Wnt/β-catenin/HNF4α feedback loop facilitates colorectal tumorigenesis and malignancy

Wnt/β-catenin/HNF4α feedback loop facilitates colorectal tumorigenesis and malignancy | 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 Research Article Wnt/β-catenin/HNF4α feedback loop facilitates colorectal tumorigenesis and malignancy Weiyu Bai, Lei Sang, Chenglu Lu, Rui Dong, Qinggang Hao, Yingru Zhang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6208041/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: The Wnt/β-catenin signaling pathway is a central regulator of colorectal cancer (CRC) development, yet its downstream targets and mechanistic contributions to tumorigenesis remain poorly defined. Hepatocyte nuclear factor 4 alpha (HNF4α), a transcription factor primarily studied in liver function and hepatocarcinogenesis, has unclear roles in CRC. This study investigates the interplay between HNF4α and Wnt/β-catenin signaling in colorectal carcinogenesis and explores its clinical relevance. Methods: Using bulk RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), in vitro and in vivo CRC models, and clinical tumor samples, we assessed HNF4α expression and its regulation by Wnt/β-catenin signaling. Transcriptional activation of HNF4α was evaluated via luciferase reporter assays and chromatin immunoprecipitation. Clinical correlations between HNF4α levels and Wnt/β-catenin activity were analyzed using immunohistochemistry, RNA sequencing, and Spearman’s rank correlation. Statistical significance was determined by Student’s t-test and ANOVA. Results: HNF4α was significantly overexpressed in CRC tissues compared to normal controls and significantly promoted tumor growth in subcutaneous xenograft models using nude mice. Mechanistically, HNF4α was transcriptionally activated by the Wnt/β-catenin/TCF7L1 axis, forming a positive feedback loop that amplified oncogenic Wnt signaling. Clinically, HNF4α expression strongly correlated with Wnt/β-catenin pathway activation in patient samples (r = 0.58, p < 0.0001). Functionally, HNF4α knockdown suppressed CRC cell proliferation and inhibited Wnt-driven tumorigenesis. Conclusions: This study identifies HNF4α as a novel downstream effector of the Wnt/β-catenin pathway and a critical driver of CRC progression. The Wnt/β-catenin/HNF4α feedback loop uncovered here provides mechanistic insights into colorectal carcinogenesis and highlights HNF4α as a potential therapeutic target. These findings may inform strategies to disrupt Wnt signaling hyperactivation in CRC. Colorectal cancer HNF4α Wnt/β-catenin TCF7L1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Colorectal cancer (CRC) is one of the most common cancers and the second leading cause of cancer deaths worldwide[1, 2]. Despite an overall decreasing incidence of the disease, early-onset CRC is rapidly rising in individuals under the age of 50 over the last two decades. While several major targets have been identified during the course of colorectal cancer development such as Ras[3], Tp53[4], and APC/β-catenin[5], the pathogenesis of CRC still remains incompletely understood. Hepatocyte Nuclear Factor 4 Alpha (HNF4α) is a transcription factor predominantly expressed in the liver, kidney, intestine and endocrine pancreas, and is necessary for liver development and function[6–8]. There is accumulating evidence showing its inhibitory role in the liver cancer. Mutations in HNF4α at G79C, F83C and M125I (Zn-finger DNA-binding domain region) have been detected in liver cancer and are believed to trigger liver tumorigenesis[9]. Previous studies have revealed the contradictory role of HNF4α in colorectal carcinogenesis[10]. While studies indicate that HNF4α promotes the occurrence of colorectal cancer[11–17], others suggest that HNF4α inhibits the occurrence and development of colorectal cancer[18–20]. The mechanisms regulating HNF4α expression in colorectal cancer are largely unknown. Further investigation of the role and the specific regulation mechanism of HNF4α in the development and progression of colorectal cancer is of great significance for establishing HNF4α as a diagnostic/prognostic marker and therapeutic target in colorectal cancer. The Wnt/β-catenin signaling pathway is a well-recognized driver of colorectal carcinogenesis, and dysregulation of this cascade occurs in 70–80% of colorectal cancers[21–24]. Activation of the Wnt pathway induces β-catenin translocation into the nucleus, where it functions as a transcriptional coactivator of TCF /LEF family factors[25–29]. While several molecules that regulate this cascade have been identified including APC, GSK3β and AXIN[23, 30–32], the role and regulation of Wnt/β-catenin activation in colorectal cancer are not completely understood. Here, we demonstrate that HNF4α is upregulated in colorectal cancer and promotes colorectal tumorigenesis. HNF4α levels positively correlate with the Wnt/β-catenin-TCF7L1 signaling pathway and was directly regulated by it. We further show that HNF4α is a positive regulator of the Wnt/β-catenin pathway and that HNF4α overexpression promotes β-catenin nuclear localization. Our results suggest that HNF4α is a potential prognostic marker for Wnt/β-catenin-related colorectal cancer and that HNF4α forms a feedback loop with Wnt/β-catenin to control colorectal tumorigenesis. Materials and methods Apc-flox-Villin-Cre mice The Apc-flox mice were obtained from Dr. Yeguang Chen lab at Tsinghua University, while the Villin-Cre(RRID:IMSR_JAX:004586) mice were provided by Professor Yonglong Wei at Yunnan University. By crossing Apc-flox heterozygous mice with Villin-Cre mice, we generated Apc (f/+) -Villin-Cre (f/+ ) mice, which spontaneously develop intestinal cancer. In contrast, Apc (f/f) -Villin-Cre (f/+) embryos were lethal. Cell culture and activator treatment CRC cell line SW480, SW620 and DLD-1 were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. For the inhibitor and activator treatment, cells (5 × 10 5 ) were seeded in 6 well plates and treated with 2uM or 5 uM SKL2001 and CHIR99021 for 12 h, 24 h, and 36 h after the cells attached to the dish. RNA interference RNA interference was performed using the plko.1 vector. To efficiently knockdown β-catenin and TCF family, the shRNAs targeting to different genes were synthetized and inserted to plko.1 vector between Age I and Eco R I. The target sequences were as follows: β-catenin shRNA: 5’-TTGTTATCAGAGGACTAAATA-3’; TCF7L1 shRNA: 5’-GCACCTACCTGCAGATGAAAT-3’; TCF1 shRNA: 5’-CAACTCTCTCTCTACGAACAT-3’; LEF1 shRNA: 5’-CCATCAGATGTCAACTCCAAA-3’; TCF4 shRNA: 5’-CCTTTCACTTCCTCCGATTAC-3’; HNF4α shRNA 1:5’-CATGTACTCCTGCAGATTTAG-3’; HNF4α shRNA 2: 5’-ATCACCAAGCAGGAAGTTATC-3’. Western blotting Western blotting Western blotting was performed as described previously[33]. Cells were lysed in RIPA buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1 mM PMSF) containing Complete Protease Inhibitor Cocktail (Roche). Cell lysates were spun down at 12,000 rpm for 10 min, and 50 µg supernatants were resolved on SDS-PAGE and blotted with the indicated antibodies. The amount of GAPDH was used as the loading control. The results were detected by an ECL-plus Western blotting detection system (Tanon-5200Multi). The primary antibodies used in this study were as follows: Rabbit anti-HNF4α (1:1000; Cat# 3113, Cell Signaling Technology, USA); Rabbit anti-TCF7L1 (1:1000; Cat# PA5-40327, Thermo Fisher Scientific, USA); Mouse anti-TCF1 (1:1000; Cat# sc-271453, Santa Cruz Biotechnology, USA);Rabbit anti-β-catenin (1:1000; Cat# A117811, GenScript, USA); Rabbit anti-Actin (1:1000; Cat# 4970, Cell Signaling Technology, USA); Mouse anti-GAPDH (1:1000; Cat# sc-166574, Santa Cruz Biotechnology, USA). Quantitative reverse transcription–polymerase chain reaction (qPCR) Total RNA was extracted using Trizol (Lablead Biotech, Beijing, China). 2 µg RNA was used as template to generate cDNAs using the ImProm-II Reverse Transcription system (TransGen Biotech, Beijing, China). qPCR reactions were carried out on an CFX96 Real-Time System. Mouse Xenograft All mice were obtained from the Animal Research and Resource Center, Yunnan University. All animal experiments were performed according to protocols approved by the Animal Care Committee of the Yunnan University (Kunming, China).For the Subcutaneous tumor models,1×10 6 SW620 cells were re-suspended in 100 µl 0.25 mg/ml Matrigel (Corning) with PBS buffer, then injected into the flanks of the nude mouse. The tumor volumes were measured from day 3 to 11 after injection. At 11 days after the injection, tumors were dissected. Tumors were then imaged and weighted. GESA analysis and correlation analysis The mRNA sequencing data of COAD, READ, BRCA, LIHC, LUAD, LUSC, PAAD and SKCM were downloaded from TCGA database ( https://portal.gdc.cancer.gov/repository ) respectively, and the merge normal tissues UCSC Xena ( https://xenabrowser.net ) mRNA Sequencing data were downloaded as control. the Pearson correlation coefficient R value and the correlation curve were calculated and drawn by Using the ggplot2 function in R 4.4.0 software. The boxplot was drawn using the simplevis package after processing the TPM as log2 (TPM + 1). The genes that are significantly(p < 0.01, r ≥ 0.3 or r ≤ − 0.3) related to HNF4α were screened by FPKM value of COAD READ and used for GSEA and KEGG enrichment analysis[34]. Bulk RNA-seq, scRNA-seq and estimate analysis The total RNA of SW480 and SW480-HNF4α cells were harvested with Trizol. Three biological replicates were used. These samples were subjected to transcriptome RNA-sequencing by Novogene[35]. The paired-end RNA-sequencing library was employed (2 × 150 bp read length) using an Illumina NovaSeq 6000 sequencer. Both raw data and clean data were provided by Novogene and the sequencing quality was assessed using FastQC. The index construction is performed using bowtie2 ( http://bowtie-bio.sourceforge.net/bowtie2/index.shtml ), the genome alignment of the clean data is conducted using TopHat2 ( https://ccb.jhu.edu/software/tophat/index.shtml ), and the transcriptome assembly is performed using Cufflinks ( https://cole-trapnell-lab.github.io/cufflinks/ ). The gene expression level was calculated according to the fragments per kilobase million reads (FPKM) method. The formula for differential analysis calculation is log2(SW480-HNF4α mean (FPKM)/SW480 control mean (FPKM)). The p-value is calculated using the t-test different expressed genes (DEGs) with |log2(foldchange)| ≥ 1, and P-value < 0.05 were significantly (DEGs). Single cell sequencing data was analyzed using the seura (v 5.1.0) R package t[36]. Luciferase Assay The dual luciferase assay was performed according to a previously reported protocol with minor modification[37]. The HNF4α promoter luciferase reporter constructs were generated by inserting 1700 bp, 800 bp or 300 bp human HNF4α promoter into the pGL3 basic vector (Promega) between XhoI and HindIII. To perform the dual luciferase reporter assay, 20,000 SW480 cells were seeded in 12-well plates and cultured overnight. Cells were transfected with 1 ug/well HNF4α promoter reporter together with 100 ng/well Renilla luciferase construct (pRL-TK RRID: Addgene_11313) using Lipofectamine 2000. 24h after transfection, the cells were lysed and cell lysates were subjected to dual reporter luciferase assays according to the manufacturer’s instructions (Promega). Tissue microarray and immunohistochemistry Tissue microarrays (TMAs) were constructed using a manual tissue microarray instrument (Beecher Instruments) equipped with a 2.0 mm punch needle, as previously described[38]. An immunohistochemical (IHC) study of the rabbit anti-human β-catenin, rabbit anti-human TCF7L1 antibody, and rabbit anti-human HNF4α antibody was carried out on formalin-fixed paraffin-embedded tissues, with a 4-µm-thick serial section of tissues, according to the manufacturer’s recommended protocol. The levels of β-catenin, TCF7L1 and HNF4α were assessed via the average of 5 count fields per patient at an original magnification of X200 on light microscopy[38]. The positive cells for HNF4α were defined as those with brown staining. The expression of β-catenin, TCF7L1 and HNF4α was scored based on staining intensity. Staining intensity was subclassified as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. The primary antibodies used in this study were as follows: Rabbit monoclonal anti-HNF4α (1:200 dilution; Cat# 3113, Cell Signaling Technology, USA); Rabbit polyclonal anti-TCF7L1 (5 µg/mL; Cat# PA5-40327, Thermo Fisher Scientific, USA); Ready-to-use rabbit monoclonal anti-β-catenin (Cat# ZA-0646, Zhongshan Golden Bridge Biotechnology, China). Immunofluorescence Mouse intestinal tissues were harvested, embedded in OCT compound, and rapidly frozen in liquid nitrogen or on dry ice. Cryosections (8–10 µm thick) were prepared using a cryostat and mounted on glass slides, followed by air-drying at room temperature for 30 minutes. The sections were fixed with 4% paraformaldehyde in PBS for 15 minutes, washed three times with PBS (5 minutes each), and permeabilized with 0.3% Triton X-100 in PBS for 10 minutes. After another set of PBS washes, sections were blocked with 5% BSA or 10% normal goat serum in PBS for 1 hour at room temperature. Primary antibodies, including HNF4α (CST #3113, 1:200) and β-Catenin (CST #37447, 1:200), were diluted in blocking solution and incubated with the sections overnight at 4°C in a humidified chamber. The next day, sections were washed three times with PBS and incubated with species-specific secondary antibodies for 1 hour at room temperature in the dark: Alexa Fluor® 488-conjugated Anti-Rabbit IgG (H + L), F(ab')₂ Fragment (CST #4412, 1:500) for HNF4α and Alexa Fluor® 594-conjugated Anti-Mouse IgG (H + L), F(ab')₂ Fragment (CST #8890, 1:500) for β-Catenin. After another round of PBS washes, nuclei were counterstained with DAPI (1 µg/mL in PBS) for 5 minutes, followed by final PBS washes. Sections were mounted with an anti-fade mounting medium and covered with a coverslip. Images were acquired using a fluorescence or confocal microscope with appropriate filter settings for Alexa Fluor® 488 (HNF4α), Alexa Fluor® 594 (β-Catenin), and DAPI. Negative controls were prepared by omitting the primary antibodies to confirm the specificity of secondary antibody staining. Statistics analysis Data were analyzed with Prism (GraphPad software RRID:SCR_002798). Statistical analyses were performed using t -tests or ANOVA. * P < 0.05 was considered statistically significant. ** P < 0.01 was considered significant. *** P 0.05 was considered not significant (ns). Results 1. HNF4α promotes colorectal carcinogenesis Our recent studies have shown that the loss of nhr-14/HNF4α in C. elegans resulted in the dysregulation of DNA damage-induced responses[39], which usually lead to cancer development[40–48]. Thus, we examined HNF4α expression levels in different cancers and matched paired normal tissues. TCGA database analysis revealed upregulation of HNF4α in Colon adenocarcinoma (COAD), Rectum adenocarcinoma (READ) and Pancreatic adenocarcinoma (PAAD) (Fig. 1 A), suggesting that HNF4α may play an important role in gastrointestinal tumorigenesis, particularly in colorectal carcinogenesis. We further analyzed HNF4α protein levels in 59 paired CRC and normal tissues and found a significant increase in HNF4α in 43 tumors (Fig. 1 B and 1 C). Immunohistochemical staining of colorectal cancer microarray also revealed remarkable upregulation of HNF4α in cancer tissues (n = 244) compared to normal colon tissues (n = 99) (Fig. 1 E). These results indicate that overexpression of HNF4α is a recurrent event in colorectal cancer and could play a significant role in this malignancy. To determine if HNF4α plays a role in the malignancy in colorectal cancer, we measured HNF4α expression levels in 26 colorectal cancer patients. We found that HNF4α was significantly higher in malignant tumors (Fig. 1 F-G) and lower levels of immune cells were present in tissues with high expression of HNF4α (Fig. 1 H-J). Through cell communication analysis, we found strong signaling communication between malignant cells (high HNF4α expression) and CD4 + T cells (Figure S1 A -S1B), and through cell correlation analysis, we found a significant positive correlation between malignant cells and T cells (Figure S1 C), indicating an interaction between malignant cells and T cells within the immune microenvironment. The strong communication may allow malignant cells to evade immune system surveillance by regulating the function of CD4 + T cells, thereby inhibiting the anti-tumor activity of CD4 + T cells and promoting their own growth and spreading. This indicates that CD4 + T cells may be activated or inhibited in specific immune states, thereby affecting the overall response of the immune system to tumors[49]. The tumor immune microenvironment is closely related to tumor proliferation and immune evasion. To investigate if HNF4α is involved in tumor immune evasion, we performed ESTIMATE analysis on single-cell sequencing data and observed that cells with high HNF4α expression had lower immune scores and ESTIMATE scores, but higher tumor purity scores and stromal scores (Figure S1 D), suggesting that HNF4α may promote malignancy in colorectal cancer. Meanwhile, the Wnt/β-catenin signaling was also enriched in malignant tumors based on Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and immune scores (Figure S1 E-S1F). 2. HNF4α expression is associated with tumor formation in colorectal cancer Previous studies have shown that HNF4α could behave as either a tumor suppressor or an oncogene, depending on the cellular and molecular context[11–17]. Thus, we investigated the function of HNF4α in colorectal cancer by stable transfection of HNF4α into SW480 or SW620 CRC cell lines. We found that ectopic expression of HNF4α significantly increased colony formation (Fig. 2 A-B). Xenograft mouse experiments also showed that overexpression of HNF4α considerably promoted the tumor growth and increased the tumor volume and weight (Fig. 2 C-F). Conversely, when HNF4α is knocked down, the tumor formation rate of colorectal cancer is significantly inhibited (Fig. 2 G-J). These findings suggest that HNF4α acts as an oncogene and could be a driver in colorectal cancer. 3. HNF4α positively correlated with the Wnt/β-catenin signaling pathway and is regulated by it To examine the mechanism by which HNF4α is upregulated in colorectal cancer, we performed GESA analysis using COAD and READ datasets from the TCGA database. Among the enriched HNF4α-correlated signaling pathway, oncogenic Wnt/β-catenin and MYC pathways were significantly and positively associated with HNF4α expression (Fig. 3A). It has been reported that the WNT/β-catenin pathway transcriptionally induces c-Myc expression to promote colorectal tumorigenesis[50]. Thus, we primarily focused on the relationship between HNF4α and the Wnt/β-catenin pathway. We also analyzed the correlation between β-catenin and HNF4α, and the result indicated that β-catenin positively associated with HNF4α expression levels in TCGA database (Fig. 3B). Then we transfected SW480 cells with shRNA-HNF4α and control shRNA and found no expression change of CTNNB1 at mRNA or protein levels between HNF4α-knockdown and control cells (data not shown). Conversely, SW480 cells after transfection with shRNA-CTNNB1 expressed significant low levels of HNF4α mRNA when compared to the cells treated with control shRNA (Fig. 3C). Moreover, we transfected shRNA-CTNNB1 into 2 other CRC cell lines (SW620 and DLD1). Western blotting analysis showed that knockdown of β-catenin significantly reduced the protein level of HNF4α in SW480, SW620 and DLD1 cell lines (Fig. 3D). These results suggest that HNF4α is regulated by the WNT /β-catenin pathway at the transcription level. To further evaluate this notion, we treated CRC cells with SKL2001, an activator of the Wnt/β-catenin pathway, at different doses for 12h. Western blotting analysis of cellular fractionation revealed significant nuclear accumulation of β-catenin and increased expression of HNF4α following SKL2001 treatment (Fig. 3E-F). In addition, we treated SW480 and SW620 cells with Chir-99021, another β-catenin activator, and found upregulation of HNF4α in both cell lines after Chir-99021treatment (Fig. 3F). These results further confirmed that β-catenin is an activator of HNF4α. As HNF4α is a downstream target of the Wnt/β-catenin pathway, we next investigated whether HNF4α mediates the Wnt/β-catenin axis in colon carcinogenesis. SW620 cells were stably transfected with β-catenin shRNA or β-catenin shRNA and HNF4α overexpression, respectively. The cells treated with vectors alone were used as the control (Fig. 3G). These cells were subcutaneously injected to nude mice. As expected, knockdown of β-catenin inhibited the tumor growth and reduced the tumor volume and weight (Fig. 3H, J). Notably, ectopic expression of HNF4α largely overrode these phenotypes caused by β-catenin knockdown (Fig. 3H, I). These findings further indicate that HNF4α is a major target of β-catenin in CRC. 4. TCF7L1 promotes HNF4α expression β-catenin is a transcription co-activator of the TCF/LEF transcription factor (TF) family. To further understand the molecular mechanisms by which Wnt/β-catenin induces HNF4α transcription, we first analyzed the expression relationship between HNF4α and TCF7 (also known as TCF-1), LEF1, TCF7L1 (also known as TCF-3), TCF7l2 (also known as TCF-4), which are TCF/LEF family members, in colorectal cancer through the TCGA database. We found that HNF4α had the highest correlation with TCF1 and TCF7L1, but not LEF1 or TCF7L2 (Fig. 4 A-D). When individually knocked down in SW480 (Fig. 4 E-F), HNF4α was reduced in TCF7L1-knockdown cells, but not in shRNA-TCF1, shRNA-TCF7L1 or shRNA-LEF1 treated cells (Fig. 4 E). These results were confirmed in two different colorectal cancer cell lines, LS174T and DLD1. Similarly, TCF7L1 knockdown decreased HNF4α expression (Fig. 4 G). Moreover, ectopic expression of TCF7L1 induced HNF4α levels, but which was abrogated by β-catenin depletion (Fig. 4 H-I). In single cell RNA sequencing data from 26 colorectal cancer, both HNF4α and TCF7L1 were highly expressed in malignant cells (Fig. 4 J-K). These results suggest that TCF7L1 is a major transcriptional factor that mediates the action of Wnt/β-catenin in response to HNF4α in colorectal cancer. 5. TCF7L1 directly binds to the − 90 ~ -80 bp region of the HNF4α promoter To investigate whether TCF7L1 can directly bind to the HNF4α promoter to regulate HNF4α transcription, we first examined the TCF7L1 consensus binding motif “CACCTGC” in the HNF4α promoter region ( https://jaspar.genereg.net/ ). As shown in Fig. 5 A, there are 10 TCF7L1 putative binding sites within a 2.0-kb HNF4α promoter. To determine which binding site(s) is required for TCF7L1-mediated HNF4α transcription, we constructed deletion mutants of the HNF4α promoter including pGL3-P HNF4α 1700bp, pGL3-P HNF4α 800bp and pGL3-P HNF4α 300bp, which contain 10, 7 and 3 TCF7L1 putative binding sites, respectively (Fig. 5 A). Luciferase reporter assays revealed that knockdown of TCF7L1 significantly reduced the basal promoter activity in three HNF4α deletion promoter mutants (Fig. 5 B), suggesting that pGL3-P HNF4α 300bp contains major TCF7L1 response motifs (Fig. 5 A). Since there are 3 TCF7L1 putative binding sites in pGL3-P HNF4α 300bp, we next mutated each site in this promoter region (Fig. 5 C). Interestingly, mutations of the 90 to 80 bp motif, which is the closest to transcription start site of the HNF4α gene, completely abrogated the pGL3-P HNF4α 300bp promoter activity, whereas mutation of the rest two sites had no significant effects on the promoter activity (Fig. 5 D). CHIP-qPCR and CHIP-seq results further corroborated this conclusion (Fig. 5 E-G). These findings suggest that TCF7L1 transcriptionally activates HNF4α primarily through binding to the first motif of the HNF4α promoter. 6. HNF4α activates WNT signaling and promotes β-catenin nuclear localization In an attempt to investigate the mechanism of HNF4α in colorectal tumorigenesis, we next performed RNA sequencing analysis between SW480-vector and SW480-HNF4a cells. The signals enriched by HNF4α overexpression were involved in cardiac system diseases, neurological diseases, tumor progression and other features, among which the most tumor-related signaling pathways were the MAPK, Hippo and Wnt signaling cascades (Fig. 6 A). The GSEA enrichment analysis results showed that the Wnt/β-catenin signaling pathway was activated after HNF4α overexpression (Fig. 6 D), which was also confirmed by TOPFlash luciferase reporter assay (Fig. 6 G). Notably, Wnt1, Wnt4, Wnt7b and Wnt11 were up-regulated in SW480-HNF4α cells when compared to SW480-vector control cells (Fig. 6 B), which were further confirmed by the qPCR assay (Fig. 6 C). However, we did not observe expression level changes of β-catenin in the cells ectopically expressing HNF4α (Fig. 6 E). As HNF4α induces expression levels of several Wnt family members, we hypothesized that HNF4α could promote β-catenin translocation from the cytoplasm to the nucleus. Cell fractionations were obtained from SW480-vector and SW480-HNF4α cells. Immunoblotting analysis revealed that the nuclear fraction of β-catenin was significantly enriched in the HNF4α overexpressing cells (Fig. 4 F). To further explore the relation between HNF4α and WNT expression, we examined HNF4α and WNTs expression levels in single cell RNA sequencing data from 26 colorectal cancer patients. We observed that high HNF4α expression was accompanied by nearly uniform high WNT family gene expression (Fig. 1 F and 6 H), which are consistent with our previous bulk RNA-seq findings and indicating that HNF4α is associated with the WNT signaling pathway and plays a crucial role in the development and progression of colorectal cancer. To further validate the transcriptional regulatory role of HNF4α on WNT family genes, we conducted comparative HNF4α ChIP-seq analyses using intestinal tissues from wild-type and HNF4α knockout mice. The experimental results demonstrated a significant reduction in peak signals at promoter regions of WNT family genes following HNF4α depletion (Fig. 6 I). These findings collectively suggest that HNF4α maintains transcriptional activation of WNT family genes through dual mechanisms: direct DNA binding and chromatin structure modulation. Collectively, our findings indicate that HNF4α and Wnt/β-catenin regulated each other and form a feedback loop to contribute to colorectal carcinogenesis. 7. The β-catenin/TCF7L1-HNF4α axis correlates with colorectal tumorigenesis To evaluate the clinical significance of β-catenin/TCF7L1-HNF4α axis in CRC, we performed IHC analysis and examined the HNF4α and β-catenin/TCF7L1 expression level in a CRC tissue array and investigated the correlation of β-catenin/ TCF7L1 with HNF4α in colorectal cancer. The results showed that the tumor tissue with high levels of β-catenin/TCF7L1 usually express high levels of HNF4α, and the tumor tissue with low levels of β-catenin/TCF7L1 usually have low levels of HNF4α (Fig. 7 A). The correlation analysis indicated that HNF4α was significantly correlated with β-catenin and TCF7L1 (Fig. 7 B and 7 C). We also tested the correlation between TCF7L1, and HNF4α in 26 colorectal cancer patients. The result indicated that HNF4α, CTNNB1 and TCF7L1 have the same expression pattern in pseudo temporal analysis (Fig. 7 D and 7 E). Taken together, these results indicated that HNF4α was correlated with the Wnt/β-catenin pathway and involved in colorectal tumorigenesis. To validate this finding in vivo, we examined the expression of HNF4α and β-catenin in the intestinal tissues of APC-deficient mice. We found strong co-localization and a similar expression pattern between HNF4α and β-catenin (Fig. 7 F), which further supports our hypothesis. To further test our hypothesis, we employed AOM/DSS Model of Colitis-associated colorectal cancer model [51], and found that AOM/DSS treatment can not only induce intestinal inflammation and colorectal cancer, but also induce highly express β-catenin and HNF4α in the intestinal tissues of treated mice (Fig. 7 G). The result implied that the β-catenin/TCF7L1-HNF4α axis plays an important role in colorectal carcinogenesis. In conclusion, our findings reveal that HNF4α is regulated by β-catenin/TCF7L1 and promotes the expression of Wnt family genes, ultimately activating the Wnt/β-catenin signaling pathway to drive tumorigenesis (Fig. 7 H). These discoveries provide novel theoretical foundations and potential therapeutic strategies for cancer prevention and treatment. Discussion CRC is a highly heterogeneous disease and the expression regulation of HNF4α isoforms in colorectal tumors exhibits significant specificity. Distinct HNF4α subtypes play divergent roles in tumorigenesis and progression[10]. Studies indicate that the loss of HNF4α is closely associated with cancer initiation and development[52–54]. For instance, Src kinase reduces the protein stability, nuclear localization, and transcriptional activity of HNF4α through phosphorylation at the Y14 site, a mechanism particularly prominent in specific isoforms[55]. During cancer progression, approximately 80% of stage III tumors demonstrate either loss of HNF4α or aberrant cytoplasmic localization, which correlates strongly with Src kinase activity. Single-cell chromatin accessibility analysis reveals that HNF4α is a key iCMS-specific transcription factor in colorectal malignant tumor cells, suggesting that HNF4α may play important roles in colorectal tumorigenesis and progression. However, the role and regulatory mechanism of HNF4α and TCF7L1 in colorectal cancer are still largely unknown. Wnt/β-catenin activation is a well-recognized driver of colorectal carcinogenesis. However, the regulation of TCF7L1, HNF4α and Wnt/β-catenin in colorectal cancer is still largely unknown. In specific CRC cell lines, β-catenin has been found to negatively regulate HNF4α expression. This observation aligns with prior studies showing that HNF4α competes with β-catenin for TCF4 binding, thereby suppressing the expression of downstream Wnt target genes and inhibiting colorectal cancer progression[19, 56]. However, some studies have also shown that HNF4α promotes the occurrence of colorectal cancer. The specific mechanism and reasons behind it are still unclear. Our results demonstrate that TCF7L1 can bind to the − 90~-80 bp region of the HNF4α promoter, thereby regulating the transcription of HNF4α and activating the Wnt/β-catenin signaling pathway, which promotes the tumorigenesis of colorectal cancer. There are four transcription factors (TCF1, LEF1, TCF7L1 and TCF7L2) in the Wnt/β-catenin signaling pathway and they exhibit significant differences in regulating target genes[57]. Compared to the other three transcription factors, TCF7L1 was the least reported in tumors. The mutual regulatory relationship between TCF7L1 and HNF4α has not been reported. These findings indicate that Wnt/β-catenin signaling can be activated by TCF7L1 and HNF4α in CRC. In conclusion, our study demonstrates that the Wnt/β-catenin-TCF7L1-HNF4α feedback loop confers colorectal tumorigenesis and malignancy and HNF4α overexpression feedback regulates Wnt expression and promotes β-catenin nuclear localization. Further investigation of the role and the specific mechanism of HNF4α in the development and progression of colorectal cancer is of great significance for establishing HNF4α as a therapeutic target in colorectal cancer. Our study provides new strategies for the treatment and prevention of colorectal cancer. Abbreviations CRC Colorectal cancer HNF4α Hepatocyte nuclear factor 4 alpha CTNNB1 Catenin beta 1 TCF1 Liver-specific transcription factor lf-b1 TCF7L1 Transcription factor 7-like 1 TCF7L2 Transcription factor 7 like 2 LEF1 Lymphoid enhancer binding factor 1 WNT3 Wnt Family Member 3 WNT4 Wnt family member 4 WNT5A Wnt family member 5A WNT5B Wnt family member 5B WNT6 Wnt family member 6 WNT9A Wnt family member 9A WNT10A Wnt family member 10A WNT11 Wnt family member 11 FPKM Fragments per kilobase million reads FBS Fetal bovine serum DEGs Different expressed genes TMAs Tissue microarrays IHC Immunohistochemical BRCA Breast invasive carcinoma LIHC Liver hepatocellular carcinoma LUAD Lung adenocarcinoma LUSC Lung squamous cell carcinoma SKCM Skin Cutaneous Melanoma COAD Colon adenocarcinoma READ Rectum adenocarcinoma PAAD Pancreatic adenocarcinoma KEGG Kyoto encyclopedia of genes and genomes TF Transcription factor Declarations Ethics approval and consent to participate All animal experiments were conducted in strict compliance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Yunnan University. Each study involving human subjects adhered to the ethical guidelines and operational protocols approved by the Ethics Committee of the School of Life Sciences at Yunnan University (Approval No. KY2023-015). Research utilizing human tissue samples followed the ethical review requirements stipulated in the "Ethical Review Measures for Biomedical Research Involving Humans" (National Health and Family Planning Commission Order No. 11, 2016), with specific approval granted under authorization number CHSRE2023023. Prior to study commencement, all participants received comprehensive study information and provided written informed consent through standardized documentation procedures. Consent for publication All authors have agreed to publish this manuscript Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. Author details 1 Yunnan Key Laboratory of Cell Metabolism and Diseases, State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China 2 Tianjin Medical University Cancer Institute and Hospital. National Clinical Research Center for Cancer. Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer. Tianjin, China 3 Medical Research Department, Qingdao Hospital, University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital), Qingdao 266071. Funding This work was supported by the National Natural Science Foundation of China (NSFC) fund (82273460, 32260167 and 81871990), the Yunnan Fundamental Research Projects (202401AS070133), Shandong Province Natural Science Foundation (ZR202111120048) and grants (KC-23234451, ZC-23236369, 2024Y014 and 202310673059) from Yunnan University. Author Contribution WYB, RD, CLL and LS: Experiments and data analysis. QGH, YRZ, RYS: Vector construction and dual-luciferase assay. JLS, WZ, YS and JWS wrote the main manuscript text. Acknowledgments We especially thank Professor Yeguang Chen for generously providing the APC-floxed mice and Dr. Jing Li for her helpful suggestion and comments on the manuscript. Data Availability CRC single-cell data and TCF7L1 ChIP-seq data can be obtained from the Gene Expression Omnibus (GEO) (GSE166555, GSE80331) . References Sinicrope, F.A., Increasing Incidence of Early-Onset Colorectal Cancer. N Engl J Med, 2022. 386 (16): p. 1547–1558. Wu, F., W. Zhang, H. Wei, H. Ma, G. Leng, and Y. Zhang, lncRNA ELFN1-AS1 promotes proliferation, migration and invasion and suppresses apoptosis in colorectal cancer cells by enhancing G6PD activity. Acta Biochim Biophys Sin (Shanghai), 2023. 55 (4): p. 649–660. Margetis, N., M. Kouloukoussa, K. Pavlou, S. Vrakas, and T. Mariolis-Sapsakos, K-ras Mutations as the Earliest Driving Force in a Subset of Colorectal Carcinomas. In Vivo, 2017. 31 (4): p. 527–542. Hassin, O. and M. Oren, Drugging p53 in cancer: one protein, many targets. Nat Rev Drug Discov, 2023. 22 (2): p. 127–144. Rosin-Arbesfeld, R., F. Townsley, and M. Bienz, The APC tumour suppressor has a nuclear export function. Nature, 2000. 406 (6799): p. 1009-12. Gunewardena, S., I. Huck, C. Walesky, D. Robarts, S. Weinman, and U. Apte, Progressive loss of hepatocyte nuclear factor 4 alpha activity in chronic liver diseases in humans. Hepatology, 2022. 76 (2): p. 372–386. Wu, H., T. Reizel, Y.J. Wang, J.L. Lapiro, B.T. Kren, J. Schug, et al., A negative reciprocal regulatory axis between cyclin D1 and HNF4α modulates cell cycle progression and metabolism in the liver. Proc Natl Acad Sci U S A, 2020. 117 (29): p. 17177–17186. Gu, W., H. Wang, X. Huang, J. Kraiczy, P.N.P. Singh, C. Ng, et al., SATB2 preserves colon stem cell identity and mediates ileum-colon conversion via enhancer remodeling. Cell Stem Cell, 2022. 29 (1): p. 101–115.e10. Taniguchi, H., A. Fujimoto, H. Kono, M. Furuta, M. Fujita, and H. Nakagawa, Loss-of-function mutations in Zn-finger DNA-binding domain of HNF4A cause aberrant transcriptional regulation in liver cancer. Oncotarget, 2018. 9 (40): p. 26144–26156. Chellappa, K., P. Deol, J.R. Evans, L.M. Vuong, G. Chen, N. Briançon, et al., Opposing roles of nuclear receptor HNF4α isoforms in colitis and colitis-associated colon cancer. Elife, 2016. 5 . Barrett, J.C., J.C. Lee, C.W. Lees, N.J. Prescott, C.A. Anderson, A. Phillips, et al., Genome-wide association study of ulcerative colitis identifies three new susceptibility loci, including the HNF4A region. Nat Genet, 2009. 41 (12): p. 1330-4. Becker, W.R., S.A. Nevins, D.C. Chen, R. Chiu, A.M. Horning, T.K. Guha, et al., Single-cell analyses define a continuum of cell state and composition changes in the malignant transformation of polyps to colorectal cancer. Nat Genet, 2022. 54 (7): p. 985–995. Darsigny, M., J.P. Babeu, E.G. Seidman, F.P. Gendron, E. Levy, J. Carrier, et al., Hepatocyte nuclear factor-4alpha promotes gut neoplasia in mice and protects against the production of reactive oxygen species. Cancer Res, 2010. 70 (22): p. 9423-33. He, Y., L. Chen, K. Chen, and Y. Sun, Immunohistochemical analysis of HNF4A and β-catenin expression to predict low-grade dysplasia in the colitis-neoplastic sequence. Acta Biochim Biophys Sin (Shanghai), 2021. 53 (1): p. 94–101. Li, S., M. Yang, S. Teng, K. Lin, Y. Wang, Y. Zhang, et al., Chromatin accessibility dynamics in colorectal cancer liver metastasis: Uncovering the liver tropism at single cell resolution. Pharmacol Res, 2023. 195 : p. 106896. Liu, Z., Y. Hu, H. Xie, K. Chen, L. Wen, W. Fu, et al., Single-Cell Chromatin Accessibility Analysis Reveals the Epigenetic Basis and Signature Transcription Factors for the Molecular Subtypes of Colorectal Cancers. Cancer Discov, 2024. 14 (6): p. 1082–1105. Rajamäki, K., A. Taira, R. Katainen, N. Välimäki, A. Kuosmanen, R.M. Plaketti, et al., Genetic and Epigenetic Characteristics of Inflammatory Bowel Disease-Associated Colorectal Cancer. Gastroenterology, 2021. 161 (2): p. 592–607. Babeu, J.P., C. Jones, S. Geha, J.C. Carrier, and F. Boudreau, P1 promoter-driven HNF4α isoforms are specifically repressed by β-catenin signaling in colorectal cancer cells. J Cell Sci, 2018. 131 (13). Vuong, L.M., K. Chellappa, J.M. Dhahbi, J.R. Deans, B. Fang, E. Bolotin, et al., Differential Effects of Hepatocyte Nuclear Factor 4α Isoforms on Tumor Growth and T-Cell Factor 4/AP-1 Interactions in Human Colorectal Cancer Cells. Mol Cell Biol, 2015. 35 (20): p. 3471-90. Saandi, T., F. Baraille, L. Derbal-Wolfrom, A.L. Cattin, F. Benahmed, E. Martin, et al., Regulation of the tumor suppressor homeogene Cdx2 by HNF4α in intestinal cancer. Oncogene, 2013. 32 (32): p. 3782-8. Fodde, R., R. Smits, and H. Clevers, APC, signal transduction and genetic instability in colorectal cancer. Nat Rev Cancer, 2001. 1 (1): p. 55–67. Bienz, M. and H. Clevers, Linking colorectal cancer to Wnt signaling. Cell, 2000. 103 (2): p. 311 − 20. Chen, S., L. Ji, Y. Wang, L. Zhang, M. Xu, Y. Su, et al., lncRNA RMST suppresses the progression of colorectal cancer by competitively binding to miR-27a-3p/RXRα axis and inactivating Wnt signaling pathway. Acta Biochim Biophys Sin (Shanghai), 2023. 55 (5): p. 726–735. Li, S., W. Han, Q. He, Y. Wang, G. Jin, and Y. Zhang, Ginsenoside Rh2 suppresses colon cancer growth by targeting the miR-150-3p/SRCIN1/Wnt axis. Acta Biochim Biophys Sin (Shanghai), 2023. 55 (4): p. 633–648. Doumpas, N., F. Lampart, M.D. Robinson, A. Lentini, C.E. Nestor, C. Cantù, et al., TCF/LEF dependent and independent transcriptional regulation of Wnt/β-catenin target genes. Embo j, 2019. 38 (2). Chen, X., J. Yang, P.M. Evans, and C. Liu, Wnt signaling: the good and the bad. Acta Biochim Biophys Sin (Shanghai), 2008. 40 (7): p. 577 − 94. Chen, Y., Z. Lu, J. Feng, Z. Chen, Z. Liu, X. Wang, et al., Novel recombinant R-spondin1 promotes hair regeneration by targeting the Wnt/β-catenin signaling pathway. Acta Biochim Biophys Sin (Shanghai), 2023. 55 (8): p. 1213–1221. Jiang, R., X. Niu, Y. Huang, and X. Wang, β-Catenin is important for cancer stem cell generation and tumorigenic activity in nasopharyngeal carcinoma. Acta Biochim Biophys Sin (Shanghai), 2016. 48 (3): p. 229 − 37. Xu, X., Y. Dai, L. Feng, H. Zhang, Y. Hu, L. Xu, et al., Knockdown of Nav1.5 inhibits cell proliferation, migration and invasion via Wnt/β-catenin signaling pathway in oral squamous cell carcinoma. Acta Biochim Biophys Sin (Shanghai), 2020. 52 (5): p. 527–535. Meng, L., R. Dong, W. Mi, K. Qin, K. Ouyang, J. Sun, et al., The ubiquitin E3 ligase APC/C(Cdc20) mediates mitotic degradation of OGT. J Biol Chem, 2024. 300 (7): p. 107448. Lee, E., A. Salic, R. Krüger, R. Heinrich, and M.W. Kirschner, The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway. PLoS Biol, 2003. 1 (1): p. E10. Tran, H., D. Bustos, R. Yeh, B. Rubinfeld, C. Lam, S. Shriver, et al., HectD1 E3 ligase modifies adenomatous polyposis coli (APC) with polyubiquitin to promote the APC-axin interaction. J Biol Chem, 2013. 288 (6): p. 3753-67. Shen, J., J. Yang, L. Sang, R. Sun, W. Bai, C. Wang, et al., PYK2 mediates the BRAF inhibitor (vermurafenib)-induced invadopodia formation and metastasis in melanomas. Cancer Biol Med, 2021. 19 (8): p. 1211-23. Bai, W., Q. Hao, Z. Zhang, B. Han, H. Xiao, D. Chang, et al., Identification of a novel inflammation-related gene signature for predicting inflammatory breast cancer survival. Genome Instability & Disease, 2023. 4 (3): p. 154–175. Hao, Q., R. Dong, W. Bai, D. Chang, X. Yao, Y. Zhang, et al., Screening for metastasis-related genes in mouse melanoma cells through sequential tail vein injection. Biophys Rep, 2024. 10 (1): p. 15–21. Hao, Y., S. Hao, E. Andersen-Nissen, W.M. Mauck, 3rd, S. Zheng, A. Butler, et al., Integrated analysis of multimodal single-cell data. Cell, 2021. 184 (13): p. 3573–3587.e29. Sun, J., H. He, S. Pillai, Y. Xiong, S. Challa, L. Xu, et al., GATA3 transcription factor abrogates Smad4 transcription factor-mediated fascin overexpression, invadopodium formation, and breast cancer cell invasion. J Biol Chem, 2013. 288 (52): p. 36971-82. Bai, W., C. Yan, Y. Yang, L. Sang, Q. Hao, X. Yao, et al., EGF/EGFR-YAP1/TEAD2 signaling upregulates STIM1 in vemurafenib resistant melanoma cells. Febs j, 2024. 291 (22): p. 4969–4983. Sang, L., R. Dong, R. Liu, Q. Hao, W. Bai, and J. Sun, Caenorhabditis elegans NHR-14/HNF4α regulates DNA damage-induced apoptosis through cooperating with cep-1/p53. Cell Commun Signal, 2022. 20 (1): p. 135. Cao, T., X. Luo, B. Zheng, Y. Deng, Y. Zhang, Y. Li, et al., Death-associated protein 3 in cell death and beyond. Genome Instability & Disease, 2024. 5 (2): p. 51–60. Zhao, Y., Z. Jiang, T. Ni, W. Jiang, K. Zhou, Y. Liu, et al., Terpenoids-enriched fraction of Celastrus orbiculatus sensitizes gemcitabine by disrupting Chk1/RAD51-mediated DNA damage response in pancreatic cancer. Genome Instability & Disease, 2021. 2 (6): p. 358–373. Pfeifer, G.P., Mechanisms of UV-induced mutations and skin cancer. Genome Instability & Disease, 2020. 1 (3): p. 99–113. Sang, L., X. Wang, W. Bai, J. Shen, Y. Zeng, and J. Sun, The role of hepatocyte nuclear factor 4α (HNF4α) in tumorigenesis. Front Oncol, 2022. 12 : p. 1011230. Yu, Y., H. Jia, T. Zhang, and W. Zhang, Advances in DNA damage response inhibitors in colorectal cancer therapy. Acta Biochim Biophys Sin (Shanghai), 2024. 56 (1): p. 15–22. Gao, Y., R. Dong, J. Yan, H. Chen, L. Sang, X. Yao, et al., Mitochondrial deoxyguanosine kinase is required for female fertility in mice. Acta Biochim Biophys Sin (Shanghai), 2024. 56 (3): p. 427–439. Lv, J., P. Gong, G. Jia, and W. Li, Targeted DNA damage repair: old mechanisms and new opportunities in clear cell renal cell carcinoma. Genome Instability & Disease, 2024. 5 (5): p. 197–209. Xiang, Z., H. Liu, and Y. Hu, DNA damage repair and cancer immunotherapy. Genome Instability & Disease, 2023. 4 (4): p. 210–226. Guo, C. and Y. Zhao, Autophagy and DNA damage repair. Genome Instability & Disease, 2020. 1 (4): p. 172–183. Li, D., W. Xu, Y. Chang, Y. Xiao, Y. He, and S. Ren, Advances in landscape and related therapeutic targets of the prostate tumor microenvironment. Acta Biochim Biophys Sin (Shanghai), 2023. 55 (6): p. 956–973. Yang, X., F. Shao, D. Guo, W. Wang, J. Wang, R. Zhu, et al., WNT/β-catenin-suppressed FTO expression increases m(6)A of c-Myc mRNA to promote tumor cell glycolysis and tumorigenesis. Cell Death Dis, 2021. 12 (5): p. 462. Parang, B., C.W. Barrett, and C.S. Williams, AOM/DSS Model of Colitis-Associated Cancer , in Gastrointestinal Physiology and Diseases: Methods and Protocols , A.I. Ivanov, Editor. 2016, Springer New York: New York, NY. p. 297–307. Chellappa, K., L. Jankova, J.M. Schnabl, S. Pan, Y. Brelivet, C.L. Fung, et al., Src tyrosine kinase phosphorylation of nuclear receptor HNF4α correlates with isoform-specific loss of HNF4α in human colon cancer. Proc Natl Acad Sci U S A, 2012. 109 (7): p. 2302-7. Tanaka, T., S. Jiang, H. Hotta, K. Takano, H. Iwanari, K. Sumi, et al., Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer. J Pathol, 2006. 208 (5): p. 662 − 72. Oshima, T., T. Kawasaki, R. Ohashi, G. Hasegawa, S. Jiang, H. Umezu, et al., Downregulated P1 promoter-driven hepatocyte nuclear factor-4alpha expression in human colorectal carcinoma is a new prognostic factor against liver metastasis. Pathol Int, 2007. 57 (2): p. 82–90. Chellappa, K., L. Jankova, J.M. Schnabl, S. Pan, Y. Brelivet, C.L.-S. Fung, et al., Src tyrosine kinase phosphorylation of nuclear receptor HNF4α correlates with isoform-specific loss of HNF4α in human colon cancer. Proceedings of the National Academy of Sciences, 2012. 109 (7): p. 2302–2307. Gougelet, A., C. Torre, P. Veber, C. Sartor, L. Bachelot, P.D. Denechaud, et al., T-cell factor 4 and β-catenin chromatin occupancies pattern zonal liver metabolism in mice. Hepatology, 2014. 59 (6): p. 2344-57. Atcha, F.A., A. Syed, B. Wu, N.P. Hoverter, N.N. Yokoyama, J.H. Ting, et al., A unique DNA binding domain converts T-cell factors into strong Wnt effectors. Mol Cell Biol, 2007. 27 (23): p. 8352-63. Additional Declarations No competing interests reported. Supplementary Files ExperimentalrawdataofWesternblot.pdf FigS1.pdf Supplementary Figure 1 Malignant cells and T cells interact with each other in the tumor immune microenvironment. A. Cell communication quantity (left) and communication intensity (right) among 12 cell types in colorectal cancer tissues; The thickness of the lines represents the strength of the communication between cells. B. Analysis of communication intensity between malignant cells and the other 12 cell types. C. Heatmap of unsupervised clustering and correlation analysis among various cell types. Numbers indicate correlation coefficients. D. ESTIMATE（tumor purity score, stromal score, immune score and ESTIMATE score）. E. KEGG analysis of high tumor purity and low tumor purity. F. Heatmap analysis of Wnt/β-catenin signaling pathway expression levels in various cell types. 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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-6208041","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":445942264,"identity":"a10d30e3-ed70-4205-87a7-29e8f510cb73","order_by":0,"name":"Weiyu Bai","email":"","orcid":"","institution":"Yunnan University","correspondingAuthor":false,"prefix":"","firstName":"Weiyu","middleName":"","lastName":"Bai","suffix":""},{"id":445942265,"identity":"0768afc3-08a8-4336-84cd-f0aa594e052e","order_by":1,"name":"Lei Sang","email":"","orcid":"","institution":"Yunnan University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Sang","suffix":""},{"id":445942266,"identity":"07d019d4-efc8-4b92-8115-7420a8347075","order_by":2,"name":"Chenglu Lu","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital, Tianjin’s Clinical Research Center for Cancer","correspondingAuthor":false,"prefix":"","firstName":"Chenglu","middleName":"","lastName":"Lu","suffix":""},{"id":445942267,"identity":"06be36da-4699-417f-9d1b-ea8ee45174a8","order_by":3,"name":"Rui Dong","email":"","orcid":"","institution":"Yunnan University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Dong","suffix":""},{"id":445942268,"identity":"368e4db9-2658-48e6-a6af-8087d41b0aa9","order_by":4,"name":"Qinggang Hao","email":"","orcid":"","institution":"Yunnan University","correspondingAuthor":false,"prefix":"","firstName":"Qinggang","middleName":"","lastName":"Hao","suffix":""},{"id":445942269,"identity":"d988588e-fec3-4d63-9e28-f3fdede7548b","order_by":5,"name":"Yingru Zhang","email":"","orcid":"","institution":"Yunnan University","correspondingAuthor":false,"prefix":"","firstName":"Yingru","middleName":"","lastName":"Zhang","suffix":""},{"id":445942273,"identity":"1b0f7cf8-97fd-4728-9e97-824316b411dd","order_by":6,"name":"Rongyuan Sun","email":"","orcid":"","institution":"Yunnan University","correspondingAuthor":false,"prefix":"","firstName":"Rongyuan","middleName":"","lastName":"Sun","suffix":""},{"id":445942275,"identity":"e2cee907-3fd4-463c-8950-0bf5b766ec46","order_by":7,"name":"Junling Shen","email":"","orcid":"","institution":"Yunnan University","correspondingAuthor":false,"prefix":"","firstName":"Junling","middleName":"","lastName":"Shen","suffix":""},{"id":445942278,"identity":"d0a500db-bfd4-40fc-a5e4-363c03c138f4","order_by":8,"name":"Wenjing Zhu","email":"","orcid":"","institution":"University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital)","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Zhu","suffix":""},{"id":445942281,"identity":"d540bec0-e717-42df-a7b8-4b37c2eba74b","order_by":9,"name":"Yan Sun","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital, Tianjin’s Clinical Research Center for Cancer","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Sun","suffix":""},{"id":445942283,"identity":"22860c36-d730-4785-9e07-963a60364f2a","order_by":10,"name":"Jianwei Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYBACxgYQWQHhSJCg5QwpWiD62kjRwjwj/Zl04bzDeQYHmA/e5mGwyyNswYwcM+mZ2w4XGxxgS7bmYUguJqxldg6bNO+2w4kbDvCYSfMwHEhsIKwF6DDeOSAt/N+I1ZJgJs3bALaFjUgt898YW/McS0+ceZjN2HKOQTJhLYY9xx/e5qmxTuw73vzwxpsKOyK0QFQ0A4MbRBsQUg8E8hCqjgilo2AUjIJRMGIBAKRlOb9Sak35AAAAAElFTkSuQmCC","orcid":"","institution":"Yunnan University","correspondingAuthor":true,"prefix":"","firstName":"Jianwei","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2025-03-12 03:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6208041/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6208041/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82056501,"identity":"0f98051e-7e3e-4173-961f-5df7a65901f7","added_by":"auto","created_at":"2025-05-06 10:37:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1109851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHNF4α is frequently upregulated in colorectal cancer and plays an oncogenic role during colorectal tumorigenesis\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eA. TCGA database analysis of HNF4α expression in different tumor types.\u003c/p\u003e\n\u003cp\u003eB. Western blot analysis of HNF4α protein level in colorectal cancer tumor (T) and corresponding adjacent normal (N) tissues.\u003c/p\u003e\n\u003cp\u003eC. Schematic diagram of HNF4α expression in 59 pairs of colorectal cancer tumor and normal tissues.\u003c/p\u003e\n\u003cp\u003eD. Quantitative analysis of Western blot results of HNF4α in colorectal cancer tissues and normal tissues using ImageJ.\u003c/p\u003e\n\u003cp\u003eE. Immunohistochemical staining of colorectal cancer tissue microarray (TMA) with an antibody against HNF4α and statistical analysis of HNF4α expression levels in the colorectal cancer TMA.\u003c/p\u003e\n\u003cp\u003eF. Cell type annotation of colorectal cancer tissue using t-SNE dimensionality reduction and Single cell sequencing\u003c/p\u003e\n\u003cp\u003eG. HNF4α expression level in the 12 types of tumor cells.\u003c/p\u003e\n\u003cp\u003eH. Relative proportions of various cell components in colorectal cancer tissues.\u003c/p\u003e\n\u003cp\u003eI. Relative proportions of various cell components in colorectal cancer tissues with high HNF4α expression.\u003c/p\u003e\n\u003cp\u003eJ. Relative proportions of various cell components in colorectal cancer tissues with low HNF4α expression.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6208041/v1/d80335affd2ed0b82bab5a0d.png"},{"id":82054220,"identity":"a40fc930-5a7b-47dc-ab1d-8a0bc9d08421","added_by":"auto","created_at":"2025-05-06 10:21:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":256070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHNF4α is involved in colorectal carcinogenesis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Effects of ectopic expression of HNF4α on colony formation in SW480 cells.\u003c/p\u003e\n\u003cp\u003eB. The quantification of colonies in SW480 control and HNF4α overexpression cells. Data are shown as mean ± SD (N = 3).\u003c/p\u003e\n\u003cp\u003eC. Western blotting analysis of HNF4α levels in control and HNF4α overexpression SW480 and SW620 cells.\u003c/p\u003e\n\u003cp\u003eD. Tumor volumes of mouse xenografts established with parental (6 mice) and HNF4α overexpressing (6 mice) SW480.\u003c/p\u003e\n\u003cp\u003eE. Growth curve reflects the effect of HNF4α overexpression on tumor growth in the xenograft mouse model. ** P\u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003eF. Effects of HNF4α overexpression on tumor weights.\u003c/p\u003e\n\u003cp\u003e** P\u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003eG. Western blotting analysis of HNF4α levels in control and HNF4α shRNA SW620 cells.\u003c/p\u003e\n\u003cp\u003eH. Tumor volumes of mouse xenografts established with parental HT29 cells (10 mice) and HNF4α knockdown HT29 cells (10 mice).\u003c/p\u003e\n\u003cp\u003eI. Growth curve reflects the effect of HNF4α knockdown on tumor growth in the xenograft mouse model. ** P\u0026lt;0.01 and *** P\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003eJ. Effects of HNF4α knockdown on tumor weights.\u003c/p\u003e\n\u003cp\u003e** P\u0026lt;0.01 and *** P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6208041/v1/806ff1ec9305d67949e1c625.png"},{"id":82056502,"identity":"c48957c3-bfba-496f-986f-6047380afe84","added_by":"auto","created_at":"2025-05-06 10:37:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":705291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHNF4α is a target of Wnt/β-catenin and mediates the Wnt/β-catenin axis in colon carcinogenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. GESA analysis of HNF4α in TCGA database, * indicates the significant correlation of HNF4α with Wnt and Myc pathways.\u003c/p\u003e\n\u003cp\u003eB. Correlations analysis between HNF4α and β-catenin mRNA level in TCGA database.\u003c/p\u003e\n\u003cp\u003eC. Q-PCR analysis of the effect of β-catenin knockdown on HNF4α mRNA level.\u003c/p\u003e\n\u003cp\u003eD. Western blotting analysis of the effect of β-catenin knockdown on HNF4α protein expression in 3 different CRC cell lines.\u003c/p\u003e\n\u003cp\u003eE. Western blotting analysis of the effect of SKL2001 on subcellular location and expression of β-catenin and HNF4α.\u003c/p\u003e\n\u003cp\u003eF. Western blotting analysis of the effect of SKL2001 and Chir-99021, 2 activators of WNT/β-catenin signaling, on HNF4α expression in SW480 and SW620 cell lines.\u003c/p\u003e\n\u003cp\u003e***p\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003eG\u003cstrong\u003e. \u003c/strong\u003eWestern blotting analysis of β-cateninand HNF4α expression in SW620 cells transfected with β-catenin shRNA, β-catenin shRNA/HNF4α.\u003c/p\u003e\n\u003cp\u003eH. Effects of SW620 control, β-catenin shRNA, HNF4α overexpression, β-catenin shRNA/HNF4α on the tumor size.\u003c/p\u003e\n\u003cp\u003eI. Effects of SW620 control, β-catenin shRNA, HNF4α overexpression,β-catenin shRNA/HNF4α on the tumor weight.\u003c/p\u003e\n\u003cp\u003e** p\u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003eJ. Effects of SW620 control, β-catenin shRNA, HNF4α overexpression, β-catenin shRNA/HNF4α on tumor growth, *** p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6208041/v1/658bbacc958ec40540029e70.png"},{"id":82055848,"identity":"d848dadb-a3bc-456b-9ecf-cbae6514c2d6","added_by":"auto","created_at":"2025-05-06 10:29:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":313102,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTCF7L1 is a major transcription factor to induce HNF4α expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-D. Correlation analysis of mRNA levels between HNF4α \u0026amp; TCF/LEF family members using TCGA database.\u003c/p\u003e\n\u003cp\u003eE. Western blotting analysis of the effects of TCF/LEF members on HNF4α expression.\u003c/p\u003e\n\u003cp\u003eF. Q-PCR analysis of the shRNA efficiency on LEF1 and TCF7L2\u003c/p\u003e\n\u003cp\u003eG. Western blotting analysis of the effect of TCF7L1 knockdown on HNF4α levels in DLD1 and LS174T cell lines.\u003c/p\u003e\n\u003cp\u003eH. Western blotting analysis of the effect of β-catenin knockdown and TCF7L1 overexpression on HNF4α expression.\u003c/p\u003e\n\u003cp\u003eI. Western blotting analysis of the effect of TCF7L1 overexpression in β-catenin knockdown cells on HNF4α expression.\u003c/p\u003e\n\u003cp\u003eJ. HNF4α expression levels in various cells of colorectal cancer tissue.\u003c/p\u003e\n\u003cp\u003eK. TCF7L1 expression levels in various cells of colorectal cancer tissue.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6208041/v1/a3b3c6f64bb9218e54c1fe7f.png"},{"id":82054225,"identity":"f1af6aa9-bfad-4aae-9853-c3baa7cbccf6","added_by":"auto","created_at":"2025-05-06 10:21:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":242029,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTCF7L1 directly binds to the HNF4\u003c/strong\u003eα\u003cstrong\u003e promoter and regulates its transcription\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. JASPAR (\u003ca href=\"https://jaspar.genereg.net/)\"\u003ehttps://jaspar.genereg.net/)\u003c/a\u003e analysis of 1.7-kb HNF4α promoter for putative TCF7L1 transcription factor binding motif(s). Graphical representation of predicted binding sites and TCF7L1 binding sequence motifs shown on the top left.\u003c/p\u003e\n\u003cp\u003eB. Luciferase activity of HNF4α promoters.\u003c/p\u003e\n\u003cp\u003eC. Mutation of each TCF7L1 binding motif in the pGL3-HNF4α-300bp promoter region.\u003c/p\u003e\n\u003cp\u003eD. Luciferase activity of three mutants of pGL3-HNF4α-300bp promoter.\u003c/p\u003e\n\u003cp\u003e***p\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003eE. In ChIP-qPCR experiments, GFP beads were used for immunoprecipitation of the HNF4α promoter sequence, followed by nucleic acid electrophoresis, in SW480-GFP and SW480-GFP-TCF7L1 cells.\u003c/p\u003e\n\u003cp\u003eF. In Chip-qPCR experiments, 1 µl of the HNF4α promoter sequence immunoprecipitated with GFP beads was used as a template for real-time quantitative PCR in SW480-GFP and SW480-GFP-TCF7L1 cells. ***p\u0026lt;0.001\u003c/p\u003e\n\u003cp\u003eG. TCF7L1 ChIP-seq analysis of binding regions in the HNF4α promoter\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6208041/v1/bda45c0120331ecc789a2242.png"},{"id":82054227,"identity":"7ec5d63d-6605-44ff-81f9-0ff6a27dc239","added_by":"auto","created_at":"2025-05-06 10:21:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":842513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHNF4α induces expression of Wnt family members and β-catenin translocation from the cytoplasm to the nucleus\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eA. KEGG pathway analysis by RNA sequencing data obtained from SW480-vector control and SW480-HNF4α cells.\u003c/p\u003e\n\u003cp\u003eB. A heat map showing expression levels of the genes involved in WNT, MAPK, Hippo signaling pathways that are regulated by HNF4α.\u003c/p\u003e\n\u003cp\u003eC. q-PCR analysis of HNF4α overexpression on Wnt family genes level.\u003c/p\u003e\n\u003cp\u003eD. GSEA analysis of Wnt/β-catenin signaling pathway in sw480 cells and HNF4α overexpression.\u003c/p\u003e\n\u003cp\u003eE. Western blot analysis of the effect of HNF4α overexpression on β-catenin level\u003c/p\u003e\n\u003cp\u003eF. Western blot analysis of the effect of HNF4α overexpression on β-catenin nuclear localization.\u003c/p\u003e\n\u003cp\u003eG. TOPFlash (reporter gene plasmid) analysis of transcriptional activity levels of Wnt signaling pathway SW480 control and SW480-HNF4αcells.\u003c/p\u003e\n\u003cp\u003eH. β-catenin and Wnts mRNA levels in various colorectal cancer tissue cells.\u003c/p\u003e\n\u003cp\u003eI. ChIP-seq analysis of the binding peak maps of HNF4α to the promoter regions of WNT family genes in the colons of wild-type and HNF4α knockoutmice.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6208041/v1/9ae91056dd5032f845cad13c.png"},{"id":82054240,"identity":"cb69b765-2d39-47f2-afaa-0334c74fcf81","added_by":"auto","created_at":"2025-05-06 10:21:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3094083,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβ-catenin/TCF7L1-HNF4\u003c/strong\u003eα\u003cstrong\u003eaxis correlate with colorectal tumorigenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Representative images of β-catenin, TCF7L1 and HNF4α immunohistochemical staining in colorectal cancer tissue\u003c/p\u003e\n\u003cp\u003eB. Correlation analysis between TCF7L1 and HNF4α level\u003c/p\u003e\n\u003cp\u003eC. Correlation analysis between β-catenin and HNF4α level\u003c/p\u003e\n\u003cp\u003eD. Pseudotime analysis of single-cell sequencing in colorectal cancer tissues.\u003c/p\u003e\n\u003cp\u003eE. Expression changes of HNF4α, β-catenin and TCF7L1 along a single-cell pseudotime trajectory. The X-axis shows pseudotime, and the Y-axis represents gene expression levels. The curves highlight potential regulatory interactions during cell state transitions, with peaks indicating key activity points.\u003c/p\u003e\n\u003cp\u003eF. Immunofluorescence detection of HNF4α and β-catenin expression in APC mutant mice.\u003c/p\u003e\n\u003cp\u003eA. Western blot analysis of β-catenin, TCF7L1 and HNF4α level in colon tissue of AOM-DSS mouse CRC model\u003c/p\u003e\n\u003cp\u003eH. A proposed model for TCF7L1-HNF4α axis regulation of wnt/β-catenin signaling in colorectal cancergenesis.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-6208041/v1/f2065850175f258161800b80.png"},{"id":82058469,"identity":"54fec406-2dcb-4b86-be64-d12f698353ba","added_by":"auto","created_at":"2025-05-06 11:01:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7783330,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6208041/v1/47911a79-182c-4913-96d2-89882917dc8b.pdf"},{"id":82054231,"identity":"64abeefb-273c-4c68-b428-d226b2e7dd3f","added_by":"auto","created_at":"2025-05-06 10:21:09","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2310925,"visible":true,"origin":"","legend":"","description":"","filename":"ExperimentalrawdataofWesternblot.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6208041/v1/a255b42e222c2f8488a02880.pdf"},{"id":82054223,"identity":"1732b4f2-c89a-40e7-974e-0d5a669e500f","added_by":"auto","created_at":"2025-05-06 10:21:08","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1093682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1 Malignant cells and T cells interact with each other in the tumor immune microenvironment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Cell communication quantity (left) and communication intensity (right) among 12 cell types in colorectal cancer tissues; The thickness of the lines represents the strength of the communication between cells.\u003c/p\u003e\n\u003cp\u003eB. Analysis of communication intensity between malignant cells and the other 12 cell types.\u003c/p\u003e\n\u003cp\u003eC. Heatmap of unsupervised clustering and correlation analysis among various cell types. Numbers indicate correlation coefficients.\u003c/p\u003e\n\u003cp\u003eD. ESTIMATE（tumor purity score, stromal score, immune score and ESTIMATE score）.\u003c/p\u003e\n\u003cp\u003eE. KEGG analysis of high tumor purity and low tumor purity.\u003c/p\u003e\n\u003cp\u003eF. Heatmap analysis of Wnt/β-catenin signaling pathway expression levels in various cell types.\u003c/p\u003e","description":"","filename":"FigS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6208041/v1/df4773569d39bea01aabe0d8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Wnt/β-catenin/HNF4α feedback loop facilitates colorectal tumorigenesis and malignancy","fulltext":[{"header":"Background","content":"\u003cp\u003eColorectal cancer (CRC) is one of the most common cancers and the second leading cause of cancer deaths worldwide[1, 2]. Despite an overall decreasing incidence of the disease, early-onset CRC is rapidly rising in individuals under the age of 50 over the last two decades. While several major targets have been identified during the course of colorectal cancer development such as Ras[3], Tp53[4], and APC/β-catenin[5], the pathogenesis of CRC still remains incompletely understood.\u003c/p\u003e \u003cp\u003eHepatocyte Nuclear Factor 4 Alpha (HNF4α) is a transcription factor predominantly expressed in the liver, kidney, intestine and endocrine pancreas, and is necessary for liver development and function[6\u0026ndash;8]. There is accumulating evidence showing its inhibitory role in the liver cancer. Mutations in HNF4α at G79C, F83C and M125I (Zn-finger DNA-binding domain region) have been detected in liver cancer and are believed to trigger liver tumorigenesis[9]. Previous studies have revealed the contradictory role of HNF4α in colorectal carcinogenesis[10]. While studies indicate that HNF4α promotes the occurrence of colorectal cancer[11\u0026ndash;17], others suggest that HNF4α inhibits the occurrence and development of colorectal cancer[18\u0026ndash;20]. The mechanisms regulating HNF4α expression in colorectal cancer are largely unknown. Further investigation of the role and the specific regulation mechanism of HNF4α in the development and progression of colorectal cancer is of great significance for establishing HNF4α as a diagnostic/prognostic marker and therapeutic target in colorectal cancer.\u003c/p\u003e \u003cp\u003eThe Wnt/β-catenin signaling pathway is a well-recognized driver of colorectal carcinogenesis, and dysregulation of this cascade occurs in 70\u0026ndash;80% of colorectal cancers[21\u0026ndash;24]. Activation of the Wnt pathway induces β-catenin translocation into the nucleus, where it functions as a transcriptional coactivator of TCF /LEF family factors[25\u0026ndash;29]. While several molecules that regulate this cascade have been identified including APC, GSK3β and AXIN[23, 30\u0026ndash;32], the role and regulation of Wnt/β-catenin activation in colorectal cancer are not completely understood.\u003c/p\u003e \u003cp\u003eHere, we demonstrate that HNF4α is upregulated in colorectal cancer and promotes colorectal tumorigenesis. HNF4α levels positively correlate with the Wnt/β-catenin-TCF7L1 signaling pathway and was directly regulated by it. We further show that HNF4α is a positive regulator of the Wnt/β-catenin pathway and that HNF4α overexpression promotes β-catenin nuclear localization. Our results suggest that HNF4α is a potential prognostic marker for Wnt/β-catenin-related colorectal cancer and that HNF4α forms a feedback loop with Wnt/β-catenin to control colorectal tumorigenesis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eApc-flox-Villin-Cre mice\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eApc-flox\u003c/em\u003e mice were obtained from Dr. Yeguang Chen lab at Tsinghua University, while the \u003cem\u003eVillin-Cre(RRID:IMSR_JAX:004586)\u003c/em\u003e mice were provided by Professor Yonglong Wei at Yunnan University. By crossing Apc-flox heterozygous mice with Villin-Cre mice, we generated \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003e(f/+)\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-Villin-Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003e(f/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e mice, which spontaneously develop intestinal cancer. In contrast, \u003cem\u003eApc\u003c/em\u003e\u003csup\u003e\u003cem\u003e(f/f)\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-Villin-Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003e(f/+)\u003c/em\u003e\u003c/sup\u003e embryos were lethal.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture and activator treatment\u003c/h3\u003e\n\u003cp\u003eCRC cell line SW480, SW620 and DLD-1 were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. For the inhibitor and activator treatment, cells (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were seeded in 6 well plates and treated with 2uM or 5 uM SKL2001 and CHIR99021 for 12 h, 24 h, and 36 h after the cells attached to the dish.\u003c/p\u003e\n\u003ch3\u003eRNA interference\u003c/h3\u003e\n\u003cp\u003eRNA interference was performed using the plko.1 vector. To efficiently knockdown β-catenin and TCF family, the shRNAs targeting to different genes were synthetized and inserted to plko.1 vector between \u003cem\u003eAge\u003c/em\u003e I and \u003cem\u003eEco\u003c/em\u003eR I. The target sequences were as follows:\u003c/p\u003e \u003cp\u003eβ-catenin shRNA: 5\u0026rsquo;-TTGTTATCAGAGGACTAAATA-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eTCF7L1 shRNA: 5\u0026rsquo;-GCACCTACCTGCAGATGAAAT-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eTCF1 shRNA: 5\u0026rsquo;-CAACTCTCTCTCTACGAACAT-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eLEF1 shRNA: 5\u0026rsquo;-CCATCAGATGTCAACTCCAAA-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eTCF4 shRNA: 5\u0026rsquo;-CCTTTCACTTCCTCCGATTAC-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eHNF4α shRNA 1:5\u0026rsquo;-CATGTACTCCTGCAGATTTAG-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eHNF4α shRNA 2: 5\u0026rsquo;-ATCACCAAGCAGGAAGTTATC-3\u0026rsquo;.\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blotting\u003c/div\u003e \u003cp\u003eWestern blotting was performed as described previously[33]. Cells were lysed in RIPA buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1 mM PMSF) containing Complete Protease Inhibitor Cocktail (Roche). Cell lysates were spun down at 12,000 rpm for 10 min, and 50 \u0026micro;g supernatants were resolved on SDS-PAGE and blotted with the indicated antibodies. The amount of GAPDH was used as the loading control. The results were detected by an ECL-plus Western blotting detection system (Tanon-5200Multi). The primary antibodies used in this study were as follows: Rabbit anti-HNF4α (1:1000; Cat# 3113, Cell Signaling Technology, USA); Rabbit anti-TCF7L1 (1:1000; Cat# PA5-40327, Thermo Fisher Scientific, USA); Mouse anti-TCF1 (1:1000; Cat# sc-271453, Santa Cruz Biotechnology, USA);Rabbit anti-β-catenin (1:1000; Cat# A117811, GenScript, USA); Rabbit anti-Actin (1:1000; Cat# 4970, Cell Signaling Technology, USA); Mouse anti-GAPDH (1:1000; Cat# sc-166574, Santa Cruz Biotechnology, USA).\u003c/p\u003e\n\u003ch3\u003eQuantitative reverse transcription–polymerase chain reaction (qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using Trizol (Lablead Biotech, Beijing, China). 2 \u0026micro;g RNA was used as template to generate cDNAs using the ImProm-II Reverse Transcription system (TransGen Biotech, Beijing, China). qPCR reactions were carried out on an CFX96 Real-Time System.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMouse Xenograft\u003c/h2\u003e \u003cp\u003eAll mice were obtained from the Animal Research and Resource Center, Yunnan University. All animal experiments were performed according to protocols approved by the Animal Care Committee of the Yunnan University (Kunming, China).For the Subcutaneous tumor models,1\u0026times;10\u003csup\u003e6\u003c/sup\u003e SW620 cells were re-suspended in 100 \u0026micro;l 0.25 mg/ml Matrigel (Corning) with PBS buffer, then injected into the flanks of the nude mouse. The tumor volumes were measured from day 3 to 11 after injection. At 11 days after the injection, tumors were dissected. Tumors were then imaged and weighted.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGESA analysis and correlation analysis\u003c/h3\u003e\n\u003cp\u003eThe mRNA sequencing data of COAD, READ, BRCA, LIHC, LUAD, LUSC, PAAD and SKCM were downloaded from TCGA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov/repository\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov/repository\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) respectively, and the merge normal tissues UCSC Xena (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://xenabrowser.net\u003c/span\u003e\u003cspan address=\"https://xenabrowser.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) mRNA Sequencing data were downloaded as control. the Pearson correlation coefficient R value and the correlation curve were calculated and drawn by Using the ggplot2 function in R 4.4.0 software. The boxplot was drawn using the simplevis package after processing the TPM as log2 (TPM\u0026thinsp;+\u0026thinsp;1). The genes that are significantly(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, r\u0026thinsp;\u0026ge;\u0026thinsp;0.3 or r\u0026thinsp;\u0026le;\u0026thinsp;\u0026minus;\u0026thinsp;0.3) related to HNF4α were screened by FPKM value of COAD READ and used for GSEA and KEGG enrichment analysis[34].\u003c/p\u003e\n\u003ch3\u003eBulk RNA-seq, scRNA-seq and estimate analysis\u003c/h3\u003e\n\u003cp\u003eThe total RNA of SW480 and SW480-HNF4α cells were harvested with Trizol. Three biological replicates were used. These samples were subjected to transcriptome RNA-sequencing by Novogene[35]. The paired-end RNA-sequencing library was employed (2 \u0026times; 150 bp read length) using an Illumina NovaSeq 6000 sequencer. Both raw data and clean data were provided by Novogene and the sequencing quality was assessed using FastQC. The index construction is performed using bowtie2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bowtie-bio.sourceforge.net/bowtie2/index.shtml\u003c/span\u003e\u003cspan address=\"http://bowtie-bio.sourceforge.net/bowtie2/index.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the genome alignment of the clean data is conducted using TopHat2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ccb.jhu.edu/software/tophat/index.shtml\u003c/span\u003e\u003cspan address=\"https://ccb.jhu.edu/software/tophat/index.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the transcriptome assembly is performed using Cufflinks (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cole-trapnell-lab.github.io/cufflinks/\u003c/span\u003e\u003cspan address=\"https://cole-trapnell-lab.github.io/cufflinks/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The gene expression level was calculated according to the fragments per kilobase million reads (FPKM) method. The formula for differential analysis calculation is log2(SW480-HNF4α mean (FPKM)/SW480 control mean (FPKM)). The p-value is calculated using the t-test different expressed genes (DEGs) with |log2(foldchange)| \u0026ge; 1, and P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were significantly (DEGs). Single cell sequencing data was analyzed using the seura (v 5.1.0) R package t[36].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase Assay\u003c/h2\u003e \u003cp\u003eThe dual luciferase assay was performed according to a previously reported protocol with minor modification[37]. The HNF4α promoter luciferase reporter constructs were generated by inserting 1700 bp, 800 bp or 300 bp human HNF4α promoter into the pGL3 basic vector (Promega) between XhoI and HindIII. To perform the dual luciferase reporter assay, 20,000 SW480 cells were seeded in 12-well plates and cultured overnight. Cells were transfected with 1 ug/well HNF4α promoter reporter together with 100 ng/well Renilla luciferase construct (pRL-TK RRID: Addgene_11313) using Lipofectamine 2000. 24h after transfection, the cells were lysed and cell lysates were subjected to dual reporter luciferase assays according to the manufacturer\u0026rsquo;s instructions (Promega).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTissue microarray and immunohistochemistry\u003c/h2\u003e \u003cp\u003eTissue microarrays (TMAs) were constructed using a manual tissue microarray instrument (Beecher Instruments) equipped with a 2.0 mm punch needle, as previously described[38]. An immunohistochemical (IHC) study of the rabbit anti-human β-catenin, rabbit anti-human TCF7L1 antibody, and rabbit anti-human HNF4α antibody was carried out on formalin-fixed paraffin-embedded tissues, with a 4-\u0026micro;m-thick serial section of tissues, according to the manufacturer\u0026rsquo;s recommended protocol. The levels of β-catenin, TCF7L1 and HNF4α were assessed via the average of 5 count fields per patient at an original magnification of X200 on light microscopy[38]. The positive cells for HNF4α were defined as those with brown staining. The expression of β-catenin, TCF7L1 and HNF4α was scored based on staining intensity. Staining intensity was subclassified as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. The primary antibodies used in this study were as follows: Rabbit monoclonal anti-HNF4α (1:200 dilution; Cat# 3113, Cell Signaling Technology, USA); Rabbit polyclonal anti-TCF7L1 (5 \u0026micro;g/mL; Cat# PA5-40327, Thermo Fisher Scientific, USA); Ready-to-use rabbit monoclonal anti-β-catenin (Cat# ZA-0646, Zhongshan Golden Bridge Biotechnology, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eMouse intestinal tissues were harvested, embedded in OCT compound, and rapidly frozen in liquid nitrogen or on dry ice. Cryosections (8\u0026ndash;10 \u0026micro;m thick) were prepared using a cryostat and mounted on glass slides, followed by air-drying at room temperature for 30 minutes. The sections were fixed with 4% paraformaldehyde in PBS for 15 minutes, washed three times with PBS (5 minutes each), and permeabilized with 0.3% Triton X-100 in PBS for 10 minutes. After another set of PBS washes, sections were blocked with 5% BSA or 10% normal goat serum in PBS for 1 hour at room temperature. Primary antibodies, including HNF4α (CST #3113, 1:200) and β-Catenin (CST #37447, 1:200), were diluted in blocking solution and incubated with the sections overnight at 4\u0026deg;C in a humidified chamber. The next day, sections were washed three times with PBS and incubated with species-specific secondary antibodies for 1 hour at room temperature in the dark: Alexa Fluor\u0026reg; 488-conjugated Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L), F(ab')₂ Fragment (CST #4412, 1:500) for HNF4α and Alexa Fluor\u0026reg; 594-conjugated Anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L), F(ab')₂ Fragment (CST #8890, 1:500) for β-Catenin. After another round of PBS washes, nuclei were counterstained with DAPI (1 \u0026micro;g/mL in PBS) for 5 minutes, followed by final PBS washes. Sections were mounted with an anti-fade mounting medium and covered with a coverslip. Images were acquired using a fluorescence or confocal microscope with appropriate filter settings for Alexa Fluor\u0026reg; 488 (HNF4α), Alexa Fluor\u0026reg; 594 (β-Catenin), and DAPI. Negative controls were prepared by omitting the primary antibodies to confirm the specificity of secondary antibody staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistics analysis\u003c/h2\u003e \u003cp\u003eData were analyzed with Prism (GraphPad software RRID:SCR_002798). Statistical analyses were performed using \u003cem\u003et\u003c/em\u003e-tests or ANOVA. * \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 was considered statistically significant. ** \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01 was considered significant. *** \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001 was considered extremely significant. \u003cem\u003eP\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05 was considered not significant (ns).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e1. HNF4\u0026alpha; promotes colorectal carcinogenesis\u003c/h2\u003e\n\u003cp\u003eOur recent studies have shown that the loss of \u003cem\u003enhr-14/HNF4\u0026alpha;\u003c/em\u003e in \u003cem\u003eC. elegans\u003c/em\u003e resulted in the dysregulation of DNA damage-induced responses[39], which usually lead to cancer development[40\u0026ndash;48]. Thus, we examined HNF4\u0026alpha; expression levels in different cancers and matched paired normal tissues. TCGA database analysis revealed upregulation of HNF4\u0026alpha; in Colon adenocarcinoma (COAD), Rectum adenocarcinoma (READ) and Pancreatic adenocarcinoma (PAAD) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), suggesting that HNF4\u0026alpha; may play an important role in gastrointestinal tumorigenesis, particularly in colorectal carcinogenesis. We further analyzed HNF4\u0026alpha; protein levels in 59 paired CRC and normal tissues and found a significant increase in HNF4\u0026alpha; in 43 tumors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Immunohistochemical staining of colorectal cancer microarray also revealed remarkable upregulation of HNF4\u0026alpha; in cancer tissues (n\u0026thinsp;=\u0026thinsp;244) compared to normal colon tissues (n\u0026thinsp;=\u0026thinsp;99) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). These results indicate that overexpression of HNF4\u0026alpha; is a recurrent event in colorectal cancer and could play a significant role in this malignancy.\u003c/p\u003e\n\u003cp\u003eTo determine if HNF4\u0026alpha; plays a role in the malignancy in colorectal cancer, we measured HNF4\u0026alpha; expression levels in 26 colorectal cancer patients. We found that HNF4\u0026alpha; was significantly higher in malignant tumors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF-G) and lower levels of immune cells were present in tissues with high expression of HNF4\u0026alpha; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eH-J). Through cell communication analysis, we found strong signaling communication between malignant cells (high HNF4\u0026alpha; expression) and CD4\u003csup\u003e+\u003c/sup\u003e T cells (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eA -S1B), and through cell correlation analysis, we found a significant positive correlation between malignant cells and T cells (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eC), indicating an interaction between malignant cells and T cells within the immune microenvironment. The strong communication may allow malignant cells to evade immune system surveillance by regulating the function of CD4\u003csup\u003e+\u003c/sup\u003e T cells, thereby inhibiting the anti-tumor activity of CD4\u003csup\u003e+\u003c/sup\u003e T cells and promoting their own growth and spreading. This indicates that CD4\u003csup\u003e+\u003c/sup\u003e T cells may be activated or inhibited in specific immune states, thereby affecting the overall response of the immune system to tumors[49]. The tumor immune microenvironment is closely related to tumor proliferation and immune evasion. To investigate if HNF4\u0026alpha; is involved in tumor immune evasion, we performed ESTIMATE analysis on single-cell sequencing data and observed that cells with high HNF4\u0026alpha; expression had lower immune scores and ESTIMATE scores, but higher tumor purity scores and stromal scores (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eD), suggesting that HNF4\u0026alpha; may promote malignancy in colorectal cancer. Meanwhile, the Wnt/\u0026beta;-catenin signaling was also enriched in malignant tumors based on Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and immune scores (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eE-S1F).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e2. HNF4\u0026alpha; expression is associated with tumor formation in colorectal cancer\u003c/h2\u003e\n\u003cp\u003ePrevious studies have shown that HNF4\u0026alpha; could behave as either a tumor suppressor or an oncogene, depending on the cellular and molecular context[11\u0026ndash;17]. Thus, we investigated the function of HNF4\u0026alpha; in colorectal cancer by stable transfection of HNF4\u0026alpha; into SW480 or SW620 CRC cell lines. We found that ectopic expression of HNF4\u0026alpha; significantly increased colony formation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Xenograft mouse experiments also showed that overexpression of HNF4\u0026alpha; considerably promoted the tumor growth and increased the tumor volume and weight (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC-F). Conversely, when HNF4\u0026alpha; is knocked down, the tumor formation rate of colorectal cancer is significantly inhibited (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG-J). These findings suggest that HNF4\u0026alpha; acts as an oncogene and could be a driver in colorectal cancer.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003e3. HNF4\u0026alpha; positively correlated with the Wnt/\u0026beta;-catenin signaling pathway and is regulated by it\u003c/h2\u003e\n\u003cp\u003eTo examine the mechanism by which HNF4\u0026alpha; is upregulated in colorectal cancer, we performed GESA analysis using COAD and READ datasets from the TCGA database. Among the enriched HNF4\u0026alpha;-correlated signaling pathway, oncogenic Wnt/\u0026beta;-catenin and MYC pathways were significantly and positively associated with HNF4\u0026alpha; expression (Fig.\u0026nbsp;3A). It has been reported that the WNT/\u0026beta;-catenin pathway transcriptionally induces c-Myc expression to promote colorectal tumorigenesis[50]. Thus, we primarily focused on the relationship between HNF4\u0026alpha; and the Wnt/\u0026beta;-catenin pathway. We also analyzed the correlation between \u0026beta;-catenin and HNF4\u0026alpha;, and the result indicated that \u0026beta;-catenin positively associated with HNF4\u0026alpha; expression levels in TCGA database (Fig.\u0026nbsp;3B). Then we transfected SW480 cells with shRNA-HNF4\u0026alpha; and control shRNA and found no expression change of CTNNB1 at mRNA or protein levels between HNF4\u0026alpha;-knockdown and control cells (data not shown). Conversely, SW480 cells after transfection with shRNA-CTNNB1 expressed significant low levels of HNF4\u0026alpha; mRNA when compared to the cells treated with control shRNA (Fig.\u0026nbsp;3C). Moreover, we transfected shRNA-CTNNB1 into 2 other CRC cell lines (SW620 and DLD1). Western blotting analysis showed that knockdown of \u0026beta;-catenin significantly reduced the protein level of HNF4\u0026alpha; in SW480, SW620 and DLD1 cell lines (Fig.\u0026nbsp;3D). These results suggest that HNF4\u0026alpha; is regulated by the WNT /\u0026beta;-catenin pathway at the transcription level.\u003c/p\u003e\n\u003cp\u003eTo further evaluate this notion, we treated CRC cells with SKL2001, an activator of the Wnt/\u0026beta;-catenin pathway, at different doses for 12h. Western blotting analysis of cellular fractionation revealed significant nuclear accumulation of \u0026beta;-catenin and increased expression of HNF4\u0026alpha; following SKL2001 treatment (Fig.\u0026nbsp;3E-F). In addition, we treated SW480 and SW620 cells with Chir-99021, another \u0026beta;-catenin activator, and found upregulation of HNF4\u0026alpha; in both cell lines after Chir-99021treatment (Fig.\u0026nbsp;3F). These results further confirmed that \u0026beta;-catenin is an activator of HNF4\u0026alpha;. As HNF4\u0026alpha; is a downstream target of the Wnt/\u0026beta;-catenin pathway, we next investigated whether HNF4\u0026alpha; mediates the Wnt/\u0026beta;-catenin axis in colon carcinogenesis. SW620 cells were stably transfected with \u0026beta;-catenin shRNA or \u0026beta;-catenin shRNA and HNF4\u0026alpha; overexpression, respectively. The cells treated with vectors alone were used as the control (Fig.\u0026nbsp;3G). These cells were subcutaneously injected to nude mice. As expected, knockdown of \u0026beta;-catenin inhibited the tumor growth and reduced the tumor volume and weight (Fig.\u0026nbsp;3H, J). Notably, ectopic expression of HNF4\u0026alpha; largely overrode these phenotypes caused by \u0026beta;-catenin knockdown (Fig.\u0026nbsp;3H, I). These findings further indicate that HNF4\u0026alpha; is a major target of \u0026beta;-catenin in CRC.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003e4. TCF7L1 promotes HNF4\u0026alpha; expression\u003c/h2\u003e\n\u003cp\u003e\u0026beta;-catenin is a transcription co-activator of the TCF/LEF transcription factor (TF) family. To further understand the molecular mechanisms by which Wnt/\u0026beta;-catenin induces HNF4\u0026alpha; transcription, we first analyzed the expression relationship between HNF4\u0026alpha; and TCF7 (also known as TCF-1), LEF1, TCF7L1 (also known as TCF-3), TCF7l2 (also known as TCF-4), which are TCF/LEF family members, in colorectal cancer through the TCGA database. We found that HNF4\u0026alpha; had the highest correlation with TCF1 and TCF7L1, but not LEF1 or TCF7L2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). When individually knocked down in SW480 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE-F), HNF4\u0026alpha; was reduced in TCF7L1-knockdown cells, but not in shRNA-TCF1, shRNA-TCF7L1 or shRNA-LEF1 treated cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). These results were confirmed in two different colorectal cancer cell lines, LS174T and DLD1. Similarly, TCF7L1 knockdown decreased HNF4\u0026alpha; expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG). Moreover, ectopic expression of TCF7L1 induced HNF4\u0026alpha; levels, but which was abrogated by \u0026beta;-catenin depletion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH-I). In single cell RNA sequencing data from 26 colorectal cancer, both HNF4\u0026alpha; and TCF7L1 were highly expressed in malignant cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eJ-K). These results suggest that TCF7L1 is a major transcriptional factor that mediates the action of Wnt/\u0026beta;-catenin in response to HNF4\u0026alpha; in colorectal cancer.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003e5. TCF7L1 directly binds to the \u0026minus;\u0026thinsp;90 ~ -80 bp region of the HNF4\u0026alpha; promoter\u003c/h2\u003e\n\u003cp\u003eTo investigate whether TCF7L1 can directly bind to the HNF4\u0026alpha; promoter to regulate HNF4\u0026alpha; transcription, we first examined the TCF7L1 consensus binding motif \u0026ldquo;CACCTGC\u0026rdquo; in the HNF4\u0026alpha; promoter region (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jaspar.genereg.net/\u003c/span\u003e\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, there are 10 TCF7L1 putative binding sites within a 2.0-kb HNF4\u0026alpha; promoter. To determine which binding site(s) is required for TCF7L1-mediated HNF4\u0026alpha; transcription, we constructed deletion mutants of the HNF4\u0026alpha; promoter including pGL3-P\u003csub\u003eHNF4\u0026alpha;\u003c/sub\u003e1700bp, pGL3-P\u003csub\u003eHNF4\u0026alpha;\u003c/sub\u003e800bp and pGL3-P\u003csub\u003eHNF4\u0026alpha;\u003c/sub\u003e300bp, which contain 10, 7 and 3 TCF7L1 putative binding sites, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Luciferase reporter assays revealed that knockdown of TCF7L1 significantly reduced the basal promoter activity in three HNF4\u0026alpha; deletion promoter mutants (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB), suggesting that pGL3-P\u003csub\u003eHNF4\u0026alpha;\u003c/sub\u003e300bp contains major TCF7L1 response motifs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Since there are 3 TCF7L1 putative binding sites in pGL3-P\u003csub\u003eHNF4\u0026alpha;\u003c/sub\u003e300bp, we next mutated each site in this promoter region (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). Interestingly, mutations of the 90 to 80 bp motif, which is the closest to transcription start site of the \u003cem\u003eHNF4\u0026alpha;\u003c/em\u003e gene, completely abrogated the pGL3-P\u003csub\u003eHNF4\u0026alpha;\u003c/sub\u003e300bp promoter activity, whereas mutation of the rest two sites had no significant effects on the promoter activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). CHIP-qPCR and CHIP-seq results further corroborated this conclusion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE-G). These findings suggest that TCF7L1 transcriptionally activates HNF4\u0026alpha; primarily through binding to the first motif of the HNF4\u0026alpha; promoter.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003e6. HNF4\u0026alpha; activates WNT signaling and promotes \u0026beta;-catenin nuclear localization\u003c/h2\u003e\n\u003cp\u003eIn an attempt to investigate the mechanism of HNF4\u0026alpha; in colorectal tumorigenesis, we next performed RNA sequencing analysis between SW480-vector and SW480-HNF4a cells. The signals enriched by HNF4\u0026alpha; overexpression were involved in cardiac system diseases, neurological diseases, tumor progression and other features, among which the most tumor-related signaling pathways were the MAPK, Hippo and Wnt signaling cascades (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). The GSEA enrichment analysis results showed that the Wnt/\u0026beta;-catenin signaling pathway was activated after HNF4\u0026alpha; overexpression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD), which was also confirmed by TOPFlash luciferase reporter assay (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG). Notably, Wnt1, Wnt4, Wnt7b and Wnt11 were up-regulated in SW480-HNF4\u0026alpha; cells when compared to SW480-vector control cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB), which were further confirmed by the qPCR assay (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). However, we did not observe expression level changes of \u0026beta;-catenin in the cells ectopically expressing HNF4\u0026alpha; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE). As HNF4\u0026alpha; induces expression levels of several Wnt family members, we hypothesized that HNF4\u0026alpha; could promote \u0026beta;-catenin translocation from the cytoplasm to the nucleus. Cell fractionations were obtained from SW480-vector and SW480-HNF4\u0026alpha; cells. Immunoblotting analysis revealed that the nuclear fraction of \u0026beta;-catenin was significantly enriched in the HNF4\u0026alpha; overexpressing cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e\n\u003cp\u003eTo further explore the relation between HNF4\u0026alpha; and WNT expression, we examined HNF4\u0026alpha; and WNTs expression levels in single cell RNA sequencing data from 26 colorectal cancer patients. We observed that high HNF4\u0026alpha; expression was accompanied by nearly uniform high WNT family gene expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eH), which are consistent with our previous bulk RNA-seq findings and indicating that HNF4\u0026alpha; is associated with the WNT signaling pathway and plays a crucial role in the development and progression of colorectal cancer.\u003c/p\u003e\n\u003cp\u003eTo further validate the transcriptional regulatory role of HNF4\u0026alpha; on WNT family genes, we conducted comparative HNF4\u0026alpha; ChIP-seq analyses using intestinal tissues from wild-type and HNF4\u0026alpha; knockout mice. The experimental results demonstrated a significant reduction in peak signals at promoter regions of WNT family genes following HNF4\u0026alpha; depletion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eI). These findings collectively suggest that HNF4\u0026alpha; maintains transcriptional activation of WNT family genes through dual mechanisms: direct DNA binding and chromatin structure modulation.\u003c/p\u003e\n\u003cp\u003eCollectively, our findings indicate that HNF4\u0026alpha; and Wnt/\u0026beta;-catenin regulated each other and form a feedback loop to contribute to colorectal carcinogenesis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003e7. The \u0026beta;-catenin/TCF7L1-HNF4\u0026alpha; axis correlates with colorectal tumorigenesis\u003c/h2\u003e\n\u003cp\u003eTo evaluate the clinical significance of \u0026beta;-catenin/TCF7L1-HNF4\u0026alpha; axis in CRC, we performed IHC analysis and examined the HNF4\u0026alpha; and \u0026beta;-catenin/TCF7L1 expression level in a CRC tissue array and investigated the correlation of \u0026beta;-catenin/ TCF7L1 with HNF4\u0026alpha; in colorectal cancer. The results showed that the tumor tissue with high levels of \u0026beta;-catenin/TCF7L1 usually express high levels of HNF4\u0026alpha;, and the tumor tissue with low levels of \u0026beta;-catenin/TCF7L1 usually have low levels of HNF4\u0026alpha; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). The correlation analysis indicated that HNF4\u0026alpha; was significantly correlated with \u0026beta;-catenin and TCF7L1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). We also tested the correlation between TCF7L1, and HNF4\u0026alpha; in 26 colorectal cancer patients. The result indicated that HNF4\u0026alpha;, CTNNB1 and TCF7L1 have the same expression pattern in pseudo temporal analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE). Taken together, these results indicated that HNF4\u0026alpha; was correlated with the Wnt/\u0026beta;-catenin pathway and involved in colorectal tumorigenesis. To validate this finding in vivo, we examined the expression of HNF4\u0026alpha; and \u0026beta;-catenin in the intestinal tissues of APC-deficient mice. We found strong co-localization and a similar expression pattern between HNF4\u0026alpha; and \u0026beta;-catenin (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF), which further supports our hypothesis. To further test our hypothesis, we employed AOM/DSS Model of Colitis-associated colorectal cancer model [51], and found that AOM/DSS treatment can not only induce intestinal inflammation and colorectal cancer, but also induce highly express \u0026beta;-catenin and HNF4\u0026alpha; in the intestinal tissues of treated mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eG). The result implied that the \u0026beta;-catenin/TCF7L1-HNF4\u0026alpha; axis plays an important role in colorectal carcinogenesis.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our findings reveal that HNF4\u0026alpha; is regulated by \u0026beta;-catenin/TCF7L1 and promotes the expression of Wnt family genes, ultimately activating the Wnt/\u0026beta;-catenin signaling pathway to drive tumorigenesis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eH). These discoveries provide novel theoretical foundations and potential therapeutic strategies for cancer prevention and treatment.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCRC is a highly heterogeneous disease and the expression regulation of HNF4α isoforms in colorectal tumors exhibits significant specificity. Distinct HNF4α subtypes play divergent roles in tumorigenesis and progression[10]. Studies indicate that the loss of HNF4α is closely associated with cancer initiation and development[52\u0026ndash;54]. For instance, Src kinase reduces the protein stability, nuclear localization, and transcriptional activity of HNF4α through phosphorylation at the Y14 site, a mechanism particularly prominent in specific isoforms[55]. During cancer progression, approximately 80% of stage III tumors demonstrate either loss of HNF4α or aberrant cytoplasmic localization, which correlates strongly with Src kinase activity. Single-cell chromatin accessibility analysis reveals that HNF4α is a key iCMS-specific transcription factor in colorectal malignant tumor cells, suggesting that HNF4α may play important roles in colorectal tumorigenesis and progression. However, the role and regulatory mechanism of HNF4α and TCF7L1 in colorectal cancer are still largely unknown.\u003c/p\u003e \u003cp\u003eWnt/β-catenin activation is a well-recognized driver of colorectal carcinogenesis. However, the regulation of TCF7L1, HNF4α and Wnt/β-catenin in colorectal cancer is still largely unknown. In specific CRC cell lines, β-catenin has been found to negatively regulate HNF4α expression. This observation aligns with prior studies showing that HNF4α competes with β-catenin for TCF4 binding, thereby suppressing the expression of downstream Wnt target genes and inhibiting colorectal cancer progression[19, 56]. However, some studies have also shown that HNF4α promotes the occurrence of colorectal cancer. The specific mechanism and reasons behind it are still unclear.\u003c/p\u003e \u003cp\u003eOur results demonstrate that TCF7L1 can bind to the \u0026minus;\u0026thinsp;90~-80 bp region of the HNF4α promoter, thereby regulating the transcription of HNF4α and activating the Wnt/β-catenin signaling pathway, which promotes the tumorigenesis of colorectal cancer. There are four transcription factors (TCF1, LEF1, TCF7L1 and TCF7L2) in the Wnt/β-catenin signaling pathway and they exhibit significant differences in regulating target genes[57]. Compared to the other three transcription factors, TCF7L1 was the least reported in tumors. The mutual regulatory relationship between TCF7L1 and HNF4α has not been reported. These findings indicate that Wnt/β-catenin signaling can be activated by TCF7L1 and HNF4α in CRC.\u003c/p\u003e \u003cp\u003eIn conclusion, our study demonstrates that the Wnt/β-catenin-TCF7L1-HNF4α feedback loop confers colorectal tumorigenesis and malignancy and HNF4α overexpression feedback regulates Wnt expression and promotes β-catenin nuclear localization. Further investigation of the role and the specific mechanism of HNF4α in the development and progression of colorectal cancer is of great significance for establishing HNF4α as a therapeutic target in colorectal cancer. Our study provides new strategies for the treatment and prevention of colorectal cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eColorectal cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHNF4α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHepatocyte nuclear factor 4 alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCTNNB1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCatenin beta 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCF1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLiver-specific transcription factor lf-b1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCF7L1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTranscription factor 7-like 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCF7L2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTranscription factor 7 like 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLEF1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLymphoid enhancer binding factor 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWNT3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWnt Family Member 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWNT4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWnt family member 4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWNT5A\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWnt family member 5A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWNT5B\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWnt family member 5B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWNT6\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWnt family member 6\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWNT9A\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWnt family member 9A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWNT10A\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWnt family member 10A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWNT11\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWnt family member 11\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFPKM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFragments per kilobase million reads\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEGs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDifferent expressed genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTMAs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTissue microarrays\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunohistochemical\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBRCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBreast invasive carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLiver hepatocellular carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLUAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLung adenocarcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLUSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLung squamous cell carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSKCM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSkin Cutaneous Melanoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCOAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eColon adenocarcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eREAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRectum adenocarcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePAAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePancreatic adenocarcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto encyclopedia of genes and genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTranscription factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted in strict compliance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Yunnan University. Each study involving human subjects adhered to the ethical guidelines and operational protocols approved by the Ethics Committee of the School of Life Sciences at Yunnan University (Approval No. KY2023-015). Research utilizing human tissue samples followed the ethical review requirements stipulated in the \"Ethical Review Measures for Biomedical Research Involving Humans\" (National Health and Family Planning Commission Order No. 11, 2016), with specific approval granted under authorization number CHSRE2023023. Prior to study commencement, all participants received comprehensive study information and provided written informed consent through standardized documentation procedures.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eAll authors have agreed to publish this manuscript\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no conflicts of interest with the contents of this article.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAuthor details\u003c/h2\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003e Yunnan Key Laboratory of Cell Metabolism and Diseases, State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China\u003c/p\u003e \u003cp\u003e \u003csup\u003e2\u003c/sup\u003e Tianjin Medical University Cancer Institute and Hospital. National Clinical Research Center for Cancer. Key Laboratory of Cancer Prevention and Therapy, Tianjin\u0026rsquo;s Clinical Research Center for Cancer. Tianjin, China\u003c/p\u003e \u003cp\u003e \u003csup\u003e3\u003c/sup\u003e Medical Research Department, Qingdao Hospital, University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital), Qingdao 266071.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (NSFC) fund (82273460, 32260167 and 81871990), the Yunnan Fundamental Research Projects (202401AS070133), Shandong Province Natural Science Foundation (ZR202111120048) and grants (KC-23234451, ZC-23236369, 2024Y014 and 202310673059) from Yunnan University.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWYB, RD, CLL and LS: Experiments and data analysis. QGH, YRZ, RYS: Vector construction and dual-luciferase assay. JLS, WZ, YS and JWS wrote the main manuscript text.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe especially thank Professor Yeguang Chen for generously providing the APC-floxed mice and Dr. Jing Li for her helpful suggestion and comments on the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eCRC single-cell data and TCF7L1 ChIP-seq data can be obtained from the Gene Expression Omnibus (GEO) (GSE166555, GSE80331) .\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Sinicrope, F.A., \u003cem\u003eIncreasing Incidence of Early-Onset Colorectal Cancer.\u003c/em\u003e N Engl J Med, 2022. \u003cb\u003e386\u003c/b\u003e(16): p. 1547\u0026ndash;1558.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wu, F., W. Zhang, H. Wei, H. Ma, G. Leng, and Y. Zhang, \u003cem\u003elncRNA ELFN1-AS1 promotes proliferation, migration and invasion and suppresses apoptosis in colorectal cancer cells by enhancing G6PD activity.\u003c/em\u003e Acta Biochim Biophys Sin (Shanghai), 2023. \u003cb\u003e55\u003c/b\u003e(4): p. 649\u0026ndash;660.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Margetis, N., M. Kouloukoussa, K. Pavlou, S. Vrakas, and T. Mariolis-Sapsakos, \u003cem\u003eK-ras Mutations as the Earliest Driving Force in a Subset of Colorectal Carcinomas.\u003c/em\u003e In Vivo, 2017. \u003cb\u003e31\u003c/b\u003e(4): p. 527\u0026ndash;542.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Hassin, O. and M. Oren, \u003cem\u003eDrugging p53 in cancer: one protein, many targets.\u003c/em\u003e Nat Rev Drug Discov, 2023. \u003cb\u003e22\u003c/b\u003e(2): p. 127\u0026ndash;144.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Rosin-Arbesfeld, R., F. Townsley, and M. Bienz, \u003cem\u003eThe APC tumour suppressor has a nuclear export function.\u003c/em\u003e Nature, 2000. \u003cb\u003e406\u003c/b\u003e(6799): p. 1009-12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Gunewardena, S., I. Huck, C. Walesky, D. Robarts, S. Weinman, and U. Apte, \u003cem\u003eProgressive loss of hepatocyte nuclear factor 4 alpha activity in chronic liver diseases in humans.\u003c/em\u003e Hepatology, 2022. \u003cb\u003e76\u003c/b\u003e(2): p. 372\u0026ndash;386.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wu, H., T. Reizel, Y.J. Wang, J.L. Lapiro, B.T. Kren, J. Schug, et al., \u003cem\u003eA negative reciprocal regulatory axis between cyclin D1 and HNF4α modulates cell cycle progression and metabolism in the liver.\u003c/em\u003e Proc Natl Acad Sci U S A, 2020. \u003cb\u003e117\u003c/b\u003e(29): p. 17177\u0026ndash;17186.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Gu, W., H. Wang, X. Huang, J. Kraiczy, P.N.P. Singh, C. Ng, et al., \u003cem\u003eSATB2 preserves colon stem cell identity and mediates ileum-colon conversion via enhancer remodeling.\u003c/em\u003e Cell Stem Cell, 2022. \u003cb\u003e29\u003c/b\u003e(1): p. 101\u0026ndash;115.e10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Taniguchi, H., A. Fujimoto, H. Kono, M. Furuta, M. Fujita, and H. Nakagawa, \u003cem\u003eLoss-of-function mutations in Zn-finger DNA-binding domain of HNF4A cause aberrant transcriptional regulation in liver cancer.\u003c/em\u003e Oncotarget, 2018. \u003cb\u003e9\u003c/b\u003e(40): p. 26144\u0026ndash;26156.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Chellappa, K., P. Deol, J.R. Evans, L.M. Vuong, G. Chen, N. Brian\u0026ccedil;on, et al., \u003cem\u003eOpposing roles of nuclear receptor HNF4α isoforms in colitis and colitis-associated colon cancer.\u003c/em\u003e Elife, 2016. \u003cb\u003e5\u003c/b\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Barrett, J.C., J.C. Lee, C.W. Lees, N.J. Prescott, C.A. Anderson, A. Phillips, et al., \u003cem\u003eGenome-wide association study of ulcerative colitis identifies three new susceptibility loci, including the HNF4A region.\u003c/em\u003e Nat Genet, 2009. \u003cb\u003e41\u003c/b\u003e(12): p. 1330-4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Becker, W.R., S.A. Nevins, D.C. Chen, R. Chiu, A.M. Horning, T.K. Guha, et al., \u003cem\u003eSingle-cell analyses define a continuum of cell state and composition changes in the malignant transformation of polyps to colorectal cancer.\u003c/em\u003e Nat Genet, 2022. \u003cb\u003e54\u003c/b\u003e(7): p. 985\u0026ndash;995.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Darsigny, M., J.P. Babeu, E.G. Seidman, F.P. Gendron, E. Levy, J. Carrier, et al., \u003cem\u003eHepatocyte nuclear factor-4alpha promotes gut neoplasia in mice and protects against the production of reactive oxygen species.\u003c/em\u003e Cancer Res, 2010. \u003cb\u003e70\u003c/b\u003e(22): p. 9423-33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e He, Y., L. Chen, K. Chen, and Y. Sun, \u003cem\u003eImmunohistochemical analysis of HNF4A and β-catenin expression to predict low-grade dysplasia in the colitis-neoplastic sequence.\u003c/em\u003e Acta Biochim Biophys Sin (Shanghai), 2021. \u003cb\u003e53\u003c/b\u003e(1): p. 94\u0026ndash;101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Li, S., M. Yang, S. Teng, K. Lin, Y. Wang, Y. Zhang, et al., \u003cem\u003eChromatin accessibility dynamics in colorectal cancer liver metastasis: Uncovering the liver tropism at single cell resolution.\u003c/em\u003e Pharmacol Res, 2023. \u003cb\u003e195\u003c/b\u003e: p. 106896.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Liu, Z., Y. Hu, H. Xie, K. Chen, L. Wen, W. Fu, et al., \u003cem\u003eSingle-Cell Chromatin Accessibility Analysis Reveals the Epigenetic Basis and Signature Transcription Factors for the Molecular Subtypes of Colorectal Cancers.\u003c/em\u003e Cancer Discov, 2024. \u003cb\u003e14\u003c/b\u003e(6): p. 1082\u0026ndash;1105.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Rajam\u0026auml;ki, K., A. Taira, R. Katainen, N. V\u0026auml;lim\u0026auml;ki, A. Kuosmanen, R.M. Plaketti, et al., \u003cem\u003eGenetic and Epigenetic Characteristics of Inflammatory Bowel Disease-Associated Colorectal Cancer.\u003c/em\u003e Gastroenterology, 2021. \u003cb\u003e161\u003c/b\u003e(2): p. 592\u0026ndash;607.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Babeu, J.P., C. Jones, S. Geha, J.C. Carrier, and F. Boudreau, \u003cem\u003eP1 promoter-driven HNF4α isoforms are specifically repressed by β-catenin signaling in colorectal cancer cells.\u003c/em\u003e J Cell Sci, 2018. \u003cb\u003e131\u003c/b\u003e(13).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Vuong, L.M., K. Chellappa, J.M. Dhahbi, J.R. Deans, B. Fang, E. Bolotin, et al., \u003cem\u003eDifferential Effects of Hepatocyte Nuclear Factor 4α Isoforms on Tumor Growth and T-Cell Factor 4/AP-1 Interactions in Human Colorectal Cancer Cells.\u003c/em\u003e Mol Cell Biol, 2015. \u003cb\u003e35\u003c/b\u003e(20): p. 3471-90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Saandi, T., F. Baraille, L. Derbal-Wolfrom, A.L. Cattin, F. Benahmed, E. Martin, et al., \u003cem\u003eRegulation of the tumor suppressor homeogene Cdx2 by HNF4α in intestinal cancer.\u003c/em\u003e Oncogene, 2013. \u003cb\u003e32\u003c/b\u003e(32): p. 3782-8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Fodde, R., R. Smits, and H. Clevers, \u003cem\u003eAPC, signal transduction and genetic instability in colorectal cancer.\u003c/em\u003e Nat Rev Cancer, 2001. \u003cb\u003e1\u003c/b\u003e(1): p. 55\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Bienz, M. and H. Clevers, \u003cem\u003eLinking colorectal cancer to Wnt signaling.\u003c/em\u003e Cell, 2000. \u003cb\u003e103\u003c/b\u003e(2): p. 311\u0026thinsp;\u0026minus;\u0026thinsp;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Chen, S., L. Ji, Y. Wang, L. Zhang, M. Xu, Y. Su, et al., \u003cem\u003elncRNA RMST suppresses the progression of colorectal cancer by competitively binding to miR-27a-3p/RXRα axis and inactivating Wnt signaling pathway.\u003c/em\u003e Acta Biochim Biophys Sin (Shanghai), 2023. \u003cb\u003e55\u003c/b\u003e(5): p. 726\u0026ndash;735.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Li, S., W. Han, Q. He, Y. Wang, G. Jin, and Y. Zhang, \u003cem\u003eGinsenoside Rh2 suppresses colon cancer growth by targeting the miR-150-3p/SRCIN1/Wnt axis.\u003c/em\u003e Acta Biochim Biophys Sin (Shanghai), 2023. \u003cb\u003e55\u003c/b\u003e(4): p. 633\u0026ndash;648.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Doumpas, N., F. Lampart, M.D. Robinson, A. Lentini, C.E. Nestor, C. Cant\u0026ugrave;, et al., \u003cem\u003eTCF/LEF dependent and independent transcriptional regulation of Wnt/β-catenin target genes.\u003c/em\u003e Embo j, 2019. \u003cb\u003e38\u003c/b\u003e(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Chen, X., J. Yang, P.M. Evans, and C. Liu, \u003cem\u003eWnt signaling: the good and the bad.\u003c/em\u003e Acta Biochim Biophys Sin (Shanghai), 2008. \u003cb\u003e40\u003c/b\u003e(7): p. 577\u0026thinsp;\u0026minus;\u0026thinsp;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Chen, Y., Z. Lu, J. Feng, Z. Chen, Z. Liu, X. Wang, et al., \u003cem\u003eNovel recombinant R-spondin1 promotes hair regeneration by targeting the Wnt/β-catenin signaling pathway.\u003c/em\u003e Acta Biochim Biophys Sin (Shanghai), 2023. \u003cb\u003e55\u003c/b\u003e(8): p. 1213\u0026ndash;1221.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Jiang, R., X. Niu, Y. Huang, and X. Wang, \u003cem\u003eβ-Catenin is important for cancer stem cell generation and tumorigenic activity in nasopharyngeal carcinoma.\u003c/em\u003e Acta Biochim Biophys Sin (Shanghai), 2016. \u003cb\u003e48\u003c/b\u003e(3): p. 229\u0026thinsp;\u0026minus;\u0026thinsp;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Xu, X., Y. Dai, L. Feng, H. Zhang, Y. Hu, L. Xu, et al., \u003cem\u003eKnockdown of Nav1.5 inhibits cell proliferation, migration and invasion via Wnt/β-catenin signaling pathway in oral squamous cell carcinoma.\u003c/em\u003e Acta Biochim Biophys Sin (Shanghai), 2020. \u003cb\u003e52\u003c/b\u003e(5): p. 527\u0026ndash;535.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Meng, L., R. Dong, W. Mi, K. Qin, K. Ouyang, J. Sun, et al., \u003cem\u003eThe ubiquitin E3 ligase APC/C(Cdc20) mediates mitotic degradation of OGT.\u003c/em\u003e J Biol Chem, 2024. \u003cb\u003e300\u003c/b\u003e(7): p. 107448.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Lee, E., A. Salic, R. Kr\u0026uuml;ger, R. Heinrich, and M.W. Kirschner, \u003cem\u003eThe roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway.\u003c/em\u003e PLoS Biol, 2003. \u003cb\u003e1\u003c/b\u003e(1): p. E10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Tran, H., D. Bustos, R. Yeh, B. Rubinfeld, C. Lam, S. Shriver, et al., \u003cem\u003eHectD1 E3 ligase modifies adenomatous polyposis coli (APC) with polyubiquitin to promote the APC-axin interaction.\u003c/em\u003e J Biol Chem, 2013. \u003cb\u003e288\u003c/b\u003e(6): p. 3753-67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Shen, J., J. Yang, L. Sang, R. Sun, W. Bai, C. Wang, et al., \u003cem\u003ePYK2 mediates the BRAF inhibitor (vermurafenib)-induced invadopodia formation and metastasis in melanomas.\u003c/em\u003e Cancer Biol Med, 2021. \u003cb\u003e19\u003c/b\u003e(8): p. 1211-23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Bai, W., Q. Hao, Z. Zhang, B. Han, H. Xiao, D. Chang, et al., \u003cem\u003eIdentification of a novel inflammation-related gene signature for predicting inflammatory breast cancer survival.\u003c/em\u003e Genome Instability \u0026amp; Disease, 2023. \u003cb\u003e4\u003c/b\u003e(3): p. 154\u0026ndash;175.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Hao, Q., R. Dong, W. Bai, D. Chang, X. Yao, Y. Zhang, et al., \u003cem\u003eScreening for metastasis-related genes in mouse melanoma cells through sequential tail vein injection.\u003c/em\u003e Biophys Rep, 2024. \u003cb\u003e10\u003c/b\u003e(1): p. 15\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Hao, Y., S. Hao, E. Andersen-Nissen, W.M. Mauck, 3rd, S. Zheng, A. Butler, et al., \u003cem\u003eIntegrated analysis of multimodal single-cell data.\u003c/em\u003e Cell, 2021. \u003cb\u003e184\u003c/b\u003e(13): p. 3573\u0026ndash;3587.e29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Sun, J., H. He, S. Pillai, Y. Xiong, S. Challa, L. Xu, et al., \u003cem\u003eGATA3 transcription factor abrogates Smad4 transcription factor-mediated fascin overexpression, invadopodium formation, and breast cancer cell invasion.\u003c/em\u003e J Biol Chem, 2013. \u003cb\u003e288\u003c/b\u003e(52): p. 36971-82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Bai, W., C. Yan, Y. Yang, L. Sang, Q. Hao, X. Yao, et al., \u003cem\u003eEGF/EGFR-YAP1/TEAD2 signaling upregulates STIM1 in vemurafenib resistant melanoma cells.\u003c/em\u003e Febs j, 2024. \u003cb\u003e291\u003c/b\u003e(22): p. 4969\u0026ndash;4983.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Sang, L., R. Dong, R. Liu, Q. Hao, W. Bai, and J. Sun, \u003cem\u003eCaenorhabditis elegans NHR-14/HNF4α regulates DNA damage-induced apoptosis through cooperating with cep-1/p53.\u003c/em\u003e Cell Commun Signal, 2022. \u003cb\u003e20\u003c/b\u003e(1): p. 135.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Cao, T., X. Luo, B. Zheng, Y. Deng, Y. Zhang, Y. Li, et al., \u003cem\u003eDeath-associated protein 3 in cell death and beyond.\u003c/em\u003e Genome Instability \u0026amp; Disease, 2024. \u003cb\u003e5\u003c/b\u003e(2): p. 51\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Zhao, Y., Z. Jiang, T. Ni, W. Jiang, K. Zhou, Y. Liu, et al., \u003cem\u003eTerpenoids-enriched fraction of Celastrus orbiculatus sensitizes gemcitabine by disrupting Chk1/RAD51-mediated DNA damage response in pancreatic cancer.\u003c/em\u003e Genome Instability \u0026amp; Disease, 2021. \u003cb\u003e2\u003c/b\u003e(6): p. 358\u0026ndash;373.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Pfeifer, G.P., \u003cem\u003eMechanisms of UV-induced mutations and skin cancer.\u003c/em\u003e Genome Instability \u0026amp; Disease, 2020. \u003cb\u003e1\u003c/b\u003e(3): p. 99\u0026ndash;113.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Sang, L., X. Wang, W. Bai, J. Shen, Y. Zeng, and J. Sun, \u003cem\u003eThe role of hepatocyte nuclear factor 4α (HNF4α) in tumorigenesis.\u003c/em\u003e Front Oncol, 2022. \u003cb\u003e12\u003c/b\u003e: p. 1011230.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Yu, Y., H. Jia, T. Zhang, and W. Zhang, \u003cem\u003eAdvances in DNA damage response inhibitors in colorectal cancer therapy.\u003c/em\u003e Acta Biochim Biophys Sin (Shanghai), 2024. \u003cb\u003e56\u003c/b\u003e(1): p. 15\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Gao, Y., R. Dong, J. Yan, H. Chen, L. Sang, X. Yao, et al., \u003cem\u003eMitochondrial deoxyguanosine kinase is required for female fertility in mice.\u003c/em\u003e Acta Biochim Biophys Sin (Shanghai), 2024. \u003cb\u003e56\u003c/b\u003e(3): p. 427\u0026ndash;439.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Lv, J., P. Gong, G. Jia, and W. Li, \u003cem\u003eTargeted DNA damage repair: old mechanisms and new opportunities in clear cell renal cell carcinoma.\u003c/em\u003e Genome Instability \u0026amp; Disease, 2024. \u003cb\u003e5\u003c/b\u003e(5): p. 197\u0026ndash;209.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Xiang, Z., H. Liu, and Y. Hu, \u003cem\u003eDNA damage repair and cancer immunotherapy.\u003c/em\u003e Genome Instability \u0026amp; Disease, 2023. \u003cb\u003e4\u003c/b\u003e(4): p. 210\u0026ndash;226.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Guo, C. and Y. Zhao, \u003cem\u003eAutophagy and DNA damage repair.\u003c/em\u003e Genome Instability \u0026amp; Disease, 2020. \u003cb\u003e1\u003c/b\u003e(4): p. 172\u0026ndash;183.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Li, D., W. Xu, Y. Chang, Y. Xiao, Y. He, and S. Ren, \u003cem\u003eAdvances in landscape and related therapeutic targets of the prostate tumor microenvironment.\u003c/em\u003e Acta Biochim Biophys Sin (Shanghai), 2023. \u003cb\u003e55\u003c/b\u003e(6): p. 956\u0026ndash;973.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Yang, X., F. Shao, D. Guo, W. Wang, J. Wang, R. Zhu, et al., \u003cem\u003eWNT/β-catenin-suppressed FTO expression increases m(6)A of c-Myc mRNA to promote tumor cell glycolysis and tumorigenesis.\u003c/em\u003e Cell Death Dis, 2021. \u003cb\u003e12\u003c/b\u003e(5): p. 462.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Parang, B., C.W. Barrett, and C.S. Williams, \u003cem\u003eAOM/DSS Model of Colitis-Associated Cancer\u003c/em\u003e, in \u003cem\u003eGastrointestinal Physiology and Diseases: Methods and Protocols\u003c/em\u003e, A.I. Ivanov, Editor. 2016, Springer New York: New York, NY. p. 297\u0026ndash;307.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Chellappa, K., L. Jankova, J.M. Schnabl, S. Pan, Y. Brelivet, C.L. Fung, et al., \u003cem\u003eSrc tyrosine kinase phosphorylation of nuclear receptor HNF4α correlates with isoform-specific loss of HNF4α in human colon cancer.\u003c/em\u003e Proc Natl Acad Sci U S A, 2012. \u003cb\u003e109\u003c/b\u003e(7): p. 2302-7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Tanaka, T., S. Jiang, H. Hotta, K. Takano, H. Iwanari, K. Sumi, et al., \u003cem\u003eDysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer.\u003c/em\u003e J Pathol, 2006. \u003cb\u003e208\u003c/b\u003e(5): p. 662\u0026thinsp;\u0026minus;\u0026thinsp;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Oshima, T., T. Kawasaki, R. Ohashi, G. Hasegawa, S. Jiang, H. Umezu, et al., \u003cem\u003eDownregulated P1 promoter-driven hepatocyte nuclear factor-4alpha expression in human colorectal carcinoma is a new prognostic factor against liver metastasis.\u003c/em\u003e Pathol Int, 2007. \u003cb\u003e57\u003c/b\u003e(2): p. 82\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Chellappa, K., L. Jankova, J.M. Schnabl, S. Pan, Y. Brelivet, C.L.-S. Fung, et al., \u003cem\u003eSrc tyrosine kinase phosphorylation of nuclear receptor HNF4α correlates with isoform-specific loss of HNF4α in human colon cancer.\u003c/em\u003e Proceedings of the National Academy of Sciences, 2012. \u003cb\u003e109\u003c/b\u003e(7): p. 2302\u0026ndash;2307.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Gougelet, A., C. Torre, P. Veber, C. Sartor, L. Bachelot, P.D. Denechaud, et al., \u003cem\u003eT-cell factor 4 and β-catenin chromatin occupancies pattern zonal liver metabolism in mice.\u003c/em\u003e Hepatology, 2014. \u003cb\u003e59\u003c/b\u003e(6): p. 2344-57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Atcha, F.A., A. Syed, B. Wu, N.P. Hoverter, N.N. Yokoyama, J.H. Ting, et al., \u003cem\u003eA unique DNA binding domain converts T-cell factors into strong Wnt effectors.\u003c/em\u003e Mol Cell Biol, 2007. \u003cb\u003e27\u003c/b\u003e(23): p. 8352-63.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Colorectal cancer, HNF4α, Wnt/β-catenin, TCF7L1","lastPublishedDoi":"10.21203/rs.3.rs-6208041/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6208041/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The Wnt/β-catenin signaling pathway is a central regulator of colorectal cancer (CRC) development, yet its downstream targets and mechanistic contributions to tumorigenesis remain poorly defined. Hepatocyte nuclear factor 4 alpha (HNF4α), a transcription factor primarily studied in liver function and hepatocarcinogenesis, has unclear roles in CRC. This study investigates the interplay between HNF4α and Wnt/β-catenin signaling in colorectal carcinogenesis and explores its clinical relevance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eUsing bulk RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), in vitro and in vivo CRC models, and clinical tumor samples, we assessed HNF4α expression and its regulation by Wnt/β-catenin signaling. Transcriptional activation of HNF4α was evaluated via luciferase reporter assays and chromatin immunoprecipitation. Clinical correlations between HNF4α levels and Wnt/β-catenin activity were analyzed using immunohistochemistry, RNA sequencing, and Spearman’s rank correlation. Statistical significance was determined by Student’s t-test and ANOVA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eHNF4α was significantly overexpressed in CRC tissues compared to normal controls and significantly promoted tumor growth in subcutaneous xenograft models using nude mice. Mechanistically, HNF4α was transcriptionally activated by the Wnt/β-catenin/TCF7L1 axis, forming a positive feedback loop that amplified oncogenic Wnt signaling. Clinically, HNF4α expression strongly correlated with Wnt/β-catenin pathway activation in patient samples (r = 0.58, p \u0026lt; 0.0001). Functionally, HNF4α knockdown suppressed CRC cell proliferation and inhibited Wnt-driven tumorigenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e This study identifies HNF4α as a novel downstream effector of the Wnt/β-catenin pathway and a critical driver of CRC progression. The Wnt/β-catenin/HNF4α feedback loop uncovered here provides mechanistic insights into colorectal carcinogenesis and highlights HNF4α as a potential therapeutic target. These findings may inform strategies to disrupt Wnt signaling hyperactivation in CRC.\u003c/p\u003e","manuscriptTitle":"Wnt/β-catenin/HNF4α feedback loop facilitates colorectal tumorigenesis and malignancy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 10:21:01","doi":"10.21203/rs.3.rs-6208041/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7bfc2e7a-05da-48b7-b810-fc5df1ad0e26","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-06T10:21:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-06 10:21:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6208041","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6208041","identity":"rs-6208041","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}