Disruption of β-Catenin Destruction Complex by Ephexin1-Axin1 Interaction Promotes Colorectal Cancer Proliferation

Disruption of β-Catenin Destruction Complex by Ephexin1-Axin1 Interaction Promotes Colorectal Cancer Proliferation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Disruption of β-Catenin Destruction Complex by Ephexin1-Axin1 Interaction Promotes Colorectal Cancer Proliferation Ho Jin You, Jeeho Kim, Young Jin Jeon, In-Youb Chang, Jung-Hee Lee This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4446931/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jan, 2025 Read the published version in Experimental & Molecular Medicine → Version 1 posted 9 You are reading this latest preprint version Abstract Wnt signaling is essential for cell growth and tumor formation, and is abnormally activated in colorectal cancer (CRC), contributing to tumor progression, but the specific role and regulatory mechanisms in tumor development are not yet clear. Here we show that Ephexin1, a guanine nucleotide exchange factor, is significantly overexpressed in CRC, correlating with increased Wnt/β-catenin pathway activity. Through comprehensive analysis, including RNA sequencing data from TCGA and functional assays, we demonstrated that Ephexin1 promotes tumor proliferation and migration by activating the Wnt/β-catenin pathway. This effect is mediated by the interaction of Ephexin1 with Axin1, a critical component of the β-catenin destruction complex, which in turn enhances stability and activity of β-catenin in signaling pathways critical for tumor development. Importantly, our findings also suggest that targeting Ephexin1 could enhance the efficacy of Wnt/β-catenin pathway inhibitors in CRC treatment. These findings highlight the potential of targeting Ephexin1 as a strategy for developing effective treatments for CRC, suggesting a novel and promising approach to therapy aimed at inhibiting cancer progression Health sciences/Diseases/Cancer/Gastrointestinal cancer/Colorectal cancer/Colon cancer Health sciences/Diseases/Cancer/Oncogenes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Wnt signaling plays a pivotal role in regulating various biological processes, including cell proliferation, differentiation, tissue regeneration, and tumorigenesis 1 , 2 , 3 . It serves primarily as a growth-stimulating factor that promotes cell proliferation 4 . This signaling pathway is notably upregulated in several cancers, with colorectal cancer (CRC) being a prime example where hyperactivation of Wnt signaling is a key contributor 5 , 6 , 7 . Despite the recognized importance of Wnt/β-catenin signaling in cancer progression, there are currently no clinically approved therapies targeting this pathway. However, the established significance of Wnt/β-catenin signaling in cancer has motivated the development of numerous therapies designed to block this pathway 8 , 9 , 10 . Wnt signaling can be categorized into two pathways: β-catenin-dependent (canonical) and β-catenin-independent (non-canonical) signaling 11 , 12 . The canonical pathway plays a critical role in regulating β-catenin levels. In the absence of Wnt ligand, β-catenin is kept at low levels through ubiquitin-dependent proteasome degradation, mediated by the β-catenin destruction complex comprising proteins such as Axin1, APC, CK1, and GSK3β 13, 14, 15, 16 . Axin1 is crucial for the complex, facilitating β-catenin phosphorylation by CK1 and GSK3β, leading to its ubiquitination by E3 ligases like SCF βTrCP and subsequent degradation 14 , 15 , 17 , 18 . Despite extensive research, the detailed molecular processes involved in tumor formation driven by Wnt/β-catenin signaling and how to effectively target this pathway are still not fully understood. Ephexin1, a member of the Dbl family of guanine nucleotide exchange factors (GEFs), plays a significant role in neurophysiological processes through its involvement in Ephrin signaling. Ephexin1 is mainly found in the developing nervous system and has minimal presence in other organs 19 , 20 . The overexpression of oncogenic K-Ras is linked to the up-regulation of Ephexin1 21, 22 , and its expression levels rise in association with the progression of lung cancer (LC), colorectal (CRC) cancers, and thyroid cancers 22 , 23 , 24 . Notably, the absence of Ephexin1 in LC and CRC leads to reduced apoptosis and migration 22 . In addition to its role in Ephrin signaling, Ephexin1 is likely to contribute to the Wnt/β-catenin signaling pathway. This relationship is reinforced by the involvement of other GEF family proteins, such as p114-RhoGEF and GEF-H1, in Wnt/β-catenin signaling 25 . Inhibition of this pathway, either through the removal of the Wnt co-receptors LRP5/LRP6 or by using sclerostin, results in reduced Ephexin1 levels 26 , 27 . These observations suggest a potential involvement of Ephexin1 in the Wnt/β-catenin signaling pathway, although its exact functional role remains to be fully elucidated. In the present study, we found that Ephexin1 is significantly overexpressed in CRC, promoting tumor growth by activating the Wnt/β-catenin pathway. Analysis of TCGA data shows a strong link between Ephexin1 expression and Wnt/β-catenin activation in CRC. The interaction of Ephexin1 with Axin1 affects β-catenin stability and Wnt signaling, indicating that Ephexin1 could be a valuable target for enhancing Wnt pathway inhibitor efficacy in CRC treatment. These findings highlight the critical role of Ephexin1 in Wnt signaling modulation and its therapeutic potential in CRC. MATERIALS AND METHODS Cell culture and transfection Normal cell lines, CCD18co and CCD841coN, were cultured in MEM medium (Invitrogen, Carlsbad, CA, USA). A549, H23, H358, H1299, H1666, HCC-827, H1650, LoVo, HCT15, and HCT116 cells were grown in RPMI-1640 medium (Invitrogen). SK-MES-1, Calu-3, Caco-2, and LS174T cells were cultured in MEM medium, while HEK293T, SW480, SW620, DLD-1, and HT-29 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen). All cell lines were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). The media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotic solution. Cells were incubated at 37°C in a 5% CO2 humidified atmosphere. Plasmids were transiently transfected into mammalian cells using TurboFect in vitro Transfection Reagent (Thermo Scientific, Waltham, MA, USA). IWR1-endo (Cat. No. S67086) was obtained from Selleckchem (Houston, TX, USA). Plasmid constructs and cloning Full-length and serial deletion constructs of human Ephexin1 have been previously described 22 . Human Axin1 was amplified from HEK293T cells by RT-PCR, using the following primers, and cloned into the pCI-neo-Flag or pCI-neo-V5 mammalian expression vectors (Promega, Madison, WI, USA). To prepare serial deletion constructs of Axin1 (ΔRGS, ΔRGS/p53, ΔRGS/p53/GSK3β, RNF11/DIX, ΔDIX, RNF11, p53, RGS, Δp53, ΔRNF11/DIX, ΔRNF11, GSK3β/β-catenin, GSK3β/β-catenin/DIX, Δp53/RNF11), the PCR products were cloned into the XhoI-NotI or XhoI-HindIII sites of the pCI-Flag vector. All constructs were verified by DNA sequencing. For the isolation of recombinant proteins, the GST-Ephexin1 construct (full-length or DH/PH domain) was previously described, and Hisx6-Axin1 (RGS or DIX domain) was cloned into the pET28a vector (Novagen). A comprehensive list of all PCR primers used in this study is provided in Supplementary Table S1 . RNAi and stable Ephexin1 knockdown cells Cells were transfected with siRNAs (40 nM) using Lipofectamine 2000 (Invitrogen). After 36 hours, the cells were trypsinized, replated, and subjected to a second round of transfection for another 36 hours. Knockdown efficiency was confirmed by western blot analysis. The sequences of Ephexin1 siRNA and shRNA have been previously described 22 , 24 . Immunoblot and immunoprecipitation analysis Cell extracts were prepared using IP150 lysis buffer (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.5% Nonidet P-40, 10% glycerol) containing protease inhibitors (1 mM Na2VO4, 10 mM NaF, 2 mM PMSF, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µg/ml pepstatin A) (Roche, Switzerland). Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes (PALL Life Sciences, USA). The membranes were then incubated with appropriate primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using the ECL chemiluminescent detection system (iNtRON Biotechnology, Korea). For the immunoprecipitation of protein complexes, cell extracts were pre-cleared with protein G-Sepharose beads (GE Healthcare) and then incubated with specific antibodies. The immune complexes were analyzed by immunoblotting using corresponding antibodies. A complete list of antibodies used can be found in Supplementary Table S2. Cell growth assay The cell growth assay was performed using the MTT assay. An equal number of HCT116 cells were seeded in triplicate in each well of 48-well plates at a density of 1x10 4 cells/0.2 ml/well. Twenty microliters of MTT solution (5.0 mg/ml) in RPMI-1640 medium was added to each well, and the plates were incubated for the indicated times at 37°C. The purple formazan crystals that formed were dissolved in 200 µl of MTT solvent (0.1% NP-40 and 4 mM HCl in isopropanol) by gentle mixing at room temperature. The optical densities of the wells were measured at 570 nm using a microplate spectrophotometer (Epoch, BioTek, Winooski, VT, USA). Soft agar colony formation assay Soft agar assays were conducted in 6-well plates, each containing a base layer of 2 ml of medium (at a final concentration of 1X) mixed with 0.6% low melting point agarose (Duchefa Biochemie, Netherlands). The plates were chilled at 4°C until the medium solidified. Subsequently, a growth layer consisting of 2 ml of 1X medium combined with 0.3% low-melting point agarose and 1 × 10^4 cells was added. Plates were again chilled at 4°C until the growth layer solidified. An additional 1 ml of 1X medium without agarose was gently layered on top of the growth layer. Cells were incubated at 37°C in a 5% CO_2 atmosphere for approximately 14–21 days. Colonies were then stained with 0.005% crystal violet (Sigma-Aldrich) and counted. Images were analyzed using an Olympus microscope (Olympus, Tokyo, Japan) and Image-Pro Plus 4.5 software (Media Cybernetics Inc., Rockville, MD, USA). The assays were performed in triplicate. Cell migration assay In vitro cell migration assays were conducted using a 24-well transwell plate with 8 µm polyethylene terephthalate membrane filters (BD Biosciences) to separate the lower and upper culture chambers. Cells were cultured until they reached sub-confluence (75%-80%) and then were serum-starved for 24 hours. After detachment with trypsin, the cells were washed with PBS, resuspended in serum-free medium, and a suspension of 2 × 10^4 cells was added to the upper chamber. Complete medium was added to the lower chamber. Cells that had not migrated were removed from the upper surface of the filters using cotton swabs. In contrast, cells that had migrated to the lower surface were fixed with 4% formaldehyde and stained with 0.2% crystal violet. Images of three random fields, magnified 10x, were captured from each membrane, and the number of migratory cells was counted. The mean of the triplicate assays for each experimental condition was calculated. Tumor formation in nude mice The mice utilized in this study were 6-week-old male BALB/c nude mice, acquired from NARA Biotech (Seoul, Korea). They were accommodated in our pathogen-free facility and managed according to standard use protocols and animal welfare regulations. HCT116 cells were harvested, resuspended in PBS, and then 1×10^6 HCT116 cells were injected subcutaneously into both the left and right flanks of the mice. Once the tumors became visible, their size was measured at 3-to-4-day intervals using micrometer calipers. Tumor volumes were calculated using the formula: volume = 0.5 × a × b^2, where 'a' and 'b' represent the larger and smaller tumor diameters, respectively. Approximately 3 weeks post-injection, the mice were humanely sacrificed, and the primary tumors were excised and immediately weighed. Immunostaining Immunohistochemistry was conducted on tissue microarrays of colorectal cancer samples. Tissue microarrays, representing cancer samples of various grades and adjacent normal tissues, were acquired from Super Bio Chips (CDA3) (Seoul, South Korea). For immunohistochemistry, heat-induced antigen retrieval was carried out using 1X antigen retrieval buffer (pH 9.0) (Abcam) at 95°C for 15 minutes. Following the quenching of endogenous peroxidase activity and blocking in a 3% H 2 O 2 solution, tissues were incubated with primary antibodies: anti-Ephexin1 (PA5-52521, Thermo Scientific), anti-Lgr5 (MA5-25644, Thermo Scientific), and anti-β-catenin (#610154, BD) overnight at 4°C. This was followed by incubation with an HRP-conjugated secondary antibody for 1 hour at room temperature and further incubation with DAB (3,3'-Diaminobenzidine) for 2 minutes. Subsequently, the slides were counterstained using Harris's hematoxylin. Staining intensity was scored from 0 to 4, and the extent of staining was scored from 0–100%. A final quantitation score for each stain was determined by multiplying the intensity and extent scores. The slides were independently analyzed by two pathologists. Proximity Ligation Assay (PLA) The Proximity Ligation Assay (PLA) was conducted on tissue microarrays of colorectal cancer of various grades and adjacent normal tissues, which were acquired from Super Bio Chips (CDA3). The assay began with heat-induced antigen retrieval using 1X antigen retrieval buffer (pH 9.0) (Abcam) at 95°C for 15 minutes, followed by blocking with Duolink™ blocking solution. Tissues were then incubated with primary anti-Ephexin1 (rabbit) and anti-Axin1 (mouse) antibodies overnight at 4°C. Subsequently, slides were incubated with anti-rabbit MINUS and anti-mouse PLUS PLA probes (Duolink™, Sigma-Aldrich) for 1 hour at 37°C. This was followed by a 30-minute incubation with ligation buffer and ligase (Duolink™, Sigma-Aldrich) at 37°C, and then amplification buffer and polymerase (Duolink™, Sigma-Aldrich) were added for a further 120 minutes at 37°C. The stained samples were analyzed using a fluorescence microscope (Nikon, Japan). Bioinformatics Analysis using The Cancer Genome Atlas (TCGA) Databases Data from The Cancer Genome Atlas (TCGA; https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga ) was downloaded using the UCSC Xena browser Data Hub ( https://xenabrowser.net/hub/ ). RNA sequencing data, measured by Illumina HiSeq and RSEM normalized, were downloaded when available. The mRNA expression data from the TCGA discovery set were transformed into a log2 scale, and correlation analyses were visualized using GraphPad Prism (GraphPad Software Inc., CA, USA). P-values between groups were calculated using Student’s t-test with GraphPad Prism. RNA sequencing analysis and GSEA Total RNA was harvested directly from cell culture plates using 1 ml of TRIzol reagent per 60 mm plate. The total RNA was isolated and treated with DNase I (Invitrogen). RNA sequencing was performed using an Illumina NovaSeq 6000™ sequencer at DNA_Link™ (Seoul, Korea). RNA-seq reads were initially mapped to the human genome GRCh37/hg19 build using Tophat version 2.0. 13 ( http://ccb.jhu.edu/software/tophat/ ). The aligned results were analyzed with Cuffdiff version 2.2. 1 ( http://cole-trapnell-lab.github.io/cufflinks/papers/ ) to calculate FPKM values and report differentially expressed genes. For library normalization and dispersion estimation, both geometric and pooled methods were utilized ( http://cole-trapnell-lab.github.io/cufflinks/cuffdiff/ ). Scatter plots and heatmaps were created using the 'heatmap' function in the 'ggplot' package in R version 3.4.1. The data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE220669. Gene Set Enrichment Analysis (GSEA) was performed using the GSEA pre-ranked module on the GSEA software (version 4.3.0), with log 2 fold change values for ranking genes. Identification of genes related to sensitivity to Wnt / β-catenin targeting agents Datasets of human cancer cell lines were obtained from The Cancer Dependency Map Project (DepMap, https://depmap.org/portal/ , version 23Q2). Data regarding responses to Wnt/β-catenin targeting agents, including ICG-001, IWR1-endo, Niclosamide, Salinomycin, WNT-C59, and XAV-939, were sourced from the drug sensitivity PRISM file (version 23Q2) 28 . RNA expression data utilized the CCLE 29 RNAseq gene expression data file (log 2 (TPM + 1). Genome-wide RNAi loss-of-function screening data were derived from two large-scale CRISPR and RNAi experiments (CERES 30 , Achilles 31 and DRIVE 32 ). Gene effects were calculated using DEMETER2 33 within DepMap. The p-values obtained from these analyses were then converted to -log 10 (p-value) to score each gene. Quantitative Real-time PCR (RT-qPCR) Total RNA was extracted from cell lysates using TriZol (Invitrogen), and 2 µg of total RNA was reverse transcribed to cDNA using an oligo dT primer and M-MuLV Reverse Transcriptase (Invitrogen). RT-qPCR analysis was performed using specific primers and the SYBR Premix Ex Taq™ kit (TaKaRa Bio, Shiga, Japan). The transcripts were detected by the CFX96 Real-Time PCR Detection System (BioRad, CA, USA). Primers used for RT-qPCR targeted Ephexin1, Wnt7a, Axin2, CXCL8, TERT, YWHAB, APC, DKK1, TCF7, Lgr5, Wnt9a, ID2, CSNK2B, PPP3CA, CyclinD1, CHD1, ROCK2, XPO1, YWHAZ, FRAT2, TBL1XR1, PRKACB, HDAC1, and β-actin. Each sample was analyzed in triplicates, and target genes were normalized relative to the reference housekeeping gene, β-actin. Relative mRNA expression levels were calculated using the comparative threshold cycle (Ct) method with β-actin as the control, according to the formula: ΔCt = Ct(β-actin) - Ct(target gene). The fold change in gene expression normalized to β-actin and relative to the control sample was calculated as as 2 − ΔΔC t . RT-qPCR primer sequences are listed in Supplementary Table S3. In vitro GST-pulldown assay Bacterially expressed GST-Ephexin1 (full-length or DH/PH domain) and GST alone were immobilized onto Glutathione Sepharose 4B beads (GE Healthcare) and incubated with bacterially expressed His\x6-Axin1 (RGS or DIX domain) fusion proteins overnight at 4°C. The GST bead-bound complexes were then washed five times with GST lysis buffer (20 mM HEPES, pH 7.6; 150 mM NaCl; 5 mM MgCl₂; 1% Triton X-100; and 5% glycerol), and bound proteins were separated by SDS-PAGE and analyzed by Western blotting using appropriate antibodies. Prediction of Ephexin1, Axin1 and APC structure For predict structures of Ephexin1(1-457aa), Axin1(1-211aa) and APC (1567-1595aa, 1716-1734aa, and 2032-2050aa), the corresponding sequences were processed using AlphaFold-Multimer ( https://github.com/deepmind/alphafold , Version 2.3.0) 34 encased in ColabFold 35 package which takes advantage of the MMseq2 server for automated MSA (Multiple Sequence Alignment) generation. The open-source PyMOL system ( https://pymol.org/2/ ) was used for visualization. Statistical analysis Data were presented as the mean ± SEM from three independent experiments. Significant differences between groups were assessed using a two-tailed paired Student's t-test or two-way ANOVA with GraphPad Prism (GraphPad Software Inc., CA, USA). Results with values of * p < 0.05, ** p < 0.01, and *** p < 0.001 were considered statistically significant Ethics statement All animal studies were reviewed and approved by the Institutional Animal Welfare and Use Committee of Chosun University School of Medicine. RESULTS Ephexin1 correlates with Wnt/β-catenin target gene expression in colorectal cancer To explore a potential link between Ephexin1 and Wnt/β-catenin signaling, we analyzed RNA sequencing data from the TCGA database, specifically looking at the expression of Ephexin1 and β-catenin target genes in CRC samples (n = 639). Our analysis revealed a statistically significant positive correlation between Ephexin1 expression and the expression of Wnt/β-catenin target genes (Fig. 1 a; Supplementary Fig. 1a, b). This correlation was specific to Wnt/β-catenin target genes, as it was not observed with non-Wnt/β-catenin target genes. This suggest that Ephexin1 selectively influences Wnt signaling in CRC. Furthermore, our research demonstrates that Ephexin1, along with β-catenin and Lgr5 (a key gene in the Wnt/β-catenin pathway), are significantly more expressed in CRC cell lines than in normal colon cells (Fig. 1 b). Immunohistochemical analysis confirmed a positive correlation between the expressions of Ephexin1, β-catenin, and Lgr5 in CRC tissues (Fig. 1 c, d). The uncontrolled proliferation of colorectal stem cells is a key mechanism in the development of CRC 36 , 37 . Given the role of Lgr5 as a key marker for colorectal stem cells 38 , 39 , we investigated the presence of Ephexin1 in Lgr5-positive stem cells specifically located in the crypt regions of the colon. Our findings confirmed that Ephexin1 is uniquely expressed in these colonic stem cells (Supplementary Fig. 2a, b). Notably, both Ephexin1 and Lgr5 were highly expressed in the same regions within CRC tissues (Supplementary Fig. 2c). Moreover, exposure to Wnt3a-conditioned medium (Wnt3a-CM), which activates the Wnt/β-catenin signaling pathway, resulted in increased Ephexin1 expression at both mRNA and protein levels (Supplementary Fig. 3). We further explored the relationship between Ephexin1 expression and sensitivity to Wnt/β-catenin inhibiting drugs. Analysis of data from the DepMap portal showed that lower Ephexin1 levels correlated with enhanced effectiveness of these inhibitors, suggesting a link between Ephexin1 expression and the efficacy of Wnt/β-catenin inhibitors (Fig. 1 e, f; Supplementary Fig. 4). Treatment with the Wnt inhibitor IWR1-endo significantly reduced growth (Fig. 1 g), anchorage-independent growth (Fig. 1 h), and migration ability (Fig. 1 i) in HCT116 cells deficient in Ephexin1, compared to control cells. Additionally, cells with diminished Ephexin1 exhibited a notable decrease in the cell proliferation marker, Ki67, following IWR1-endo treatment (Fig. 1 j). These findings suggest that Ephexin1 plays a vital role in controlling Wnt/β-catenin signaling in CRC, underscoring its potential as a target for therapy. Ephexin1 influences on Wnt / β-catenin signaling pathway To investigate the role of Ephexin1 in the Wnt/β-catenin signaling pathway, we conducted RNA-Seq analysis on HCT116 cells with and without Ephexin1 knockdown. Our bioinformatics analysis identified 326 genes that were differentially expressed (Fig. 2 a). We then focused on statistically significant gene changes, using ontology and interaction network analysis of these differentially expressed genes (DEGs), along with functional enrichment analysis. These analyses demonstrated that the absence of Ephexin1 affects genes associated with Wnt signaling (Fig. 2 b, c; Supplementary Fig. 5a). Specifically, genes normally activated by Wnt signaling were found to be suppressed in Ephexin1 knockdown HCT116 cells, whereas genes typically inhibited by Wnt signaling, along with genes activated by apoptosis, were increased (Fig. 2 d). Further, Gene Set Enrichment Analysis (GSEA) and heatmap data revealed a direct correlation between Ephexin1 levels and the activation of Wnt/β-catenin target genes (Fig. 2 e, f). Through RT-qPCR analysis, we validated significant differences in the expression of Wnt/β-catenin target genes in Ephexin1-deficient cells compared to control cells (Fig. 2 g, Supplementary Fig. 5b). These results indicate that Ephexin1 plays a critical role in facilitating the positive regulation of genes downstream of the Wnt/β-catenin signaling pathway. Ephexin1 enhances the transcriptional activity of β-catenin in the Wnt signaling pathway The primary role of canonical Wnt/β-catenin signaling pathway is to regulate gene expression by allowing β-catenin to function as a transcription factor 40 . Based on this, we hypothesized that Ephexin1 influences the transcriptional activity of β-catenin. We tested this by reducing Ephexin1 in HCT116 and HEK293T cells. Phosphorylation of β-catenin leads to its degradation, while non-phosphorylation at these sites promotes stabilization 15 , 41 . Our results showed that Ephexin1 depletion increased phosphorylated β-catenin levels and decreased non-phosphorylated β-catenin, total β-catenin, and the expression of its target genes, such as cyclinD1 and c-myc. Conversely, Ephexin1 overexpression increased β-catenin, cyclinD1, c-myc, and Lgr5 levels (Fig. 3 a; Supplementary Fig. 6a, b). Moreover, Ephexin1 knockdown significantly reduced the Wnt3a-CM-mediated increase in β-catenin levels (Fig. 3 b). To further explore the effect of Ephexin1 on the transcriptional activity of β-catenin, we conducted a TOP-Flash Luciferase reporter assay. In both HCT116 and HEK293T cells, simultaneous overexpression of Ephexin1 and β-catenin significantly enhanced transcriptional activity of β-catenin more than overexpression of either β-catenin or Ephexin1 alone (Fig. 3 c, Supplementary Fig. 6c). Additionally, depleting Ephexin1 significantly reduced the TOP-Flash Luciferase reporter activity induced by Wnt3a-CM (Fig. 3 d, Supplementary Fig. 6d), whereas overexpressing Flag-tagged Ephexin1 markedly increased it (Fig. 3 e, Supplementary Fig. 6e). Furthermore, the treatment with Wnt3a-CM significantly increased the mRNA levels of TCF7, cyclinD1, and Lgr5, which are key target genes of the Wnt/β-catenin signaling pathway, in control groups, but this effect was not observed in Ephexin1-deficient HCT116 cells. On the other hand, the expression of the β-catenin gene, which is not directly targeted by Wnt/β-catenin signaling, remained unchanged (Fig. 3 f). These findings collectively indicate that Ephexin1 plays a crucial role in enhancing the activity of β-catenin as a transcription factor. Ephexin1 interacts with the β-catenin destruction complex through binding to Axin1 Considering the role of Ephexin1 in regulating transcriptional activity of β-catenin, we hypothesized a potential interaction between Ephexin1 and the β-catenin destruction complex. To test this hypothesis, we employed both yeast two-hybrid assays and co-immunoprecipitation techniques. In the yeast two-hybrid assay, we discovered an interaction between Ephexin1 and Axin1, a key component of the β-catenin destruction complex (Supplementary Fig. 7a-c). This interaction was confirmed in HEK293T cells through co-immunoprecipitation, where Ephexin1 was found to bind not only with Axin1 but also with other elements of the destruction complex, such as β-catenin, TCF4, CK1, GSK3β, and APC (Fig. 4 a; Supplementary Fig. 7d, e). Importantly, we observed that the interaction between Ephexin1 and Axin1 became stronger with increased duration of Wnt-3a CM treatment (Fig. 4 b). To identify which part of Ephexin1 interacts with Axin1, we examined five different Ephexin1 mutants, each missing a specific domain of Ephexin1. Our findings indicated that Axin1 attaches to the DH/PH domain of Ephexin1, spanning amino acids 273 to 601. In contrast, the RR and SH3 domains of Ephexin1 showed no affinity for binding to Axin1 (Fig. 4 c, d). To further determine the specific domain of Axin1 involved in binding to Ephexin1, we generated and analyzed six Flag-tagged Axin1 mutants. This analysis revealed that the RGS domain (1-221aa) of Axin1 was crucial for binding to V5-tagged Ephexin1 (Fig. 4 e, f). For direct binding confirmation between Ephexin1 and Axin1, we performed GST pulldown assays using recombinant proteins of GST-Ephexin1 (either full-length or containing the DH/PH domain) and His x6 -Axin1 (RGS or DIX domain). The GST pulldown assays confirmed a direct interaction between the DH/PH domain of Ephexin1 and the RGS domain of Axin1, without interaction with the DIX domain of Axin1 (Fig. 4 g, Supplementary Fig. 8). To further verify the interaction between Ephexin1 and Axin1, we used AlphaFold-multimer to predict how these proteins bind together 34 , 42 , 43 , supporting our immunoprecipitation results. A precise analysis of the binding domain of Ephexin1 to Axin1 was necessary for AlphaFold-multimer accurate predictions, leading to the generation of seven Ephexin1 mutants with specific deletions in the DH/PH domain. We observed that DH domain, but not PH domain, of Ephexin1 is required for its interaction with Axin1 (Supplementary Fig. 9a, b). To accurately predict the stable structure of Ephexin1, we utilized the RR/DH domain (1-457 amino acids) of Ephexin1 and the RGS domain (1-211 amino acids) of Axin1 in AlphaFold-multimer modeling. This approach yielded predictions of a high-quality interaction between RR/DH domain of Ephexin1 and RGS domain of Axin1, as evidenced by sequence coverage, the predicted Local Distance Difference Test (pLDDT) confidence value, and predicted alignment error (PAE) (Supplementary Fig. 10a-c). Further structural analysis indicated that the DH domain of Ephexin1 interacts with RGS domain of Axin1 through the formation of four hydrogen bonds, underscoring the specificity and strength of this interaction (Fig. 4 h, i). Ephexin1 regulates β-catenin degradation through modulation of the Axin1 interaction network Considering the function of Axin1 in the Wnt signaling pathway as a scaffold protein that facilitates the proteasomal degradation of β-catenin 41 , we hypothesized that the interaction of Ephexin1 with Axin1 could influence degradation pathway of β-catenin via ubiquitination mechanisms. To investigate this hypothesis, we conducted immunoprecipitation experiments on Axin1 in HEK293T cells under conditions of both Ephexin1 deficiency and overexpression. Our findings indicate that the absence of Ephexin1 amplifies the interactions between Axin1 and key proteins in the Wnt signaling pathway, such as β-catenin, CK1, and GSK3β (Fig. 5 a), whereas overexpressing Ephexin1 diminishes these interactions (Fig. 5 b). The phosphorylation of β-catenin by CK1 and GSK3β is essential for its ubiquitination and subsequent degradation via the SCF βTrCP complex 14 , 15 , 44 , 45 . Interestingly, while overexpression of Axin1 increases the interaction of β-catenin with CK1 and GSK3β, this effect is mitigated by Ephexin1 overexpression (Fig. 5 c, Supplementary Fig. 11). The adenomatous polyposis coli (APC), a crucial scaffolding component, collaborates with Axin1 to promote the ubiquitination of β-catenin by the E3 ligase SCF βTrCP 46 . Notably, the RGS domain of Axin1 attaches to three distinct regions on APC, at amino acids 1567–1595, 1716–1736, and 2032-2050 47 . Given our previous findings that Ephexin1 binds to RGS domain of Axin1 (Fig. 4 ), this suggest a competitive relationship between Ephexin1 and APC for RGS domain of Axin1. To explore this hypothesis further, we employed AlphaFold-multimer for predictive modeling. Our findings, aligning with existing research, confirmed the interaction of Axin1 with APC through AlphaFold-multimer predictions (Fig. 5 d, Supplementary Fig. 12a-c). Importantly, our predictive models indicate that the binding of Ephexin1 to Axin1 could interfere with the interaction between Axin1 and APC (Fig. 5 e, f; Supplementary Fig. 12d-f). To validate these findings, we performed experiments using immunoprecipitation assays involving all three proteins. These experiments demonstrated that increasing Ephexin1 levels significantly decreased the interaction between Axin1 and APC, as well as between Axin1 and SCF βTrCP . Conversely, reducing Ephexin1 levels led to an increase in these interactions (Fig. 5 g, h). Moreover, in HCT116 cells lacking Ephexin1, treatment with Wnt3a-CM caused a smaller reduction in the Axin1-APC interaction compared to the control cells, indicating that the absence of Ephexin1 allows for a more stable Axin1-APC connection (Fig. 5 i). Also, analysis of β-catenin from Ephexin1-deficient HCT116 cells showed more interactions with components of the destruction complex and higher levels of β-catenin poly-ubiquitination (Fig. 5 j). These results indicate that Ephexin1 is essential for β-catenin stability, as it binds to Axin1, thereby inhibiting the activity of the β-catenin destruction complex and reducing β-catenin ubiquitination, which is crucial for its degradation. Ephexin1 depletion enhances apoptotic and tumor suppressive effects of Wnt Inhibition in CRC To explore the therapeutic potential of Ephexin1 in CRC, we evaluated the impact of Ephexin1 depletion on the effectiveness of a Wnt inhibitor. Initially, we assessed apoptotic responses in various CRC cell lines treated with IWR-endo and discovered that most cells underwent apoptosis following IWR1-endo treatment. However, HCT116 cells did not undergo apoptosis with IWR1-endo treatment (Supplementary Fig. 13a), indicating resistance to this treatment. Further investigation using IWR1-endo on both control and Ephexin1-depleted HCT116 cells, followed by apoptosis measurement through AnnexinV staining, revealed that Ephexin1 depletion significantly increased apoptosis in HCT116 cells treated with IWR1-endo (Fig. 6 a). To determine if these effects were related to the Wnt signaling pathway, we analyzed β-catenin stability and target gene expression in both control and Ephexin1-depleted HCT116 cells. After IWR1-endo treatment, Ephexin1-depleted HCT116 cells showed a more pronounced decrease in β-catenin and its target gene cyclinD1 compared to control cells (Supplementary Fig. 13b, c). Supporting this observation, treatment of Ephexin1-depleted HCT116 cells with IWR1-endo further increased β-catenin ubiquitination (Fig. 6 b). Next, to assess the ability of Ephexin1 to counteract Wnt inhibitor resistance in xenograft models, we administered IWR1-Endo to mice with tumors derived from either control HCT116 cells or Ephexin1-deficient HCT116 cells. The treatment was applied once every 3 to 4 days for a total of 33 days. While a slight reduction in tumor volume was observed in the control group, tumors derived from Ephexin1-deficient HCT116 cells exhibited a significant decrease in both volume and weight following IWR1-Endo treatment (Fig. 6 c-e). Immunohistochemical analysis of the tumor tissues further confirmed that the depletion of Ephexin1, combined with IWR1-endo treatment, results in decreased levels of Ki-67 and β-catenin (Fig. 6 f), as well as lower levels of β-catenin target genes (Fig. 6 g), compared to controls. These findings indicate that Ephexin1 absence could enhance the tumor-suppressive effects of Wnt pathway inhibitors in CRC, presenting a promising strategy for tumor suppression. The interaction of Ephexin1 and Axin1 has clinical relevance in CRC To Investigate the Role of Ephexin1 overexpression in CRC development, we generated transgenic (TG) mice that overexpress the mouse Ephexin1(mEphexin1) gene (Supplementary Fig. 14). In mouse embryonic fibroblast (MEF) cells derived from these genetically modified mice, treatment with Wnt3a-CM significantly enhanced LRP6 phosphorylation and β-catenin accumulation compared to the control group (Fig. 7 a). Given the established role of AOM (Azoxyemethane) and DSS (Dextran Sodium Sulfate) in inducing CRC through Wnt/β-catenin signaling activation 48 , 49 , 50 , we initiated colon cancer in both wild-type and mEphexin1 TG mice using AOM/DSS treatment over two weeks. The mEphexin1 TG mice exhibited an increased size and number of colon tumors compared to controls. Furthermore, both Ki67 and β-catenin levels were elevated in the colon tissues of the mEphexin1 TG mice, with a notable increase in nuclear β-catenin localization (Fig. 7 b, c). A long-term survival analysis revealed that mEphexin1 overexpression significantly decreased the survival rate of the mice (Fig. 7 d). To assess the clinical relevance of Ephexin1 alongside β-catenin and Axin1 in human CRC, we examined tissue microarrays of colorectal tissues encompassing normal tissues, carcinomas of varying grades, and metastatic tumors. The expression levels of Ephexin1, β-catenin, and Axin1 were markedly higher in CRC tissues compared to normal tissues. Notably, these levels increased progressively with the tumor grade and the presence of metastatic tumors (Fig. 7 e, f). Proximity Ligation Assay (PLA) was employed to examine the interactions between Ephexin1 and Axin1 and the quantitative changes in β-catenin in CRC patient samples. PLA foci indicating Ephexin1 and Axin1 interactions were markedly higher in cancer tissues than in normal tissues and significantly increased with tumor grade and metastasis (Fig. 7 g, h). Notably, the PLA foci scores for Ephexin1 and Axin1 correlated positively with β-catenin immunohistochemistry scores, and patients with high levels of Ephexin1-Axin1 binding exhibited poorer prognoses (Fig. 7 i, j). These findings emphasize the importance of the interaction between Ephexin1 and Axin1, suggesting that inhibiting this interaction could be a promising strategy for the development of anticancer drugs targeting Wnt/β-catenin active CRC. DISCUSSION The Wnt/β-catenin signaling pathway is crucial in cancer development and progression, making it a key subject of research in CRC. Our study reveals that Ephexin1, significantly overexpressed in CRC, activates the Wnt/β-catenin signaling pathway. Furthermore, we highlight the critical role of Ephexin1 in promoting tumor growth through the activation of the Wnt/β-catenin pathway. This discovery enhances our understanding of the mechanisms underlying wnt signaling dysregulation, which plays a crucial role in the progression of CRC, and introduces new possibilities for therapeutic interventions. Ephexin1, traditionally known for its role in neurophysiological processes and minimal expression outside the nervous system 19 , 22 , 24 , has emerged as a pivotal player in CRC through its overexpression and interaction with the Wnt/β-catenin signaling pathway. Our findings, corroborated by TCGA data analysis, establish a significant correlation between Ephexin1 expression and the activation of Wnt/β-catenin target genes in CRC. This relationship underscores the contribution of Ephexin1 to the hyperactive Wnt signaling observed in CRC, driving the uncontrolled proliferation of colorectal stem cells and tumor growth. The unique impact of Ephexin1 on Wnt/β-catenin target genes, not observed in the non-target genes of Wnt/β-catenin signaling pathway, underscores its specific role in regulating this signaling pathway in CRC. This effect is further substantiated by our immunohistochemical analyses and RNA sequencing data, which show a higher expression of Ephexin1, β-catenin, and Lgr5 in CRC cell lines and tissues compared to normal colon cells. These observations suggest that Ephexin1 not only facilitates Wnt/β-catenin signaling but also enhances the transcriptional activity of β-catenin, thus promoting oncogenic gene expression. The canonical Wnt/β-catenin pathway, a key regulator of cellular proliferation and differentiation, is frequently dysregulated in various cancers, including CRC 5 , 6 , 7 . Our study explores the underlying mechanisms of Ephexin1 by demonstrating its interaction with Axin1, a key component of the β-catenin destruction complex. This interaction plays a vital role in controlling the stability of β-catenin and, consequently, the activity of the Wnt/β-catenin signaling pathway. The binding of Ephexin1 to Axin1, particularly when Wnt signaling is enhanced, indicates a novel regulatory mechanism. This mechanism suggests Ephexin1 could prevent the degradation of β-catenin, thus maintaining its oncogenic potential. The specificity of this interaction, delineated through various experimental approaches including yeast two-hybrid assays, co-immunoprecipitation, GST pulldown assays, and structural predictions based on AlphaFold2 models, highlights the potential of targeting the Ephexin1-Axin1 interaction as a therapeutic strategy. Given the oncogenic nature of Ephexin1 and the tumor-suppressive function of Axin1, the overexpression of Ephexin1 in CRC suggest that it could counteract the effect of Axin1, thereby promoting cancer progression. Discovering that CRC cells lacking Ephexin1 exhibit increased sensitivity to inhibitors of the Wnt/β-catenin pathway highlights the potential benefits of targeting Ephexin1 for therapeutic purposes. By exploiting this sensitivity, we can potentially overcome the resistance faced by treatments targeting the Wnt pathway, offering a new approach to CRC treatment. Moreover, developing treatments that interfere with the interaction between Ephexin1 and Axin1 could provide an advanced way to control the stability of β-catenin and the activity of Wnt signaling in CRC. Our findings also emphasize the clinical significance of Ephexin1 and its interaction with Axin1 in CRC progression. The association between Ephexin1-Axin1 binding and worse patient outcomes, as evidenced by tissue microarray analysis and proximity ligation assays, highlights the critical need for further research into targeting this interaction as a therapeutic strategy. Disrupting this interaction could restore the β-catenin degradation pathway, consequently reducing Wnt signaling and its cancer-promoting role in CRC. Therefore, Ephexin1 emerges as a potential biomarker for tracking cancer progression and an attractive target for developing new therapies specific to CRC. While clinical trials are evaluating Wnt-targeted inhibitors like Vantictumab, Ipafricept, and Cetuximab 51 , 52 , 53 , none have received FDA approval yet. The difficulty in creating effective therapies that target the Wnt/β-catenin pathway could be due to its essential role in maintaining tissue homeostasis 54 . The reported side effects of these therapies, such as bone toxicity, hair loss, skin rashes, and gastrointestinal issues 55 , 56 , 57 , 58 , emphasize the necessity for targeting Wnt/β-catenin signaling in a more specific and safer manner. Focusing on anticancer drugs that target Ephexin1, which is mainly found in brain tissue but significantly increased in in LC and CRC 19 , 20 , 22 , 24 , could offer specificity and effeteness against cancer cells with minimal impact on normal cells. Given the complexity of the Wnt pathway, with its numerous components and regulatory mechanisms, future research should focus on ensuring that targeting Ephexin1 does not disrupt normal cell functions or produce adverse effects. Intestinal cells renew every five days through the Wnt/β-catenin signaling pathway, crucial for tissue equilibrium and stem cell regeneration in the intestinal crypts 2 , 3 , 59 . These stem cells facilitate surface growth, but their excessive proliferation may lead to CRC 60 , 61 . Approximately 80% of CRCs result from mutations in Wnt/β-catenin signaling genes 62 , 63 , 64 , and around 37% involving K-Ras oncogene mutations 65 , 66 . These mutations cause deregulation of the Wnt and Ras pathways, leading to uncontrolled stem cell growth and cancer 2 , 67 , 68 . Our findings indicate that Ephexin1, which is markedly increased in cancer stem cells in CRC patients, is influenced by K-Ras mutations 22 , 24 and impacts the Wnt/β-catenin pathway, potentially leading to unregulated stem cell proliferation. We are continuing research to further explore this effect. In summary, this study elucidates the pivotal role of Ephexin1 in CRC by demonstrating its overexpression and its direct influence on the Wnt/β-catenin signaling pathway. The interaction of Ephexin1 with Axin1 modulates stability of β-catenin, suggesting a novel mechanism for Wnt pathway dysregulation in CRC (Supplementary Fig. 15). Developing drugs that specifically inhibit Ephexin1 or its interaction with Axin1 represents a new strategy in CRC therapy, particularly for Wnt-driven CRC. This approach, potentially in combination with existing Wnt pathway inhibitors, could improve treatment outcomes. Declarations COMPETING INTERESTS The authors declare no competing interests. FUNDING This work is supported by National Research Foundation of Korea (NRF) grants (NRF-2022R1C1C2004575 and NRF-2022R1A5A2030454), funded by the Korea government. AUTHOR CONTRIBUTIONS JK, JHL, and HJY designed the study. JK, YJJ, and IYC performed the experiments, collected data, and performed statistical analysis. JH analyzed TCGA data set. IYC performed the immunohistochemistry staining and the pathological analysis. JK and YJJ carried out animal experiment. JK. YJJ, JY, JHL and HJY wrote the manuscript. ACKNOWLEDGEMENTS We thank all participants of this study for their invaluable devotion. We also thank Chosun university of Cancer Mutation Research Center for providing research equipment and technical support for this study. 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Additional Declarations (Not answered) Supplementary Files Sup.Ephexin1andwntsignalingEMM.docx Cite Share Download PDF Status: Published Journal Publication published 01 Jan, 2025 Read the published version in Experimental & Molecular Medicine → Version 1 posted Editorial decision: revise 20 Jun, 2024 Review # 2 received at journal 19 Jun, 2024 Review # 1 received at journal 12 Jun, 2024 Reviewer # 2 agreed at journal 12 Jun, 2024 Reviewer # 1 agreed at journal 27 May, 2024 Reviewers invited by journal 27 May, 2024 Submission checks completed at journal 20 May, 2024 First submitted to journal 20 May, 2024 Editor assigned by journal 20 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-4446931","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":307122816,"identity":"3ba89992-dbf3-416b-80c6-a0051d3f76a3","order_by":0,"name":"Ho Jin You","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACA4YDQLKCIQEuIkGcljOkaQECxjZStJgznjF8zDvPLo+f/4zZA4YaOwbJ2Qfwa7FsOGNszLstuVhyRo65AcOxZAZpvgT8WgwOnDGT5t12IHHDDR4zCQa2AwxyPAQcBtEyB6jl/Bmgln9Ea2kAajmQYybB2HaAQZqwlmPFhnOOJSfOnJFWJpHYl8wj2UNIy43DGx+8qbFL7Oc/vE3iwzc7OYkzBLQwSJwwQHASGBgIOQsI+NsfEFY0CkbBKBgFIxsAAP65QFsoUlpBAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-0530-4017","institution":"Chosun University","correspondingAuthor":true,"prefix":"","firstName":"Ho","middleName":"Jin","lastName":"You","suffix":""},{"id":307122817,"identity":"1a11cdc6-0cc0-4c91-a728-448ecdddeff2","order_by":1,"name":"Jeeho Kim","email":"","orcid":"https://orcid.org/0000-0001-6869-3606","institution":"Chosun University","correspondingAuthor":false,"prefix":"","firstName":"Jeeho","middleName":"","lastName":"Kim","suffix":""},{"id":307122818,"identity":"d58e15f3-4799-4bf1-8ea1-4faf7664bd9f","order_by":2,"name":"Young Jin Jeon","email":"","orcid":"","institution":"Chosun University","correspondingAuthor":false,"prefix":"","firstName":"Young","middleName":"Jin","lastName":"Jeon","suffix":""},{"id":307122819,"identity":"0e6d72a5-dcd9-4774-a9a0-31a519872bd4","order_by":3,"name":"In-Youb Chang","email":"","orcid":"","institution":"Chosun University","correspondingAuthor":false,"prefix":"","firstName":"In-Youb","middleName":"","lastName":"Chang","suffix":""},{"id":307122820,"identity":"426ede07-c524-4089-b919-8f2b577293f1","order_by":4,"name":"Jung-Hee Lee","email":"","orcid":"","institution":"Chosun University","correspondingAuthor":false,"prefix":"","firstName":"Jung-Hee","middleName":"","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2024-05-20 06:10:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4446931/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4446931/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s12276-024-01381-1","type":"published","date":"2025-01-01T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57935642,"identity":"4a2f3287-d560-4e9f-be58-8bb45bb0d0cb","added_by":"auto","created_at":"2024-06-07 17:07:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":795089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEphexin1 and Wnt / β-catenin signaling are related in colorectal cancer. a\u003c/strong\u003e Analysis of Ephexin1 and Wnt/β-catenin target gene expression, along with Ephexin1 and non-Wnt target gene expression, in the TCGA-COAD and COADREAD cohorts (n = 639). \u003cstrong\u003eb \u003c/strong\u003eWestern blot analysis of Ephexin1, b-catenin, and Lgr5 expression in normal and cancerous lung cell lines. \u003cstrong\u003ec\u003c/strong\u003e Immunohistochemical staining for Ephexin1, b-catenin, and Lgr5 in cancerous (n = 40) and corresponding normal tissue (n = 10). Scale bar = 100 mm. Immunohistochemistry (IHC) scores are presented as mean ± SEM, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.0001 (Student’s t-test). \u003cstrong\u003ed\u003c/strong\u003e A correlation of IHC levels between Ephexin1 and b-catenin, Ephexin1 and Lgr5, and b-catenin and Lgr5. Correlated analyses are presented as mean Spearman r, and \u003cem\u003ep\u003c/em\u003e values are for a two-tailed Student’s \u003cem\u003et\u003c/em\u003e test. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003ee\u003c/strong\u003e Volcano plot illustrating the relationship between the sensitivity to Wnt / β-catenin inhibitors and Ephexin1 expression levels, based on RNAi screening data from DepMap. \u003cstrong\u003ef \u003c/strong\u003eAnalysis of\u003cstrong\u003e \u003c/strong\u003eIWR1-endo drug (10 mM) sensitivity according to Ephexin1 mRNA expression levels in lung cancer cell lines (n = 60). \u003cstrong\u003eg\u003c/strong\u003e The effect of Ephexin1 knockdown on cell proliferation in control and IWR1-endo (80 mM) treated HCT116 cells. Data are presented as mean ± SEM. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, two-way ANOVA. \u003cstrong\u003eh, i\u003c/strong\u003e The effect of Ephexin1 knockdown on anchorage-independent growth (\u003cstrong\u003eh\u003c/strong\u003e) and migration ability (\u003cstrong\u003ei\u003c/strong\u003e) in HCT116 cells, both untreated (control) and treated with IWR1-endo (80 mM). Representative images are shown. Scale bar = 100 mm. Data are presented as mean ± SEM. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.0001, two-tailed Student’s t-test. \u003cstrong\u003ej\u003c/strong\u003eThe impact of reducing Ephexin1 on the staining levels of the Ki67 proliferation marker in control and IWR-1 endo-treated HCT116 cells. Representative images are shown. Scale bar = 100 mm. Data are presented as mean ±SEM. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.0001, two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure1Ephexin1andwntEMM1.png","url":"https://assets-eu.researchsquare.com/files/rs-4446931/v1/587f2d748579d7d3e9b644f3.png"},{"id":57935643,"identity":"57cc5c8d-4abb-4ff8-8af1-7ff19a8a4597","added_by":"auto","created_at":"2024-06-07 17:07:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":229745,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEphexin1 enhances the Wnt/b-catenin signaling pathway and regulates Wnt / b-catenin target genes. a\u003c/strong\u003eHeatmap showing the differentially expressed genes in control and Ephexin1-deficient HCT116 cells. Red and green indicate high and low mRNA expression levels, respectively. \u003cstrong\u003eb, c \u003c/strong\u003eRNA-seq datasets generated from control and Ephexin1-depleted HCT116 cells were analyzed to identify differentially expressed genes (DEGs), including both up- and down-regulated genes. Gene ontology analysis of differentially expressed genes in KEGG and GO terms (\u003cstrong\u003eb\u003c/strong\u003e). Analysis of the interaction network of differentially expressed genes (DEGs) using ClueGO (\u003ca href=\"https://apps.cytoscape.org/apps/cluego\"\u003ehttps://apps.cytoscape.org/apps/cluego\u003c/a\u003e) (\u003cstrong\u003ec\u003c/strong\u003e). \u003cstrong\u003ed\u003c/strong\u003e Volcano plot showing the differentially expressed genes (with FDR ≤ 0.05 and a fold change ≥ 2), including Wnt-target genes, non-Wnt target genes, and apoptosis-related genes, in HCT116 cells depleted of Ephexin1 compared to control cells. Ephexin1 knockdown results in the downregulation of 199 genes and the upregulation of 127 genes compared to the control. Blue and red circles indicate the positions of these target genes. \u003cstrong\u003ee \u003c/strong\u003eGene Set Enrichment Analysis (GSEA) identified differentially expressed genes targeted by the Wnt signaling pathway and the β-catenin nuclear pathway between control and Ephexin1-depleted HCT116 cells. \u003cstrong\u003ef\u003c/strong\u003e Heatmap showing Wnt/β-catenin target genes downregulated and upregulated by Ephexin1 knockdown in HCT116 cells. \u003cstrong\u003eg\u003c/strong\u003e RT-qPCR analysis comparing the expression of specified Wnt target genes in control versus Ephexin1-depleted HCT116 cells. Values represent relative expression normalized to b-actin mRNA ±SEM. *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001 compared with control. \u003cem\u003ep\u003c/em\u003e values are for a two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Figure1Ephexin1andwntEMM2.png","url":"https://assets-eu.researchsquare.com/files/rs-4446931/v1/aa138cfbe736c3006bafafbc.png"},{"id":57935279,"identity":"55b1f23d-b388-45ba-9031-3baba7d85683","added_by":"auto","created_at":"2024-06-07 16:59:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":100450,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEphexin1 increases the transcriptional activity of b-catenin. a\u003c/strong\u003e HCT116 and HEK293T cells were transiently transfected with either control siRNA or Ephexin1 siRNAs. Cell lysates were then subjected to Western blot analysis with the indicated antibodies. \u003cstrong\u003eb \u003c/strong\u003eControl and Ephexin1-depleted HCT116 cells were treated with Wnt3a-CM for the indicated periods of time. Cell lysates were analyzed by Western blot with the indicated antibodies. \u003cstrong\u003ec\u003c/strong\u003e TOP-Flash luciferase activity was analyzed in HCT116 cells after transfection with Flag-tagged β-catenin and/or V5-tagged Ephexin1. \u003cstrong\u003ed\u003c/strong\u003e The luciferase activities of TOP-Flash and FOP-Flash were measured in control and Ephexin1-depleted HCT116 cells after treatment with Wnt3a-CM. \u003cstrong\u003ee\u003c/strong\u003e The luciferase activities of TOP-Flash and FOP-Flash were measured in control and Flag-Ephexin1-overexpressing HCT116 cells following treatment with Wnt3a-CM. \u003cstrong\u003ef\u003c/strong\u003e Control and Ephexin1-depleted HCT116 cells were treated with Wnt3a-CM for the indicated periods of time, and the selected transcripts were analyzed using RT-qPCR. Values represent relative expression normalized to b-actin mRNA ± SEM. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared with control. \u003cem\u003ep\u003c/em\u003e values are for a two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Figure1Ephexin1andwntEMM3.png","url":"https://assets-eu.researchsquare.com/files/rs-4446931/v1/7d71dbbe780ee0b8bd0991fe.png"},{"id":57935282,"identity":"87349b49-8980-422c-94ae-964639454e2f","added_by":"auto","created_at":"2024-06-07 16:59:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":807364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEphexin1 interacts with the RGS of Axin1 via the DH domain. a \u003c/strong\u003eHEK293T cells were immunoprecipitated with an anti-Flag antibody after transient transfection with Flag-tagged Ephexin1 and immunoblotted with the indicated antibodies. \u003cstrong\u003eb\u003c/strong\u003eCo-immunoprecipitation was performed with an anti-Flag antibody on protein extracts from HEK293T cell that had been co-transfected with proteins Ephexin1 (Flag-tagged) and Axin1 (V5-tagged), following their treatment with Wnt3a-CM for the indicated time durations. Western blot analysis was then performed with the indicated antibodies. \u003cstrong\u003ec\u003c/strong\u003e Schematic representation of the full-length and a series of deletion mutants of Ephexin1. A summary of the degree to which each interacts with Axin1 is shown on the right. \u003cstrong\u003ed\u003c/strong\u003eProtein extracts from HEK293T cells, co-transfected with either V5-tagged full-length or mutant Ephexin1 along with Flag-tagged Axin1, were immunoprecipitated with anti-Flag antibody and subjected to western blot analysis with the indicated antibodies. \u003cstrong\u003ee\u003c/strong\u003eSchematic representation of full length and a series of deletion mutants of Axin1. A summary of the degree to which each interacts with Ephexin1 is shown to the right. \u003cstrong\u003ef\u003c/strong\u003e Lysates from HEK293T cells, co-transfected with V5-tagged Ephexin1 and either full-length or mutant Flag-tagged Axin1, were subjected to immunoprecipitation using anti-Flag antibody. Western blot analysis was then performed with V5 and Flag antibodies. \u003cstrong\u003eg\u003c/strong\u003e \u003cem\u003eIn vitro\u003c/em\u003e GST-pulldown assay was utilized to investigate the binding between the GST-DH/PH domain of Ephexin1 and either the recombinant Hisx6-tagged Axin1-RGS (1-221aa) or the Axin1-DIX (780-862aa) domains. GST alone was used as a negative control. \u003cstrong\u003eh\u003c/strong\u003e Using the AlphaFold-multimer, the most accurate predictions for the protein structures of Ephexin1_RR-DH (1-457 amino acids) in blue and Axin1_RGS (1-211 amino acids) in yellow were generated and subsequently visualized with the PyMol software. \u003cstrong\u003ei\u003c/strong\u003e Hydrogen bonding sites within the predicted protein structures of Ephexin1_RR-DH (1-457aa) and Axin1_RGS (1-211aa) were analyzed using ChimeraX software (\u003ca href=\"https://www.rbvi.ucsf.edu/chimerax/\"\u003ehttps://www.rbvi.ucsf.edu/chimerax/\u003c/a\u003e), and the visualization was performed using PyMOL software (\u003ca href=\"https://pymol.org/2/\"\u003ehttps://pymol.org/2/\u003c/a\u003e).\u003c/p\u003e","description":"","filename":"Figure1Ephexin1andwntEMM4.png","url":"https://assets-eu.researchsquare.com/files/rs-4446931/v1/aea16395761867d151de8879.png"},{"id":57935281,"identity":"835e8d21-1fa6-40b3-9026-1d1a30f4c150","added_by":"auto","created_at":"2024-06-07 16:59:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":557534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction of Ephexin1 with Axin1 inhibits β-catenin ubiquitination. a\u003c/strong\u003eLysates from control and Ephexin1-depleted HEK293T cells were immunoprecipitated with an anti-Axin1 antibody and subjected to western blot analysis with the indicated antibodies. \u003cstrong\u003eb \u003c/strong\u003eLysates from HEK293T cells, co-transfected with V5-tagged Ephexin1 and Flag-tagged Axin1, were immunoprecipitated with an anti-Axin1 antibody and subjected to western blot analysis with the indicated antibodies. \u003cstrong\u003ec\u003c/strong\u003e Lysates from HEK293T cells, either transfected with V5-Axin1 alone or co-transfected with Flag-Ephexin1 and V5-Axin1, were immunoprecipitated with an anti-β-catenin antibody and subjected to Western blot analysis using the indicated antibodies. \u003cstrong\u003ed\u003c/strong\u003e Using AlphaFold-multimer, the best model for predicting the protein binding structure of the RGS domain of Axin1 (1-211aa) in blue, along with the Axin1 binding sites 1, 2, and 3 (1567-1595aa in yellow, 1716-1734aa in red, 2032-2050aa in green), was generated and subsequently visualized with PyMol software. \u003cstrong\u003ee\u003c/strong\u003e Using AlphaFold-multimer, the most accurate predictions for the protein binding structures of the RGS domain of Axin1 (1-211aa) in blue, Axin1 binding sites 1, 2, and 3 on APC (1567-1595aa in yellow, 1716-1734aa in red, 2032-2050aa in green), and the RR-DH domain of Ephexin1 (1-457aa) in orange were generated and subsequently visualized with PyMol software. \u003cstrong\u003ef\u003c/strong\u003e Comparison of the Predicted Alignment Errors (PAE) for the best models of the Axin1/APC complex and the Ephexin1/Axin1/APC complex, as determined by AlphaFold-multimer. \u003cstrong\u003eg\u003c/strong\u003eLysates from HEK293T cells, either transfected with Flag-Axin1 alone or co-transfected with Flag-Axin1 and V5-Ephexin1, were immunoprecipitated with an anti-Flag antibody and subjected to Western blot analysis using the indicated antibodies. \u003cstrong\u003eh\u003c/strong\u003e Lysates from control and Ephexin1-depleted HCT116 cells were immunoprecipitated with an anti-Axin1 antibody and subjected to western blot analysis with the indicated antibodies. \u003cstrong\u003ei\u003c/strong\u003e Control and Ephexin1-deficient HCT116 cells were treated with Wnt3a-CM or left untreated. Cell lysates were then immunoprecipitated using an anti-Axin1 antibody, followed by immunoblotting with the indicated antibodies. \u003cstrong\u003ej\u003c/strong\u003eLysates from control and Ephexin1-depleted HCT116 cells were immunoprecipitated with an anti-β-catenin antibody and subjected to western blot analysis with the indicated antibodies.\u003c/p\u003e","description":"","filename":"Figure1Ephexin1andwntEMM5.png","url":"https://assets-eu.researchsquare.com/files/rs-4446931/v1/04fbaa65b2aff38c54a225dc.png"},{"id":57935284,"identity":"6018e5df-3b14-4f22-b50c-5ad15292cfd0","added_by":"auto","created_at":"2024-06-07 16:59:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1281653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEphexin1 deficiency enhances sensitivity to Wnt inhibitor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Control and Ephexin1-deficient HCT116 cells were either treated with IWR1-endo (80 mM) or left untreated for 12 hours, followed by staining with Annexin V (green) for immunohistochemical analysis. Data are presented as mean ± SEM. ns, not significant; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, two-tailed Student’s t-test. \u003cstrong\u003eb\u003c/strong\u003e Control and Ephexin1-deficient HCT116 cells were treated with Wnt3a-CM or left untreated. Cell lysates were then immunoprecipitated using an anti-b-catenin antibody, followed by immunoblotting with the indicated antibodies. \u003cstrong\u003ec \u003c/strong\u003eControl and Ephexin1-depleted HCT116 cells were subcutaneously inoculated into BALB/c nude mice (n = 4). IWR1-endo (5 mg/g) was administered every 3 or 4 days starting 17 days after transplantation. \u003cstrong\u003ed\u003c/strong\u003e The recorded tumor volumes for each group at the indicated times are shown. Data are presented as mean ± SEM. ns, not significant; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, two-way ANOVA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e The average tumor weight for each group at the endpoint of the experiment is presented. Data are presented as mean ± SEM. ns, not significant; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, two-tailed Student’s t-test. \u003cstrong\u003ef\u003c/strong\u003e H\u0026amp;E staining and immunohistochemistry analysis of Ki67 and β-catenin for each group at the endpoint of the experiment are presented. Scale bar = 100 mm. Quantification of the Ki67 and b-catenin staining for each group is shown. Data are presented as mean ± SEM. **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, two-tailed Student’s t-test. \u003cstrong\u003eg\u003c/strong\u003e The mRNA levels of Wnt / β-catenin target genes in HCT116 xenograft tumors obtained in (\u003cstrong\u003ec\u003c/strong\u003e) were analyzed by qRT-PCR analysis.\u003c/p\u003e","description":"","filename":"Figure1Ephexin1andwntEMM6.png","url":"https://assets-eu.researchsquare.com/files/rs-4446931/v1/545bc0c808721d378dd5e6df.png"},{"id":57935285,"identity":"e450233d-4f49-4811-9fc8-9752430eb4a5","added_by":"auto","created_at":"2024-06-07 16:59:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2482112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction of Ephexin1 and Axin1 is associated with poor prognosis of CRC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eWestern blot analysis was conducted to observe the time-dependent effects of Wnt3a-CM treatment on MEF cells from WT (wild type) and mEphexin1 TG (transgenic) mice. \u003cstrong\u003eb\u003c/strong\u003e H\u0026amp;E and Ki67 staining of colorectal tissue after induction of inflammatory CRC by AOM/DSS administration for 2 weeks in WT and mEphexin1 TG mice. Arrows indicate areas of CRC. Scale bar = 500 mm. \u003cstrong\u003e\u0026nbsp;c\u003c/strong\u003eThe average tumor size and number of tumors for each group were presented. Data are presented as mean ± SEM. ns, not significant; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, two-tailed Student’s t-test. \u003cstrong\u003ed\u003c/strong\u003e Kaplan-Meier survival curves are shown for WT and mEphexin1 TG mice (n = 11). \u003cem\u003ep\u003c/em\u003e-values are from a log-rank test. \u003cstrong\u003ee \u003c/strong\u003eImmunohistochemistry staining was used to evaluate Ephexin1, b-catenin, and Lgr5 expression in normal, grade I/II, grade III/IV, and metastatic CRC tissues, along with their corresponding normal tissues. Hematoxylin was used as the counterstain. Scale bar = 100 mm. \u003cstrong\u003ef\u003c/strong\u003eExpression scores for Ephexin1, b-catenin, and Lgr5 in a CRC tissue microarray containing 9 cases of normal mucosa tissues, 2 cases of grade I, 9 cases of grade II, 15 cases of grade III, 12 cases of grade IV, and 10 cases of metastatic tumor tissues. Data are presented as mean ±SEM. \u003cem\u003ep\u003c/em\u003e-values are from a Mann-Whitney test. ns, not significant; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eg\u003c/strong\u003e A proximity ligation assay (PLA) was performed to identify interactions between Ephexin1 and Axin1 (red) in grade I (n = 2), grade II/III (n = 24), and grade IV (n = 12), and metastatic (n = 10) colorectal cancer tissues and their corresponding normal tissues (n = 9). DAPI (blue) was used as the counterstain to visualize nuclei. Scale bar = 100 mm. \u003cstrong\u003eh \u003c/strong\u003eQuantification of PLA data shown in (\u003cstrong\u003eg\u003c/strong\u003e). Data are presented as mean ±SEM. \u003cem\u003ep\u003c/em\u003e-values are from a Mann-Whitney test. ns, not significant; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003ei\u003c/strong\u003e Correlation analysis between PLA (Ephexin1-Axin1) and b-catenin expression in CRC tissue. Correlated analyses are presented as mean Spearman r, and \u003cem\u003ep\u003c/em\u003e values are for a two-tailed Student’s \u003cem\u003et\u003c/em\u003etest. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003ej\u003c/strong\u003e Kaplan-Meier graph representing overall survival rates for patients with Ephexin1 and Axin1 interaction in CRC. High Ephexin1-Axin1 PLA, n = 23; low Ephexin1-Axin1 PLA, n = 18. \u003cem\u003ep\u003c/em\u003e-values are from a log-rank test.\u003c/p\u003e","description":"","filename":"Figure1Ephexin1andwntEMM7.png","url":"https://assets-eu.researchsquare.com/files/rs-4446931/v1/08b9bd81219507cc74ee5f23.png"},{"id":72734905,"identity":"2d84c657-e5d0-48eb-9c87-bedaf6beaf7b","added_by":"auto","created_at":"2025-01-01 08:06:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7196793,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4446931/v1/dd678563-8ff0-4323-bf2f-4cabf02ca585.pdf"},{"id":57935286,"identity":"aa373a11-5a8a-4c86-a841-e9469b30b744","added_by":"auto","created_at":"2024-06-07 16:59:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16371801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Sup.Ephexin1andwntsignalingEMM.docx","url":"https://assets-eu.researchsquare.com/files/rs-4446931/v1/f9f6e1fa0edf60a80d51400f.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Disruption of β-Catenin Destruction Complex by Ephexin1-Axin1 Interaction Promotes Colorectal Cancer Proliferation","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eWnt signaling plays a pivotal role in regulating various biological processes, including cell proliferation, differentiation, tissue regeneration, and tumorigenesis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. It serves primarily as a growth-stimulating factor that promotes cell proliferation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This signaling pathway is notably upregulated in several cancers, with colorectal cancer (CRC) being a prime example where hyperactivation of Wnt signaling is a key contributor\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Despite the recognized importance of Wnt/β-catenin signaling in cancer progression, there are currently no clinically approved therapies targeting this pathway. However, the established significance of Wnt/β-catenin signaling in cancer has motivated the development of numerous therapies designed to block this pathway\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWnt signaling can be categorized into two pathways: β-catenin-dependent (canonical) and β-catenin-independent (non-canonical) signaling\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The canonical pathway plays a critical role in regulating β-catenin levels. In the absence of Wnt ligand, β-catenin is kept at low levels through ubiquitin-dependent proteasome degradation, mediated by the β-catenin destruction complex comprising proteins such as Axin1, APC, CK1, and GSK3β\u003csup\u003e13, 14, 15, 16\u003c/sup\u003e. Axin1 is crucial for the complex, facilitating β-catenin phosphorylation by CK1 and GSK3β, leading to its ubiquitination by E3 ligases like SCF\u003csup\u003eβTrCP\u003c/sup\u003e and subsequent degradation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Despite extensive research, the detailed molecular processes involved in tumor formation driven by Wnt/β-catenin signaling and how to effectively target this pathway are still not fully understood.\u003c/p\u003e \u003cp\u003eEphexin1, a member of the Dbl family of guanine nucleotide exchange factors (GEFs), plays a significant role in neurophysiological processes through its involvement in Ephrin signaling. Ephexin1 is mainly found in the developing nervous system and has minimal presence in other organs\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The overexpression of oncogenic K-Ras is linked to the up-regulation of Ephexin1\u003csup\u003e21, 22\u003c/sup\u003e, and its expression levels rise in association with the progression of lung cancer (LC), colorectal (CRC) cancers, and thyroid cancers\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Notably, the absence of Ephexin1 in LC and CRC leads to reduced apoptosis and migration\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In addition to its role in Ephrin signaling, Ephexin1 is likely to contribute to the Wnt/β-catenin signaling pathway. This relationship is reinforced by the involvement of other GEF family proteins, such as p114-RhoGEF and GEF-H1, in Wnt/β-catenin signaling\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Inhibition of this pathway, either through the removal of the Wnt co-receptors LRP5/LRP6 or by using sclerostin, results in reduced Ephexin1 levels\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. These observations suggest a potential involvement of Ephexin1 in the Wnt/β-catenin signaling pathway, although its exact functional role remains to be fully elucidated.\u003c/p\u003e \u003cp\u003eIn the present study, we found that Ephexin1 is significantly overexpressed in CRC, promoting tumor growth by activating the Wnt/β-catenin pathway. Analysis of TCGA data shows a strong link between Ephexin1 expression and Wnt/β-catenin activation in CRC. The interaction of Ephexin1 with Axin1 affects β-catenin stability and Wnt signaling, indicating that Ephexin1 could be a valuable target for enhancing Wnt pathway inhibitor efficacy in CRC treatment. These findings highlight the critical role of Ephexin1 in Wnt signaling modulation and its therapeutic potential in CRC.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and transfection\u003c/h2\u003e \u003cp\u003eNormal cell lines, CCD18co and CCD841coN, were cultured in MEM medium (Invitrogen, Carlsbad, CA, USA). A549, H23, H358, H1299, H1666, HCC-827, H1650, LoVo, HCT15, and HCT116 cells were grown in RPMI-1640 medium (Invitrogen). SK-MES-1, Calu-3, Caco-2, and LS174T cells were cultured in MEM medium, while HEK293T, SW480, SW620, DLD-1, and HT-29 cells were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) (Invitrogen). All cell lines were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). The media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotic solution. Cells were incubated at 37\u0026deg;C in a 5% CO2 humidified atmosphere. Plasmids were transiently transfected into mammalian cells using TurboFect in vitro Transfection Reagent (Thermo Scientific, Waltham, MA, USA). IWR1-endo (Cat. No. S67086) was obtained from Selleckchem (Houston, TX, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid constructs and cloning\u003c/h2\u003e \u003cp\u003eFull-length and serial deletion constructs of human Ephexin1 have been previously described\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Human Axin1 was amplified from HEK293T cells by RT-PCR, using the following primers, and cloned into the pCI-neo-Flag or pCI-neo-V5 mammalian expression vectors (Promega, Madison, WI, USA). To prepare serial deletion constructs of Axin1 (ΔRGS, ΔRGS/p53, ΔRGS/p53/GSK3β, RNF11/DIX, ΔDIX, RNF11, p53, RGS, Δp53, ΔRNF11/DIX, ΔRNF11, GSK3β/β-catenin, GSK3β/β-catenin/DIX, Δp53/RNF11), the PCR products were cloned into the XhoI-NotI or XhoI-HindIII sites of the pCI-Flag vector. All constructs were verified by DNA sequencing. For the isolation of recombinant proteins, the GST-Ephexin1 construct (full-length or DH/PH domain) was previously described, and Hisx6-Axin1 (RGS or DIX domain) was cloned into the pET28a vector (Novagen). A comprehensive list of all PCR primers used in this study is provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNAi and stable Ephexin1 knockdown cells\u003c/h2\u003e \u003cp\u003eCells were transfected with siRNAs (40 nM) using Lipofectamine 2000 (Invitrogen). After 36 hours, the cells were trypsinized, replated, and subjected to a second round of transfection for another 36 hours. Knockdown efficiency was confirmed by western blot analysis. The sequences of Ephexin1 siRNA and shRNA have been previously described\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblot and immunoprecipitation analysis\u003c/h2\u003e \u003cp\u003eCell extracts were prepared using IP150 lysis buffer (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.5% Nonidet P-40, 10% glycerol) containing protease inhibitors (1 mM Na2VO4, 10 mM NaF, 2 mM PMSF, 5 \u0026micro;g/ml leupeptin, 10 \u0026micro;g/ml aprotinin, 1 \u0026micro;g/ml pepstatin A) (Roche, Switzerland). Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes (PALL Life Sciences, USA). The membranes were then incubated with appropriate primary antibodies overnight at 4\u0026deg;C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using the ECL chemiluminescent detection system (iNtRON Biotechnology, Korea). For the immunoprecipitation of protein complexes, cell extracts were pre-cleared with protein G-Sepharose beads (GE Healthcare) and then incubated with specific antibodies. The immune complexes were analyzed by immunoblotting using corresponding antibodies. A complete list of antibodies used can be found in Supplementary Table S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell growth assay\u003c/h2\u003e \u003cp\u003eThe cell growth assay was performed using the MTT assay. An equal number of HCT116 cells were seeded in triplicate in each well of 48-well plates at a density of 1x10\u003csup\u003e4\u003c/sup\u003e cells/0.2 ml/well. Twenty microliters of MTT solution (5.0 mg/ml) in RPMI-1640 medium was added to each well, and the plates were incubated for the indicated times at 37\u0026deg;C. The purple formazan crystals that formed were dissolved in 200 \u0026micro;l of MTT solvent (0.1% NP-40 and 4 mM HCl in isopropanol) by gentle mixing at room temperature. The optical densities of the wells were measured at 570 nm using a microplate spectrophotometer (Epoch, BioTek, Winooski, VT, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSoft agar colony formation assay\u003c/h2\u003e \u003cp\u003eSoft agar assays were conducted in 6-well plates, each containing a base layer of 2 ml of medium (at a final concentration of 1X) mixed with 0.6% low melting point agarose (Duchefa Biochemie, Netherlands). The plates were chilled at 4\u0026deg;C until the medium solidified. Subsequently, a growth layer consisting of 2 ml of 1X medium combined with 0.3% low-melting point agarose and 1 \u0026times; 10^4 cells was added. Plates were again chilled at 4\u0026deg;C until the growth layer solidified. An additional 1 ml of 1X medium without agarose was gently layered on top of the growth layer. Cells were incubated at 37\u0026deg;C in a 5% CO_2 atmosphere for approximately 14\u0026ndash;21 days. Colonies were then stained with 0.005% crystal violet (Sigma-Aldrich) and counted. Images were analyzed using an Olympus microscope (Olympus, Tokyo, Japan) and Image-Pro Plus 4.5 software (Media Cybernetics Inc., Rockville, MD, USA). The assays were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell migration assay\u003c/h2\u003e \u003cp\u003eIn vitro cell migration assays were conducted using a 24-well transwell plate with 8 \u0026micro;m polyethylene terephthalate membrane filters (BD Biosciences) to separate the lower and upper culture chambers. Cells were cultured until they reached sub-confluence (75%-80%) and then were serum-starved for 24 hours. After detachment with trypsin, the cells were washed with PBS, resuspended in serum-free medium, and a suspension of 2 \u0026times; 10^4 cells was added to the upper chamber. Complete medium was added to the lower chamber. Cells that had not migrated were removed from the upper surface of the filters using cotton swabs. In contrast, cells that had migrated to the lower surface were fixed with 4% formaldehyde and stained with 0.2% crystal violet. Images of three random fields, magnified 10x, were captured from each membrane, and the number of migratory cells was counted. The mean of the triplicate assays for each experimental condition was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTumor formation in nude mice\u003c/h2\u003e \u003cp\u003eThe mice utilized in this study were 6-week-old male BALB/c nude mice, acquired from NARA Biotech (Seoul, Korea). They were accommodated in our pathogen-free facility and managed according to standard use protocols and animal welfare regulations. HCT116 cells were harvested, resuspended in PBS, and then 1\u0026times;10^6 HCT116 cells were injected subcutaneously into both the left and right flanks of the mice. Once the tumors became visible, their size was measured at 3-to-4-day intervals using micrometer calipers. Tumor volumes were calculated using the formula: volume\u0026thinsp;=\u0026thinsp;0.5 \u0026times; a \u0026times; b^2, where 'a' and 'b' represent the larger and smaller tumor diameters, respectively. Approximately 3 weeks post-injection, the mice were humanely sacrificed, and the primary tumors were excised and immediately weighed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunostaining\u003c/h2\u003e \u003cp\u003eImmunohistochemistry was conducted on tissue microarrays of colorectal cancer samples. Tissue microarrays, representing cancer samples of various grades and adjacent normal tissues, were acquired from Super Bio Chips (CDA3) (Seoul, South Korea). For immunohistochemistry, heat-induced antigen retrieval was carried out using 1X antigen retrieval buffer (pH 9.0) (Abcam) at 95\u0026deg;C for 15 minutes. Following the quenching of endogenous peroxidase activity and blocking in a 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution, tissues were incubated with primary antibodies: anti-Ephexin1 (PA5-52521, Thermo Scientific), anti-Lgr5 (MA5-25644, Thermo Scientific), and anti-β-catenin (#610154, BD) overnight at 4\u0026deg;C. This was followed by incubation with an HRP-conjugated secondary antibody for 1 hour at room temperature and further incubation with DAB (3,3'-Diaminobenzidine) for 2 minutes. Subsequently, the slides were counterstained using Harris's hematoxylin. Staining intensity was scored from 0 to 4, and the extent of staining was scored from 0\u0026ndash;100%. A final quantitation score for each stain was determined by multiplying the intensity and extent scores. The slides were independently analyzed by two pathologists.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProximity Ligation Assay (PLA)\u003c/h2\u003e \u003cp\u003eThe Proximity Ligation Assay (PLA) was conducted on tissue microarrays of colorectal cancer of various grades and adjacent normal tissues, which were acquired from Super Bio Chips (CDA3). The assay began with heat-induced antigen retrieval using 1X antigen retrieval buffer (pH 9.0) (Abcam) at 95\u0026deg;C for 15 minutes, followed by blocking with Duolink\u0026trade; blocking solution. Tissues were then incubated with primary anti-Ephexin1 (rabbit) and anti-Axin1 (mouse) antibodies overnight at 4\u0026deg;C. Subsequently, slides were incubated with anti-rabbit MINUS and anti-mouse PLUS PLA probes (Duolink\u0026trade;, Sigma-Aldrich) for 1 hour at 37\u0026deg;C. This was followed by a 30-minute incubation with ligation buffer and ligase (Duolink\u0026trade;, Sigma-Aldrich) at 37\u0026deg;C, and then amplification buffer and polymerase (Duolink\u0026trade;, Sigma-Aldrich) were added for a further 120 minutes at 37\u0026deg;C. The stained samples were analyzed using a fluorescence microscope (Nikon, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics Analysis using The Cancer Genome Atlas (TCGA) Databases\u003c/h2\u003e \u003cp\u003eData from The Cancer Genome Atlas (TCGA; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga\u003c/span\u003e\u003cspan address=\"https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was downloaded using the UCSC Xena browser Data Hub (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://xenabrowser.net/hub/\u003c/span\u003e\u003cspan address=\"https://xenabrowser.net/hub/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). RNA sequencing data, measured by Illumina HiSeq and RSEM normalized, were downloaded when available. The mRNA expression data from the TCGA discovery set were transformed into a log2 scale, and correlation analyses were visualized using GraphPad Prism (GraphPad Software Inc., CA, USA). P-values between groups were calculated using Student\u0026rsquo;s t-test with GraphPad Prism.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing analysis and GSEA\u003c/h2\u003e \u003cp\u003eTotal RNA was harvested directly from cell culture plates using 1 ml of TRIzol reagent per 60 mm plate. The total RNA was isolated and treated with DNase I (Invitrogen). RNA sequencing was performed using an Illumina NovaSeq 6000\u0026trade; sequencer at DNA_Link\u0026trade; (Seoul, Korea). RNA-seq reads were initially mapped to the human genome GRCh37/hg19 build using Tophat version 2.0. 13 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ccb.jhu.edu/software/tophat/\u003c/span\u003e\u003cspan address=\"http://ccb.jhu.edu/software/tophat/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The aligned results were analyzed with Cuffdiff version 2.2. 1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cole-trapnell-lab.github.io/cufflinks/papers/\u003c/span\u003e\u003cspan address=\"http://cole-trapnell-lab.github.io/cufflinks/papers/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to calculate FPKM values and report differentially expressed genes. For library normalization and dispersion estimation, both geometric and pooled methods were utilized (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cole-trapnell-lab.github.io/cufflinks/cuffdiff/\u003c/span\u003e\u003cspan address=\"http://cole-trapnell-lab.github.io/cufflinks/cuffdiff/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Scatter plots and heatmaps were created using the 'heatmap' function in the 'ggplot' package in R version 3.4.1. The data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE220669. Gene Set Enrichment Analysis (GSEA) was performed using the GSEA pre-ranked module on the GSEA software (version 4.3.0), with log\u003csub\u003e2\u003c/sub\u003e fold change values for ranking genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of genes related to sensitivity to Wnt / β-catenin targeting agents\u003c/h2\u003e \u003cp\u003eDatasets of human cancer cell lines were obtained from The Cancer Dependency Map Project (DepMap, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://depmap.org/portal/\u003c/span\u003e\u003cspan address=\"https://depmap.org/portal/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, version 23Q2). Data regarding responses to Wnt/β-catenin targeting agents, including ICG-001, IWR1-endo, Niclosamide, Salinomycin, WNT-C59, and XAV-939, were sourced from the drug sensitivity PRISM file (version 23Q2)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. RNA expression data utilized the CCLE\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e RNAseq gene expression data file (log\u003csub\u003e2\u003c/sub\u003e(TPM\u0026thinsp;+\u0026thinsp;1). Genome-wide RNAi loss-of-function screening data were derived from two large-scale CRISPR and RNAi experiments (CERES\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, Achilles\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and DRIVE\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e). Gene effects were calculated using DEMETER2\u003csup\u003e33\u003c/sup\u003e within DepMap. The p-values obtained from these analyses were then converted to -log\u003csub\u003e10\u003c/sub\u003e (p-value) to score each gene.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Real-time PCR (RT-qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cell lysates using TriZol (Invitrogen), and 2 \u0026micro;g of total RNA was reverse transcribed to cDNA using an oligo dT primer and M-MuLV Reverse Transcriptase (Invitrogen). RT-qPCR analysis was performed using specific primers and the SYBR Premix Ex Taq\u0026trade; kit (TaKaRa Bio, Shiga, Japan). The transcripts were detected by the CFX96 Real-Time PCR Detection System (BioRad, CA, USA). Primers used for RT-qPCR targeted Ephexin1, Wnt7a, Axin2, CXCL8, TERT, YWHAB, APC, DKK1, TCF7, Lgr5, Wnt9a, ID2, CSNK2B, PPP3CA, CyclinD1, CHD1, ROCK2, XPO1, YWHAZ, FRAT2, TBL1XR1, PRKACB, HDAC1, and β-actin. Each sample was analyzed in triplicates, and target genes were normalized relative to the reference housekeeping gene, β-actin. Relative mRNA expression levels were calculated using the comparative threshold cycle (Ct) method with β-actin as the control, according to the formula: ΔCt\u0026thinsp;=\u0026thinsp;Ct(β-actin) - Ct(target gene). The fold change in gene expression normalized to β-actin and relative to the control sample was calculated as as 2\u003csup\u003e\u0026minus;\u003c/sup\u003eΔΔC\u003csub\u003et\u003c/sub\u003e. RT-qPCR primer sequences are listed in Supplementary Table S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro GST-pulldown assay\u003c/h2\u003e \u003cp\u003eBacterially expressed GST-Ephexin1 (full-length or DH/PH domain) and GST alone were immobilized onto Glutathione Sepharose 4B beads (GE Healthcare) and incubated with bacterially expressed His\\x6-Axin1 (RGS or DIX domain) fusion proteins overnight at 4\u0026deg;C. The GST bead-bound complexes were then washed five times with GST lysis buffer (20 mM HEPES, pH 7.6; 150 mM NaCl; 5 mM MgCl₂; 1% Triton X-100; and 5% glycerol), and bound proteins were separated by SDS-PAGE and analyzed by Western blotting using appropriate antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePrediction of Ephexin1, Axin1 and APC structure\u003c/h2\u003e \u003cp\u003eFor predict structures of Ephexin1(1-457aa), Axin1(1-211aa) and APC (1567-1595aa, 1716-1734aa, and 2032-2050aa), the corresponding sequences were processed using AlphaFold-Multimer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/deepmind/alphafold\u003c/span\u003e\u003cspan address=\"https://github.com/deepmind/alphafold\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, Version 2.3.0)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e encased in ColabFold\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e package which takes advantage of the MMseq2 server for automated MSA (Multiple Sequence Alignment) generation. The open-source PyMOL system (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pymol.org/2/\u003c/span\u003e\u003cspan address=\"https://pymol.org/2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used for visualization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from three independent experiments. Significant differences between groups were assessed using a two-tailed paired Student's t-test or two-way ANOVA with GraphPad Prism (GraphPad Software Inc., CA, USA). Results with values of *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and *** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 were considered statistically significant\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e All animal studies were reviewed and approved by the Institutional Animal Welfare and Use Committee of Chosun University School of Medicine.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eEphexin1 correlates with Wnt/β-catenin target gene expression in colorectal cancer\u003c/h2\u003e \u003cp\u003eTo explore a potential link between Ephexin1 and Wnt/β-catenin signaling, we analyzed RNA sequencing data from the TCGA database, specifically looking at the expression of Ephexin1 and β-catenin target genes in CRC samples (n\u0026thinsp;=\u0026thinsp;639). Our analysis revealed a statistically significant positive correlation between Ephexin1 expression and the expression of Wnt/β-catenin target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea; Supplementary Fig.\u0026nbsp;1a, b). This correlation was specific to Wnt/β-catenin target genes, as it was not observed with non-Wnt/β-catenin target genes. This suggest that Ephexin1 selectively influences Wnt signaling in CRC. Furthermore, our research demonstrates that Ephexin1, along with β-catenin and Lgr5 (a key gene in the Wnt/β-catenin pathway), are significantly more expressed in CRC cell lines than in normal colon cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Immunohistochemical analysis confirmed a positive correlation between the expressions of Ephexin1, β-catenin, and Lgr5 in CRC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe uncontrolled proliferation of colorectal stem cells is a key mechanism in the development of CRC \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Given the role of Lgr5 as a key marker for colorectal stem cells\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e,\u003c/p\u003e \u003cp\u003ewe investigated the presence of Ephexin1 in Lgr5-positive stem cells specifically located in the crypt regions of the colon. Our findings confirmed that Ephexin1 is uniquely expressed in these colonic stem cells (Supplementary Fig.\u0026nbsp;2a, b). Notably, both Ephexin1 and Lgr5 were highly expressed in the same regions within CRC tissues (Supplementary Fig.\u0026nbsp;2c). Moreover, exposure to Wnt3a-conditioned medium (Wnt3a-CM), which activates the Wnt/β-catenin signaling pathway, resulted in increased Ephexin1 expression at both mRNA and protein levels (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eWe further explored the relationship between Ephexin1 expression and sensitivity to Wnt/β-catenin inhibiting drugs. Analysis of data from the DepMap portal showed that lower Ephexin1 levels correlated with enhanced effectiveness of these inhibitors, suggesting a link between Ephexin1 expression and the efficacy of Wnt/β-catenin inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f; Supplementary Fig.\u0026nbsp;4). Treatment with the Wnt inhibitor IWR1-endo significantly reduced growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), anchorage-independent growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh), and migration ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei) in HCT116 cells deficient in Ephexin1, compared to control cells. Additionally, cells with diminished Ephexin1 exhibited a notable decrease in the cell proliferation marker, Ki67, following IWR1-endo treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej). These findings suggest that Ephexin1 plays a vital role in controlling Wnt/β-catenin signaling in CRC, underscoring its potential as a target for therapy.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eEphexin1 influences on Wnt / β-catenin signaling pathway\u003c/h2\u003e \u003cp\u003eTo investigate the role of Ephexin1 in the Wnt/β-catenin signaling pathway, we conducted RNA-Seq analysis on HCT116 cells with and without Ephexin1 knockdown. Our bioinformatics analysis identified 326 genes that were differentially expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). We then focused on statistically significant gene changes, using ontology and interaction network analysis of these differentially expressed genes (DEGs), along with functional enrichment analysis. These analyses demonstrated that the absence of Ephexin1 affects genes associated with Wnt signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c; Supplementary Fig.\u0026nbsp;5a). Specifically, genes normally activated by Wnt signaling were found to be suppressed in Ephexin1 knockdown HCT116 cells, whereas genes typically inhibited by Wnt signaling, along with genes activated by apoptosis, were increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Further, Gene Set Enrichment Analysis (GSEA) and heatmap data revealed a direct correlation between Ephexin1 levels and the activation of Wnt/β-catenin target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f). Through RT-qPCR analysis, we validated significant differences in the expression of Wnt/β-catenin target genes in Ephexin1-deficient cells compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg, Supplementary Fig.\u0026nbsp;5b). These results indicate that Ephexin1 plays a critical role in facilitating the positive regulation of genes downstream of the Wnt/β-catenin signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eEphexin1 enhances the transcriptional activity of β-catenin in the Wnt signaling pathway\u003c/h2\u003e \u003cp\u003eThe primary role of canonical Wnt/β-catenin signaling pathway is to regulate gene expression by allowing β-catenin to function as a transcription factor\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Based on this, we hypothesized that Ephexin1 influences the transcriptional activity of β-catenin. We tested this by reducing Ephexin1 in HCT116 and HEK293T cells. Phosphorylation of β-catenin leads to its degradation, while non-phosphorylation at these sites promotes stabilization\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Our results showed that Ephexin1 depletion increased phosphorylated β-catenin levels and decreased non-phosphorylated β-catenin, total β-catenin, and the expression of its target genes, such as cyclinD1 and c-myc. Conversely, Ephexin1 overexpression increased β-catenin, cyclinD1, c-myc, and Lgr5 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea; Supplementary Fig.\u0026nbsp;6a, b). Moreover, Ephexin1 knockdown significantly reduced the Wnt3a-CM-mediated increase in β-catenin levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore the effect of Ephexin1 on the transcriptional activity of β-catenin, we conducted a TOP-Flash Luciferase reporter assay. In both HCT116 and HEK293T cells, simultaneous overexpression of Ephexin1 and β-catenin significantly enhanced transcriptional activity of β-catenin more than overexpression of either β-catenin or Ephexin1 alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, Supplementary Fig.\u0026nbsp;6c). Additionally, depleting Ephexin1 significantly reduced the TOP-Flash Luciferase reporter activity induced by Wnt3a-CM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, Supplementary Fig.\u0026nbsp;6d), whereas overexpressing Flag-tagged Ephexin1 markedly increased it (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, Supplementary Fig.\u0026nbsp;6e). Furthermore, the treatment with Wnt3a-CM significantly increased the mRNA levels of TCF7, cyclinD1, and Lgr5, which are key target genes of the Wnt/β-catenin signaling pathway, in control groups, but this effect was not observed in Ephexin1-deficient HCT116 cells. On the other hand, the expression of the β-catenin gene, which is not directly targeted by Wnt/β-catenin signaling, remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). These findings collectively indicate that Ephexin1 plays a crucial role in enhancing the activity of β-catenin as a transcription factor.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eEphexin1 interacts with the β-catenin destruction complex through binding to Axin1\u003c/h2\u003e \u003cp\u003eConsidering the role of Ephexin1 in regulating transcriptional activity of β-catenin, we hypothesized a potential interaction between Ephexin1 and the β-catenin destruction complex. To test this hypothesis, we employed both yeast two-hybrid assays and co-immunoprecipitation techniques. In the yeast two-hybrid assay, we discovered an interaction between Ephexin1 and Axin1, a key component of the β-catenin destruction complex (Supplementary Fig.\u0026nbsp;7a-c). This interaction was confirmed in HEK293T cells through co-immunoprecipitation, where Ephexin1 was found to bind not only with Axin1 but also with other elements of the destruction complex, such as β-catenin, TCF4, CK1, GSK3β, and APC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea; Supplementary Fig.\u0026nbsp;7d, e). Importantly, we observed that the interaction between Ephexin1 and Axin1 became stronger with increased duration of Wnt-3a CM treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify which part of Ephexin1 interacts with Axin1, we examined five different Ephexin1 mutants, each missing a specific domain of Ephexin1. Our findings indicated that Axin1 attaches to the DH/PH domain of Ephexin1, spanning amino acids 273 to 601. In contrast, the RR and SH3 domains of Ephexin1 showed no affinity for binding to Axin1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). To further determine the specific domain of Axin1 involved in binding to Ephexin1, we generated and analyzed six Flag-tagged Axin1 mutants. This analysis revealed that the RGS domain (1-221aa) of Axin1 was crucial for binding to V5-tagged Ephexin1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f). For direct binding confirmation between Ephexin1 and Axin1, we performed GST pulldown assays using recombinant proteins of GST-Ephexin1 (either full-length or containing the DH/PH domain) and His\u003csub\u003ex6\u003c/sub\u003e-Axin1 (RGS or DIX domain). The GST pulldown assays confirmed a direct interaction between the DH/PH domain of Ephexin1 and the RGS domain of Axin1, without interaction with the DIX domain of Axin1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, Supplementary Fig.\u0026nbsp;8).\u003c/p\u003e \u003cp\u003eTo further verify the interaction between Ephexin1 and Axin1, we used AlphaFold-multimer to predict how these proteins bind together\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, supporting our immunoprecipitation results. A precise analysis of the binding domain of Ephexin1 to Axin1 was necessary for AlphaFold-multimer accurate predictions, leading to the generation of seven Ephexin1 mutants with specific deletions in the DH/PH domain. We observed that DH domain, but not PH domain, of Ephexin1 is required for its interaction with Axin1 (Supplementary Fig.\u0026nbsp;9a, b).\u003c/p\u003e \u003cp\u003eTo accurately predict the stable structure of Ephexin1, we utilized the RR/DH domain (1-457 amino acids) of Ephexin1 and the RGS domain (1-211 amino acids) of Axin1 in AlphaFold-multimer modeling. This approach yielded predictions of a high-quality interaction between RR/DH domain of Ephexin1 and RGS domain of Axin1, as evidenced by sequence coverage, the predicted Local Distance Difference Test (pLDDT) confidence value, and predicted alignment error (PAE) (Supplementary Fig.\u0026nbsp;10a-c). Further structural analysis indicated that the DH domain of Ephexin1 interacts with RGS domain of Axin1 through the formation of four hydrogen bonds, underscoring the specificity and strength of this interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh, i).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eEphexin1 regulates β-catenin degradation through modulation of the Axin1 interaction network\u003c/h2\u003e \u003cp\u003eConsidering the function of Axin1 in the Wnt signaling pathway as a scaffold protein that facilitates the proteasomal degradation of β-catenin\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, we hypothesized that the interaction of Ephexin1 with Axin1 could influence degradation pathway of β-catenin via ubiquitination mechanisms. To investigate this hypothesis, we conducted immunoprecipitation experiments on Axin1 in HEK293T cells under conditions of both Ephexin1 deficiency and overexpression. Our findings indicate that the absence of Ephexin1 amplifies the interactions between Axin1 and key proteins in the Wnt signaling pathway, such as β-catenin, CK1, and GSK3β (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), whereas overexpressing Ephexin1 diminishes these interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The phosphorylation of β-catenin by CK1 and GSK3β is essential for its ubiquitination and subsequent degradation via the SCF\u003csup\u003eβTrCP\u003c/sup\u003e complex\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Interestingly, while overexpression of Axin1 increases the interaction of β-catenin with CK1 and GSK3β, this effect is mitigated by Ephexin1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, Supplementary Fig.\u0026nbsp;11).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe adenomatous polyposis coli (APC), a crucial scaffolding component, collaborates with Axin1 to promote the ubiquitination of β-catenin by the E3 ligase SCF\u003csup\u003eβTrCP \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Notably, the RGS domain of Axin1 attaches to three distinct regions on APC, at amino acids 1567\u0026ndash;1595, 1716\u0026ndash;1736, and 2032-2050\u003csup\u003e47\u003c/sup\u003e. Given our previous findings that Ephexin1 binds to RGS domain of Axin1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), this suggest a competitive relationship between Ephexin1 and APC for RGS domain of Axin1. To explore this hypothesis further, we employed AlphaFold-multimer for predictive modeling. Our findings, aligning with existing research, confirmed the interaction of Axin1 with APC through AlphaFold-multimer predictions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, Supplementary Fig.\u0026nbsp;12a-c). Importantly, our predictive models indicate that the binding of Ephexin1 to Axin1 could interfere with the interaction between Axin1 and APC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, f; Supplementary Fig.\u0026nbsp;12d-f). To validate these findings, we performed experiments using immunoprecipitation assays involving all three proteins. These experiments demonstrated that increasing Ephexin1 levels significantly decreased the interaction between Axin1 and APC, as well as between Axin1 and SCF\u003csup\u003eβTrCP\u003c/sup\u003e. Conversely, reducing Ephexin1 levels led to an increase in these interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, h). Moreover, in HCT116 cells lacking Ephexin1, treatment with Wnt3a-CM caused a smaller reduction in the Axin1-APC interaction compared to the control cells, indicating that the absence of Ephexin1 allows for a more stable Axin1-APC connection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). Also, analysis of β-catenin from Ephexin1-deficient HCT116 cells showed more interactions with components of the destruction complex and higher levels of β-catenin poly-ubiquitination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej). These results indicate that Ephexin1 is essential for β-catenin stability, as it binds to Axin1, thereby inhibiting the activity of the β-catenin destruction complex and reducing β-catenin ubiquitination, which is crucial for its degradation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eEphexin1 depletion enhances apoptotic and tumor suppressive effects of Wnt Inhibition in CRC\u003c/h2\u003e \u003cp\u003eTo explore the therapeutic potential of Ephexin1 in CRC, we evaluated the impact of Ephexin1 depletion on the effectiveness of a Wnt inhibitor. Initially, we assessed apoptotic responses in various CRC cell lines treated with IWR-endo and discovered that most cells underwent apoptosis following IWR1-endo treatment. However, HCT116 cells did not undergo apoptosis with IWR1-endo treatment (Supplementary Fig.\u0026nbsp;13a), indicating resistance to this treatment. Further investigation using IWR1-endo on both control and Ephexin1-depleted HCT116 cells, followed by apoptosis measurement through AnnexinV staining, revealed that Ephexin1 depletion significantly increased apoptosis in HCT116 cells treated with IWR1-endo (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). To determine if these effects were related to the Wnt signaling pathway, we analyzed β-catenin stability and target gene expression in both control and Ephexin1-depleted HCT116 cells. After IWR1-endo treatment, Ephexin1-depleted HCT116 cells showed a more pronounced decrease in β-catenin and its target gene cyclinD1 compared to control cells (Supplementary Fig.\u0026nbsp;13b, c). Supporting this observation, treatment of Ephexin1-depleted HCT116 cells with IWR1-endo further increased β-catenin ubiquitination (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, to assess the ability of Ephexin1 to counteract Wnt inhibitor resistance in xenograft models, we administered IWR1-Endo to mice with tumors derived from either control HCT116 cells or Ephexin1-deficient HCT116 cells. The treatment was applied once every 3 to 4 days for a total of 33 days. While a slight reduction in tumor volume was observed in the control group, tumors derived from Ephexin1-deficient HCT116 cells exhibited a significant decrease in both volume and weight following IWR1-Endo treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-e). Immunohistochemical analysis of the tumor tissues further confirmed that the depletion of Ephexin1, combined with IWR1-endo treatment, results in decreased levels of Ki-67 and β-catenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), as well as lower levels of β-catenin target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg), compared to controls. These findings indicate that Ephexin1 absence could enhance the tumor-suppressive effects of Wnt pathway inhibitors in CRC, presenting a promising strategy for tumor suppression.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eThe interaction of Ephexin1 and Axin1 has clinical relevance in CRC\u003c/h2\u003e \u003cp\u003eTo Investigate the Role of Ephexin1 overexpression in CRC development, we generated transgenic (TG) mice that overexpress the mouse Ephexin1(mEphexin1) gene (Supplementary Fig.\u0026nbsp;14). In mouse embryonic fibroblast (MEF) cells derived from these genetically modified mice, treatment with Wnt3a-CM significantly enhanced LRP6 phosphorylation and β-catenin accumulation compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Given the established role of AOM (Azoxyemethane) and DSS (Dextran Sodium Sulfate) in inducing CRC through Wnt/β-catenin signaling activation\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, we initiated colon cancer in both wild-type and mEphexin1 TG mice using AOM/DSS treatment over two weeks. The mEphexin1 TG mice exhibited an increased size and number of colon tumors compared to controls. Furthermore, both Ki67 and β-catenin levels were elevated in the colon tissues of the mEphexin1 TG mice, with a notable increase in nuclear β-catenin localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, c). A long-term survival analysis revealed that mEphexin1 overexpression significantly decreased the survival rate of the mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the clinical relevance of Ephexin1 alongside β-catenin and Axin1 in human CRC, we examined tissue microarrays of colorectal tissues encompassing normal tissues, carcinomas of varying grades, and metastatic tumors. The expression levels of Ephexin1, β-catenin, and Axin1 were markedly higher in CRC tissues compared to normal tissues. Notably, these levels increased progressively with the tumor grade and the presence of metastatic tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, f). Proximity Ligation Assay (PLA) was employed to examine the interactions between Ephexin1 and Axin1 and the quantitative changes in β-catenin in CRC patient samples. PLA foci indicating Ephexin1 and Axin1 interactions were markedly higher in cancer tissues than in normal tissues and significantly increased with tumor grade and metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg, h). Notably, the PLA foci scores for Ephexin1 and Axin1 correlated positively with β-catenin immunohistochemistry scores, and patients with high levels of Ephexin1-Axin1 binding exhibited poorer prognoses (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei, j). These findings emphasize the importance of the interaction between Ephexin1 and Axin1, suggesting that inhibiting this interaction could be a promising strategy for the development of anticancer drugs targeting Wnt/β-catenin active CRC.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe Wnt/β-catenin signaling pathway is crucial in cancer development and progression, making it a key subject of research in CRC. Our study reveals that Ephexin1, significantly overexpressed in CRC, activates the Wnt/β-catenin signaling pathway. Furthermore, we highlight the critical role of Ephexin1 in promoting tumor growth through the activation of the Wnt/β-catenin pathway. This discovery enhances our understanding of the mechanisms underlying wnt signaling dysregulation, which plays a crucial role in the progression of CRC, and introduces new possibilities for therapeutic interventions.\u003c/p\u003e \u003cp\u003eEphexin1, traditionally known for its role in neurophysiological processes and minimal expression outside the nervous system\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, has emerged as a pivotal player in CRC through its overexpression and interaction with the Wnt/β-catenin signaling pathway. Our findings, corroborated by TCGA data analysis, establish a significant correlation between Ephexin1 expression and the activation of Wnt/β-catenin target genes in CRC. This relationship underscores the contribution of Ephexin1 to the hyperactive Wnt signaling observed in CRC, driving the uncontrolled proliferation of colorectal stem cells and tumor growth. The unique impact of Ephexin1 on Wnt/β-catenin target genes, not observed in the non-target genes of Wnt/β-catenin signaling pathway, underscores its specific role in regulating this signaling pathway in CRC. This effect is further substantiated by our immunohistochemical analyses and RNA sequencing data, which show a higher expression of Ephexin1, β-catenin, and Lgr5 in CRC cell lines and tissues compared to normal colon cells. These observations suggest that Ephexin1 not only facilitates Wnt/β-catenin signaling but also enhances the transcriptional activity of β-catenin, thus promoting oncogenic gene expression.\u003c/p\u003e \u003cp\u003eThe canonical Wnt/β-catenin pathway, a key regulator of cellular proliferation and differentiation, is frequently dysregulated in various cancers, including CRC\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Our study explores the underlying mechanisms of Ephexin1 by demonstrating its interaction with Axin1, a key component of the β-catenin destruction complex. This interaction plays a vital role in controlling the stability of β-catenin and, consequently, the activity of the Wnt/β-catenin signaling pathway. The binding of Ephexin1 to Axin1, particularly when Wnt signaling is enhanced, indicates a novel regulatory mechanism. This mechanism suggests Ephexin1 could prevent the degradation of β-catenin, thus maintaining its oncogenic potential. The specificity of this interaction, delineated through various experimental approaches including yeast two-hybrid assays, co-immunoprecipitation, GST pulldown assays, and structural predictions based on AlphaFold2 models, highlights the potential of targeting the Ephexin1-Axin1 interaction as a therapeutic strategy. Given the oncogenic nature of Ephexin1 and the tumor-suppressive function of Axin1, the overexpression of Ephexin1 in CRC suggest that it could counteract the effect of Axin1, thereby promoting cancer progression.\u003c/p\u003e \u003cp\u003eDiscovering that CRC cells lacking Ephexin1 exhibit increased sensitivity to inhibitors of the Wnt/β-catenin pathway highlights the potential benefits of targeting Ephexin1 for therapeutic purposes. By exploiting this sensitivity, we can potentially overcome the resistance faced by treatments targeting the Wnt pathway, offering a new approach to CRC treatment. Moreover, developing treatments that interfere with the interaction between Ephexin1 and Axin1 could provide an advanced way to control the stability of β-catenin and the activity of Wnt signaling in CRC. Our findings also emphasize the clinical significance of Ephexin1 and its interaction with Axin1 in CRC progression. The association between Ephexin1-Axin1 binding and worse patient outcomes, as evidenced by tissue microarray analysis and proximity ligation assays, highlights the critical need for further research into targeting this interaction as a therapeutic strategy. Disrupting this interaction could restore the β-catenin degradation pathway, consequently reducing Wnt signaling and its cancer-promoting role in CRC. Therefore, Ephexin1 emerges as a potential biomarker for tracking cancer progression and an attractive target for developing new therapies specific to CRC. While clinical trials are evaluating Wnt-targeted inhibitors like Vantictumab, Ipafricept, and Cetuximab \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, none have received FDA approval yet. The difficulty in creating effective therapies that target the Wnt/β-catenin pathway could be due to its essential role in maintaining tissue homeostasis \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. The reported side effects of these therapies, such as bone toxicity, hair loss, skin rashes, and gastrointestinal issues\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, emphasize the necessity for targeting Wnt/β-catenin signaling in a more specific and safer manner. Focusing on anticancer drugs that target Ephexin1, which is mainly found in brain tissue but significantly increased in in LC and CRC\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, could offer specificity and effeteness against cancer cells with minimal impact on normal cells. Given the complexity of the Wnt pathway, with its numerous components and regulatory mechanisms, future research should focus on ensuring that targeting Ephexin1 does not disrupt normal cell functions or produce adverse effects.\u003c/p\u003e \u003cp\u003eIntestinal cells renew every five days through the Wnt/β-catenin signaling pathway, crucial for tissue equilibrium and stem cell regeneration in the intestinal crypts\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. These stem cells facilitate surface growth, but their excessive proliferation may lead to CRC\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Approximately 80% of CRCs result from mutations in Wnt/β-catenin signaling genes\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, and around 37% involving K-Ras oncogene mutations\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. These mutations cause deregulation of the Wnt and Ras pathways, leading to uncontrolled stem cell growth and cancer\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Our findings indicate that Ephexin1, which is markedly increased in cancer stem cells in CRC patients, is influenced by K-Ras mutations\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and impacts the Wnt/β-catenin pathway, potentially leading to unregulated stem cell proliferation. We are continuing research to further explore this effect.\u003c/p\u003e \u003cp\u003eIn summary, this study elucidates the pivotal role of Ephexin1 in CRC by demonstrating its overexpression and its direct influence on the Wnt/β-catenin signaling pathway. The interaction of Ephexin1 with Axin1 modulates stability of β-catenin, suggesting a novel mechanism for Wnt pathway dysregulation in CRC (Supplementary Fig.\u0026nbsp;15). Developing drugs that specifically inhibit Ephexin1 or its interaction with Axin1 represents a new strategy in CRC therapy, particularly for Wnt-driven CRC. This approach, potentially in combination with existing Wnt pathway inhibitors, could improve treatment outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis work is supported by National Research Foundation of Korea (NRF) grants (NRF-2022R1C1C2004575 and NRF-2022R1A5A2030454), funded by the Korea government.\u003c/p\u003e\u003ch2\u003eAUTHOR CONTRIBUTIONS\u003c/h2\u003e \u003cp\u003eJK, JHL, and HJY designed the study. JK, YJJ, and IYC performed the experiments, collected data, and performed statistical analysis. JH analyzed TCGA data set. IYC performed the immunohistochemistry staining and the pathological analysis. JK and YJJ carried out animal experiment. JK. YJJ, JY, JHL and HJY wrote the manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eWe thank all participants of this study for their invaluable devotion. We also thank Chosun university of Cancer Mutation Research Center for providing research equipment and technical support for this study.\u003c/p\u003e\n\u003ch3\u003eDATA AVAILABILITY\u003c/h3\u003e\n\u003cp\u003eThe RNA-seq data were deposited in the NCBI Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE220669.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAcebron SP, Karaulanov E, Berger BS, Huang YL, Niehrs C. Mitotic wnt signaling promotes protein stabilization and regulates cell size. Mol Cell 2014, 54(4): 663\u0026ndash;674.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClevers H, Loh KM, Nusse R. Stem cell signaling. 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Cancer Res 2016, 76(2): 305\u0026ndash;318.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4446931/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4446931/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWnt signaling is essential for cell growth and tumor formation, and is abnormally activated in colorectal cancer (CRC), contributing to tumor progression, but the specific role and regulatory mechanisms in tumor development are not yet clear. Here we show that Ephexin1, a guanine nucleotide exchange factor, is significantly overexpressed in CRC, correlating with increased Wnt/β-catenin pathway activity. Through comprehensive analysis, including RNA sequencing data from TCGA and functional assays, we demonstrated that Ephexin1 promotes tumor proliferation and migration by activating the Wnt/β-catenin pathway. This effect is mediated by the interaction of Ephexin1 with Axin1, a critical component of the β-catenin destruction complex, which in turn enhances stability and activity of β-catenin in signaling pathways critical for tumor development. Importantly, our findings also suggest that targeting Ephexin1 could enhance the efficacy of Wnt/β-catenin pathway inhibitors in CRC treatment. These findings highlight the potential of targeting Ephexin1 as a strategy for developing effective treatments for CRC, suggesting a novel and promising approach to therapy aimed at inhibiting cancer progression\u003c/p\u003e","manuscriptTitle":"Disruption of β-Catenin Destruction Complex by Ephexin1-Axin1 Interaction Promotes Colorectal Cancer Proliferation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-07 16:59:26","doi":"10.21203/rs.3.rs-4446931/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-06-20T23:30:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-06-19T08:07:40+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-06-13T01:51:25+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-06-12T04:01:11+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-05-28T01:30:19+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-05-27T07:44:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-20T23:58:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2024-05-20T06:06:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-20T06:06:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"48deecf8-4a01-4f2a-9dcb-68d2395c358d","owner":[],"postedDate":"June 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":32437111,"name":"Health sciences/Diseases/Cancer/Gastrointestinal cancer/Colorectal cancer/Colon cancer"},{"id":32437112,"name":"Health sciences/Diseases/Cancer/Oncogenes"}],"tags":[],"updatedAt":"2025-01-01T08:06:14+00:00","versionOfRecord":{"articleIdentity":"rs-4446931","link":"https://doi.org/10.1038/s12276-024-01381-1","journal":{"identity":"experimental-and-molecular-medicine","isVorOnly":false,"title":"Experimental \u0026 Molecular Medicine"},"publishedOn":"2025-01-01 05:00:00","publishedOnDateReadable":"January 1st, 2025"},"versionCreatedAt":"2024-06-07 16:59:26","video":"","vorDoi":"10.1038/s12276-024-01381-1","vorDoiUrl":"https://doi.org/10.1038/s12276-024-01381-1","workflowStages":[]},"version":"v1","identity":"rs-4446931","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4446931","identity":"rs-4446931","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}