com/files/rs-5586218/v1/1d17a3e7472b5460c0f82291.tif"},{"id":72732331,"identity":"5df63594-1fb8-4ba6-b09c-3f2a7b7fda74","added_by":"auto","created_at":"2025-01-01 07:02:16","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2711962,"visible":true,"origin":"","legend":"unedited blot","description":"","filename":"uneditedwb.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5586218/v1/fd748fde2e9c8c600c0e4bee.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Lactylation affects p53 Nuclear Translocation to Promote Colorectal Cancer Progression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer is the third most common cancer worldwide and the second leading cause of cancer death\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The development of such as diet, disease, genetics, and age, but the exact causes and mechanisms of its development are not fully understood\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Metabolic reprogramming, which refers to alterations in metabolism or nutrient supply, is a hallmark of cancer\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Lactate is one of the most consistently upregulated metabolites, while glucose is consistently downregulated in various cancers\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, a phenomenon known as the \"Warburg effect\"\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Growing evidence suggests that lactate plays a crucial role in metabolic activities such as cell fate, embryonic development, inflammation and immune responses, and tumorigenesis, and it regulates gene expression by modifying histone lysine residues\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, the relationship between lactylation and non-histone proteins remains unclear in the pathogenesis of colorectal cancer.\u003c/p\u003e \u003cp\u003eThe p53 protein is a tumor suppressor protein that plays a key role in many cellular processes, including cell division, maintaining genomic stability, apoptosis, autophagy, and immune responses\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The multi-modular structure of p53 facilitates various covalent modifications, including phosphorylation, ubiquitination, acetylation, methylation, sumoylation, neddylation, glycosylation, hydroxylation, and β-hydroxybutyrylation\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These covalent modifications can alter the activity and function of p53 to a certain extent\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, so understanding the mechanisms of p53 regulation may offer new avenues for cancer treatment strategies to enhance the tumor suppressor function of p53 in cancer cells.\u003c/p\u003e \u003cp\u003eIn this study, we found that lactylation levels are closely related to the biological functions of colorectal cancer cells, and we demonstrated the impact of lactylation on the nuclear translocation and stability of the p53 protein. We then discovered that p53 protein lactylation exists in both colorectal cancer tissues and cells. We performed LC-MS/MS analysis and identified K291 and K370 as the most abundant sites of p53 protein lactylation modification in HCT116 cells. By mutating lysine (K) at positions 291 and 370 to arginine (R), we found that lactylation at these two sites promotes proliferation and migration of colorectal cancer cells and affects the nuclear translocation of the p53 protein. Taken together, our findings suggest that p53 protein lactylation can promote proliferation and metastasis of colorectal cancer cells, and targeting p53 protein lactylation may be an effective strategy for preventing the development of colorectal cancer.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eLactate and lactylation levels are elevated in colorectal cancer tissues and cell lines\u003c/h2\u003e\n\u003cp\u003eLactate and lactylation play important roles in regulating tumorigenesis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, but their specific roles in the development of colorectal cancer remain unclear. We collected colorectal cancer tissue and performed HE staining. Compared to adjacent tissues, colorectal cancer tissues showed signs of carcinogenesis, including disordered glandular structure and nuclear translocation of cancer cells (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). To verify the characteristics of aerobic glycolysis in tumors, we used lactate kit to measure the lactate level in colorectal cancer tissues. The results showed that the lactate level in colorectal cancer tissues was significantly higher than that in adjacent tissues (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), indicating strong glycolysis in colorectal cancer cells. To simulate the characteristics of aerobic glycolysis of colorectal cancer cells in vitro, HCT116 cells were divided into four groups according to different treatments: low glucose group (5 mM glucose), high glucose group (25 mM glucose), LA group (20 mM lactate), and Nala group (20 mM sodium lactate). The results showed that the intracellular lactate level was significantly increased in the high glucose group, LA group, and Nala group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Subsequently, HCT116 cells were treated with glycolysis inhibitor 2-deoxy-D-glucose (2-DG) or LDHA inhibitor oxamate. The results showed that the intracellular lactate level was significantly reduced (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results suggest that colorectal cancer cells have active aerobic glycolysis metabolism and high lactate levels.\u003c/p\u003e\n\u003cp\u003eTo investigate the association between protein lactylation and colorectal cancer, we observed that high glucose treatment significantly elevated pan-lactylation levels in HCT116 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). Conversely, treatment with the glycolysis inhibitors 2-DG or oxamate resulted in a decrease in pan-lactylation levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). Furthermore, treatment with LA and Nala, which promote lactate, led to an increase in pan-lactylation in HCT116 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). Importantly, pan-lactylation levels were significantly higher in colorectal cancer tissues compared to adjacent tissues (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eH). These findings strongly suggest that elevated lactylation is a characteristic of both colorectal cancer cells and tissues, and it is likely associated with glycolytic metabolism.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eHyperlactylation of colorectal cancer cells in vitro promotes cell proliferation and metastasis\u003c/h3\u003e\n\u003cp\u003eRecent studies have consistently reported a positive correlation between elevated lactylation and disease progression in cancer\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Yu et al. found that elevated histone lactylation (H3K18la) levels were observed in human ocular melanoma tissues and cell lines, proving to be an adverse prognostic factor for patients with ocular melanoma\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In non-small cell lung cancer (NSCLC), histone lactylation is enriched on promoters of metabolic-related genes, thereby promoting NSCLC proliferation and migration\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, the impact of protein hyperlactylation on the biological functions of colorectal cancer cells remains unclear.\u003c/p\u003e\n\u003cp\u003eHere, we found that high glucose treatment increased the proliferative capacity of HCT116 cells, while low glucose treatment or oxamate treatment decreased the proliferative capacity of HCT116 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, both LA treatment and Nala treatment did not enhance the proliferative capacity of HCT116 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). High glucose treatment increased the wound healing rate of HCT116 cells at 24 h, while oxamate treatment decreased the wound healing rate of HCT116 cells at 24 h (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). In cell migration and invasion experiments, we found that high glucose treatment increased the number of migrating cells in HCT116 cells at 14 h, while oxamate treatment or 2-DG treatment showed a contrast outcome (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). High glucose treatment increased the number of invading cells in HCT116 cells at 18 h, while oxamate treatment or 2-DG treatment showed a contrast outcome (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). Regrettably, the number of migrating cells and invading cells in the LA and Nala treatment groups did not change significantly (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG). This suggests that under conditions of high lactylation, the proliferation, migration, and invasion abilities of colorectal cancer cells are significantly enhanced.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eLactylation affects the nuclear translocation and stability of p53 protein\u003c/h3\u003e\n\u003cp\u003eRecently, Zong, Z. et al. evaluated the TCGA breast cancer dataset and found that patients carrying wild-type p53 with high serum lactate levels showed lower p53 signaling pathway scores, suggesting that lactylation in tumors may inhibit p53 function. Colorectal cancer HCT116 cells highly express wild-type p53 and have high lactylation levels\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, so we hypothesized whether lactylation might affect the function of the p53 protein in colorectal cancer cells. The p53 protein acts as a transcription factor in tumor cells and plays a powerful role in the nucleus \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In human colorectal cancer tissue, we observed significantly higher p53 levels compared to adjacent tissue, consistent with previous reports (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eH). We investigated its subcellular localization in cells. In cells treated with low glucose, p53 was primarily nuclear. Conversely, p53 predominantly localized to the cytoplasm in cells treated with high glucose (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). This pattern was also observed in cells treated with oxamate or 2-DG (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). However, this phenomenon was not observed in cells treated with LA and Nala (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). This suggests that the mechanism by which lactylation promotes proliferation and metastasis of colorectal cancer cells may be to reduce the expression of the p53 protein in the nucleus, thereby reducing its function as a transcription factor, and ultimately promoting the development and metastasis of colorectal cancer.\u003c/p\u003e\n\u003cp\u003eNext, we further investigated the effect of lactylation on the stability of the p53 protein. Cycloheximide (CHX) is a protein synthesis inhibitor that works by binding to the 80S ribosome and blocking the translocation of tRNA, thereby inhibiting protein synthesis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Therefore, we used CHX to treat HCT116 cells to ensure that no new proteins were produced, so that we could observe the degradation of the p53 protein. The results showed that the p53 protein degradation rate was slower in cells treated with low glucose or oxamate, while the p53 protein degradation rate was faster in cells treated with high glucose (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). Since lactylation can affect the degradation rate of the p53 protein, we further treated HCT116 cells with MG-132 to observe whether lactylation-mediated degradation of the p53 protein occurs through the proteasome pathway. MG-132 is a proteasome inhibitor that effectively blocks the proteolytic activity of the 26S proteasome complex\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The results showed that after 8 h of treatment with MG-132, the p53 protein content was higher in cells treated with high glucose, while the p53 protein content was lower in cells treated with low glucose or oxamte (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF). However, this phenomenon was not observed in cells treated with LA or Nala (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH). These results suggest that lactylation may reduce the stability of the p53 protein through the proteasome pathway, thereby reducing its function as a tumor suppressor protein.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003ep53 lactylation levels are elevated in colorectal cancer tissues and cell lines\u003c/h3\u003e\n\u003cp\u003eGiven the possibility of various post-translational modifications occurring on lysine residues of p53\u003csup\u003e30\u003c/sup\u003e and the previously observed high levels of lactylation in colorectal cancer tissues and cells, we predicted that p53 protein is subject to lactylation. We first observed co-localization of p53 protein and lactylation in both colorectal cancer tissues and cells (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Subsequently, we used co-immunoprecipitation experiments to confirm the presence of p53 lactylation. Our results indicated the presence of p53 lactylation in a portion of colorectal cancer tissues (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). We then observed the same phenomenon in colorectal cancer cells. Results showed that the level of p53 protein lactylation was significantly increased in HCT116 cells treated with high glucose (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD), while the opposite result was observed in the Oxamate and 2-DG treatment groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). Lactate and sodium lactate also increased the level of p53 protein lactylation in HCT116 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e\n\u003cp\u003eLactylation is a new form of acylation modification that may share similar regulatory enzymes with other lysine acylation modifications\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. p300 is currently the most common \"writer\" in lactylation research\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. We detected the level of p300 in HCT116 cells and found that the level of p300 protein was slightly increased in cells treated with high glucose, but the level of p300 protein was significantly reduced in the Oxamate and 2-DG treatment groups (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH). To further validate whether p300 mediates p53 lactylation, we treated cells with the p300 inhibitor (C646) and found that p53 lactylation was significantly inhibited (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI). We therefore deduce that p53 lactylation is mediated by p300.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003ep53 lactylation promotes colorectal cancer cell proliferation and migration\u003c/h3\u003e\n\u003cp\u003ep53 is a well-characterized tumor suppressor protein with six major domains: two intrinsically disordered N-terminal transactivation domains (TADs), a proline-rich domain (PRD), a central DNA-binding domain (DBD), a tetramerization domain (TD), and an intrinsically disordered C-terminal regulatory domain (CTD)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. To identify the specific sites of p53 lactylation, we performed LC-MS/MS analysis, revealing abundant lactylation modifications on multiple lysine residues within the p53 protein in HCT116 cells (Figure S2A). Notably, K291 and K370 emerged as the most prominent sites of lactylation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eTo investigate the functional significance of p53 K291 and K370 lactylation in colorectal cancer cells, we overexpressed Flag-tagged wild-type p53 (WT) and Flag-tagged mutant p53 (K291R/K370R) in HCT116 and H1299 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). The mutant, with lysine (K) residues at positions 291 and 370 replaced by arginine (R), mimicked the de-lactylated state. H1299 cells, deficient in p53, served as a control to eliminate potential variations in endogenous p53 levels. Immunoprecipitation experiments confirmed a significant reduction in p53 lactylation in the mutant group (K291R/K370R) compared to the wild-type group (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD), aligning with the LC-MS/MS findings.\u003c/p\u003e\n\u003cp\u003eFurther functional analysis revealed that p53 K291 and K370 lactylation significantly promoted tumor cell proliferation (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF) and migration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF), but had no effect on cell invasion (Figures S2B and S2C). These observations suggest that lactylation at K291 and K370 may play a critical role in regulating the proliferative and migratory capabilities of tumor cells.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003ep53 lactylation affects nuclear translocation of p53 protein\u003c/h2\u003e\n\u003cp\u003eOur previous findings indicated that lactylation influences the nuclear translocation and stability of the p53 protein. We aimed to further investigate whether lactylation at K291/K370 sites affects p53 protein function. Immunofluorescence results showed that lactylated proteins were widely distributed in both the nucleus and cytoplasm. However, in the WT group, Flag-p53 expression was higher in the cytoplasm than in the nucleus. In contrast, Flag-p53 expression was significantly increased in the nucleus of the mutant group (K291R/K370R) cells (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). To clarify this phenomenon, we performed nuclear-cytoplasmic separation on transfected and pretreated cells. The results revealed that, compared to the WT group, Flag-p53 expression was significantly increased in the nucleus and decreased in the cytoplasm of the mutant group (K291R/K370R) cells (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e\n\u003cp\u003eNext, to investigate the impact of K291/K370 lactylation on p53 protein stability, we treated transfected cells with CHX. The results showed that K291/K370 lactylation did not affect the degradation rate of p53 protein (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF). Similarly, after treating transfected cells with MG-132, we observed that despite the inhibition of the proteasome pathway, Flag-p53 expression was not reduced in the mutant group (K291R/K370R) cells (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eH). This indicates that lactylation at K291 and K370 does not affect p53 protein stability.\u003c/p\u003e\n\u003cp\u003eK291 is located at the end of the DBD region of the p53 protein, which is the region responsible for p53 binding to DNA\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. K370 is located at the CTD region of the p53 protein, which is a regulatory domain containing nuclear export signals and nuclear localization signals\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. This domain is crucial for the function of p53 as a transcription factor in the nucleus and for exporting p53 to the cytoplasm for degradation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Our results show that lactylation at K291 and K370 promotes the accumulation of p53 in the nucleus of tumor cells while reducing its expression in the cytoplasm (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eI). This suggests that lactylation at K291 and K370 may inhibit the tumor suppressor activity of p53 as a transcription factor by this mechanism, thereby promoting tumor cell proliferation and migration.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSince the discovery of the \u0026ldquo;Warburg effect\u0026rdquo;, researchers have been interested in the following questions: (1) why cancer cells prefer aerobic glycolysis over oxidative phosphorylation; (2) how cancer cells utilize lactate metabolism; (3) the potential of targeting lactate metabolic pathways for cancer treatment\u003csup\u003e39\u003c/sup\u003e. Zhang et al. used mass spectrometry analysis to identify a 72.021 Da mass shift on lysine residues in human breast cancer cells, confirming the existence of histone lactylation modification\u003csup\u003e11\u003c/sup\u003e. This discovery opened up new insights into lactate metabolism. With the advancement of mass spectrometry technology, Wan et al. mined publicly available human MeltomeAtlas datasets and found that lactylation modifications are also widespread on human non-histone proteins, particularly prevalent on glycolytic enzymes\u003csup\u003e40\u003c/sup\u003e. This discovery ushered in a new era of research on protein lactylation modifications. While histone lactylation primarily plays a significant role at the transcriptional level, the function of non-histone lactylation remains largely unknown.\u003c/p\u003e\n\u003cp\u003eP53, known as the \u0026quot;guardian of the genome\u0026quot;, plays a crucial role in maintaining genomic stability and preventing tumor development\u003csup\u003e41\u003c/sup\u003e. Dysregulation of p53 can lead to disruptions in cell division, genomic stability, apoptosis, autophagy, and immune responses, ultimately increasing the risk of cancer\u003csup\u003e42\u003c/sup\u003e. Precise regulation of p53 is therefore essential for safeguarding genomic integrity and preventing tumorigenesis. Mechanisms regulating p53 function are multifaceted, with post-translational modifications (PTMs) of p53 being the most widespread and effective\u003csup\u003e17\u003c/sup\u003e. p53 can act as an upstream regulator of aerobic glycolysis\u003csup\u003e43, 44\u003c/sup\u003e. Researchers have combined the expression levels of six proteins (PTEN, p53, GLUT1, PKM2, LDHA, and MCT4) into a scoring system and categorized colorectal cancer patients into three Warburg subtypes (low/medium/high) based on their scores. They found that colorectal cancer patients with Warburg-high subtype tumors had poorer overall survival\u003csup\u003e44\u003c/sup\u003e. These findings suggest a complex regulatory relationship between glycolysis, lactate metabolism, and p53 in colorectal cancer.\u003c/p\u003e\n\u003cp\u003eIn this study, we observed high expression of lactylation and p53 in colorectal cancer tissues and cells. These lactate-mediated proteins undergo lactylation modifications, which subsequently promote proliferation and metastasis of colorectal cancer cells. We further discovered that p53 is lactylated by p300 and demonstrated that p53 is specifically lactylated at its DBD K291 and CTD K370 sites. Interestingly, our findings indicate that lactylation of p53 at K291 and K370 promotes proliferation and migration of colorectal cancer cells by affecting p53 nuclear translocation. The DBD region is crucial for the transcriptional activity of p53, while the CTD region regulates subcellular localization\u0026nbsp;of p53\u003csup\u003e45\u003c/sup\u003e. Based on this, we hypothesize that lactylation may affect transcriptional activity of p53, thereby promoting tumorigenesis. Recent studies have shown that AARS1 can catalyze lactylation of K120 and K139 in the DBD of p53, leading to impaired DNA binding, weakened liquid-liquid phase separation, and reduced transcriptional activity\u003csup\u003e26\u003c/sup\u003e. This research conclusion validates our hypothesis and is highly consistent with our findings. Moreover, we discovered that lactylation can increase p53 degradation through the proteasome pathway, affecting p53 stability. Unfortunately, this phenomenon was not observed in subsequent studies on p53 lactylation sites. We speculate that this discrepancy might be attributed to the existence of other abundant PTMs in the DBD and CTD regions, which, together with lactylation modifications, collectively regulate p53 stability.\u003c/p\u003e\n\u003cp\u003eOverall, our study reveals a significant role of p53 lactylation in colorectal cancer progression. While lactylation of p53 at K291 and K370 does not affect p53 stability, it strongly influences p53 nuclear translocation. The specific mechanism involves reducing p53 accumulation in the nucleus, thereby lowering its transcriptional activity. Our research sheds light on a novel regulatory mechanism linking lactate to p53 function, potentially paving the way for new strategies in colorectal cancer prevention and treatment.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture and treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH1299 and the colorectal cancer cell lines HCT116 were purchased from Procell. Unless otherwise stated, cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 \u0026ordm;C in 5% (v/v) CO\u003csub\u003e2\u003c/sub\u003e. In follow-up experiments, cells were usually treated with glucose at a concentration of 5 or 25 mM for 24 h,\u0026nbsp;Lactic acid\u0026nbsp;(Sigma, 50-21-5) at a concentration of 25 mM for 24 h, Sodium lactate(Sigma, 867-56-1) at a concentration of 25 mM for 24 h, oxamate (Macklin, S818460-5g) at a concentration of 20 mM for 24 h, 2-Deoxy-D-glucose (MCE, 154-17-6) at a concentration of 20 mM for 24 h, Cycloheximide (MKBio, MS0035) at a concentration of 10\u0026nbsp;\u0026mu;M,\u0026nbsp;MG-132 (MCE, HY-13259) at a concentration of 10\u0026nbsp;\u0026mu;M.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman tissue sample\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman tissue samples of colorectal cancer were obtained from the Fourth Affiliated Hospital of Jiangsu University (all confirmed by the Department of Pathology). The patients had not received any prior treatment and complete clinical data was available. This study was approved by the Ethics Committee of the Fourth Affiliated Hospital of Jiangsu University and informed consent was obtained from all patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of lactate levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe LA Assay Kit (Jiancheng, A019-2-1) was used to determine the content of lactate in tissues and cell lysates. According to the manufacturer\u0026apos;s instructions, the sample, chromogenic reagent, and enzyme reaction solution were incubated at 37\u0026deg;C for 10 min, followed by the addition of the stop solution. Lactate content was determined by absorbance measurement at 530 nm. Each experiment was performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCK-8 assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were digested, resuspended, and seeded at a density of 2 x 10\u003csup\u003e3\u003c/sup\u003e cells per well in a 96-well plate. After incubation at 37\u0026deg;C for 24 hours, the supernatant was removed, and the cells were washed once with PBS. Different culture media were then added, and the cells were incubated for varying durations. At 0, 24, 48, and 72 hours, the culture medium was removed, and the cells were washed once with PBS. 10% CCK-8 solution in culture medium was then added to each well, and the plates were incubated at 37\u0026deg;C for 30 minutes. Finally, the optical density (OD) values were measured at 450 nm using a microplate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound healing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were digested, resuspended, and seeded at a density of 6 x 10\u003csup\u003e4\u003c/sup\u003e cells per well in a 6-well plate. Once the cells reached 90% confluence, three vertical lines were uniformly drawn on the cells using a sterile 200 \u0026mu;L pipette tip. The culture medium was then removed, and the cells were washed twice with PBS. Different culture media containing 1% FBS were added, and the cells were incubated at 37\u0026deg;C. Microscopic images were captured at 0 and 24 h, and the cell migration was quantified using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranswell assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTranswell assays were performed using a 24-well Transwell system (Corning). Cells were pretreated, digested, resuspended in serum-free media, and seeded at a density of 4 x 10\u003csup\u003e4\u003c/sup\u003e cells per well in the upper chamber. 600 \u0026mu;L of culture medium containing 10% FBS was added to the lower chamber. After incubation for 14 hours, cells that migrated to the lower chamber were stained with crystal violet. The migrated cells were then imaged and counted. For invasion assays, a diluted Matrigel solution was pre-coated in the upper chamber before seeding the cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCoimmunoprecipitation (Co‑IP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tissue or cell lysates were incubated with p53 antibody (Santa Cruz, DO-1) for 2 h at 4 \u0026ordm;C. Forty microliters of protein A/G beads (Santa Cruz, sc-2003) was prewashed and then incubated with beads for another 2 h. After full washing, After full washing with IP lysate (Servicebio, G2038), 5x protein buffer (Biosharp, BL502A) was added and denatured at 99\u0026ordm;C for 5 min. Proteins were processed by Western blot using the corresponding antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue and cell lysates were prepared with RIPA lysis buffer (Beyotime, P0013B), and the protein concentrations were determined using a BCA assay kit (Beyotime, P0012S). The same amount of protein (30 \u0026mu;g) was separated by 10% SDS-PAGE and transferredonto PVDF membranes (Millipore, USA). The membranes were blocked with sealing fluid, followed by incubation with primary antibodies overnight at 4 \u0026ordm;C and secondary antibodies for 1 h at 37 \u0026ordm;C. The signals were detected using an ECL kit (Biosharp, BL520B) and quantified with ImageJ software. The following primary antibodies were used: Pan-Kla (PTM-1401, diluted 1:1000); p53 (Santa Cruz, DO-1, diluted 1:2000); p53 (Proteintech, 60283-2-Ig, diluted 1:1000) ; p300 (CST, D1M7C, diluted 1:500); \u0026beta; Actin (Proteintech, 20,536\u0026ndash;1-AP, diluted 1:2000); DYKDDDDK tag Monoclonal antibody (Proteintech, 66008-4-Ig, diluted 1:100).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were fixed overnight with 4% paraformaldehyde. The cells were treated with 0.3% TritonX-100 at room temperature for 10 min. Then cells were blocked with 5% goat serum for 1 h, followed by incubation with primary antibodies at 4 \u0026ordm;C overnight. Then the cells were washed carefully and incubated with secondary antibody combinations for 1 h. Images were taken by fluorescence microscope (Zeiss, Germany). The following primary antibodies were used: p53 (Santa Cruz,\u0026nbsp;DO-1,\u0026nbsp;diluted 1:200); Pan-Kla (PTM-1401, diluted 1:100);\u0026nbsp;DYKDDDDK tag Monoclonal antibody (Proteintech,\u0026nbsp;66008-4-Ig, diluted 1:100). The following secondary antibodies were used: DyLight 488 Conjugated AffiniPure Goat Anti-rabbit IgG (H+L) (Boster, BA1127); CY3 Conjugated AffiniPure Goat Anti-mouse IgG (H+L) (Boster, BA1031). All secondary antibodies were diluted 1:500.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH1299 and HCT116 cells were seeded in 12-well plates at over 60% confluence and used for plasmid transfection. Mutants of p53 K291R and K370R with flag p53 WT or flag were constructed and cloned, which were purchased from Shanghai Genechem Co, Ltd. Transfected into H1299 and HCT116 cells with Lipofectamine\u0026trade;3000 (Invitrogen, L3000015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePan antibody‑based PTM enrichment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHCT116 cells were treated with either 25 mM glucose hypoxia or 25 mM lactate for 24 hours. Cells were collected and lysated with IP lysate, lysates were incubated with p53 antibody (Santa Cruz, DO-1) for 2 h at 4 \u0026ordm;C. Then pre-washed protein A/G beads were incubated at 4 \u0026ordm;C for 2 h. After full washing with IP lysate (Servicebio, G2038), 5x protein buffer (Biosharp, BL502A) was added and denatured at 99\u0026ordm;C for 5 min. LC-MS /MS analysis was performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC‒MS/MS analysis and database search\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLC-MS/MS analysis was performed with support from BiotechPack. Samples were subjected to SDS-PAGE followed by staining with Coomassie Brilliant Blue and destaining until complete. After enzymatic digestion, the peptides were desalted using self-packed desalting columns and the solvent was evaporated using a vacuum concentrator at 45\u0026deg;C. LC-MS/MS analysis was then performed, and the resulting raw spectra were searched against a target protein database using Byonic.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData analysis and graph generation were performed using GraphPad Prism 8.0. Quantitative data are presented as mean \u0026plusmn; standard deviation. For comparisons between multiple groups, one-way ANOVA was employed, followed by LSD-t test for pairwise comparisons. For comparisons between groups within a randomized block design, two-way ANOVA was used. The differences at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthical Approval\u003c/strong\u003e \u003cp\u003eThe methods were performed in accordance with relevant guidelines and regulations and approved by the Institutional Animal Care and Use Committee of Jiangsu University (UJS-IACUC-AP-20222030702).\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by National Natural Science Foundation of China (32270964). Jiangsu Social Development Project (BE2022779).\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eJie Ma and Shengjun Wang designed the experiments. Yao Dai and Wenxin Da performed the experiments and data analysis. Yao Dai wrote the manuscript. Yan Zhang and Pengtao Bao provided technical support. Bo Shen and Deqiang Wang provided material support. Wei Zhu provided advice and comments. Jie Ma organized and supervised the study. All the authors critically reviewed the manuscript and approved the submitted version.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eData are available on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMorgan E, Arnold M, Gini A, Lorenzoni V, Cabasag CJ, Laversanne M, \u003cem\u003eet al.\u003c/em\u003e Global burden of colorectal cancer in 2020 and 2040: incidence and mortality estimates from GLOBOCAN. Gut 2023, 72(2): 338\u0026ndash;344.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSedlak JC, Yilmaz \u0026Ouml; H, Roper J. Metabolism and Colorectal Cancer. Annu Rev Pathol 2023, 18: 467\u0026ndash;492.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia L, Oyang L, Lin J, Tan S, Han Y, Wu N, \u003cem\u003eet al.\u003c/em\u003e The cancer metabolic reprogramming and immune response. Mol Cancer 2021, 20(1): 28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReznik E, Luna A, Aksoy BA, Liu EM, La K, Ostrovnaya I, \u003cem\u003eet al.\u003c/em\u003e A Landscape of Metabolic Variation across Tumor Types. Cell Syst 2018, 6(3): 301\u0026ndash;313.e303.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X, Yang Y, Zhang B, Lin X, Fu X, An Y, \u003cem\u003eet al.\u003c/em\u003e Lactate metabolism in human health and disease. Signal Transduct Target Ther 2022, 7(1): 305.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang T, Ye Z, Li Z, Jing DS, Fan GX, Liu MQ, \u003cem\u003eet al.\u003c/em\u003e Lactate-induced protein lactylation: A bridge between epigenetics and metabolic reprogramming in cancer. Cell Prolif 2023, 56(10): e13478.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRacker E, Spector M. Warburg effect revisited: merger of biochemistry and molecular biology. Science 1981, 213(4505): 303\u0026ndash;307.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Zhang Y, Li W, Zhou X. Lactylation, an emerging hallmark of metabolic reprogramming: Current progress and open challenges. Front Cell Dev Biol 2022, 10: 972020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDou X, Fu Q, Long Q, Liu S, Zou Y, Fu D, \u003cem\u003eet al.\u003c/em\u003e PDK4-dependent hypercatabolism and lactate production of senescent cells promotes cancer malignancy. Nat Metab 2023, 5(11): 1887\u0026ndash;1910.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang C, Xue L, Zhu W, Liu L, Zhang S, Luo D. Lactate from glycolysis regulates inflammatory macrophage polarization in breast cancer. Cancer Immunol Immunother 2023, 72(6): 1917\u0026ndash;1932.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang D, Tang Z, Huang H, Zhou G, Cui C, Weng Y, \u003cem\u003eet al.\u003c/em\u003e Metabolic regulation of gene expression by histone lactylation. Nature 2019, 574(7779): 575\u0026ndash;580.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTimofeev O. Editorial: Mutant p53 in Cancer Progression and Personalized Therapeutic Treatments. Front Oncol 2021, 11: 740578.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHassin O, Oren M. Drugging p53 in cancer: one protein, many targets. Nat Rev Drug Discov 2023, 22(2): 127\u0026ndash;144.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD'Orazi G. p53 Function and Dysfunction in Human Health and Diseases. Biomolecules 2023, 13(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Hou N, Chen B, Kan C, Han F, Zhang J, \u003cem\u003eet al.\u003c/em\u003e Post-Translational Modifications of p53 in Ferroptosis: Novel Pharmacological Targets for Cancer Therapy. Front Pharmacol 2022, 13: 908772.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarques MA, de Andrade GC, Silva JL, de Oliveira GAP. Protein of a thousand faces: The tumor-suppressive and oncogenic responses of p53. Front Mol Biosci 2022, 9: 944955.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen J, Wang D. Deciphering the PTM codes of the tumor suppressor p53. J Mol Cell Biol 2022, 13(11): 774\u0026ndash;785.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Su Z, Tavana O, Gu W. Understanding the complexity of p53 in a new era of tumor suppression. Cancer Cell 2024, 42(6): 946\u0026ndash;967.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarx C, Sonnemann J, Beyer M, Maddocks ODK, Lilla S, Hauzenberger I, \u003cem\u003eet al.\u003c/em\u003e Mechanistic insights into p53-regulated cytotoxicity of combined entinostat and irinotecan against colorectal cancer cells. Mol Oncol 2021, 15(12): 3404\u0026ndash;3429.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang H, Zou X, Yang S, Zhang A, Li N, Ma Z. Identification of lactylation related model to predict prognostic, tumor infiltrating immunocytes and response of immunotherapy in gastric cancer. Front Immunol 2023, 14: 1149989.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu X, Yang J, Xu J, Pan H, Wang W, Yu X, \u003cem\u003eet al.\u003c/em\u003e Histone lactylation: from tumor lactate metabolism to epigenetic regulation. Int J Biol Sci 2024, 20(5): 1833\u0026ndash;1854.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu J, Chai P, Xie M, Ge S, Ruan J, Fan X, \u003cem\u003eet al.\u003c/em\u003e Histone lactylation drives oncogenesis by facilitating m(6)A reader protein YTHDF2 expression in ocular melanoma. Genome Biol 2021, 22(1): 85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLv X, Lv Y, Dai X. Lactate, histone lactylation and cancer hallmarks. Expert Rev Mol Med 2023, 25: e7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Z, Yan C, Ma J, Peng P, Ren X, Cai S, \u003cem\u003eet al.\u003c/em\u003e Lactylome analysis suggests lactylation-dependent mechanisms of metabolic adaptation in hepatocellular carcinoma. Nat Metab 2023, 5(1): 61\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang J, Huang D, Jiang Y, Hou J, Tian M, Li J, \u003cem\u003eet al.\u003c/em\u003e Lactate Modulates Cellular Metabolism Through Histone Lactylation-Mediated Gene Expression in Non-Small Cell Lung Cancer. Front Oncol 2021, 11: 647559.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZong Z, Xie F, Wang S, Wu X, Zhang Z, Yang B, \u003cem\u003eet al.\u003c/em\u003e Alanyl-tRNA synthetase, AARS1, is a lactate sensor and lactyltransferase that lactylates p53 and contributes to tumorigenesis. Cell 2024, 187(10): 2375\u0026ndash;2392.e2333.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEngeland K. Cell cycle regulation: p53-p21-RB signaling. Cell Death Differ 2022, 29(5): 946\u0026ndash;960.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Cai Z, Vaites LP, Shen N, Mitchell DC, Huttlin EL, \u003cem\u003eet al.\u003c/em\u003e Proteome-wide mapping of short-lived proteins in human cells. Mol Cell 2021, 81(22): 4722\u0026ndash;4735.e4725.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKisselev AF. Site-Specific Proteasome Inhibitors. Biomolecules 2021, 12(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKon N, Churchill M, Li H, Mukherjee S, Manfredi JJ, Gu W. Robust p53 Stabilization Is Dispensable for Its Activation and Tumor Suppressor Function. Cancer Res 2021, 81(4): 935\u0026ndash;944.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Fan W, Li N, Ma Y, Yao M, Wang G, \u003cem\u003eet al.\u003c/em\u003e YY1 lactylation in microglia promotes angiogenesis through transcription activation-mediated upregulation of FGF2. Genome Biol 2023, 24(1): 87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang K, Fan M, Wang X, Xu J, Wang Y, Tu F, \u003cem\u003eet al.\u003c/em\u003e Lactate promotes macrophage HMGB1 lactylation, acetylation, and exosomal release in polymicrobial sepsis. Cell Death Differ 2022, 29(1): 133\u0026ndash;146.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan M, Yang K, Wang X, Chen L, Gill PS, Ha T, \u003cem\u003eet al.\u003c/em\u003e Lactate promotes endothelial-to-mesenchymal transition via Snail1 lactylation after myocardial infarction. Sci Adv 2023, 9(5): eadc9465.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi F, Si W, Xia L, Yin D, Wei T, Tao M, \u003cem\u003eet al.\u003c/em\u003e Positive feedback regulation between glycolysis and histone lactylation drives oncogenesis in pancreatic ductal adenocarcinoma. Mol Cancer 2024, 23(1): 90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHo TLF, Lee MY, Goh HC, Ng GYN, Lee JJH, Kannan S, \u003cem\u003eet al.\u003c/em\u003e Domain-specific p53 mutants activate EGFR by distinct mechanisms exposing tissue-independent therapeutic vulnerabilities. Nat Commun 2023, 14(1): 1726.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan CW, Lee HN, Jeong MS, Park SY, Jang SB. Structural basis of the p53 DNA binding domain and PUMA complex. Biochem Biophys Res Commun 2021, 548: 39\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar A, Kumar P, Kumari S, Uversky VN, Giri R. Folding and structural polymorphism of p53 C-terminal domain: One peptide with many conformations. Arch Biochem Biophys 2020, 684: 108342.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaptenko O, Tong DR, Manfredi J, Prives C. The Tail That Wags the Dog: How the Disordered C-Terminal Domain Controls the Transcriptional Activities of the p53 Tumor-Suppressor Protein. Trends Biochem Sci 2016, 41(12): 1022\u0026ndash;1034.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaupel P, Multhoff G. Revisiting the Warburg effect: historical dogma versus current understanding. J Physiol 2021, 599(6): 1745\u0026ndash;1757.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan N, Wang N, Yu S, Zhang H, Tang S, Wang D, \u003cem\u003eet al.\u003c/em\u003e Cyclic immonium ion of lactyllysine reveals widespread lactylation in the human proteome. Nat Methods 2022, 19(7): 854\u0026ndash;864.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang H, Guo M, Wei H, Chen Y. Targeting p53 pathways: mechanisms, structures, and advances in therapy. Signal Transduct Target Ther 2023, 8(1): 92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee SC, Lin KH, Balogh A, Norman DD, Bavaria M, Kuo B, \u003cem\u003eet al.\u003c/em\u003e Dysregulation of lysophospholipid signaling by p53 in malignant cells and the tumor microenvironment. Cell Signal 2021, 78: 109850.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMolinari F, Frattini M. Functions and Regulation of the PTEN Gene in Colorectal Cancer. Front Oncol 2013, 3: 326.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang M, Zhang Z, Lou Q, Zhang X, Yin F, Yin Y, \u003cem\u003eet al.\u003c/em\u003e SIRT1/P53 pathway is involved in the Arsenic induced aerobic glycolysis in hepatocytes L-02 cells. Environ Sci Pollut Res Int 2023, 30(29): 73799\u0026ndash;73811.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLevine AJ. p53: 800 million years of evolution and 40 years of discovery. Nat Rev Cancer 2020, 20(8): 471\u0026ndash;480.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5586218/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5586218/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLysine lactylation is a post-translational modification that connects lactate metabolism with protein function. Our study identifies lysine lactylation of p53 in colorectal cancer tissues and cells. This modification results in increased cytoplasmic accumulation and reduced nuclear accumulation of p53, along with enhanced protein degradation via the proteasome pathway. These changes collectively promote the proliferation, migration, and invasion of colorectal cancer cells. Specifically, we observe enrichment of lactate groups at lysine 291 within the p53 DNA-binding domain and lysine 370 in its C-terminal regulatory domain. Mutating these lysine residues to arginine decreased cytoplasmic accumulation and increased nuclear localization of p53, thereby inhibiting colorectal cancer cells proliferation and migration. Our findings suggest that p53 lactylation contributes to tumorigenesis by modulating its nuclear translocation.\u003c/p\u003e","manuscriptTitle":"Lactylation affects p53 Nuclear Translocation to Promote Colorectal Cancer Progression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-01 06:54:10","doi":"10.21203/rs.3.rs-5586218/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d40164cd-e81c-43c8-820e-5b0eb53f771b","owner":[],"postedDate":"January 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42171524,"name":"Biological sciences/Cancer/Cancer metabolism"},{"id":42171525,"name":"Biological sciences/Diseases"}],"tags":[],"updatedAt":"2025-08-27T15:31:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-01 06:54:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5586218","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5586218","identity":"rs-5586218","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}