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 described22. 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 described22, 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 1x104 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% H2O2 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 log2 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 CCLE29 RNAseq gene expression data file (log2(TPM + 1). Genome-wide RNAi loss-of-function screening data were derived from two large-scale CRISPR and RNAi experiments (CERES30, Achilles31 and DRIVE32). Gene effects were calculated using DEMETER233 within DepMap. The p-values obtained from these analyses were then converted to -log10 (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−ΔΔCt. 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 ColabFold35 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.