Transcription factor ONECUT3 regulates HDAC6/HIF-1α activity to promote the Warburg effect and tumor growth in colorectal cancer

Transcription factor ONECUT3 regulates HDAC6/HIF-1α activity to promote the Warburg effect and tumor growth in colorectal cancer | 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 Transcription factor ONECUT3 regulates HDAC6/HIF-1α activity to promote the Warburg effect and tumor growth in colorectal cancer Junli Xue, Junli Xue, Hao Wang, Weihan Li, Kexin He, Shan Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4296431/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Mar, 2025 Read the published version in Cell Death & Disease → Version 1 posted 8 You are reading this latest preprint version Abstract The Warburg effect, also known as aerobic glycolysis, plays a crucial role in the onset and progression of colorectal cancer, although its mechanism remains unclear. Bioinformatics analysis of public databases and verification of clinical specimens revealed that the transcription factor ONECUT3 is a key regulator related to the Warburg effect in colorectal cancer. Functionally, genetic silencing of ONECUT3 reverses the Warburg effect and blunts tumor growth. Importantly, ONECUT3 promotes tumor growth in a glycolysis-dependent manner. Mechanistically, ONECUT3 does not directly alter the expression of hypoxia-inducible factor 1α (HIF-1α), but rather inhibits the acetylation of HIF-1α via histone deacetylase 6 (HDAC6). This inhibition leads to increased transcriptional activity of HIF-1α, ultimately upregulating various glycolysis-related genes downstream of HIF-1α, thereby promoting the Warburg effect in colorectal cancer and facilitating tumor growth. Our study provides evidence for the mechanism of the Warburg effect in colorectal cancer, suggesting that ONECUT3 could be a potential new target for colorectal cancer treatment. Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer/Colon cancer Health sciences/Diseases/Cancer/Cancer metabolism the Warburg effect colorectal cancer ONECUT3 HIF-1α HDAC6 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Colorectal cancer (CRC) is a prevalent form of cancer, ranking third in terms of incidence and second in terms of cancer-related mortality worldwide. Despite advancements in diagnosis and treatment methods, the incidence and mortality rates of colorectal cancer continue to rise, particularly in developing countries (including China) and among young adults (< 50 years old). It is projected that by 2040, there will be 3.2 million new cases and 1.6 million deaths from CRC 1 . This poses a significant threat to human survival and imposes a substantial economic burden. Therefore, deeper understanding of the specific molecular mechanisms involved in CRC and developing targeted drugs could potentially improve the disease prognosis. Unlike normal tissue, tumor cells undergo metabolic reprogramming, which is a significant hallmark of cancer 2, 3 . Even in the presence of sufficient oxygen supply, tumor cells predominantly metabolize glucose through glycolysis, resulting in lactate accumulation. This phenomenon, known as aerobic glycolysis, is commonly referred to as the Warburg effect 4 . Numerous studies have demonstrated that the Warburg effect remodels the tumor microenvironment and plays a crucial role in the initiation, progression, metastasis, and prognosis of colon cancer 5-8 . Research has confirmed that transcription factors can regulate tumor glycolysis through various mechanisms, including modulation of the glycolysis phase, expression of related genes, and regulation of key enzyme activities involved in glycolysis 9-12 . The process of glycolysis is primarily regulated by hypoxia-inducible factor 1α (HIF-1α). Under hypoxic conditions, HIF-1α forms a heterodimer with HIF-1β, which translocates to the nucleus and transcriptionally activates downstream target genes (e.g., GLUT1, etc.). The regulation of HIF-1α in colorectal cancer involves common mutated genes (APC, RAS, TP53) and epigenetic modifications 5, 13-17 . However, the specific mechanism underlying the regulation of HIF-1α in colorectal cancer is still unclear. The ONECUT family, which consists of ONECUT1 (HNF6), ONECUT2, and ONECUT3, possesses a single “CUT” DNA binding domain and an atypical homologous domain, and functions as translational factors 18 . The three members of the ONECUT family, which are highly conserved from Drosophila to humans, play a crucial role in regulating the development of various tissues derived from the ectoderm or endoderm, as well as activating the expression of numerous gene families 19, 20 . It has been reported that ONECUT1 promotes colon cancer cell proliferation and liver metastasis by enhancing cell adhesion ability and inhibiting apoptosis 21 . In gastric cancer, the upregulation of ONECUT2 expression promotes cell migration, invasion, epithelial-mesenchymal transition, and tumor growth 22 . Similarly, elevated ONECUT2 expression is associated with lymph node metastasis 23 , and poorer prognosis in CRC 24 . However, as a new member of the ONECUT family 25 , there has been relatively limited research on the relationship between ONECUT3 and cancer, particularly in terms of glucose metabolism reprogramming. In this study, we demonstrated that ONECUT3 mediates HIF-1α deacetylation through histone deacetylase 6 (HDAC6), leading to the activation of HIF-1α transcription and its downstream glycolysis genes, thereby enhancing the Warburg effect and promoting tumor growth in CRC. The ONECUT3-HDAC6-HIF-1α axis is anticipated to serve as a potential therapeutic target for modulating aerobic glycolysis in colorectal cancer. 2. Results 2.1 ONECUT3 is a key transcription factor related to glycolysis in colorectal cancer To elucidate the genes associated with aerobic glycolysis in colorectal cancer, mRNA data from the COAD subset of the TCGA database were analyzed. In the TCGA-COAD database, colon cancer samples were categorized into two groups based on the expression level of glycolysis-related genes: the high-glycolysis group and the low-glycolysis group (Figure 1A). A total of 400 differentially expressed genes (DEGs) were identified using a significance level of Log 2 |Fold change| ≥1 and p<0.05. Among these, 127 genes were up-regulated in the high-glycolysis group, including 11 transcription factors (Figure 1B-C). Among the 11 transcription factors, ONECUT3 (Figure 1D) was identified as the most significant DEG. Therefore, ONECUT3 was selected as the key gene for investigating the glycolytic pathway in colon cancer. Subsequently, immunohistochemical staining was performed to determine the expression of ONECUT3 in both colon cancer tissues and paracancerous tissues. The results revealed a significant increase in ONECUT3 expression in colon cancer tissues (Figure 1E), suggesting a potential regulatory role of ONECUT3 in colon cancer through glycolysis. 2.2 Regulation of glycolytic metabolism in colorectal cancer cells by ONECUT3 Initially, we detected the mRNA and protein expression of ONECUT3 in six colon cancer cell lines (CACO2, COLO205, HT29, LOVO, SW480, SW620) and a normal colon epithelial cell (NCM460) (Figure 2A-B). The expression of ONECUT3 was highest in HT29 and LOVO cell lines, while lowest in SW620 and COLO205 cell lines. Subsequently, ONECUT3 was knocked down in the high-expressing cell lines (HT29 and LOVO) and overexpressed in the low-expressing cell lines (SW620 and COLO205), as depicted in Figure 2C and 2E. We conducted an extracellular acidification rate assay in colon cancer cells to examine the regulatory role of ONECUT3 in glycolysis. The results demonstrated a significant decrease in the extracellular acidification rate (ECAR), an indicator of glycolysis, following ONECUT3 knockdown (Figure 2D), and a significant increase after ONECUT3 overexpression in colon cancer cells (Figure 2F). These findings indicate that ONECUT3 promotes the glycolytic metabolism of colon cancer cells. 2.3 ONECUT3 promotes the growth of colorectal cancer cells in a Warburg effect-dependent manner We investigated whether ONECUT3 could influence the proliferation of colon cancer cells through the Warburg effect, which is known to enhance tumor cell proliferation. In the colony-forming assay, knockdown of ONECUT3 significantly inhibited the proliferation of HT29 and LOVO cell lines (Figure 3A), while overexpression of ONECUT3 significantly promoted the proliferation of SW620 and COLO205 cell lines (Figure 3C). The promotion of proliferation by ONECUT3 overexpression was largely abolished by the glycolysis inhibitor 2-Deoxy-D-Glucose (2-DG) (Figure 3D). We used a nude mouse subcutaneous transplantation tumor model to verify the effect of ONECUT3 on tumor growth in vivo. Likewise, knockdown of ONECUT3 significantly reduced the weight of the tumors, indicating a significant inhibition of colon cancer growth by ONECUT3 (Figure 3B). These results indicated that ONECUT3 promoted tumor growth on a Warburg effect dependent manner. 2.4 ONECUT3 enhances the transcriptional activity of HIF-1α and regulates the expression of its downstream glycolytic enzymes To determine if transcription factor ONECUT3 can directly regulate the transcription of glycolytic genes, we utilized online databases including JASPAR, ALGGEN, and hTFtarget to predict the genes that are regulated by ONECUT3. No glycolysis-related genes were found. Subsequently, RNA sequencing was employed to analyze the changes in the transcriptome resulting from ONECUT3 knockdown in colon cells. The results of Gene Set Enrichment Analysis (GSEA) indicated the close involvement of ONECUT3 in glycolytic and hypoxic signaling pathways (Figure 4A). Subsequently, the expression of key genes in glycolytic and hypoxic signaling pathways was assessed at both the RNA and protein levels. Cytological experiments confirmed that the expression of GLUT1, ALDOA, and PKM2 was reduced after ONECUT3 knockdown (Figure 4B-C). These enzymes are downstream targets of HIF-1α and play a crucial role in the glycolytic pathway. Given that HIF-1α is a key regulator of glycolytic signaling, it is hypothesized that ONECUT3 may be involved in the regulation of glycolytic metabolism through HIF-1α. We measured the expression level of HIF-1α after knocking down or overexpressing ONECUT3. However, the subsequent experimental results showed that knockdown or overexpressing ONECUT3 in colon cancer cells did not affect the mRNA or protein expression of HIF-1α (Figure 4D-G). Therefore, we investigated whether ONECUT3 affects the expression of downstream genes of HIF-1α by regulating its transcriptional activity. Experiments using the HIF-1 alpha Transcription Factor Assay kit revealed that knocking down ONECUT3 significantly suppressed HIF-1α transcriptional activity (Figure 4H), while overexpressing ONECUT3 enhanced HIF-1α transcriptional activity (Figure 4I). Since both ONECUT3 and HIF-1α are transcription factors, we hypothesized that they can directly bind and interact with each other in the nucleus. However, the Co-IP assay did not provide evidence of a direct interaction between ONECUT3 and HIF-1α (Figure 4J). This suggests that ONECUT3 may enhance the transcriptional activity of HIF-1α and influence the expression of multiple downstream glycolytic enzymes, but not through direct interaction with HIF-1α. 2.5 ONECUT3 regulates HIF-1α transcriptional activity through HDAC6 To elucidate the interaction mechanism between ONECUT3 and HIF-1α, we initially screened 438 genes regulated by ONECUT3 using RNA-seq analysis (Figure 5A). Subsequently, we identified 539 proteins that interact with HIF-1α through Co-IP and mass spectrometry. By performing a Venn analysis (Figure 5B), we obtained five genes (NAA10, HNF4A, TCEB2, HDAC6, and ELAVL1) that were present in both groups. Previous studies have shown that inhibiting HDAC6 selectively can decrease HIF-1-mediated transcription in hypoxic conditions. Additionally, HDAC inhibitors can suppress tumor angiogenesis and the expression of HIF-1α protein 26, 27 . Based on these findings, we hypothesized that ONECUT3 may regulate the transcriptional activity of HIF-1α by modulating HDAC6. Supporting our hypothesis, knockdown of ONECUT3 significantly reduced HDAC6 expression in both qRT-PCR and Western Blot analyses (Figure 5C-D). Additional investigations using ChIP-PCR and luciferase reporter gene assays revealed that ONECUT3 binds to the HDAC6 promoter region and directly regulates its transcription in colon cancer cells (Figure 5E-F). 2.6 ONECUT3 facilitates the deacetylation of HIF-1α and enhances its transcriptional activity through HDAC6 To validate the involvement of HDAC6 in the regulation of HIF-1α transcriptional activity by ONECUT3, we performed knockdown experiments for ONECUT3 or HDAC6 in colon cancer cells. Subsequently, HIF-1α was enriched through immunoprecipitation (IP). Post-translational modifications are known to affect the activity of HIF-1α, with acetylation leading to decreased stability 28 . In normoxic conditions, the hydroxylation of two proline residues and the acetylation of a lysine residue on the oxygen-dependent degradation domain (ODDD) of HIF-1α facilitate its binding to the pVHL E3 ligase complex, resulting in its degradation through the ubiquitin-proteasome pathway 29 . The acetylation level of HIF-1α was determined using a panacetylation antibody. Consistent expression of HIF-1α resulted in a significant increase in its acetylation level following knockdown of ONECUT3 or HDAC6 (Figure 6A). Conversely, overexpression of ONECUT3 led to a significant reduction in the acetylation level of HIF-1α (Figure 6B). Additionally, the facilitative effect of ONECUT3 overexpression on HIF-1α deacetylation could be reversed by using ACY241 to inhibit HDAC6 (Figure 6B). Notably, the inhibition of HDAC6 significantly eliminated the enhanced HIF-1α transcriptional activity in ONECUT3 overexpressing cells (Figure 6C). These results indicate that the regulatory impact of ONECUT3 on HIF-1α deacetylation and transcriptional activity is mediated by HDAC6. 2.7 Expression of ONECUT3 in colorectal cancer and its clinical significance To enhance clinical relevance, we conducted immunohistochemical staining on microarrays of colorectal cancer patients from the Renji Hospital cohort (Figure 7A) to examine the expression of ONECUT3, HIF-1A, HDAC6, and glycolysis-related proteins. The expression of ONECUT3 showed a positive correlation with certain glycolysis-related proteins (ALDOA, TPI1) and HIF-α (P < 0.05). Additionally, both HIF-α and HDAC6 exhibited a positive correlation with the expression of glycolysis-related proteins (ALDOA, TPI1, GLUT1, and ENO1) (P < 0.05). The expression of HIF-α and HDAC6 showed a positive correlation (P < 0.05) (Figure 7B). We conducted a correlation analysis between ONECUT3 and clinicopathologic parameters. The results showed a significant correlation between the expression of ONECUT3 and microsatellite status, tumor diameter, and Ki-67 level. However, there was no correlation with TNM stage, KRAS mutation, vascular invasion, perineural invasion, histological grading, and tumor site. Specifically, higher ONECUT3 expression was associated with microsatellite instability status, larger tumor diameters, and higher Ki-67 levels (Figure 7E-G). In the survival analysis, there was no association between ONECUT3 expression and overall survival (OS) (P = 0.6207) or disease-free survival (DFS) (P = 0.4178) (Figure 7C and 7D). 3. Discussion The Warburg effect promotes the development of various malignant tumors, but the specific mechanism of regulating the Warburg effect in colorectal cancer is still unclear. Our research has discovered through in vivo and in vitro experiments that ONECUT3 can regulate the Warburg effect in colorectal cancer, and has elucidated the specific mechanism by which ONECUT3 regulates the transcriptional activity of HIF1α by HDAC6. Considering the limited development of targeted therapies for colorectal cancer, the discovery of ONECUT3 may become a potential target for the treatment of colorectal cancer. The ONECUT3 gene is situated on human chromosome 19p13.3 and codes for the protein transcription factor ONECUT3. ONECUT3, a homolog of ONECUT1, is crucial in zebrafish for early-stage bile duct development, neuronal differentiation, endodermal differentiation of the human pancreas, and hearing development in Drosophila 18, 30-33 . Our study has presented novel evidence regarding the role of ONECUT3 in the aerobic glycolysis of colorectal cancer. The study focused on ONECUT3, which exhibited the highest upregulation in the high glycolytic group in the TCGA database. And a significant upregulation of ONECUT3 expression in colon cancer tissues compared to paracancerous tissues. Subsequent experiments demonstrated that knockdown of ONECUT3 suppressed the proliferation and glycolysis of colon cancer cells, whereas overexpression of ONECUT3 promoted the proliferation and glycolysis of tumor cells. Additionally, the glycolysis inhibitor 2-DG could counteract the proliferative effect of ONECUT3 overexpression on colon cancer cells. Our clinical study also identified a positive correlation between ONECUT3 expression and MSI. Given that CRC patients with MSI respond better to immunotherapy, further investigation into the potential correlation between ONECUT3 and immunotherapy sensitivity is warranted. In recent years, there have been limited breakthroughs in immunotherapy for colorectal cancer, and addressing how to enhance its effectiveness remains an unresolved issue. In addition to developing new immunotherapy targets, exploring novel combination strategies is another approach to enhance immunotherapy. ONECUT3, identified as a regulator of the Warburg effect in colorectal cancer in this study, may be combined with immunotherapy in the future to enhance its efficacy and address the current challenges in immunotherapy for colorectal cancer. In the exploration of mechanisms, GSEA enrichment analysis revealed a close association between ONECUT3 and the hypoxia signaling pathway. Colorectal cancer microarray immunohistochemistry results confirmed a positive correlation between ONECUT3 and HIF-1α, as well as its downstream glycolysis-related proteins. Cytological experiments further validated that knockdown of ONECUT3 could suppress the expression of several key enzymes in the glycolytic pathway downstream of HIF-1α. HIF-1α is widely recognized as a crucial effector molecule enabling cells to adapt to hypoxic stress and regulating tumor glycolysis metabolism 9 . HIF-1α plays a significant role in regulating tumor cell glycolysis via transcriptional regulation of the expression of various metabolic enzymes in glycolysis pathway. This pathway includes glucose transporters 1 and 3 (GLUT1/3), pyruvic acid dehydrogenase 1 (PDK-1), lactate dehydrogenase A (LDH-A), and pyruvate kinase M2 (PKM-2). Upregulation of HIF-1α expression during tumor development can enhance the Warburg effect 34 . Based on the significance of HIF-1α in aerobic glycolysis, it was hypothesized that ONECUT3 could potentially regulate tumor glycolytic metabolism via HIF-1α. However, cellular assays showed that both knockdown and overexpression of ONECUT3 did not affect the expression of HIF-1α at the mRNA and protein levels. Further investigation demonstrated that ONECUT3 regulates the transcriptional activity of HIF-1α. However, Co-IP assays indicated that there was no direct interaction between ONECUT3 and HIF-1α. Subsequently, we identified five genes (NAA10, HNF4A, TCEB2, HDAC6, and ELAVL1) that directly interacted with HIF-1α and regulated by ONECUT3. HIF-1α, as a classic regulatory factor in signaling pathways, has been extensively researched. However, there has been limited progress in clinical therapy research targeting HIF-1α, particularly in colorectal cancer. The discovery of ONECUT3 and its regulatory effect on HIF-1α holds potential research value for expanding the study of new drugs targeting HIF-1α. HDAC6, a member of class IIB histone deacetylases, primarily targets α-microtubulin, affecting cytoskeleton and cell mobility 35 . Its overexpression is linked to tumorigenesis and can serve as a prognostic marker. Due to its significant involvement in tumors, HDAC6 has emerged as a potential target for the development of antitumor drugs 36 . Inhibition of HDAC6 selectively reduced HIF-1-mediated transcription under hypoxia 26 . HDAC6 contains two catalytic domains, CD1 and CD2, which are both robust lysine deacetylase 35 , and deacetylase activity of HDAC6 contributed to the stabilization of HIF-1α and its transcriptional activity 37 . Therefore, we identified HDAC6 as a potential downstream factor of ONECUT3. Molecular biology experiments showed that knockdown of ONECUT3 significantly inhibited HDAC6 expression and that ONECUT3 directly regulated the transcriptional expression of HDAC6. The knockdown of ONECUT3 or HDAC6 expression in colon cancer cells significantly increased the acetylation level of HIF-1α. The overexpression of ONECUT3 significantly reduced the acetylation level of HIF-1α, resulting in incresed transcriptional activity of HIF-1α. The inhibition of HDAC6 significantly weakened the regulation of HIF-1α acetylation and transcriptional activity by overexpression of ONECUT3. Due to the high expression of HDAC family members in various malignant tumors and diverse carcinogenic mechanisms, the U.S. Food and Drug Administration (FDA) has approved HDAC inhibitors (HDACIs) for T-cell lymphoma and multiple melanoma therapies. Currently, various HDACIs have been used in combination with other anti-tumor drugs in CRC clinical trials, showing promise in combined therapy 38 . For instance, at the 2023 European Society for Medical Oncology (ESMO) conference, the Cancer Prevention and Treatment Center of Sun Yat-sen University reported a new treatment approach for CRC using Sintilimab in combination with Bevacizumab and Camrelizumab, which preliminarily explored the application of HDACIs with anti-vascular therapy and immunotherapy in colorectal cancer 39 . Our study found that HDAC6 is involved in regulating the Warburg effect in CRC by OC3, and this result may provide new evidence for the application of HDAC inhibitors in colorectal cancer. This study still has limitations: (1) The mechanism research is not deep enough, especially the molecular mechanism research, which needs further in-depth exploration in subsequent work; (2) The clinical sample size is small, making it difficult to represent the entire clinical population. In conclusion, the up-regulated expression of ONECUT3 in colorectal cancer promotes tumor growth by enhancing the transcriptional activity of HIF-1α through HDAC6-mediated deacetylation. This leads to the initiation of glycolytic gene transcription and enhancement of the Warburg effect in colorectal cancer. Targeting ONECUT3 and its associated signaling pathway may hold promise for colorectal cancer therapy. 4. Methods 4.1 Bioinformatics analysis COAD mRNA data were downloaded from the official website of TCGA. Unsupervised clustering was performed on the TCGA-COAD data to select a classification that includes glycolysis-related genes for hierarchical clustering 40 . ConsensusClusterPlus hierarchical clustering was used to classify colon cancer samples into low-glycolytic and high-glycolytic groups. The limma package of R was used to screen for differentially expressed genes in the two groups. Log2 |Fold change| ≥ 1 and a significance level of p < 0.05 were used as the cut-off value to identify genes that were up-regulated in the high-glycolysis group. The up-regulated genes were compared with the transcription factor subset (MsigDB: TF) of the Molecular Characterization Database for further analysis. The JASPAR database (http://jaspardev.genereg.net/), ALGGENdatabase (http://alggen.lsi.upc.es/home.html), and hTFtarget database (http://bioinfo.life.hust.edu.cn/hTFtarget) were used to predict the potential target genes regulated by ONECUT3. 4.2 Cell culture and reagents We obtained human colon cancer cell lines CACO2, LOVO, SW480, SW620 from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China), COLO205, HT29 and the colon immortalized cell line NCM460 from ATCC (American type culture collection). The cells were cultured in suggested standard medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 500 units/mL of penicillin, and 200 μg/mL of streptomycin. The cells were maintained at 37°C with 5% CO 2 . To create a hypoxic environment, the cells were cultured in a hypoxia incubator with an atmosphere containing 1% O 2 , 94% N 2 , and 5% CO 2 . The reagents used in this study included 2-DG (Sigma-Aldrich, D8375). 4.3 Clinical samples and IHC staining We utilized paraffin-embedded tissue microarrays, which consisted of cancer tissues from 141 patients who underwent surgeries at Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, for immunohistochemical analysis. All patients included in this study were pathologically diagnosed with colorectal cancer and had complete clinicopathological characteristics. None of these patients received any preoperative antitumor therapy, such as chemotherapy or radiotherapy. The specimens were collected with informed consent from all patients and approved by the ethics committee of Renji Hospital (KY2021-120-B). Immunohistochemical (IHC) staining was conducted as the previously described 41 . The primary antibodies used for IHC staining included ONECUT3 (1:200, abcam, ab181450), HDAC6 (1:200, Immunoway, YT2118), ALDOA (1:100, proteintech, 11217-1-AP), ENO1 (1:2000, proteintech, 11204-1-AP), TPI1 (1:100, proteintech, 10713-1-AP), GLUT1 (1:200, CST, D3J3A/12939S), and HIF-1α (1:100, proteintech, 20960-1-AP). The scoring was assessed by two investigators who were blinded to the clinical information, based on the staining intensity. 4.4 Quantitative real-time polymerase chain reaction (qRT-PCR) TRIzol total RNA isolation reagent (share-bio, Shanghai, China) was used to extract total RNA from the indicated cells. The quality and quantity of RNA were determined using a Nanodrop™ spectrophotometer (NanoDrop products, Wilmington, CA). Then, 1 μg of total RNA was reverse transcribed into complementary DNA (cDNA) using All-in-One First-Strand Synthesis Master Mix (with dsDNase) (share-bio, Shanghai, China). Subsequently, the cDNA product was amplified by PCR on the ViiA 7 Real-Time PCR System (Thermo Scientific) to analyze mRNA expression. The 2*Universal SYBR Green qPCR Premix (share-bio, Shanghai, China) and specific primers were used for qPCR. 18sRNA was used as an internal control. The PCR primer sequences used in this study are listed in supplementary Table 1. Relative quantification was performed using the comparative 2–ΔΔCt method. 4.5 Western blot analysis and co-immunoprecipitation Whole-cell protein lysates were obtained using IP lysis buffer (share-bio, Shanghai, China) supplemented with a protease and phosphatase inhibitor (share-bio, Shanghai, China). The cell lysates were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently electrophoretically transferred onto PVDF membranes. The membranes were blocked with 5% defatted milk for 1 h at room temperature (RT), hybridized with primary antibodies overnight at 4 °C, and then incubated with HRP-conjugated secondary antibodies at RT for 60 min. β-actin antibody was used as a loading control. Immunoblots were developed using the Basic Luminol Chemiluminescent Kit (share-bio, Shanghai, China) and the ChemiDoc Touch image system (Bio-Rad). The antibodies used were listed as follows: ONECUT3 (1:500, Abcam, ab181450), HIF1α (1:1000, Abcam, ab2185), GLUT1 (1:1000, Proteintech, 21829-1-AP), ALDOA (1:10000, Proteintech,11217-1-AP), HDAC6 (1: 10000, Abcam, ab133493), and β-actin (1: 5000, Abcam, ab6276). For co-immunoprecipitation, protein lysates obtained as described above were incubated with Pierce Anti-HA Magnetic Beads (Thermo Fisher Scientific, USA, #88836) or Pierce Protein-A/G Magnetic Beads (Thermo Fisher Scientific, USA, #88803), which were already incubated with anti-HIF1α (2 µg, Abcam, ab308433) or anti-IgG (as a negative control, 2 µg, Abcam, ab200699) for 15 min at RT, with rotation for 30 min at RT. The immuno-complexes were washed three times with TBS-T or PBS-T and then resuspended in 1 × SDS-PAGE sample buffer for western blotting analysis. 4.6 Lentivirus production and transfection To achieve overexpression, plasmids expressing HA-tagged ONECUT3 were constructed by Shanghai Generay Biotech Co., Ltd. The cDNAs encoding full-length human ONECUT3 (NM_001080488.2) with HA tag were synthesized and inserted into the pCDH-CMV-MCS-EF1-Puro vector (Generay, Shanghai, China). For knockdown, lentiviral siRNA negative control and siRNA oligonucleotides targeting human ONECUT3 were designed and synthesized by Genepharma (Shanghai, China). The sequences for the siRNA were as follows: siONECUT3-1: 5’-CGCTGATCGCCATCTTCAAGGAGAA-3’; HDAC6 siRNA sequence: 5’-GGACAACATGGAGGAGGACAATGTA-3’. 293T packaging cells were used to produce lentivirus, which was subsequently transfected into target cell lines with 6 µg/ml polybrene for 24 h. Transfected cells used for overexpression or knockdown, as well as their control cells, were selected with 5 µg/ml puromycin for 2 weeks. The overexpression or knockdown efficiency of ONECUT3 was assessed using qRT-PCR and western blotting. 4.7 siRNA targeting ONECUT3 or HDAC6 ONECUT3 or HDAC6 specific siRNA and non-targeting control (NTC) siRNA were purchased from Genepharma (Shanghai, China). According to the instructions of jetPRIME® in vitro DNA & siRNA transfection reagent (Polyplus, Shanghai, China), dilute ONECUT3 or HDAC6 specific siRNA (25 nM) or NTC siRNA (25 nM) into 200 µL of jetPRIME® buffer, then add 4 µL jetPRIME® reagent, incubate for 10 to 15 min at RT, add the transfection mix to the cells in serum containing medium dropwise, and incubate the plate at 37 °C. As mentioned earlier, transfection efficiency was determined by quantitative PCR (qPCR) and immunoblot analysis. Relevant experiments were conducted between 24 and 36 hours after siRNA introduction. 4.8 Colony formation assay Different types of cells (1 × 10 3 cells per plate) were seeded in 6-well plates and incubated for approximately 14 days. After the experiments, the formed colonies were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, and stained with 0.2% crystal violet for 30 min. Colonies larger than 100 μm in diameter were counted for each plate. 4.9 Extracellular acidification rate (ECAR) and oxygen consumption ratio (OCR) assays The Seahorse XF96 Flux Analyzer (Seahorse Bioscience, Billerica, Massachusetts, USA) was used to measure the real-time extracellular acidification rate (ECAR) of CRC cells in vitro, following the manufacturer’s instructions. Briefly, CRC cells were seeded at a density of 2-3 × 10 4 cells per well in an XF96-well plate and allowed to attach overnight. Cells were incubated in non-buffered media under basal conditions for 1 hour. Subsequently, they were sequentially injected with 10 mM glucose, 1 mM mitochondrial poison (oligomycin, Sigma-Aldrich, Saint Louis, Missouri, USA), and 80 mM glycolysis inhibitor (2-deoxyglucose, 2-DG, Sigma-Aldrich). ECAR measurement was normalized by total protein content, as demonstrated by the BCA assay. The experimental data was processed using the Seahorse XF96 Wave software. 4.10 Subcutaneous xenograft model 2 × 10 6 cells (ONECUT3 knockdown colon cancer cells, and their control cells), resuspended in 100 μl of PBS, were subcutaneously injected into the backs of male Balb/c nude mice (5-6 weeks old, five mice per group). After 5 weeks of subcutaneous inoculation, the experiment was terminated, and the mice were euthanized. The tumors were then collected, weighed, and fixed in formalin. All animal studies were approved by the Animal Care and Use Committee of Shanghai East Hospital, Tongji University School of Medicine. The mice received humane care in accordance with the criteria outlined in the Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences and published by the National Institutes of Health. 4.11 HIF-1α activity measurement We quantified HIF-1α activity by using the HIF-1 alpha Transcription Factor Assay Kit (Abcam, ab133104) following the manufacturer’s instructions, after extracting the nuclear fraction. Briefly, processed colorectal cancer cells was collected and the nuclear and cytoplasmic protein were extracted by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0027) . The samples were added to the wells of the HIF-1α transcription factor plate and then incubated overnight at 4°C. Diluted HIF-1α primary antibody was added to each well and incubated at room temperature for 1 hour. Subsequently, diluted goat anti-rabbit HRP conjugate was added to each well and incubated at room temperature for 1 hour. After adding the stop solution, the HIF-α DNA binding activity level was measured at 450 nm using a microplate reader. 4.12 Chromatin immunoprecipitation (ChIP) assays ChIP assay was were carried out using the EZ-Magna ChIP Assay Kit according to the manufacturer’s protocols (Millipore, 17-10086). Briefly, cells were cross-linked with 1% PFA/PBS at room temperature for 10 minutes. Then, unreacted PFA was eliminated using a 10-fold concentration of glycine. Subsequently, samples were sonicated in lysis buffer to obtain DNA fragments ranging from 200 to 1,000 bp. Immunoprecipitation was performed using 5 μg of ONECUT3 or IgG antibodies. Primers targeting the promoter region of the HDAC6 gene were used for quantitative RT-PCR. The results were presented as the relative mRNA expression, calculated by comparing the delta CT values of ONECUT3-specific antibodies with IgG antibodies. 4.13 Luciferase reporter assay To evaluate the activity of the HDAC6 gene promoter, processed cells and control cells were seeded into 96-well plates and co-transfected with a PTRF luciferase reporter plasmid. The plasmid contained a tandem repeat of the PTRF transcriptional response element, while the Renilla control reporter served as an internal control. After 48 hours, the cells were lysed, and the enzymatic activity of luciferase and Renilla was measured using the Dual-Luciferase Assay kit (Promega, E1910) following the manufacturer’s protocol. 4.14 Statistical analysis We conducted statistical analyses using SPSS 19.0 for Windows (IBM Corporation) and GraphPad Prism 7 software (San Diego, CA). The results were reported as mean ± standard deviation (SD) and compared using a two-tailed, unpaired Student’s t-test or one-way ANOVA. p-value of <0.05 was considered statistically significant. Declarations Acknowledgements The authors would like to thank State Key Laboratory of Systems Medicine for Cancer for providing instrumentation and some reagents related to this work. Conflict of Interest The authors declare no potential conflicts of interest. Author Contributions S.J., R.L. and J.X. were in charge of overall direction and planning designed the experiments. R.H., H.W., and W.L. conducted the experiments and statistical analyses. K.H. and S.Z. contributed to sample preparation. R.H. led the writing of the manuscript. All authors provided critical feedback and contributed to shaping the research, analysis, and manuscript. Ethics Approval and Consent to Participate The study was approved by the ethics committee of Shanghai Jiaotong University (KY2021-120-B). Funding This study was funded by a grant from the Natural Science Foundation of China (82260562, 82372922), the Academic Leaders Training Program of Pudong Health Bureau of Shanghai (PWRd2021-09), the CSCO research funding (Y-xsk2021-0003, Y-2022HER2AZMS-0181), the Science and Technology Commission of Shanghai Municipality (19DZ1910502), and the Top-level Clinical Discipline Project of Shanghai Pudong (PWYgf2021-07). Data Availability Statement Data available within the article. References Morgan E, Arnold M, Gini A, et al. Global burden of colorectal cancer in 2020 and 2040: incidence and mortality estimates from GLOBOCAN. Gut 2023;72:338-344. Vallee A, Lecarpentier Y, Guillevin R, et al. Aerobic Glycolysis Hypothesis Through WNT/Beta-Catenin Pathway in Exudative Age-Related Macular Degeneration. J Mol Neurosci 2017;62:368-379. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646-74. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 2009;324:1029-33. Zhong X, He X, Wang Y, et al. Warburg effect in colorectal cancer: the emerging roles in tumor microenvironment and therapeutic implications. J Hematol Oncol 2022;15:160. Lu S, Han L, Hu X, et al. N6-methyladenosine reader IMP2 stabilizes the ZFAS1/OLA1 axis and activates the Warburg effect: implication in colorectal cancer. J Hematol Oncol 2021;14:188. Zhao G, Yuan H, Li Q, et al. DDX39B drives colorectal cancer progression by promoting the stability and nuclear translocation of PKM2. Signal Transduct Target Ther 2022;7:275. Jing Z, Liu Q, He X, et al. NCAPD3 enhances Warburg effect through c-myc and E2F1 and promotes the occurrence and progression of colorectal cancer. J Exp Clin Cancer Res 2022;41:198. Li L, Liang Y, Kang L, et al. Transcriptional Regulation of the Warburg Effect in Cancer by SIX1. Cancer Cell 2018;33:368-385 e7. Sukonina V, Ma H, Zhang W, et al. FOXK1 and FOXK2 regulate aerobic glycolysis. Nature 2019;566:279-283. Wiel C, Le Gal K, Ibrahim MX, et al. BACH1 Stabilization by Antioxidants Stimulates Lung Cancer Metastasis. Cell 2019;178:330-345 e22. Cui J, Shi M, Xie D, et al. FOXM1 promotes the warburg effect and pancreatic cancer progression via transactivation of LDHA expression. Clin Cancer Res 2014;20:2595-606. Gomes AS, Ramos H, Soares J, et al. p53 and glucose metabolism: an orchestra to be directed in cancer therapy. Pharmacol Res 2018;131:75-86. Berkers CR, Maddocks OD, Cheung EC, et al. Metabolic regulation by p53 family members. Cell Metab 2013;18:617-33. Zhang L, Shay JW. Multiple Roles of APC and its Therapeutic Implications in Colorectal Cancer. J Natl Cancer Inst 2017;109. Cha PH, Hwang JH, Kwak DK, et al. APC loss induces Warburg effect via increased PKM2 transcription in colorectal cancer. Br J Cancer 2021;124:634-644. Sun L, Zhang H, Gao P. Metabolic reprogramming and epigenetic modifications on the path to cancer. Protein Cell 2022;13:877-919. van der Raadt J, van Gestel SHC, Nadif Kasri N, et al. ONECUT transcription factors induce neuronal characteristics and remodel chromatin accessibility. Nucleic Acids Res 2019;47:5587-5602. Laudadio I, Manfroid I, Achouri Y, et al. A feedback loop between the liver-enriched transcription factor network and miR-122 controls hepatocyte differentiation. Gastroenterology 2012;142:119-29. Sapkota D, Chintala H, Wu F, et al. Onecut1 and Onecut2 redundantly regulate early retinal cell fates during development. Proc Natl Acad Sci U S A 2014;111:E4086-95. Jiang K, Jiao Y, Liu Y, et al. HNF6 promotes tumor growth in colorectal cancer and enhances liver metastasis in mouse model. J Cell Physiol 2019;234:3675-3684. Wang GH, Zhou YM, Yu Z, et al. Up-regulated ONECUT2 and down-regulated SST promote gastric cell migration, invasion, epithelial-mesenchymal transition and tumor growth in gastric cancer. Eur Rev Med Pharmacol Sci 2020;24:9378-9390. Sun Y, Shen S, Liu X, et al. MiR-429 inhibits cells growth and invasion and regulates EMT-related marker genes by targeting Onecut2 in colorectal carcinoma. Mol Cell Biochem 2014;390:19-30. Rankovic B, Zidar N, Zlajpah M, et al. Epithelial-Mesenchymal Transition-Related MicroRNAs and Their Target Genes in Colorectal Cancerogenesis. J Clin Med 2019;8. Vanhorenbeeck V, Jacquemin P, Lemaigre FP, et al. OC-3, a novel mammalian member of the ONECUT class of transcription factors. Biochem Biophys Res Commun 2002;292:848-54. Schoepflin ZR, Shapiro IM, Risbud MV. Class I and IIa HDACs Mediate HIF-1alpha Stability Through PHD2-Dependent Mechanism, While HDAC6, a Class IIb Member, Promotes HIF-1alpha Transcriptional Activity in Nucleus Pulposus Cells of the Intervertebral Disc. J Bone Miner Res 2016;31:1287-99. Qian DZ, Kachhap SK, Collis SJ, et al. Class II histone deacetylases are associated with VHL-independent regulation of hypoxia-inducible factor 1 alpha. Cancer Res 2006;66:8814-21. Lee JW, Bae SH, Jeong JW, et al. Hypoxia-inducible factor (HIF-1)alpha: its protein stability and biological functions. Exp Mol Med 2004;36:1-12. Ke Q, Costa M. Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol 2006;70:1469-80. Matthews RP, Lorent K, Pack M. Transcription factor onecut3 regulates intrahepatic biliary development in zebrafish. Dev Dyn 2008;237:124-31. Keder A, Tardieu C, Malong L, et al. Homeostatic maintenance and age-related functional decline in the Drosophila ear. Sci Rep 2020;10:7431. Ito R, Kimura A, Hirose Y, et al. Elucidation of HHEX in pancreatic endoderm differentiation using a human iPSC differentiation model. Sci Rep 2023;13:8659. Francius C, Clotman F. Dynamic expression of the Onecut transcription factors HNF-6, OC-2 and OC-3 during spinal motor neuron development. Neuroscience 2010;165:116-29. Soni S, Padwad YS. HIF-1 in cancer therapy: two decade long story of a transcription factor. Acta Oncol 2017;56:503-515. Ho TCS, Chan AHY, Ganesan A. Thirty Years of HDAC Inhibitors: 2020 Insight and Hindsight. J Med Chem 2020;63:12460-12484. Aldana-Masangkay GI, Sakamoto KM. The role of HDAC6 in cancer. J Biomed Biotechnol 2011;2011:875824. Ryu HW, Won HR, Lee DH, et al. HDAC6 regulates sensitivity to cell death in response to stress and post-stress recovery. Cell Stress Chaperones 2017;22:253-261. Garmpis N, Damaskos C, Garmpi A, et al. Histone Deacetylases and their Inhibitors in Colorectal Cancer Therapy: Current Evidence and Future Considerations. Curr Med Chem 2022;29:2979-2994. Wang F, Jin Y, Wang M, et al. Combined anti-PD-1, HDAC inhibitor and anti-VEGF for MSS/pMMR colorectal cancer: a randomized phase 2 trial. Nat Med 2024. Spranger S, Bao R, Gajewski TF. Melanoma-intrinsic beta-catenin signalling prevents anti-tumour immunity. Nature 2015;523:231-5. Peng W, Huang W, Ge X, et al. Type Igamma phosphatidylinositol phosphate kinase promotes tumor growth by facilitating Warburg effect in colorectal cancer. EBioMedicine 2019;44:375-386. Supplemental Table 1 Supplemental Table 1 isnot available with this version. Additional Declarations (Not answered) Cite Share Download PDF Status: Published Journal Publication published 03 Mar, 2025 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 12 Aug, 2024 Review # 2 received at journal 08 Jul, 2024 Reviewer # 2 agreed at journal 03 Jul, 2024 Reviewer # 1 agreed at journal 16 May, 2024 Reviewers invited by journal 12 May, 2024 Submission checks completed at journal 22 Apr, 2024 First submitted to journal 20 Apr, 2024 Editor assigned by journal 20 Apr, 2024 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. 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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-4296431","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":301604952,"identity":"f57e811e-a869-4133-882e-558fc96afe97","order_by":0,"name":"Junli Xue","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-0564-5284","institution":"Shanghai East Hospital","correspondingAuthor":true,"prefix":"","firstName":"Junli","middleName":"","lastName":"Xue","suffix":""},{"id":301604953,"identity":"adf3007f-8bf2-4b68-8438-b6a502683da0","order_by":1,"name":"Junli Xue","email":"","orcid":"","institution":"[email protected]","correspondingAuthor":false,"prefix":"","firstName":"Junli","middleName":"","lastName":"Xue","suffix":""},{"id":301604954,"identity":"72b26490-12e3-43ff-bea0-a3d38fd0646e","order_by":2,"name":"Hao Wang","email":"","orcid":"","institution":"Shanghai East Hospital, School of Medicine, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Wang","suffix":""},{"id":301604955,"identity":"25c69041-a5c3-42da-ba41-19310640722b","order_by":3,"name":"Weihan Li","email":"","orcid":"","institution":"Shanghai East Hospital, School of Medicine, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Weihan","middleName":"","lastName":"Li","suffix":""},{"id":301604956,"identity":"c72d77bf-be5d-477d-8111-702a9312736e","order_by":4,"name":"Kexin He","email":"","orcid":"","institution":"Shanghai East Hospital, School of Medicine, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Kexin","middleName":"","lastName":"He","suffix":""},{"id":301604957,"identity":"cc03428f-1073-40db-94ba-09ee8f1f2800","order_by":5,"name":"Shan Zhang","email":"","orcid":"","institution":"Renji Hospital, School of Medicine, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Shan","middleName":"","lastName":"Zhang","suffix":""},{"id":301604958,"identity":"481b4f30-6351-4095-b256-6d9d2c85b504","order_by":6,"name":"Shu-Heng Jiang","email":"","orcid":"https://orcid.org/0000-0001-8516-6234","institution":"State Key Laboratory of Systems Medicine for Cancer","correspondingAuthor":false,"prefix":"","firstName":"Shu-Heng","middleName":"","lastName":"Jiang","suffix":""},{"id":301604959,"identity":"c14f0eea-fba5-4df7-87d5-39b9b2cb2f4f","order_by":7,"name":"Rongkun Li Rongkun Li","email":"","orcid":"","institution":"Shanghai Jiao Tong University doctor","correspondingAuthor":false,"prefix":"","firstName":"Rongkun","middleName":"Li Rongkun","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-04-20 07:55:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4296431/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4296431/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-025-07457-8","type":"published","date":"2025-03-03T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57037691,"identity":"c4d0d9f3-66e2-4b50-9a32-ccc4fd27c766","added_by":"auto","created_at":"2024-05-23 18:58:46","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6066077,"visible":true,"origin":"","legend":"\u003cp\u003eA. Based on the known glycolysis-related gene clusters, TCGA-COAD samples were hierarchically clustered into high-glycolysis and low-glycolysis groups; B. Screening for differentially expressed genes in the two groups, 127 genes were upregulated in the high glycolytic samples; C. The 127 upregulated genes were intersected with MsigDB: TF to obtain 11 transcription factors; D. Ranking of log\u003csub\u003e2\u003c/sub\u003e|FC| values of 11 transcription factors; E. Immunohistochemical results showed that ONECUT3 protein expression was significantly higher in colon cancer tissues than in paracancerous tissues (scale bar: 50 μm).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4296431/v1/762d229059a01d9313e4d357.jpg"},{"id":57037161,"identity":"4928c2f0-b647-47d4-850c-84b4ffc5e108","added_by":"auto","created_at":"2024-05-23 18:42:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2718870,"visible":true,"origin":"","legend":"\u003cp\u003eA-B. qRT-PCR (A) and Western Blot (B) to detect ONECUT3 expression in colon cancer cell lines (CACO2, COLO205, HT29, LOVO, SW480, SW620) and normal colon epithelial line NCM460; C. ONECUT3 was knocked down in HT29 and LOVO and the knockdown efficiency was verified; D. The results of Seahorse XF Bioanalyzer assay showed that knockdown of ONECUT3 significantly reduced the ECAR of colon cancer cells; E. ONECUT3 was overexpressed in SW620 and COLO205 and the overexpression efficiency was verified; F. The results of Seahorse XF Bioanalyzer showed that overexpression of ONECUT3 significantly enhanced the ECAR of colon cancer cells. (OC3: ONECUT3; ECAR: extracellular acidification rate)\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4296431/v1/be9c30ef8ab1874ab4071cbc.jpg"},{"id":57037163,"identity":"6b958cda-fd68-42cd-8e5c-a0b20160669d","added_by":"auto","created_at":"2024-05-23 18:42:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4667841,"visible":true,"origin":"","legend":"\u003cp\u003eA. colony-forming assay showed that knockdown of ONECUT3 significantly inhibited the proliferation of HT29 and LOVO cell lines; B. Subcutaneous xenograft model showed that knockdown of ONECUT3 significantly inhibited tumor growth; C. colony-forming assay showed that overexpression of ONECUT3 significantly promoted the proliferation of SW620 and COLO205 cell lines; D. colony-forming assay showed that the glycolysis inhibitor 2-DG largely eliminated the proliferative effect of ONECUT3 overexpression on colon cancer cells. (OC3: ONECUT3; **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ns: no significance).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4296431/v1/86ae90ab6ff93e59e91892ce.jpg"},{"id":57037445,"identity":"4d3c445e-a111-4f20-aadb-3ddfc427ae4c","added_by":"auto","created_at":"2024-05-23 18:50:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3478141,"visible":true,"origin":"","legend":"\u003cp\u003eA. After knockdown of ONECUT3 in HT29 cells, GSEA enrichment of transcriptome sequencing showed that \u003cem\u003eONECUT3\u003c/em\u003eexpression was positively correlated with GLYCOLYSIS and HYPOXIA; B-C. qRT-PCR (B) and Western Blot (C) showed that knockdown of ONECUT3 significantly inhibited the mRNA and protein expression of GLUT1, ALDOA and PKM2, which were downstream glycolysis-related enzymes of HIF-1α; D-E. qRT-PCR (D) and Western Blot (E) showed that knockdown of ONECUT3 did not affect HIF-1α expression; H-I. HIF-1 alpha Transcription Factor Assay kit showed that knockdown of ONECUT3 in colon cancer cells decreased HIF-1α transcriptional activity (H) and overexpression of ONECUT3 increased HIF-1α transcriptional activity (I); J. Co-IP assay showed that there was no direct interaction between ONECUT3 and HIF-1α. (OC3: ONECUT3; **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ns: no significance).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4296431/v1/7338fa61deb554270385f022.jpg"},{"id":57037165,"identity":"79e36953-2aad-4bdb-9040-f807fafcf2f3","added_by":"auto","created_at":"2024-05-23 18:42:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2178113,"visible":true,"origin":"","legend":"\u003cp\u003eA. Volcano plot showed differentially expressed genes (DEGs) caused by ONECUT3 knockdown; B. 539 proteins interacting with HIF-1α were identified by Co-IP and mass spectrometry, and intersected with ONECUT3-regulated genes (n=438). Five genes (NAA10, HNF4A, TCEB2, HDAC6 and ELAVL1) were identified; C. qRT-PCR results showed that knockdown of ONECUT3 downregulated mRNA expression of HDAC6 in HT29 and LOVO cells; D. Western Blot results showed that protein expression of HDAC6 was also decreased after ONECUT3 knockdown; E. ChIP-PCR results showed that ONECUT3 bound to the promoter region of HDAC6 gene; F. Luciferase reporter gene assay demonstrated that ONECUT3 transcriptionally regulated the HDAC6 gene. (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ns: no significance).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4296431/v1/ef30ff13019c16922e5f9bee.jpg"},{"id":57037447,"identity":"5ea36131-565e-4143-8804-dcf9cce3b47b","added_by":"auto","created_at":"2024-05-23 18:50:46","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1157084,"visible":true,"origin":"","legend":"\u003cp\u003eA. Western Blot showed that HIF-1α acetylation level by detection of pan-acetylation antibody significantly increased after knockdown of ONECUT3 or HDAC6 in HT29 cells; B. Westren Blot showed that HIF-1α acetylation level significantly reduced after overexpression of ONECUT3 in SW620 cells, and HDAC6 inhibitor (ACY241) attenuated the regulatory effect of ONECUT3-overexpression on HIF-1α deacetylation; C. After inhibition of HDAC6 in ONECUT3-overexpression cells, transcriptional activity of HIF-1α was not upregulated. (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ns: no significance).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4296431/v1/900111b5a6b5a10dac4655b1.jpg"},{"id":57037168,"identity":"50621e9c-bc3d-4918-abe7-3a40175d43c5","added_by":"auto","created_at":"2024-05-23 18:42:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4642941,"visible":true,"origin":"","legend":"\u003cp\u003eA. In immunohistochemical staining of colorectal cancer pathology tissue microarrays (n=141), ONECUT3 expression could be shown as negative (-), positive (+, ++, +++); B. Correlation analysis of ONECUT3 with the expression of HIF-1a, HDAC6 and glycolysis-related proteins (ALDOA, TPI1, GLUT1, and ENO1) in histopathology microarrays; C-D. Overall survival analysis of ONECUT3 (C), disease-free survival analysis of ONECUT3; E-G. expression of ONECUT3 was significantly correlated with microsatellite status (E), tumor diameter (F), and Ki-67 level (G). (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4296431/v1/d04705030b3c983d306de3ae.png"},{"id":77678332,"identity":"7d69c5e8-bd35-49ae-8fd9-c40f0676a6bc","added_by":"auto","created_at":"2025-03-04 08:09:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23477646,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4296431/v1/4ed93e99-e1a0-4970-b406-851a59f0c646.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Transcription factor ONECUT3 regulates HDAC6/HIF-1α activity to promote the Warburg effect and tumor growth in colorectal cancer","fulltext":[{"header":"1.\tIntroduction","content":"\u003cp\u003eColorectal cancer (CRC) is a prevalent form of cancer, ranking third in terms of incidence and second in terms of cancer-related mortality worldwide. Despite advancements in diagnosis and treatment methods, the incidence and mortality rates of colorectal cancer continue to rise, particularly in developing countries (including China) and among young adults (\u0026lt; 50 years old). It is projected that by 2040, there will be 3.2 million new cases and 1.6 million deaths from CRC\u003csup\u003e1\u003c/sup\u003e. This poses a significant threat to human survival and imposes a substantial economic burden. Therefore, deeper understanding of the specific molecular mechanisms involved in CRC and developing targeted drugs could potentially improve the disease prognosis.\u003c/p\u003e\n\u003cp\u003eUnlike normal tissue, tumor cells undergo metabolic reprogramming, which is a significant hallmark of cancer\u003csup\u003e2, 3\u003c/sup\u003e.\u0026nbsp;Even in the presence of sufficient oxygen supply, tumor cells predominantly metabolize glucose through glycolysis, resulting in lactate accumulation. This phenomenon, known as aerobic glycolysis, is commonly referred to as the Warburg effect\u003csup\u003e4\u003c/sup\u003e.\u0026nbsp;Numerous studies have demonstrated that the Warburg effect remodels the tumor microenvironment and plays a crucial role in the initiation, progression, metastasis, and prognosis of colon cancer\u003csup\u003e5-8\u003c/sup\u003e. Research has confirmed that transcription factors can regulate tumor glycolysis through various mechanisms, including modulation of the glycolysis phase, expression of related genes, and regulation of key enzyme activities involved in glycolysis\u003csup\u003e9-12\u003c/sup\u003e. The process of glycolysis is primarily regulated by hypoxia-inducible factor 1\u0026alpha; (HIF-1\u0026alpha;). Under hypoxic conditions, HIF-1\u0026alpha; forms a heterodimer with HIF-1\u0026beta;, which translocates to the nucleus and transcriptionally activates downstream target genes (e.g., GLUT1, etc.). The regulation of HIF-1\u0026alpha; in colorectal cancer involves common mutated genes (APC, RAS, TP53) and epigenetic modifications\u003csup\u003e5, 13-17\u003c/sup\u003e. However, the specific mechanism underlying the regulation of HIF-1\u0026alpha; in colorectal cancer is still unclear.\u003c/p\u003e\n\u003cp\u003eThe ONECUT family, which consists of ONECUT1 (HNF6), ONECUT2, and ONECUT3, possesses a single \u0026ldquo;CUT\u0026rdquo; DNA binding domain and an atypical homologous domain, and functions as translational factors\u003csup\u003e18\u003c/sup\u003e. The three members of the ONECUT family, which are highly conserved from Drosophila to humans, play a crucial role in regulating the development of various tissues derived from the ectoderm or endoderm, as well as activating the expression of numerous gene families\u003csup\u003e19, 20\u003c/sup\u003e. It has been reported that ONECUT1 promotes colon cancer cell proliferation and liver metastasis by enhancing cell adhesion ability and inhibiting apoptosis\u003csup\u003e21\u003c/sup\u003e. In gastric cancer, the upregulation of ONECUT2 expression promotes cell migration, invasion, epithelial-mesenchymal transition, and tumor growth\u003csup\u003e22\u003c/sup\u003e. Similarly, elevated ONECUT2 expression is associated with lymph node metastasis\u003csup\u003e23\u003c/sup\u003e, and poorer prognosis in CRC\u003csup\u003e24\u003c/sup\u003e. However, as a new member of the ONECUT family\u003csup\u003e25\u003c/sup\u003e, there has been relatively limited research on the relationship between ONECUT3 and cancer, particularly in terms of glucose metabolism reprogramming.\u003c/p\u003e\n\u003cp\u003eIn this study, we demonstrated that ONECUT3 mediates HIF-1\u0026alpha; deacetylation through histone deacetylase 6 (HDAC6), leading to the activation of HIF-1\u0026alpha; transcription and its downstream glycolysis genes, thereby enhancing the Warburg effect and promoting tumor growth in CRC. The ONECUT3-HDAC6-HIF-1\u0026alpha; axis is anticipated to serve as a potential therapeutic target for modulating aerobic glycolysis in colorectal cancer.\u003c/p\u003e"},{"header":"2.\tResults","content":"\u003cp\u003e\u003cstrong\u003e2.1 ONECUT3 is a key transcription factor related to glycolysis in colorectal cancer\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the genes associated with aerobic glycolysis in colorectal cancer, mRNA data from the COAD subset of the TCGA database were analyzed.\u0026nbsp;In the TCGA-COAD database, colon cancer samples were categorized into two groups based on the expression level of glycolysis-related genes: the high-glycolysis group and the low-glycolysis group (Figure 1A). A total of 400 differentially expressed genes (DEGs) were identified using a significance level of Log\u003csub\u003e2\u003c/sub\u003e|Fold change| \u0026ge;1 and p\u0026lt;0.05. Among these, 127 genes were up-regulated in the high-glycolysis group, including 11 transcription factors (Figure 1B-C). Among the 11 transcription factors, ONECUT3 (Figure 1D) was identified as the most significant DEG. Therefore, ONECUT3 was selected as the key gene for investigating the glycolytic pathway in colon cancer. Subsequently, immunohistochemical staining was performed to determine the expression of ONECUT3 in both colon cancer tissues and paracancerous tissues. The results revealed a significant increase in ONECUT3 expression in colon cancer tissues (Figure 1E), suggesting a potential regulatory role of ONECUT3 in colon cancer through glycolysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Regulation of glycolytic metabolism in colorectal cancer cells by ONECUT3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInitially, we detected the mRNA and protein expression of ONECUT3 in six colon cancer cell lines (CACO2, COLO205, HT29, LOVO, SW480, SW620) and a normal colon epithelial cell (NCM460) (Figure 2A-B). The expression of ONECUT3 was highest in HT29 and LOVO cell lines, while lowest in SW620 and COLO205 cell lines. Subsequently, ONECUT3 was knocked down in the high-expressing cell lines (HT29 and LOVO) and overexpressed in the low-expressing cell lines (SW620 and COLO205), as depicted in Figure 2C and 2E. We conducted an extracellular acidification rate assay in colon cancer cells to examine the regulatory role of ONECUT3 in glycolysis. The results demonstrated a significant decrease in the extracellular acidification rate (ECAR), an indicator of glycolysis, following ONECUT3 knockdown (Figure 2D), and a significant increase after ONECUT3 overexpression in colon cancer cells (Figure 2F). These findings indicate that ONECUT3 promotes the glycolytic metabolism of colon cancer cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 ONECUT3 promotes the growth of colorectal cancer cells in a Warburg effect-dependent manner\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated whether\u0026nbsp;ONECUT3\u0026nbsp;could influence the proliferation of colon cancer cells through the Warburg effect, which is known to enhance tumor cell proliferation. In the colony-forming assay, knockdown of ONECUT3 significantly inhibited the proliferation of HT29 and LOVO cell lines (Figure 3A), while overexpression of ONECUT3 significantly promoted the proliferation of SW620 and COLO205 cell lines (Figure 3C). The promotion of proliferation by ONECUT3 overexpression was largely abolished by the glycolysis inhibitor 2-Deoxy-D-Glucose (2-DG) (Figure 3D). We used a nude mouse subcutaneous transplantation tumor model to verify the effect of ONECUT3 on tumor growth in vivo. Likewise, knockdown of ONECUT3 significantly reduced the weight of the tumors, indicating a significant inhibition of colon cancer growth by ONECUT3 (Figure 3B). These results indicated that ONECUT3 promoted tumor growth on a Warburg effect dependent manner.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 ONECUT3 enhances the transcriptional activity of HIF-1\u0026alpha; and regulates the expression of its downstream glycolytic enzymes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine if transcription factor ONECUT3 can directly regulate the transcription of glycolytic genes, we utilized online databases including JASPAR, ALGGEN, and hTFtarget to predict the genes that are regulated by ONECUT3. No glycolysis-related genes were found. Subsequently, RNA sequencing was employed to analyze the changes in the transcriptome resulting from ONECUT3 knockdown in colon cells. The results of Gene Set Enrichment Analysis (GSEA) indicated the close involvement of ONECUT3 in glycolytic and hypoxic signaling pathways (Figure 4A). Subsequently, the expression of key genes in glycolytic and hypoxic signaling pathways was assessed at both the RNA and protein levels. Cytological experiments confirmed that the expression of GLUT1, ALDOA, and PKM2 was reduced after ONECUT3 knockdown (Figure 4B-C). These enzymes are downstream targets of HIF-1\u0026alpha; and play a crucial role in the glycolytic pathway. Given that HIF-1\u0026alpha; is a key regulator of glycolytic signaling, it is hypothesized that ONECUT3 may be involved in the regulation of glycolytic metabolism through HIF-1\u0026alpha;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe measured the expression level of HIF-1\u0026alpha; after knocking down or overexpressing ONECUT3. However, the subsequent experimental results showed that knockdown or overexpressing ONECUT3 in colon cancer cells did not affect the mRNA or protein expression of HIF-1\u0026alpha; (Figure 4D-G). Therefore, we investigated whether ONECUT3 affects the expression of downstream genes of HIF-1\u0026alpha; by regulating its transcriptional activity. Experiments using the HIF-1 alpha Transcription Factor Assay kit revealed that knocking down ONECUT3 significantly suppressed HIF-1\u0026alpha; transcriptional activity (Figure 4H), while overexpressing ONECUT3 enhanced HIF-1\u0026alpha; transcriptional activity (Figure 4I). Since both ONECUT3 and HIF-1\u0026alpha; are transcription factors, we hypothesized that they can directly bind and interact with each other in the nucleus. However, the Co-IP assay did not provide evidence of a direct interaction between ONECUT3 and HIF-1\u0026alpha; (Figure 4J). This suggests that ONECUT3 may enhance the transcriptional activity of HIF-1\u0026alpha; and influence the expression of multiple downstream glycolytic enzymes, but not through direct interaction with HIF-1\u0026alpha;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 ONECUT3 regulates HIF-1\u0026alpha; transcriptional activity through HDAC6\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the interaction mechanism between ONECUT3 and HIF-1\u0026alpha;, we initially screened 438 genes regulated by ONECUT3 using RNA-seq analysis (Figure 5A). Subsequently, we identified 539 proteins that interact with HIF-1\u0026alpha; through Co-IP and mass spectrometry. By performing a Venn analysis (Figure 5B), we obtained five genes (NAA10, HNF4A, TCEB2, HDAC6, and ELAVL1) that were present in both groups. Previous studies have shown that inhibiting HDAC6 selectively can decrease HIF-1-mediated transcription in hypoxic conditions. Additionally, HDAC inhibitors can suppress tumor angiogenesis and the expression of HIF-1\u0026alpha; protein\u003csup\u003e26, 27\u003c/sup\u003e. Based on these findings, we hypothesized that ONECUT3 may regulate the transcriptional activity of HIF-1\u0026alpha; by modulating HDAC6. Supporting our hypothesis, knockdown of ONECUT3 significantly reduced HDAC6 expression in both qRT-PCR and Western Blot analyses (Figure 5C-D). Additional investigations using ChIP-PCR and luciferase reporter gene assays revealed that ONECUT3 binds to the HDAC6 promoter region and directly regulates its transcription in colon cancer cells (Figure 5E-F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 ONECUT3 facilitates the deacetylation of HIF-1\u0026alpha; and enhances its transcriptional activity through HDAC6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the involvement of HDAC6 in the regulation of HIF-1\u0026alpha; transcriptional activity by ONECUT3, we performed knockdown experiments for ONECUT3 or HDAC6 in colon cancer cells. Subsequently, HIF-1\u0026alpha; was enriched through immunoprecipitation (IP). Post-translational modifications are known to affect the activity of HIF-1\u0026alpha;, with acetylation leading to decreased stability\u003csup\u003e28\u003c/sup\u003e. In normoxic conditions, the hydroxylation of two proline residues and the acetylation of a lysine residue on the oxygen-dependent degradation domain (ODDD) of HIF-1\u0026alpha; facilitate its binding to the pVHL E3 ligase complex, resulting in its degradation through the ubiquitin-proteasome pathway\u003csup\u003e29\u003c/sup\u003e. The acetylation level of HIF-1\u0026alpha; was determined using a panacetylation antibody. Consistent expression of HIF-1\u0026alpha; resulted in a significant increase in its acetylation level following knockdown of ONECUT3 or HDAC6 (Figure 6A). Conversely, overexpression of ONECUT3 led to a significant reduction in the acetylation level of HIF-1\u0026alpha; (Figure 6B). Additionally, the facilitative effect of ONECUT3 overexpression on HIF-1\u0026alpha; deacetylation could be reversed by using ACY241 to inhibit HDAC6 (Figure 6B). Notably, the inhibition of HDAC6 significantly eliminated the enhanced HIF-1\u0026alpha; transcriptional activity in ONECUT3 overexpressing cells (Figure 6C). These results indicate that the regulatory impact of ONECUT3 on HIF-1\u0026alpha; deacetylation and transcriptional activity is mediated by HDAC6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Expression of ONECUT3 in colorectal cancer and its clinical significance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo enhance clinical relevance, we conducted immunohistochemical staining on microarrays of colorectal cancer patients from the Renji Hospital cohort (Figure 7A) to examine the expression of ONECUT3, HIF-1A, HDAC6, and glycolysis-related proteins. The expression of ONECUT3 showed a positive correlation with certain glycolysis-related proteins (ALDOA, TPI1) and HIF-\u0026alpha; (P \u0026lt; 0.05). Additionally, both HIF-\u0026alpha; and HDAC6 exhibited a positive correlation with the expression of glycolysis-related proteins (ALDOA, TPI1, GLUT1, and ENO1) (P \u0026lt; 0.05). The expression of HIF-\u0026alpha; and HDAC6 showed a positive correlation (P \u0026lt; 0.05) (Figure 7B). We conducted a correlation analysis between ONECUT3 and clinicopathologic parameters. The results showed a significant correlation between the expression of ONECUT3 and microsatellite status, tumor diameter, and Ki-67 level. However, there was no correlation with TNM stage, KRAS mutation, vascular invasion, perineural invasion, histological grading, and tumor site. Specifically, higher ONECUT3 expression was associated with microsatellite instability status, larger tumor diameters, and higher Ki-67 levels (Figure 7E-G). In the survival analysis, there was no association between ONECUT3 expression and overall survival (OS) (P = 0.6207) or disease-free survival (DFS) (P = 0.4178) (Figure 7C and 7D).\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe Warburg effect promotes the development of various malignant tumors, but the specific mechanism of regulating the Warburg effect in colorectal cancer is still unclear. Our research has discovered through in vivo and in vitro experiments that ONECUT3 can regulate the Warburg effect in colorectal cancer, and has elucidated the specific mechanism by which ONECUT3 regulates the transcriptional activity of HIF1α by HDAC6. Considering the limited development of targeted therapies for colorectal cancer, the discovery of ONECUT3 may become a potential target for the treatment of colorectal cancer.\u003c/p\u003e\n\u003cp\u003eThe ONECUT3 gene is situated on human chromosome 19p13.3 and codes for the protein transcription factor ONECUT3. ONECUT3, a homolog of ONECUT1, is crucial in zebrafish for early-stage bile duct development, neuronal differentiation, endodermal differentiation of the human pancreas, and hearing development in Drosophila\u003csup\u003e18, 30-33\u003c/sup\u003e. Our study has presented novel evidence regarding the role of ONECUT3 in the aerobic glycolysis of colorectal cancer. The study focused on ONECUT3, which exhibited the highest upregulation in the high glycolytic group in the TCGA database. And a significant upregulation of ONECUT3 expression in colon cancer tissues compared to paracancerous tissues. Subsequent experiments demonstrated that knockdown of ONECUT3 suppressed the proliferation and glycolysis of colon cancer cells, whereas overexpression of ONECUT3 promoted the proliferation and glycolysis of tumor cells. Additionally, the glycolysis inhibitor 2-DG could counteract the proliferative effect of ONECUT3 overexpression on colon cancer cells. Our clinical study also identified a positive correlation between ONECUT3 expression and MSI. Given that CRC patients with MSI respond better to immunotherapy, further investigation into the potential correlation between ONECUT3 and immunotherapy sensitivity is warranted. In recent years, there have been limited breakthroughs in immunotherapy for colorectal cancer, and addressing how to enhance its effectiveness remains an unresolved issue. In addition to developing new immunotherapy targets, exploring novel combination strategies is another approach to enhance immunotherapy. ONECUT3, identified as a regulator of the Warburg effect in colorectal cancer in this study, may be combined with immunotherapy in the future to enhance its efficacy and address the current challenges in immunotherapy for colorectal cancer.\u003c/p\u003e\n\u003cp\u003eIn the exploration of mechanisms, GSEA enrichment analysis revealed a close association between ONECUT3 and the hypoxia signaling pathway. Colorectal cancer microarray immunohistochemistry results confirmed a positive correlation between ONECUT3 and HIF-1α, as well as its downstream glycolysis-related proteins. Cytological experiments further validated that knockdown of ONECUT3 could suppress the expression of several key enzymes in the glycolytic pathway downstream of HIF-1α.\u0026nbsp;HIF-1α is widely recognized as a crucial effector molecule enabling cells to adapt to hypoxic stress and regulating tumor glycolysis metabolism\u0026nbsp;\u003csup\u003e9\u003c/sup\u003e. HIF-1α plays a significant role in regulating tumor cell glycolysis via transcriptional regulation of the expression of various metabolic enzymes in glycolysis pathway. This pathway includes glucose transporters 1 and 3 (GLUT1/3), pyruvic acid dehydrogenase 1 (PDK-1), lactate dehydrogenase A (LDH-A), and pyruvate kinase M2 (PKM-2). Upregulation of HIF-1α expression during tumor development can enhance the Warburg effect\u003csup\u003e34\u003c/sup\u003e. Based on the significance of HIF-1α in aerobic glycolysis, it was hypothesized that ONECUT3 could potentially regulate tumor glycolytic metabolism via HIF-1α. However, cellular assays showed that both knockdown and overexpression of ONECUT3 did not affect the expression of HIF-1α at the mRNA and protein levels. Further investigation demonstrated that ONECUT3 regulates the transcriptional activity of HIF-1α. However, Co-IP assays indicated that there was no direct interaction between ONECUT3 and HIF-1α. Subsequently, we identified five genes (NAA10, HNF4A, TCEB2, HDAC6, and ELAVL1) that directly interacted with HIF-1α and regulated by ONECUT3. HIF-1α, as a classic regulatory factor in signaling pathways, has been extensively researched. However, there has been limited progress in clinical therapy research targeting HIF-1α, particularly in colorectal cancer. The discovery of ONECUT3 and its regulatory effect on HIF-1α holds potential research value for expanding the study of new drugs targeting HIF-1α.\u003c/p\u003e\n\u003cp\u003eHDAC6, a member of class IIB histone deacetylases, primarily targets α-microtubulin, affecting cytoskeleton and cell mobility\u003csup\u003e35\u003c/sup\u003e. Its overexpression is linked to tumorigenesis and can serve as a prognostic marker. Due to its significant involvement in tumors, HDAC6 has emerged as a potential target for the development of antitumor drugs\u003csup\u003e36\u003c/sup\u003e.\u0026nbsp;Inhibition of HDAC6 selectively reduced HIF-1-mediated transcription under hypoxia\u003csup\u003e26\u003c/sup\u003e. HDAC6 contains two catalytic domains, CD1 and CD2, which are both robust lysine deacetylase\u003csup\u003e35\u003c/sup\u003e, and deacetylase activity of HDAC6 contributed to the stabilization of HIF-1α and its transcriptional activity\u003csup\u003e37\u003c/sup\u003e. Therefore, we identified HDAC6 as a potential downstream factor of ONECUT3. Molecular biology experiments showed that knockdown of ONECUT3 significantly inhibited HDAC6 expression and that ONECUT3 directly regulated the transcriptional expression of HDAC6. The knockdown of ONECUT3 or HDAC6 expression in colon cancer cells significantly increased the acetylation level of HIF-1α. The overexpression of ONECUT3 significantly reduced the acetylation level of HIF-1α, resulting in incresed transcriptional activity of HIF-1α. The inhibition of HDAC6 significantly weakened the regulation of HIF-1α acetylation and transcriptional activity by overexpression of ONECUT3. Due to the high expression of HDAC family members in various malignant tumors and diverse carcinogenic mechanisms, the U.S. Food and Drug Administration (FDA) has approved HDAC inhibitors (HDACIs) for T-cell lymphoma and multiple melanoma therapies. Currently, various HDACIs have been used in combination with other anti-tumor drugs in CRC clinical trials, showing promise in combined therapy\u003csup\u003e38\u003c/sup\u003e. For instance, at the 2023 European Society for Medical Oncology (ESMO) conference, the Cancer Prevention and Treatment Center of Sun Yat-sen University reported a new treatment approach for CRC using Sintilimab in combination with Bevacizumab and Camrelizumab, which preliminarily explored the application of HDACIs with anti-vascular therapy and immunotherapy in colorectal cancer\u003csup\u003e39\u003c/sup\u003e. Our study found that HDAC6 is involved in regulating the Warburg effect in CRC by OC3, and this result may provide new evidence for the application of HDAC inhibitors in colorectal cancer.\u003c/p\u003e\n\u003cp\u003eThis study still has limitations:\u0026nbsp;(1)\u0026nbsp;The mechanism research is not deep enough, especially the molecular mechanism research, which needs further in-depth exploration in subsequent work;\u0026nbsp;(2)\u0026nbsp;The clinical sample size is small, making it difficult to represent the entire clinical population.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, the up-regulated expression of ONECUT3 in colorectal cancer promotes tumor growth by enhancing the transcriptional activity of HIF-1α through HDAC6-mediated deacetylation. This leads to the initiation of glycolytic gene transcription and enhancement of the Warburg effect in colorectal cancer. Targeting ONECUT3 and its associated signaling pathway may hold promise for colorectal cancer therapy.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cp\u003e\u003cstrong\u003e4.1 Bioinformatics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCOAD mRNA data were downloaded from the official website of TCGA. Unsupervised clustering was performed on the TCGA-COAD data to select a classification that includes glycolysis-related genes for hierarchical clustering\u003csup\u003e40\u003c/sup\u003e. ConsensusClusterPlus hierarchical clustering was used to classify colon cancer samples into low-glycolytic and high-glycolytic groups. The limma package of R was used to screen for differentially expressed genes in the two groups. Log2 |Fold change| \u0026ge; 1 and a significance level of p \u0026lt; 0.05 were used as the cut-off value to identify genes that were up-regulated in the high-glycolysis group. The up-regulated genes were compared with the transcription factor subset (MsigDB: TF) of the Molecular Characterization Database for further analysis. The JASPAR database (http://jaspardev.genereg.net/), ALGGENdatabase (http://alggen.lsi.upc.es/home.html), and hTFtarget database (http://bioinfo.life.hust.edu.cn/hTFtarget) were used to predict the potential target genes regulated by ONECUT3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2 Cell culture and reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe obtained human colon cancer cell lines CACO2, LOVO, SW480, SW620 from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China),\u0026nbsp;COLO205, HT29 and the colon immortalized cell line NCM460 from ATCC (American type culture collection). The cells were cultured in suggested standard medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 500 units/mL of penicillin, and 200 \u0026mu;g/mL of streptomycin. The cells were maintained at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. To create a hypoxic environment, the cells were cultured in a hypoxia incubator with an atmosphere containing 1% O\u003csub\u003e2\u003c/sub\u003e, 94% N\u003csub\u003e2\u003c/sub\u003e, and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u0026nbsp;The reagents used in this study included 2-DG (Sigma-Aldrich, D8375).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 Clinical samples and IHC staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe utilized paraffin-embedded tissue microarrays, which consisted of cancer tissues from 141 patients who underwent surgeries at Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, for immunohistochemical analysis. All patients included in this study were pathologically diagnosed with colorectal cancer and had complete clinicopathological characteristics.\u0026nbsp;None of these patients received any preoperative antitumor therapy, such as chemotherapy or radiotherapy. The specimens were collected with informed consent from all patients and approved by the ethics committee of Renji Hospital (KY2021-120-B). Immunohistochemical (IHC) staining was conducted as the previously described\u0026nbsp;\u003csup\u003e41\u003c/sup\u003e. The primary antibodies used for IHC staining included ONECUT3 (1:200, abcam, ab181450), HDAC6 (1:200, Immunoway, YT2118), ALDOA (1:100, proteintech, 11217-1-AP), ENO1 (1:2000, proteintech, 11204-1-AP), TPI1 (1:100, proteintech, 10713-1-AP), GLUT1 (1:200, CST, D3J3A/12939S), and HIF-1\u0026alpha; (1:100, proteintech, 20960-1-AP).\u0026nbsp;The scoring was assessed by two investigators who were blinded to the clinical information, based on the staining intensity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4 Quantitative real-time polymerase chain reaction (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTRIzol total RNA isolation reagent (share-bio, Shanghai, China) was used to extract total RNA from the indicated cells. The quality and quantity of RNA were determined using a Nanodrop\u0026trade; spectrophotometer (NanoDrop products, Wilmington, CA). Then, 1 \u0026mu;g of total RNA was reverse transcribed into complementary DNA (cDNA) using All-in-One First-Strand Synthesis Master Mix (with dsDNase) (share-bio, Shanghai, China). Subsequently, the cDNA product was amplified by PCR on the ViiA 7 Real-Time PCR System (Thermo Scientific) to analyze mRNA expression. The 2*Universal SYBR Green qPCR Premix (share-bio, Shanghai, China) and specific primers were used for qPCR. 18sRNA was used as an internal control. The PCR primer sequences used in this study are listed in supplementary Table 1. Relative quantification was performed using\u0026nbsp;the comparative 2\u0026ndash;\u0026Delta;\u0026Delta;Ct method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eWestern blot analysis and co-immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole-cell protein lysates were obtained using IP lysis buffer (share-bio, Shanghai, China) supplemented with a protease and phosphatase inhibitor (share-bio, Shanghai, China).\u0026nbsp;The cell lysates were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently electrophoretically transferred onto PVDF membranes.\u0026nbsp;The membranes were blocked with 5% defatted milk for 1 h at room temperature (RT), hybridized with primary antibodies overnight at 4 \u0026deg;C, and then incubated with HRP-conjugated secondary antibodies at RT for 60 min.\u0026nbsp;\u0026beta;-actin antibody was used as a loading control.\u0026nbsp;Immunoblots were developed using the Basic Luminol Chemiluminescent Kit (share-bio, Shanghai, China) and the ChemiDoc Touch image system (Bio-Rad). The antibodies used were listed as follows: ONECUT3 (1:500, Abcam, ab181450), HIF1\u0026alpha; (1:1000, Abcam, ab2185), GLUT1 (1:1000, Proteintech, 21829-1-AP), ALDOA (1:10000, Proteintech,11217-1-AP), HDAC6 (1: 10000, Abcam, ab133493), and \u0026beta;-actin (1: 5000, Abcam, ab6276).\u0026nbsp;For co-immunoprecipitation, protein lysates obtained as described above were incubated with\u0026nbsp;Pierce Anti-HA Magnetic Beads (Thermo Fisher Scientific, USA, #88836) or Pierce Protein-A/G Magnetic Beads (Thermo Fisher Scientific, USA, #88803), which were already incubated with anti-HIF1\u0026alpha; (2 \u0026micro;g, Abcam, ab308433) or anti-IgG (as a negative control, 2 \u0026micro;g, Abcam, ab200699) for 15 min at RT, with rotation for 30 min at RT. The immuno-complexes were washed three times with TBS-T or PBS-T and then resuspended in 1 \u0026times; SDS-PAGE sample buffer for western blotting analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.6 Lentivirus production and transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo achieve overexpression, plasmids expressing HA-tagged ONECUT3 were constructed by Shanghai Generay Biotech Co., Ltd. The cDNAs encoding full-length human ONECUT3 (NM_001080488.2) with HA tag were synthesized and inserted into the pCDH-CMV-MCS-EF1-Puro vector (Generay, Shanghai, China). For knockdown, lentiviral siRNA negative control and siRNA oligonucleotides targeting human ONECUT3 were designed and synthesized by Genepharma (Shanghai, China). The sequences for the siRNA were as follows: siONECUT3-1:\u0026nbsp;5\u0026rsquo;-CGCTGATCGCCATCTTCAAGGAGAA-3\u0026rsquo;; HDAC6 siRNA sequence: 5\u0026rsquo;-GGACAACATGGAGGAGGACAATGTA-3\u0026rsquo;. 293T packaging cells were used to produce lentivirus, which was subsequently transfected into target cell lines with 6 \u0026micro;g/ml polybrene for 24 h. Transfected cells used for overexpression or knockdown, as well as their control cells, were selected with 5 \u0026micro;g/ml puromycin for 2 weeks. The overexpression or knockdown efficiency of ONECUT3 was assessed using qRT-PCR and western blotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.7 siRNA targeting ONECUT3 or HDAC6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eONECUT3 or HDAC6 specific siRNA and non-targeting control (NTC) siRNA were purchased from Genepharma (Shanghai, China). According to the instructions of jetPRIME\u0026reg; in vitro DNA \u0026amp; siRNA transfection reagent (Polyplus, Shanghai, China), dilute ONECUT3 or HDAC6 specific siRNA (25 nM) or NTC siRNA (25 nM) into 200 \u0026micro;L of jetPRIME\u0026reg; buffer, then add 4 \u0026micro;L jetPRIME\u0026reg; reagent, incubate for 10 to 15 min at RT, add the transfection mix to the cells in serum containing medium dropwise, and incubate the plate at 37 \u0026deg;C. As mentioned earlier, transfection efficiency was determined by quantitative PCR (qPCR) and immunoblot analysis. Relevant experiments were conducted between 24 and 36 hours after siRNA introduction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.8 Colony formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent types of cells (1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per plate) were seeded in 6-well plates and incubated for approximately 14 days. After the experiments, the formed colonies were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, and stained with 0.2% crystal violet for 30 min. Colonies larger than 100 \u0026mu;m in diameter were counted for each plate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.9 Extracellular acidification rate (ECAR) and oxygen consumption ratio (OCR) assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Seahorse XF96 Flux Analyzer (Seahorse Bioscience, Billerica, Massachusetts, USA) was used to measure the real-time extracellular acidification rate (ECAR) of CRC cells in vitro, following the manufacturer\u0026rsquo;s instructions. Briefly, CRC cells were seeded at a density of 2-3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in an XF96-well plate and allowed to attach overnight. Cells were incubated in non-buffered media under basal conditions for 1 hour. Subsequently, they were sequentially injected with 10 mM glucose, 1 mM mitochondrial poison (oligomycin, Sigma-Aldrich, Saint Louis, Missouri, USA), and 80 mM glycolysis inhibitor (2-deoxyglucose, 2-DG, Sigma-Aldrich). ECAR measurement was normalized by total protein content, as demonstrated by the BCA assay. The experimental data was processed using the Seahorse XF96 Wave software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.10 Subcutaneous xenograft model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells (ONECUT3 knockdown colon cancer cells, and their control cells), resuspended in 100 \u0026mu;l of PBS, were subcutaneously injected into the backs of male Balb/c nude mice (5-6 weeks old, five mice per group). After 5 weeks of subcutaneous inoculation, the experiment was terminated, and the mice were euthanized. The tumors were then collected, weighed, and fixed in formalin. All animal studies were approved by the Animal Care and Use Committee of Shanghai East Hospital, Tongji University School of Medicine. The mice received humane care in accordance with the criteria outlined in the Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences and published by the National Institutes of Health.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.11 HIF-1\u0026alpha; activity measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe quantified HIF-1\u0026alpha; activity by using the HIF-1 alpha Transcription Factor Assay Kit (Abcam, ab133104) following the manufacturer\u0026rsquo;s instructions, after extracting the nuclear fraction. Briefly, processed colorectal cancer cells was collected and the nuclear and cytoplasmic protein were extracted by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0027) . The samples were added to the wells of the HIF-1\u0026alpha; transcription factor plate and then incubated overnight at 4\u0026deg;C. Diluted HIF-1\u0026alpha; primary antibody was added to each well and incubated at room temperature for 1 hour. Subsequently, diluted goat anti-rabbit HRP conjugate was added to each well and incubated at room temperature for 1 hour. After adding the stop solution, the HIF-\u0026alpha; DNA binding activity level was measured at 450 nm using a microplate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.12 Chromatin immunoprecipitation (ChIP) assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChIP assay was were carried out using the EZ-Magna ChIP Assay Kit according to the manufacturer\u0026rsquo;s protocols (Millipore, 17-10086). Briefly, cells were cross-linked with 1% PFA/PBS at room temperature for 10 minutes. Then, unreacted PFA was eliminated using a 10-fold concentration of glycine. Subsequently, samples were sonicated in lysis buffer to obtain DNA fragments ranging from 200 to 1,000 bp. Immunoprecipitation was performed using 5 \u0026mu;g of ONECUT3 or IgG antibodies. Primers targeting the promoter region of the HDAC6 gene were used for quantitative RT-PCR. The results were presented as the relative mRNA expression, calculated by comparing the delta CT values of ONECUT3-specific antibodies with IgG antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.13 Luciferase reporter assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the activity of the HDAC6 gene promoter, processed cells and control cells were seeded into 96-well plates and co-transfected with a PTRF luciferase reporter plasmid. The plasmid contained a tandem repeat of the PTRF transcriptional response element, while the Renilla control reporter served as an internal control. After 48 hours, the cells were lysed, and the enzymatic activity of luciferase and Renilla was measured using the Dual-Luciferase Assay kit (Promega, E1910) following the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.14 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted statistical analyses using SPSS 19.0 for Windows (IBM Corporation) and GraphPad Prism 7 software (San Diego, CA). The results were reported as mean \u0026plusmn; standard deviation (SD) and compared using a two-tailed, unpaired Student\u0026rsquo;s t-test or one-way ANOVA. p-value of \u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank State Key Laboratory of Systems Medicine for Cancer for providing instrumentation and some reagents related to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.J., R.L. and J.X. were in charge of overall direction and planning designed the experiments. R.H., H.W., and W.L. conducted the experiments and statistical analyses. K.H. and S.Z. contributed to sample preparation. R.H. led the writing of the manuscript. All authors provided critical feedback and contributed to shaping the research, analysis, and manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the ethics committee of Shanghai Jiaotong University (KY2021-120-B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by a grant from the Natural Science Foundation of China (82260562, 82372922), the Academic Leaders Training Program of Pudong Health Bureau of Shanghai (PWRd2021-09), the CSCO research funding (Y-xsk2021-0003, Y-2022HER2AZMS-0181), the Science and Technology Commission of Shanghai Municipality (19DZ1910502), and the Top-level Clinical Discipline Project of Shanghai Pudong (PWYgf2021-07).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData available within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMorgan E, Arnold M, Gini A, et al. 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Br J Cancer 2021;124:634-644.\u003c/li\u003e\n\u003cli\u003eSun L, Zhang H, Gao P. Metabolic reprogramming and epigenetic modifications on the path to cancer. Protein Cell 2022;13:877-919.\u003c/li\u003e\n\u003cli\u003evan der Raadt J, van Gestel SHC, Nadif Kasri N, et al. ONECUT transcription factors induce neuronal characteristics and remodel chromatin accessibility. Nucleic Acids Res 2019;47:5587-5602.\u003c/li\u003e\n\u003cli\u003eLaudadio I, Manfroid I, Achouri Y, et al. A feedback loop between the liver-enriched transcription factor network and miR-122 controls hepatocyte differentiation. Gastroenterology 2012;142:119-29.\u003c/li\u003e\n\u003cli\u003eSapkota D, Chintala H, Wu F, et al. Onecut1 and Onecut2 redundantly regulate early retinal cell fates during development. Proc Natl Acad Sci U S A 2014;111:E4086-95.\u003c/li\u003e\n\u003cli\u003eJiang K, Jiao Y, Liu Y, et al. HNF6 promotes tumor growth in colorectal cancer and enhances liver metastasis in mouse model. 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Homeostatic maintenance and age-related functional decline in the Drosophila ear. Sci Rep 2020;10:7431.\u003c/li\u003e\n\u003cli\u003eIto R, Kimura A, Hirose Y, et al. Elucidation of HHEX in pancreatic endoderm differentiation using a human iPSC differentiation model. Sci Rep 2023;13:8659.\u003c/li\u003e\n\u003cli\u003eFrancius C, Clotman F. Dynamic expression of the Onecut transcription factors HNF-6, OC-2 and OC-3 during spinal motor neuron development. Neuroscience 2010;165:116-29.\u003c/li\u003e\n\u003cli\u003eSoni S, Padwad YS. HIF-1 in cancer therapy: two decade long story of a transcription factor. Acta Oncol 2017;56:503-515.\u003c/li\u003e\n\u003cli\u003eHo TCS, Chan AHY, Ganesan A. Thirty Years of HDAC Inhibitors: 2020 Insight and Hindsight. J Med Chem 2020;63:12460-12484.\u003c/li\u003e\n\u003cli\u003eAldana-Masangkay GI, Sakamoto KM. The role of HDAC6 in cancer. J Biomed Biotechnol 2011;2011:875824.\u003c/li\u003e\n\u003cli\u003eRyu HW, Won HR, Lee DH, et al. HDAC6 regulates sensitivity to cell death in response to stress and post-stress recovery. Cell Stress Chaperones 2017;22:253-261.\u003c/li\u003e\n\u003cli\u003eGarmpis N, Damaskos C, Garmpi A, et al. Histone Deacetylases and their Inhibitors in Colorectal Cancer Therapy: Current Evidence and Future Considerations. Curr Med Chem 2022;29:2979-2994.\u003c/li\u003e\n\u003cli\u003eWang F, Jin Y, Wang M, et al. Combined anti-PD-1, HDAC inhibitor and anti-VEGF for MSS/pMMR colorectal cancer: a randomized phase 2 trial. Nat Med 2024.\u003c/li\u003e\n\u003cli\u003eSpranger S, Bao R, Gajewski TF. Melanoma-intrinsic beta-catenin signalling prevents anti-tumour immunity. Nature 2015;523:231-5.\u003c/li\u003e\n\u003cli\u003ePeng W, Huang W, Ge X, et al. Type Igamma phosphatidylinositol phosphate kinase promotes tumor growth by facilitating Warburg effect in colorectal cancer. EBioMedicine 2019;44:375-386.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplemental Table 1","content":"\u003cp\u003eSupplemental Table 1 isnot available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"the Warburg effect, colorectal cancer, ONECUT3, HIF-1α, HDAC6","lastPublishedDoi":"10.21203/rs.3.rs-4296431/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4296431/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The Warburg effect, also known as aerobic glycolysis, plays a crucial role in the onset and progression of colorectal cancer, although its mechanism remains unclear. Bioinformatics analysis of public databases and verification of clinical specimens revealed that the transcription factor ONECUT3 is a key regulator related to the Warburg effect in colorectal cancer. Functionally, genetic silencing of ONECUT3 reverses the Warburg effect and blunts tumor growth. Importantly, ONECUT3 promotes tumor growth in a glycolysis-dependent manner. Mechanistically, ONECUT3 does not directly alter the expression of hypoxia-inducible factor 1α (HIF-1α), but rather inhibits the acetylation of HIF-1α via histone deacetylase 6 (HDAC6). This inhibition leads to increased transcriptional activity of HIF-1α, ultimately upregulating various glycolysis-related genes downstream of HIF-1α, thereby promoting the Warburg effect in colorectal cancer and facilitating tumor growth. Our study provides evidence for the mechanism of the Warburg effect in colorectal cancer, suggesting that ONECUT3 could be a potential new target for colorectal cancer treatment.","manuscriptTitle":"Transcription factor ONECUT3 regulates HDAC6/HIF-1α activity to promote the Warburg effect and tumor growth in colorectal cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-23 18:42:42","doi":"10.21203/rs.3.rs-4296431/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-08-12T11:18:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-07-08T04:24:47+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-03T08:51:49+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-05-16T08:27:07+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-05-12T20:27:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-22T10:24:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2024-04-20T07:53:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-20T07:53:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7e80fb9e-03c8-4bb0-a3d6-522b5ce4a376","owner":[],"postedDate":"May 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31821483,"name":"Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer/Colon cancer"},{"id":31821484,"name":"Health sciences/Diseases/Cancer/Cancer metabolism"}],"tags":[],"updatedAt":"2025-03-04T08:09:41+00:00","versionOfRecord":{"articleIdentity":"rs-4296431","link":"https://doi.org/10.1038/s41419-025-07457-8","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-03-03 05:00:00","publishedOnDateReadable":"March 3rd, 2025"},"versionCreatedAt":"2024-05-23 18:42:42","video":"","vorDoi":"10.1038/s41419-025-07457-8","vorDoiUrl":"https://doi.org/10.1038/s41419-025-07457-8","workflowStages":[]},"version":"v1","identity":"rs-4296431","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4296431","identity":"rs-4296431","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}