Interaction between MUC1 and β-catenin promotes transformation of adenomas to adenocarcinomas in Familial Adenomatous Polyposis

Interaction between MUC1 and β-catenin promotes transformation of adenomas to adenocarcinomas in Familial Adenomatous Polyposis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Interaction between MUC1 and β-catenin promotes transformation of adenomas to adenocarcinomas in Familial Adenomatous Polyposis Yinan Li, Wenjun Shi, Pei Luo, Xianshuo Cheng, Jun Yang, Yunfeng Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5722397/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose This study aims to elucidate the mechanisms of interaction between MUC1 and β-catenin in the transformation from adenoma to adenocarcinoma in FAP, providing insights for potential interventions in this progression and establishing a scientific basis for the prevention and treatment of FAP. Methods (1)Bioinformatics and Clinical Specimen Analysis to investigate the differential expression of MUC1 and β-catenin and their association with clinical characteristics, including tumor staging, grading, metastasis, and survival outcomes in CRC. (2)Confirmation of MUC1-β-catenin Complexes using Alphafold, PyMol, immunofluorescence, and co-immunoprecipitation techniques. (3)Regulation of β-catenin through inhibitors, siRNA, and lentivirus to modulate β-catenin levels and assess MUC1 expression. (4)Biological behavior observation to evaluate changes in the biological behavior of APC mutant cells following MUC1 knockdown or overexpression. Results (1)Both MUC1 and β-catenin are overexpressed in colorectal cancer, with high expression correlating with poor prognosis. (2)In APC mutant colorectal cell lines, MUC1 knockdown inhibits cell proliferation, migration, and invasion, while MUC1 overexpression enhances these behaviors. (3)Both in vitro and in vivo models, β-catenin regulates MUC1 expression and interacts with it. Modulating MUC1 or β-catenin influences the binding or dissociation of MUC1-β-catenin complexes, impacting downstream signaling pathways. Conclusion In FAP patients, APC gene mutations impair β-catenin degradation within the WNT signaling pathway. Consequently, MUC1 is recruited around free β-catenin, forming a stable complex that promotes the progression from adenoma to adenocarcinoma by activating the WNT/β-catenin pathways. Inhibiting the interaction between MUC1 and β-catenin may offer significant potential for the prevention and treatment of FAP. Familial Adenomatous Polyposis (FAP) adenomas adenocarcinoma MUC1 β-catenin protein interactions Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1. INTRODUCTION As per the oncological data published by the World Health Organization (WHO), in the year 2023, colorectal carcinoma was designated as the third most frequently occurring malignancy and the second leading cause of cancer-related fatalities on a global scale 1 . However, the situation is more severe in China, where both the incidence and mortality rates are second highest in CRC, with an increasing trend year by year 23 . In the etiology of colorectal cancer, a combination of genetic predispositions and environmental factors is typically implicated in the disease's pathogenesis and prevalence 4 5 . An estimated one-third of people with colorectal cancer had a hereditary susceptibility, according to incomplete statistics 6 . Familial Adenomatous Polyposis (FAP), a syndrome inherited in an autosomal dominant pattern, is significantly associated with an elevated risk of colorectal cancer due to deleterious alterations in the APC tumor suppressor gene 7 . Notably, approximately 60% of sporadic colorectal cancer cases also exhibit mutations in the APC gene 8 . Nevertheless, it is still unknown how precisely these mutations promote the development of colorectal cancer from adenoma to adenocarcinoma 9 . Therefore, a thorough investigation into the changes in key molecules resulting from APC gene mutations is essential not only for elucidating the progression of FAP from adenoma to adenocarcinoma but also for enhancing our understanding of the developmental mechanisms underlying sporadic colorectal cancer 10 . Multiple adenomatous polyps in the gut and a high level of familial clusterin are hallmarks of FAP, a devastating genetic condition. Since Crisp first reported this condition (later named FAP) in 1882 11 , the typical pathogenesis of FAP has been in-depth documented. FAP patients frequently have the illness early in life, and as they age, they may acquire hundreds to thousands of colon and rectal polyps 12 . If left untreated, there is a significant risk that these polyps will progress to adenocarcinoma between the ages of 30 and 40, posing a serious threat to the patient's life 13 . Currently, there are still no effective treatments available for FAP 14 . In the 1970s, advancements in whole genome sequencing technology revealed that most FAP patients have mutations in the APC gene 15 16 . The APC gene, classified as a tumor suppressor, is situated within the 5q21-22 band of the human genome, encompassing roughly 108 kilobase pairs, and translates into a protein comprising 2,843 amino acid residues 17 . Notably, 15-exon of APC gene is the largest known exon in the human genome, accounting for 77% of the coding region and representing the most frequently mutated area 18 . The APC protein plays several critical roles within cells, including maintaining cell adhesion, participating in transcriptional activation, promoting cell migration, and regulating apoptosis 19 . Under normal conditions, β-catenin, which is necessary for cellular metabolism and embryonic development, is degraded by the "destruction complex" made up of APC, GSK-3β, Axin, etc. 20 . From 1020aa to 2075aa, the APC protein's three 15-amino acid repeat sequences, seven 20-amino acid repeat sequences, and SAMP repeat sequences are regarded as essential structural domains that interact with β-catenin 21 – 23 . APC mutations in FAP patients result in aberrant folding of the APC protein's region that should bind to β-catenin, which inhibits the “destruction complex” 9 . Consequently, The canonical β-catenin degradation pathway, which involves ubiquitination and proteasomal degradation, is typically impaired, leading to an anomalous accumulation of β-catenin within the cellular cytoplasm. After entering the nucleus, excess β-catenin attaches itself to the transcription factor TCF4 to create a complex that abnormally promotes the transcription of downstream target genes in the Wnt signaling pathway 24 25 . This activation causes uncontrolled mitotic signals, contributing to the development of FAP adenomas. Research has indicated that colorectal adenomas exhibit elevated β-catenin expression. MUC1 is also significantly expressed in adenocarcinoma, in addition to β-catenin being overexpressed 26 , 27 . Furthermore, as polyp adenomas progress to adenocarcinoma, MUC1 expression tends to increase 28 . We hypothesize that MUC1 could exert a significant influence in the neoplastic evolution of the FAP process. The MUC1 gene, mapped to the 1q22 locus on chromosome 1 and comprising seven exons and six introns, encodes the MUC1 protein. This transmembrane glycoprotein, characterized by its extensive glycosylation, possesses a molecular mass of approximately 500 kilodaltons 29 . Two peptide segments, MUC1-C and MUC1-N, make up the MUC1 protein and are joined by non-covalent links 30 . MUC1 functions as a physical barrier to stop pathogen invasion in normal cells by being polarized and dispersed on the luminal surface of epithelial cells in a highly glycosylated state 31 . Nevertheless, MUC1 expression is more than 100 times more in malignant tumor cells than in healthy cells. According to observations, over 70% of colorectal cancer cells have high MUC1 expression 32 , along with reduced glycosylation and a lack of apical polarity. MUC1 controls a number of proteins involved in tumor growth by interacting with oncogenes, transcription factors, and co-activators. These interactions cause immune evasion, drug resistance, invasion, proliferation, epithelial-mesenchymal transition, and stemness 33 . Based on our earlier studies, we discovered that MUC1 and β-catenin are strongly related, especially when APC mutations are present. In addition to being associated with the development of FAP adenomas and the advancement of adenocarcinoma, aberrant β-catenin accumulation may also affect MUC1 function. In light of the dearth of useful research models for FAP, we intend to look more closely at the relationship and interaction between MUC1 and β-catenin in APC mutant CRC cells. In the context of APC gene alterations, we specifically want to investigate their functions and processes in the development of adenocarcinoma from FAP adenomas. 2. MATERIALS AND METHODS 2.1 Bioinformatics analysis Using TCGAbiolinks (version 2.22.1) with the GDCquery function, MUC1 and β-catenin expression levels were downloaded from the TCGA database ( https://portal.gdc.cancer.gov ) together with clinical correlations in colorectal cancer (TCGA-COAD and TCGA-READ). R (version 4.3.2) was used for analysis. Of the 620 samples, the HMUC1 group consisted of the top three-quarters of patients with elevated MUC1 expression, while the LMUC1 group consisted of the remaining one-quarter. Similarly, the HCTNNB1 group was defined as the top one-quarter of patients with high β-catenin expression, while the LCTNNB1 group was defined as the remaining three-quarters. 2.2 PPI analysis and docking modeling of MUC1 and β-catenin protein The proteins that might interact with MUC1 were predicted using the STRING database ( https://cn.stringdb.org/ ). Using PyMOL (Version 3.1.3) and the AlphaFold website ( https://alphafold.ebi.ac.uk/ ), molecular docking between MUC1 and β-catenin proteins was carried out. 2.3 Clinical specimens Specimens of primary colorectal carcinoma and corresponding adjacent non-neoplastic tissues were procured from a cohort of ten individuals undergoing surgical intervention at the Department of Colorectal Surgery, Third Affiliated Hospital of Kunming Medical University. The investigation was sanctioned by the Ethics Committee of the Third Affiliated Hospital of Kunming Medical University, with all participants furnishing written informed consent. 2.4 Cell culture Cell lines NCM460, HT29, SW620, and DLD-1 were cultivated in DMEM/F-12 media (Gibco, USA), whilst RKO cells were cultured in RPMI 1640 medium (Gibco, USA), with 10% fetal bovine serum (FBS) (NEWZERUM, China). Cells were cultured in an incubator at 37°C with 5% CO2 . 2.5 Usage of β-catenin inhibitor, gene knockdown and overexpression MSAB (MCE, China), a β-catenin inhibitor, was used at a concentration of 10 µM for 48 hours. The sequences of siRNAs used were 5’-UUCUCCGAACGUGUCACGUTT-3’(si-NC), 5’-GUUCAGUGCCCAGCUCUACTT-3’(si-MUC1),5’-GGGAGTGGTTTAGGCTATTTG-3’(si-CTNNB1). Following the manufacturer's instructions, all si-RNAs (GenePharma, China) were transfected into the HT29 and SW620 cell lines using Lipofectamine 2000 reagent (Thermo, USA). Lentivovirus (GenePharma, China) was used to produce MUC1 gene overexpression. Each well of a 6-well plate (NEST) contained around 1×10 5 cells, incubated with 2 mL of complete media for 24 hours. The system was then supplemented with 1 µL of polybrene and 10 µL of virus. The cells were moved to puromycin-containing regular media after a 24-hour period. The effectiveness of inhibition, knockdown, and overexpression was assessed using qRT-PCR, Western blotting, and immunofluorescence. 2.6 Western blot After collecting the treated cells and tissue samples, RIPA lysis buffer (Beyotime, China) enhanced with a protease and phosphatase inhibitor cocktail (Beyotime, China) was used to extract the total protein. After being separated with SDS-PAGE loading buffer (Solarbio, China), the proteins were transferred to PVDF membranes with a thickness of 0.45 µm (Millipore, USA). After 30 minutes of room temperature blocking with a protein-free fast blocking solution (Epizyme, China), the membrane was incubated in diluted primary antibodies for about 10 hours at 4°C, then underwent exposure treatment after being cleaned with TBST and incubated in diluted secondary antibodies for two hours at a room temperature. The antibodies used were as follows: β-actin (1:50000, Proteintech, China), β-catenin (1:10000, Proteintech, China), MUC1 (1:5000, Abcam, USA), phospho-GSK3 β(Ser9) (1:500, Abmart, China), GSK3 β(1:1000, MCE, China), NF-κB (1:500, MCE, China), TCF4 (1:500, MCE, China) and APC (1:500, SANTA CRUZ, USA). An antibody diluent buffer (Epizyme, China) was used to dilute each antibody to the proper amounts. 2.7 qRT-PCR Following the manufacturer's instructions, protein was recovered from the treated cells and tissue samples using a total RNA extraction kit (Vazyme, China). With β-actin acting as an internal reference, reverse transcription was carried out to transform RNA into cDNA using the iScript™ cDNA Synthesis Kit (Bio-Rad, China). The iTaq Universal SYBR Green Supermix (Bio-Rad, China) was used to determine the relative RNA levels. Sangon Biotech (Shanghai, China) supplied all of the primers, and here are their sequences: β-actin(human)5’-AGAGCTACGAGCTGCCTGAC-3’,MUC1(human)5’-ACCTACCATCCTATGAGCGAGTA-3’,β-catenin(human)5’-AGGTGCTATCTGTCTGCTCTAGT-3’,β-actin(mouse)5’-GATTACTGCTCTGGCTCCTAGC-3’,β-catenin(mouse)5’-GTTCGCCTTCATTATGGACTGCC-3’, and MUC1(mouse) 5’-AGTGCCTCTGACGTGAAGTCAC-3’. 2.8 Immunofluorescence assay After xenograft tumor paraffin slices were removed from naked mice, they underwent antigen retrieval, serum blocking, and an overnight primary antibody incubation at 4°C. The slices were then treated for 60 minutes at room temperature, shielded from light, with DAPI and a photolabeled secondary antibody. Using a fluorescent microscope, pictures were taken. Following fixation with 4% paraformaldehyde, the procedures for cell samples were the same as those for tissue samples. The following antibodies were used: MUC1 (1:200, Abcam, USA), and β-catenin (1:200, Proteintech, China). An antibody diluent buffer (Beyotime, China) was used to dilute each antibody in the proper amounts. 2.9 Co-Immunoprecipitation (Co-IP) assay For the analysis of cellular interactions post-treatment, an Immunoprecipitation Kit utilizing Protein A + G Agarose Gel (Beyotime, China) was employed on cells cultured in 10 cm tissue culture dishes (NEST). Cells were harvested using a cell scraper and subsequently lysed in an Immunoprecipitation (IP) lysis buffer, supplemented with a comprehensive mixture of protease and phosphatase inhibitors (Beyotime, China). The protein lysates were diluted to a final concentration of 10 mg/mL and incubated with either an equal volume of mouse IgG isotype control antibody or 5 µL of a β-catenin-specific antibody (1,500 µg/mL, Proteintech, China). This incubation was performed overnight at 4°C with continuous mixing on a rotatory platform. Subsequently, 40 µL of the antibody-conjugated beads were introduced to the reaction mixture and further incubated for 4 hours at 4°C on a rotatory mixer. Ultimately, the precipitated protein complexes were denatured using 5×SDS loading buffer in preparation for subsequent Western blot analysis. 2.10 Cell viability assay Cell viability assessment was conducted utilizing the Cell Counting Kit-8 (CCK-8) assay (Abbkine, China). HT29 and SW620 cells, post-treatment, were seeded into 96-well microplates (NEST) at a concentration of 1×10 3 cells per well and incubated overnight. Subsequent to the incubation period, each well was supplemented with the CCK-8 reagent and allowed to react for a duration of one hour. The optical density of the wells was subsequently quantified at a wavelength of 450 nm using a spectrophotometric plate reader. 2.11 Colony formation assay For the formation of cellular colonies, HT29 and SW620 cells, post-treatment, were inoculated into 6-well culture plates (NEST) at a seeding density of 500 cells per well. After a three-week incubation period, the colonies were immobilized with a 4% paraformaldehyde solution for 30 minutes, followed by staining with a 0.1% crystal violet solution (Beyotime, China) for an additional 30 minutes. Quantitative analysis of the colonies was performed using FIJI image processing software (version 1.0). 2.12 Transwell assay A 24-well plate was put into a Transwell chamber (BIOFIL, China) with 500 µL of complete medium supplied to each well. After that, 200 µL of serum-free media with 2×10 5 cells per well was added, and the mixture was incubated for 24 to 72 hours at 37°C with 5% CO2. Following incubation, the chamber was taken out and given two gentle PBS washes. A cotton swab was used to gently clean cells from the top chamber that did not move through the chamber. After 30 minutes of 4% paraformaldehyde fixation, the cells were stained for an additional 30 minutes with 0.1% crystal violet. Images were taken using a microscope when the chamber was turned upside down onto a glass slide. For statistical analysis, FIJI software (version 1.0) was used. 2.13 Wound healing assay The cells were scraped using a 10 µL sterile pipette and grown in serum-free media for 48 hours once the cell density in the 6-well plate exceeded 90%. Using a microscope, pictures of the scratch region were taken at 0 hours, 24 hours, and 48 hours. To assess the percentage decrease in the scratch area at 48 hours compared to the original measurement at 0 hours, the scratch area was examined using FIJI software (version 1.0). 2.14 Xenograft animal model The Institution of Medical Biology of the Chinese Academy of Medical Sciences, Peking Union Medical College, provided the BALB/c nude mice, four-week-old male. For in vivo research, mice were given subcutaneous injections of 5×106 HT29 or SW620 cells in 100 µL of serum-free growth media under their armpits. Every day, body weight and tumor volume (length and breadth) were noted. The following formula was used to determine tumor volumes: tumor volume = length×width×width / 2. The mice were killed two weeks later, and the tumors were removed. The xenografts were separated into three sections for qRT-PCR, Western blot, HE staining, and immunohistochemical staining in order to further examine the stimulating impact of MUC1 in vivo. 2.15 Statistical analysis The data was analyzed by GraphPad Prism (Version 10.2.0), which included One-Way ANOVA, correlation analysis, and t-tests. The threshold for statistical significance was set at p < 0.05. 3. RESULTS 3.1 MUC1 and β-catenin are abnormally highly expressed in colorectal cancer, and patients with both high expression have a worse prognosis. We used the TCGA database to examine the mRNA levels of MUC1 and β-catenin in tumors and their corresponding normal tissues in order to compare these proteins expression between tumor and normal tissues in colorectal cancer (Fig. 1 A-B). Higher expression of either MUC1 or β-catenin was linked to patient age, tumor stage, and TNM stage, according to our study of the association between these proteins and patient clinical features (Fig. 1 C-D). The survival curves for both markers did not, however, differ significantly between the groups with high and low expression. The survival curves of the HMUC1_HCTNNB1 group and the LMUC1_LCTNNB1 group differed significantly when we classified patients according to expression levels, designating the top three-quarters of MUC1 expression as the HMUC1 group and the remaining one-quarter as the LMUC1 group, and the top one-quarter of β-catenin expression as the HCTNNB1 group and the remaining three-quarters as the LCTNNB1 group (Fig. 1 E). Interestingly, there was no discernible difference in the survival curves between the HMUC1_HCTNNB1 and LMUC1_LCTNNB1 groups when we set the median as the threshold for high and low expression of MUC1 and β-catenin. We suggest that whereas MUC1 elevation is less common and weaker in colorectal cancer, β-catenin elevation is more common and much greater than baseline levels. MUC1 expression may have an impact on survival outcomes only once it surpasses a certain threshold. Additionally, the HMUC1_HCTNNB1 group is linked to microsatellite instability (MSI) (Fig. 1 F), but not to tumor mutational burden (TMB) (Fig. 1 H) or neoantigen levels (Fig. 1 G). Furthermore, qPCR and Western blot analysis were used to confirm these findings in our patient cohort using four typical matched colorectal cancer samples and nearby normal frozen tissues (Fig. 1 I-Q). These findings lead us to hypothesize that MUC1 and β-catenin overexpression may be important in influencing colorectal cancer behavior. 3.2 In APC mutant colorectal cell lines HT29 and SW620, knockdown of MUC1 inhibits the proliferation, migration, and invasion of tumor cells. We used siRNA to knock down MUC1 in APC mutant colorectal cell lines HT29 and SW620 in order to ascertain the function of MUC1 in colorectal cancer. Western blot (Fig. 2 A-C), qRT-PCR (Fig. 2 D-E), and immunofluorescence (Fig. 2 F-H) were used to evaluate the effectiveness of knockdown. The Cell Counting Kit-8 (CCK-8) test (Fig. 3 A-B) and colony formation assays (Fig. 3 C-F) showed that MUC1 knockdown in HT29 and SW620 cells led to decreased proliferation in comparison to control cells. Furthermore, it was shown by transwell assays (Fig. 3 G-K) and wound healing assays (Fig. 3 M-P) that MUC1 knockdown markedly reduced the HT29 and SW620 cells' capacity for migration, invasion, and wound healing. In conclusion, the si-MUC1 group markedly reduced the capacity of APC mutant HT29 and SW620 cells to proliferate, migrate, and invade. 3.3 In APC mutant colorectal cell lines HT29 and SW620, overexpression of MUC1 promotes the proliferation, migration, and invasion of tumor cells. In contrast, we used lentivirus or control lentivirus to infect APC mutant colorectal cell lines HT29 and SW620 in order to stably overexpress MUC1 and examine its function in colorectal cancer. Western blot (Fig. 4 B-D), qRT-PCR (Fig. 4 E-F), fluorescence and bright field imaging (Fig. 4 A), and immunofluorescence (Fig. 4 G-I) were used to demonstrate the overexpression efficiency. The Cell Counting Kit-8 (CCK-8) test (Fig. 5 A-B) and colony formation assays (Fig. 5 C-F) demonstrated that HT29 and SW620 cells overexpressing MUC1 proliferated more than control cells. Additionally, MUC1 overexpression markedly enhanced the migration, invasion, and wound healing capacities of HT29 and SW620 cells, as shown by transwell assays (Fig. 5 G-K) and wound healing tests (Fig. 5 M-P). In summary, the oe-MUC1 group significantly improved the APC mutant HT29 and SW620 cells' capacities for invasion, migration, and proliferation. 3.4 In APC mutant colorectal cell lines HT29 and SW620, β-catenin regulates the expression of MUC1 mRNA and protein. As is well known, mutations in the APC gene cause aberrant accumulation of β-catenin in the nucleus and cytoplasm and alter complex function in individuals with Familial Adenomatous Polyposis (FAP). This leads to a number of molecular alterations that encourage the growth of tumors and impair regular cellular function. We looked at five cell lines: the normal intestinal epithelial cell line (NCM460), APC mutant cell lines (HT29 and SW620), and APC non-mutant cell lines (DLD-1 and RKO) in order to determine the precise function of MUC1 in the formation of FAP. Western blot examination revealed that APC mutant cells had much greater levels of MUC1 and β-catenin expression than normal cells, while APC non-mutant cells had significantly lower levels of these proteins than normal cells (Fig. 6 A). This implies that, in the setting of APC mutations, MUC1 could play a role in the development of FAP. We hypothesize that MUC1 may contribute to the transition from FAP adenoma to adenocarcinoma because of its elevated expression levels, which may impact patients' clinical prognoses. Our earlier investigation showed a substantial link between MUC1 and β-catenin. By suppressing, knocking down, or overexpressing β-catenin, we were able to identify changes in MUC1 at both the mRNA and protein levels, which helped us elucidate the precise link between them. First, the transcription and translation levels of MUC1 were significantly downregulated when we treated HT29 and SW620 cells with the β-catenin inhibitor MSAB at a dose of 10 µM for 48 hours (Fig. 6 B-C, H). Second, we noticed the similar downregulation of MUC1 (Fig. 6 D-E, H) as with MSAB therapy when β-catenin was knocked down using siRNA. On the other hand, transfection with a plasmid to overexpress β-catenin seems to increase MUC1 expression (Fig. 6 F-H). 3.5 In APC mutant colorectal cell lines HT29 and SW620, MUC1 and β-catenin exhibit a phenomenon of protein interaction. We used the STRING database ( https://cn.stringdb.org/ ) to identify possible proteins that may interact with MUC1 in order to further investigate the link between MUC1 and β-catenin proteins. CTNNB1, MUC4, MUC6, MUC5AC, EGFR, ERBB2, SRC, LGALS3, ICAM1, and SIGLEC1 were among the noteworthy possibilities (Fig. 7 A). Furthermore, we used PyMOL (Version 3.1.3) and the Alphafold website ( https://alphafold.ebi.ac.uk/ ) to perform molecular docking experiments between MUC1 and β-catenin. A 2D molecular docking picture (Fig. 7 B), a 3D molecular docking diagram (Fig. 7 C), and an examination of the development of salt bridges, hydrogen bonds, and hydrophobic interactions between the AB chains were among the findings. The binding energy of the AB chains was − 13.7 kcal/mol (Fig. 7 D). The tail of MUC1's intracellular portion, known as MUC1-C, can wrap around the 7–12 repeat sequences of the stable region of β-catenin to create a U-shaped domain. This implies that their stability could be aided by the development of the MUC1-β-catenin complex. Moreover, MUC1 and β-catenin protein binding was shown by immunofluorescence tests in APC mutant cells (HT29 and SW620). MUC1 is shown by red fluorescence, β-catenin by green fluorescence, and DAPI indicating nuclear localization by blue fluorescence. When MUC1 and β-catenin are co-localized, their binding is shown by yellow or orange fluorescence (Fig. 8 A-B). Notably, we saw possible binding events in the nucleus or regions near the nuclear membrane in addition to the binding of these two proteins in the cytoplasm (Fig. 8 Ae-Be). TIn APC mutant colorectal cancer cells, the overabundance of β-catenin may trigger downstream signaling pathways by generating MUC1-β-catenin complexes that reach the nucleus through nuclear pores, a behavior that has never been seen before. Furthermore, MUC1 and β-catenin co-immunoprecipitation tests were conducted on five cell lines: APC non-mutant cell lines (DLD-1 and RKO), APC mutant cell lines (HT29 and SW620), and normal intestinal epithelial cell lines (NCM460). MUC1 and β-catenin interacted in the APC mutant cell lines (HT29 and SW620) (Fig. 8 C), but not in the APC wild-type cell lines (DLD-1, RKO) or normal intestinal epithelial cell line (NCM460) (Fig. 8 D). 3.6 In APC mutant colorectal cell lines HT29 and SW620, the regulation of MUC1 or β-catenin expression can promote or inhibit the binding or dissociation of MUC1-β-catenin complexes, leading to alterations in key molecules within downstream signaling pathways. We evaluated the complex's expression levels by immunoprecipitation after MUC1 knockdown or overexpression (Fig. 9 A), treatment with the β-catenin inhibitor MSAB, or β-catenin knockdown in order to better understand how MUC1 and β-catenin affect the MUC1-β-catenin complex. According to the experimental findings, downregulating the expression of either MUC1 or β-catenin prevents the MUC1-β-catenin complex from forming and inhibits the production of important molecules in the downstream Wnt signaling pathways (Fig. 9 B). On the other hand, the opposite outcome occurs when MUC1 expression is increased. 3.7 In vivo, overexpression of MUC1 promotes the growth of xenograft tumors and increase the expression of MUC1 and β-catenin, facilitating the formation of MUC1-β-catenin complexes. MUC1 overexpression (oe-MUC1) dramatically increases the tumor formation capacity of APC mutant cell lines HT29 and SW620, as shown by xenograft tumors removed from BALB/c nude mice (Fig. 10 A, F). The tumors in the oe-MUC1 groups are noticeably bigger than those in the oe-NC groups, according to the examination of tumor development curves (Fig. 10 B, G), tumor volume (Fig. 10 C, H), and tumor weight (Fig. 10 D, I). Interestingly, the mice's body weights (D, I) are identical. The xenograft tumors' overexpression of MUC1 and β-catenin is confirmed by Western blot analysis (Fig. 10 K, N-Q), qRT-PCR (Fig. 10 R-U), immunohistochemical tests (Fig. 11 A-E), and immunofluorometric assays (Fig. 11 F-G). It's interesting to note that whereas MUC1 overexpression in vivo dramatically raises β-catenin expression, this impact is less noticeable in vitro. On the other hand, we also saw that MUC1 and β-catenin interacted in vivo (Fig. 12 A-B). 4. DISCUSSION Mutations in the APC gene cause abnormal β-catenin breakdown, which causes it to accumulate in intestinal epithelial cells and is the hallmark of Familial Adenomatous Polyposis (FAP). The development of adenomas from these cells is largely dependent on this accumulation. Genes that support the development of adenocarcinoma are altered when β-catenin levels fluctuate quantitatively, causing qualitative changes in downstream signaling pathways. According to our research, colorectal cancer (CRC) tissues overexpress both MUC1 and β-catenin, which is associated with a worse prognosis. MUC1 knockdown dramatically reduced tumor cell migration, invasion, and proliferation in APC mutant cell lines including HT29 and SW620, but overexpression had the reverse impact. These findings highlight β-catenin's regulatory function in MUC1 expression and imply that their connection is essential for WNT/β-catenin signaling pathway activation. The bidirectional control of β-catenin degradation is crucial to the traditional WNT signaling pathway. Disrupting aberrant WNT/β-catenin signaling and stopping tumor growth can be achieved by blocking β-catenin's transfer from the cytoplasm to the nucleus 34 . We explicitly highlight how MUC1 and β-catenin interact, precisely how the MUC1 protein (amino acids 1187–1245) binds to the β-catenin armadillo repeat region. The complexes co-localize in the cytoplasm, which is where this interaction mostly takes place. Furthermore, immunofluorescence research has shown that these complexes also bind to nuclear regions and the nuclear envelope. It is noteworthy that MUC1 overexpression facilitates the nuclear translocation of β-catenin by improving the synthesis of the MUC1-β-catenin complex, extending its half-life, and increasing its stability 35 . These mechanisms contribute to the conversion of adenomas into adenocarcinomas in FAP by activating the WNT/β-catenin signaling pathways. All things considered, our study emphasizes how important the MUC1-β-catenin complex is to the development of adenocarcinoma in FAP from adenoma. A possible therapeutic approach for the prevention and treatment of FAP and related colorectal malignancies may be to target this connection. In order to prevent FAP from progressing to more advanced stages of cancer, additional research is necessary to investigate the potential of MUC1 and β-catenin as therapeutic targets and to devise ways that could interfere with their interaction. Declarations CONFLICT OF INTEREST STATEMENT The authors have no conflict of interest. ETHICS STATEMENT Ethical approval was granted by the Ethics Committee of the Third Affiliated Hospital of Kunming Medical University. FUNDING INFORMATION Key Laboratory of Cell Therapy Technology Transformation Medicine of Yunnan Province, Grant/Award Number: 2015DG034. Graduate Education Innovation Fund of Kunming Medical University in 2024, Grant/Award Number: 2024S330. Author Contribution Yinan Li: conceptualization (equal), data curation (equal), formal analysis (equal), methodology (equal), writing original draft (equal). Wenjun Shi: conceptualization (equal), writing review and editing (equal). Pei Luo: conceptualization (equal), writing review and editing (equal). Xianshuo Cheng: conceptualization (equal), writing review and editing (equal). Jun Yang: methodology (equal). Yunfeng Li: methodology (equal). Linghan Tian: writing review and editing (equal), Nanlu Ren: clinical sample collection (equal), Jian Dong: conceptualization (equal), funding acquisition (equal), project administration (equal), supervision (equal), writing review and editing (equal). ACKNOWLEDGMENTS The authors acknowledge Xin Cai, Gen Pei, Jie Du, Lan Wang for excellent technical support. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. References R.L. Siegel, N.S. Wagle, A. Cercek, R.A. Smith, A. Jemal, Colorectal cancer statistics, 2023. CA Cancer J. Clin. 73 , 233–254 (2023). 10.3322/caac.21772 N. Tomita, H. Ishida, K. Tanakaya, T. Yamaguchi, K. Kumamoto, T. Tanaka, T. Hinoi, Y. Miyakura, H. Hasegawa, T. Takayama et al., Japanese Society for Cancer of the Colon and Rectum (JSCCR) guidelines 2020 for the Clinical Practice of Hereditary Colorectal Cancer. Int. J. Clin. 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J. 70 , 731–743 (2023). 10.1507/endocrj.EJ21-0787 J. Milara, B. Ballester, P. Montero, J. Escriva, E. Artigues, M. Alos, A. Pastor-Clerigues, E. Morcillo, J. Cortijo, MUC1 intracellular bioactivation mediates lung fibrosis. THORAX. 75 , 132–142 (2020). 10.1136/thoraxjnl-2018-212735 D. Shi, X.X. Xi, Regulation of MUC6 Methylation Correlates with Progression of Gastric Cancer. Yonsei Med. J. 62 , 1005–1015 (2021). 10.3349/ymj.2021.62.11.1005 F. Maleki, F. Rezazadeh, K. Varmira, MUC1-Targeted Radiopharmaceuticals in Cancer Imaging and Therapy. Mol. Pharm. 18 , 1842–1861 (2021). 10.1021/acs.molpharmaceut.0c01249 W. Li, Y. Han, C. Sun, X. Li, J. Zheng, J. Che, X. Yao, D. Kufe, Novel insights into the roles and therapeutic implications of MUC1 oncoprotein via regulating proteins and non-coding RNAs in cancer. Theranostics. 12 , 999–1011 (2022). 10.7150/thno.63654 E. Fredericks, G. Dealtry, S. Roux, (2018). beta-Catenin Regulation in Sporadic Colorectal Carcinogenesis: Not as Simple as APC. Can J Gastroenterol Hepatol 2018 , 4379673. 10.1155/2018/4379673 A.M. Zhang, X.H. Chi, Z.Q. Bo, X.F. Huang, J. Zhang, MUC1 gene silencing inhibits proliferation, invasion, and migration while promoting apoptosis of oral squamous cell carcinoma cells. Biosci. Rep. 39 (2019). 10.1042/BSR20182193 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5722397","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":398401560,"identity":"307d59e6-5cec-49b5-b984-f599684e70d9","order_by":0,"name":"Yinan Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAo0lEQVRIiWNgGAWjYBACPiCWYGCw4eHnbyBSCxtES5qM5IwDpGk5bGPQkECsFoncgzc+1JznMWA4wPjhYw5RWvKSLWccu81jztzALDlzG1FacsykeRtu81g2HGBj5iVByzkegwMJpGk5QIoWnjfGQL8k80jOONhMnF/42XMMgSFmZ8/P33zww0ditDAIJMBYjA3EqAdZc4BIhaNgFIyCUTByAQACQy+JjRLRdAAAAABJRU5ErkJggg==","orcid":"","institution":"Yunnan Cancer Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yinan","middleName":"","lastName":"Li","suffix":""},{"id":398401561,"identity":"01db14a1-65d3-494f-a8eb-85852bfaef6e","order_by":1,"name":"Wenjun Shi","email":"","orcid":"","institution":"First People's Hospital of Yunnan Province","correspondingAuthor":false,"prefix":"","firstName":"Wenjun","middleName":"","lastName":"Shi","suffix":""},{"id":398401562,"identity":"ddf3e339-de73-497f-876a-7d57d49c71c8","order_by":2,"name":"Pei Luo","email":"","orcid":"","institution":"Yunnan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pei","middleName":"","lastName":"Luo","suffix":""},{"id":398401563,"identity":"fb3c0276-6375-4847-86b5-023027d0f440","order_by":3,"name":"Xianshuo Cheng","email":"","orcid":"","institution":"Yunnan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xianshuo","middleName":"","lastName":"Cheng","suffix":""},{"id":398401564,"identity":"f9a9803a-3fc6-4ce5-a649-b66f9156dbca","order_by":4,"name":"Jun Yang","email":"","orcid":"","institution":"First Affiliated Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Yang","suffix":""},{"id":398401565,"identity":"0e83f8c4-6a34-496e-9c80-8eb388b660e1","order_by":5,"name":"Yunfeng Li","email":"","orcid":"","institution":"Yunnan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yunfeng","middleName":"","lastName":"Li","suffix":""},{"id":398401566,"identity":"1a595f6e-4fb0-4859-afa1-80cceb82ebc5","order_by":6,"name":"Linghan Tian","email":"","orcid":"","institution":"Yunnan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Linghan","middleName":"","lastName":"Tian","suffix":""},{"id":398401567,"identity":"57bc606e-7711-495c-9af3-4b0044b08a23","order_by":7,"name":"Nanlu Ren","email":"","orcid":"","institution":"Yunnan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Nanlu","middleName":"","lastName":"Ren","suffix":""},{"id":398401568,"identity":"3a0b47bf-af74-4e75-8231-30017a6e21bd","order_by":8,"name":"Jian Dong","email":"","orcid":"","institution":"Yunnan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Dong","suffix":""}],"badges":[],"createdAt":"2024-12-27 15:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5722397/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5722397/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73315328,"identity":"b56dcdb0-6680-4acf-a2db-292d38a4b1e0","added_by":"auto","created_at":"2025-01-08 19:55:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":797244,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between the Expression Levels of MUC1 and β-Catenin Genes and Clinical Characteristics, Overall Survival, MSI, Neoantigen, and TMB in the TCGA Database (TCGA-COAD and TCGA-READ). MUC1 (A) and β-catenin (B) expression levels are much greater in colorectal cancer than in healthy tissues. TNM stage, tumor stage, and patient age are all correlated with high expression of MUC1 (C) and β-catenin (D). Individuals who expressed more MUC1 and β-catenin had lower overall survival (E), which is linked to microsatellite instability (MSI) (F) but not to tumor mutational burden (TMB) (H) or neoantigen levels (G). The mRNA (I-P) and protein (Q) expression levels of MUC1 and β-catenin were greater in cancerous tissues than in adjacent non-cancerous tissues in five pairs of colorectal cancer (CRC) adjacent and malignant tissues.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5722397/v1/03a5bee2432d5391f575b82a.png"},{"id":73315330,"identity":"0d9ac820-e76f-4270-951a-107183476306","added_by":"auto","created_at":"2025-01-08 19:55:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":302523,"visible":true,"origin":"","legend":"\u003cp\u003eVerification of si-MUC1 interference efficiency. Western blot (A-C), qRT-PCR (D-E), and immunofluorescence (F-H) were used to confirm the knockdown effectiveness of MUC1 after siRNA infection in HT29 and SW620 cells in comparison to the negative control siRNA.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5722397/v1/24b2470b5e5b80d0013c3c11.png"},{"id":73315335,"identity":"a66499e0-59a1-4049-b8e7-8da766c23d04","added_by":"auto","created_at":"2025-01-08 19:55:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":792535,"visible":true,"origin":"","legend":"\u003cp\u003eThe influence of MUC1 knockdown on APC mutant colorectal cell lines. Transwell tests (G-K), colony formation assays (C-F), wound healing assays (M-P), and CCK-8 assays (A-B) showed that MUC1 knockdown markedly reduced the HT29 and SW620 cells' capacity for invasion, migration, proliferation, and wound healing.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5722397/v1/7e0ff54475684169459a606f.png"},{"id":73316108,"identity":"845e5d40-acae-4e58-be8f-9e9a88cad7cd","added_by":"auto","created_at":"2025-01-08 20:03:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":471406,"visible":true,"origin":"","legend":"\u003cp\u003eVerification of oe-MUC1 interference efficiency. After lentivirus or control lentivirus infection, fluorescence and bright field imaging (A), Western blot (B-D), qRT-PCR (E-F), and immunofluorescence (G-I) were used to confirm the overexpression effectiveness of MUC1 in HT29 and SW620 cells.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5722397/v1/1534247291c89f849ce88b42.png"},{"id":73316114,"identity":"e583c998-0bb6-4b7b-b1d7-b42ef107e091","added_by":"auto","created_at":"2025-01-08 20:03:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":859469,"visible":true,"origin":"","legend":"\u003cp\u003eThe influence of MUC1 overexpression on APC mutation colorectal cell lines. MUC1 overexpression dramatically improved the proliferation, migration, invasion, and wound healing capacities of HT29 and SW620 cells, as shown by CCK-8 assays (A-B), colony formation assays (C-F), transwell assays (G-K), and wound healing assays (M-P).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5722397/v1/d907b828ae6889b7119c012c.png"},{"id":73316110,"identity":"5461934d-47d8-459f-b6bb-7055b7804bfa","added_by":"auto","created_at":"2025-01-08 20:03:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":696598,"visible":true,"origin":"","legend":"\u003cp\u003eIn APC mutant cell lines HT29 and SW620, β-catenin affects the expression of MUC1 RNA and protein. The expression of MUC1 (A) and β-catenin was evaluated by Western blot analysis in the APC wild-type cell lines (DLD-1, RKO), APC mutant cell lines (HT29, SW620), and normal intestinal epithelial cell line (NCM460). MUC1 transcription (B-G) and translation (H) may be inhibited or promoted by the application of the β-catenin inhibitor MSAB (B-C), siRNA infection (D-E) to decrease β-catenin, or plasmid transfection (F-G) to overexpress β-catenin.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5722397/v1/1531e95ef86ef60ac62b98b7.png"},{"id":73315351,"identity":"88087513-c6e8-49b6-aafb-a9774c62ba2a","added_by":"auto","created_at":"2025-01-08 19:55:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":416474,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of protein interaction between MUC1 and β-catenin. Using the STRING database (https://cn.stringdb.org/), we analyzed the protein-protein interaction network (PPI) between MUC1 and β-catenin (A). Alphafold and PyMOL were used in molecular docking experiments to examine the interaction between the β-catenin and MUC1 proteins. Among the findings were a 2D and 3D molecular docking diagram (B and C), as well as an examination of the development of salt bridges, hydrophobic interactions, and hydrogen bonds between the AB chains, with a binding energy of -13.7 kcal/mol (D).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5722397/v1/6e5c1ab0abed452c4897a969.png"},{"id":73316111,"identity":"53859c4b-d577-4ac8-be2c-2122fd4e19ee","added_by":"auto","created_at":"2025-01-08 20:03:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1334755,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction between MUC1 and β-catenin in APC mutant cell lines HT29 and SW620. The APC mutant cell lines HT29 (A) and SW620 (B) were used for the co-immunoprecipitation of MUC1 and β-catenin, which was then examined by western blot. Furthermore, immunofluorescence tests demonstrated that MUC1 and β-catenin interacted in these APC mutant cell lines (HT29 and SW620) (C), although neither the normal intestinal epithelial cell line (NCM460) nor the APC wild-type cell lines (DLD-1, RKO) (D) showed this connection.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5722397/v1/27a0cf103b13041566c36746.png"},{"id":73315360,"identity":"9dec70f2-8def-41f0-b5a4-919e786e3cf8","added_by":"auto","created_at":"2025-01-08 19:55:04","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":406885,"visible":true,"origin":"","legend":"\u003cp\u003eIn APC mutant cell line HT29, the interaction between MUC1 and beta promotes the activation of Wnt signaling pathways. MUC1 knockdown (A) decreases MUC1-β-catenin binding, which inhibits downstream signals in the Wnt signaling pathway (B). On the other hand, MUC1 overexpression makes an opposite effect.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5722397/v1/d8dae32f0d841dbc48c5e3f8.png"},{"id":73315347,"identity":"a51ceca6-20ca-4d28-8ab1-411f14e93dd6","added_by":"auto","created_at":"2025-01-08 19:55:03","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":548100,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of MUC1 promotets tumorigenicity in BALB/c nude mice. Comparison of xenograft tumors removed between oe-NC and oe-MUC1 in HT29 (A) and SW620 (B) groups, as well as BALB/c nude mice. The tumor (B, G) and tumor volume (C, H) growth curves of the xenograft tumors removed from naked mice. Nude mice's body weight (D, I). xenograft tumor weights (E, J) removed from naked mice. The overexpression of MUC1 and β-catenin extracted from naked mice (K, N-Q) is estimated by Western Blot. A substantial positive association was found between MUC1 and β-catenin protein, according to correlation analysis (L, M). The overexpression of MUC1 and β-catenin mRNA extracted from naked mice (R-U) is estimated by RT-PCR.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-5722397/v1/a2e4eaf9e2082ae2962543c7.png"},{"id":73316116,"identity":"05d5fbd8-74a9-4edb-923e-7da88b9396b1","added_by":"auto","created_at":"2025-01-08 20:03:04","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":971050,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression MUC1 could increase the expression of β-catenin in vivo. Cell proliferation, MUC1, and β-catenin expression in the oe-MUC1 overexpression and oe-NC groups were assessed using H\u0026amp;E and immunohistochemistry (A-E) analysis. The oe-MUC1 group had considerably greater levels of MUC1 and β-catenin than the oe-NC group, according to immunofluorometric tests (F-G).\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-5722397/v1/f6380cdced5ce945217ff46b.png"},{"id":73315346,"identity":"fb2fbef3-3960-4ea6-845e-2052951efc8a","added_by":"auto","created_at":"2025-01-08 19:55:03","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":919852,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction of MUC1 and β-catenin in vivo. The relationship between MUC1 and β-catenin is seen in representative immunofluorescence pictures of xenograft tumors from the oe-MUC1 group that were removed from naked mice that had been injected with HT29 (A) and SW620 (B) cell lines.\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-5722397/v1/9c51641f5e36695ab871c0f9.png"},{"id":73583864,"identity":"434e4205-ef39-45f1-a8e0-381befe3d5d6","added_by":"auto","created_at":"2025-01-12 05:01:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9731610,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5722397/v1/5e741558-d025-4d37-a4de-84e4e69f0b53.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Interaction between MUC1 and β-catenin promotes transformation of adenomas to adenocarcinomas in Familial Adenomatous Polyposis","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eAs per the oncological data published by the World Health Organization (WHO), in the year 2023, colorectal carcinoma was designated as the third most frequently occurring malignancy and the second leading cause of cancer-related fatalities on a global scale\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. However, the situation is more severe in China, where both the incidence and mortality rates are second highest in CRC, with an increasing trend year by year\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In the etiology of colorectal cancer, a combination of genetic predispositions and environmental factors is typically implicated in the disease's pathogenesis and prevalence\u003csup\u003e4 5\u003c/sup\u003e. An estimated one-third of people with colorectal cancer had a hereditary susceptibility, according to incomplete statistics\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFamilial Adenomatous Polyposis (FAP), a syndrome inherited in an autosomal dominant pattern, is significantly associated with an elevated risk of colorectal cancer due to deleterious alterations in the APC tumor suppressor gene \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Notably, approximately 60% of sporadic colorectal cancer cases also exhibit mutations in the APC gene\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Nevertheless, it is still unknown how precisely these mutations promote the development of colorectal cancer from adenoma to adenocarcinoma\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Therefore, a thorough investigation into the changes in key molecules resulting from APC gene mutations is essential not only for elucidating the progression of FAP from adenoma to adenocarcinoma but also for enhancing our understanding of the developmental mechanisms underlying sporadic colorectal cancer \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMultiple adenomatous polyps in the gut and a high level of familial clusterin are hallmarks of FAP, a devastating genetic condition. Since Crisp first reported this condition (later named FAP) in 1882\u003csup\u003e11\u003c/sup\u003e, the typical pathogenesis of FAP has been in-depth documented. FAP patients frequently have the illness early in life, and as they age, they may acquire hundreds to thousands of colon and rectal polyps\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. If left untreated, there is a significant risk that these polyps will progress to adenocarcinoma between the ages of 30 and 40, posing a serious threat to the patient's life \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Currently, there are still no effective treatments available for FAP\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the 1970s, advancements in whole genome sequencing technology revealed that most FAP patients have mutations in the APC gene\u003csup\u003e15 16\u003c/sup\u003e. The APC gene, classified as a tumor suppressor, is situated within the 5q21-22 band of the human genome, encompassing roughly 108 kilobase pairs, and translates into a protein comprising 2,843 amino acid residues\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Notably, 15-exon of APC gene is the largest known exon in the human genome, accounting for 77% of the coding region and representing the most frequently mutated area\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The APC protein plays several critical roles within cells, including maintaining cell adhesion, participating in transcriptional activation, promoting cell migration, and regulating apoptosis\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUnder normal conditions, β-catenin, which is necessary for cellular metabolism and embryonic development, is degraded by the \"destruction complex\" made up of APC, GSK-3β, Axin, etc.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. From 1020aa to 2075aa, the APC protein's three 15-amino acid repeat sequences, seven 20-amino acid repeat sequences, and SAMP repeat sequences are regarded as essential structural domains that interact with β-catenin\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. APC mutations in FAP patients result in aberrant folding of the APC protein's region that should bind to β-catenin, which inhibits the \u0026ldquo;destruction complex\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Consequently, The canonical β-catenin degradation pathway, which involves ubiquitination and proteasomal degradation, is typically impaired, leading to an anomalous accumulation of β-catenin within the cellular cytoplasm. After entering the nucleus, excess β-catenin attaches itself to the transcription factor TCF4 to create a complex that abnormally promotes the transcription of downstream target genes in the Wnt signaling pathway \u003csup\u003e24 25\u003c/sup\u003e. This activation causes uncontrolled mitotic signals, contributing to the development of FAP adenomas.\u003c/p\u003e \u003cp\u003eResearch has indicated that colorectal adenomas exhibit elevated β-catenin expression. MUC1 is also significantly expressed in adenocarcinoma, in addition to β-catenin being overexpressed\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Furthermore, as polyp adenomas progress to adenocarcinoma, MUC1 expression tends to increase\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We hypothesize that MUC1 could exert a significant influence in the neoplastic evolution of the FAP process.\u003c/p\u003e \u003cp\u003eThe MUC1 gene, mapped to the 1q22 locus on chromosome 1 and comprising seven exons and six introns, encodes the MUC1 protein. This transmembrane glycoprotein, characterized by its extensive glycosylation, possesses a molecular mass of approximately 500 kilodaltons\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Two peptide segments, MUC1-C and MUC1-N, make up the MUC1 protein and are joined by non-covalent links\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. MUC1 functions as a physical barrier to stop pathogen invasion in normal cells by being polarized and dispersed on the luminal surface of epithelial cells in a highly glycosylated state\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Nevertheless, MUC1 expression is more than 100 times more in malignant tumor cells than in healthy cells. According to observations, over 70% of colorectal cancer cells have high MUC1 expression\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, along with reduced glycosylation and a lack of apical polarity. MUC1 controls a number of proteins involved in tumor growth by interacting with oncogenes, transcription factors, and co-activators. These interactions cause immune evasion, drug resistance, invasion, proliferation, epithelial-mesenchymal transition, and stemness\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on our earlier studies, we discovered that MUC1 and β-catenin are strongly related, especially when APC mutations are present. In addition to being associated with the development of FAP adenomas and the advancement of adenocarcinoma, aberrant β-catenin accumulation may also affect MUC1 function. In light of the dearth of useful research models for FAP, we intend to look more closely at the relationship and interaction between MUC1 and β-catenin in APC mutant CRC cells. In the context of APC gene alterations, we specifically want to investigate their functions and processes in the development of adenocarcinoma from FAP adenomas.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Bioinformatics analysis\u003c/h2\u003e \u003cp\u003eUsing TCGAbiolinks (version 2.22.1) with the GDCquery function, MUC1 and β-catenin expression levels were downloaded from the TCGA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) together with clinical correlations in colorectal cancer (TCGA-COAD and TCGA-READ). R (version 4.3.2) was used for analysis. Of the 620 samples, the HMUC1 group consisted of the top three-quarters of patients with elevated MUC1 expression, while the LMUC1 group consisted of the remaining one-quarter. Similarly, the HCTNNB1 group was defined as the top one-quarter of patients with high β-catenin expression, while the LCTNNB1 group was defined as the remaining three-quarters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 PPI analysis and docking modeling of MUC1 and β-catenin protein\u003c/h2\u003e \u003cp\u003eThe proteins that might interact with MUC1 were predicted using the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.stringdb.org/\u003c/span\u003e\u003cspan address=\"https://cn.stringdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Using PyMOL (Version 3.1.3) and the AlphaFold website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk/\u003c/span\u003e\u003cspan address=\"https://alphafold.ebi.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), molecular docking between MUC1 and β-catenin proteins was carried out.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Clinical specimens\u003c/h2\u003e \u003cp\u003eSpecimens of primary colorectal carcinoma and corresponding adjacent non-neoplastic tissues were procured from a cohort of ten individuals undergoing surgical intervention at the Department of Colorectal Surgery, Third Affiliated Hospital of Kunming Medical University. The investigation was sanctioned by the Ethics Committee of the Third Affiliated Hospital of Kunming Medical University, with all participants furnishing written informed consent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell culture\u003c/h2\u003e \u003cp\u003eCell lines NCM460, HT29, SW620, and DLD-1 were cultivated in DMEM/F-12 media (Gibco, USA), whilst RKO cells were cultured in RPMI 1640 medium (Gibco, USA), with 10% fetal bovine serum (FBS) (NEWZERUM, China). Cells were cultured in an incubator at 37\u0026deg;C with 5% CO2 .\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Usage of β-catenin inhibitor, gene knockdown and overexpression\u003c/h2\u003e \u003cp\u003eMSAB (MCE, China), a β-catenin inhibitor, was used at a concentration of 10 \u0026micro;M for 48 hours. The sequences of siRNAs used were 5\u0026rsquo;-UUCUCCGAACGUGUCACGUTT-3\u0026rsquo;(si-NC), 5\u0026rsquo;-GUUCAGUGCCCAGCUCUACTT-3\u0026rsquo;(si-MUC1),5\u0026rsquo;-GGGAGTGGTTTAGGCTATTTG-3\u0026rsquo;(si-CTNNB1). Following the manufacturer's instructions, all si-RNAs (GenePharma, China) were transfected into the HT29 and SW620 cell lines using Lipofectamine 2000 reagent (Thermo, USA). Lentivovirus (GenePharma, China) was used to produce MUC1 gene overexpression. Each well of a 6-well plate (NEST) contained around 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells, incubated with 2 mL of complete media for 24 hours. The system was then supplemented with 1 \u0026micro;L of polybrene and 10 \u0026micro;L of virus. The cells were moved to puromycin-containing regular media after a 24-hour period. The effectiveness of inhibition, knockdown, and overexpression was assessed using qRT-PCR, Western blotting, and immunofluorescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Western blot\u003c/h2\u003e \u003cp\u003eAfter collecting the treated cells and tissue samples, RIPA lysis buffer (Beyotime, China) enhanced with a protease and phosphatase inhibitor cocktail (Beyotime, China) was used to extract the total protein. After being separated with SDS-PAGE loading buffer (Solarbio, China), the proteins were transferred to PVDF membranes with a thickness of 0.45 \u0026micro;m (Millipore, USA). After 30 minutes of room temperature blocking with a protein-free fast blocking solution (Epizyme, China), the membrane was incubated in diluted primary antibodies for about 10 hours at 4\u0026deg;C, then underwent exposure treatment after being cleaned with TBST and incubated in diluted secondary antibodies for two hours at a room temperature. The antibodies used were as follows: β-actin (1:50000, Proteintech, China), β-catenin (1:10000, Proteintech, China), MUC1 (1:5000, Abcam, USA), phospho-GSK3 β(Ser9) (1:500, Abmart, China), GSK3 β(1:1000, MCE, China), NF-κB (1:500, MCE, China), TCF4 (1:500, MCE, China) and APC (1:500, SANTA CRUZ, USA). An antibody diluent buffer (Epizyme, China) was used to dilute each antibody to the proper amounts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 qRT-PCR\u003c/h2\u003e \u003cp\u003eFollowing the manufacturer's instructions, protein was recovered from the treated cells and tissue samples using a total RNA extraction kit (Vazyme, China). With β-actin acting as an internal reference, reverse transcription was carried out to transform RNA into cDNA using the iScript\u0026trade; cDNA Synthesis Kit (Bio-Rad, China). The iTaq Universal SYBR Green Supermix (Bio-Rad, China) was used to determine the relative RNA levels. Sangon Biotech (Shanghai, China) supplied all of the primers, and here are their sequences: β-actin(human)5\u0026rsquo;-AGAGCTACGAGCTGCCTGAC-3\u0026rsquo;,MUC1(human)5\u0026rsquo;-ACCTACCATCCTATGAGCGAGTA-3\u0026rsquo;,β-catenin(human)5\u0026rsquo;-AGGTGCTATCTGTCTGCTCTAGT-3\u0026rsquo;,β-actin(mouse)5\u0026rsquo;-GATTACTGCTCTGGCTCCTAGC-3\u0026rsquo;,β-catenin(mouse)5\u0026rsquo;-GTTCGCCTTCATTATGGACTGCC-3\u0026rsquo;, and MUC1(mouse) 5\u0026rsquo;-AGTGCCTCTGACGTGAAGTCAC-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Immunofluorescence assay\u003c/h2\u003e \u003cp\u003eAfter xenograft tumor paraffin slices were removed from naked mice, they underwent antigen retrieval, serum blocking, and an overnight primary antibody incubation at 4\u0026deg;C. The slices were then treated for 60 minutes at room temperature, shielded from light, with DAPI and a photolabeled secondary antibody. Using a fluorescent microscope, pictures were taken. Following fixation with 4% paraformaldehyde, the procedures for cell samples were the same as those for tissue samples. The following antibodies were used: MUC1 (1:200, Abcam, USA), and β-catenin (1:200, Proteintech, China). An antibody diluent buffer (Beyotime, China) was used to dilute each antibody in the proper amounts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Co-Immunoprecipitation (Co-IP) assay\u003c/h2\u003e \u003cp\u003eFor the analysis of cellular interactions post-treatment, an Immunoprecipitation Kit utilizing Protein A\u0026thinsp;+\u0026thinsp;G Agarose Gel (Beyotime, China) was employed on cells cultured in 10 cm tissue culture dishes (NEST). Cells were harvested using a cell scraper and subsequently lysed in an Immunoprecipitation (IP) lysis buffer, supplemented with a comprehensive mixture of protease and phosphatase inhibitors (Beyotime, China). The protein lysates were diluted to a final concentration of 10 mg/mL and incubated with either an equal volume of mouse IgG isotype control antibody or 5 \u0026micro;L of a β-catenin-specific antibody (1,500 \u0026micro;g/mL, Proteintech, China). This incubation was performed overnight at 4\u0026deg;C with continuous mixing on a rotatory platform. Subsequently, 40 \u0026micro;L of the antibody-conjugated beads were introduced to the reaction mixture and further incubated for 4 hours at 4\u0026deg;C on a rotatory mixer. Ultimately, the precipitated protein complexes were denatured using 5\u0026times;SDS loading buffer in preparation for subsequent Western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Cell viability assay\u003c/h2\u003e \u003cp\u003eCell viability assessment was conducted utilizing the Cell Counting Kit-8 (CCK-8) assay (Abbkine, China). HT29 and SW620 cells, post-treatment, were seeded into 96-well microplates (NEST) at a concentration of 1\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well and incubated overnight. Subsequent to the incubation period, each well was supplemented with the CCK-8 reagent and allowed to react for a duration of one hour. The optical density of the wells was subsequently quantified at a wavelength of 450 nm using a spectrophotometric plate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Colony formation assay\u003c/h2\u003e \u003cp\u003eFor the formation of cellular colonies, HT29 and SW620 cells, post-treatment, were inoculated into 6-well culture plates (NEST) at a seeding density of 500 cells per well. After a three-week incubation period, the colonies were immobilized with a 4% paraformaldehyde solution for 30 minutes, followed by staining with a 0.1% crystal violet solution (Beyotime, China) for an additional 30 minutes. Quantitative analysis of the colonies was performed using FIJI image processing software (version 1.0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Transwell assay\u003c/h2\u003e \u003cp\u003eA 24-well plate was put into a Transwell chamber (BIOFIL, China) with 500 \u0026micro;L of complete medium supplied to each well. After that, 200 \u0026micro;L of serum-free media with 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well was added, and the mixture was incubated for 24 to 72 hours at 37\u0026deg;C with 5% CO2. Following incubation, the chamber was taken out and given two gentle PBS washes. A cotton swab was used to gently clean cells from the top chamber that did not move through the chamber. After 30 minutes of 4% paraformaldehyde fixation, the cells were stained for an additional 30 minutes with 0.1% crystal violet. Images were taken using a microscope when the chamber was turned upside down onto a glass slide. For statistical analysis, FIJI software (version 1.0) was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Wound healing assay\u003c/h2\u003e \u003cp\u003eThe cells were scraped using a 10 \u0026micro;L sterile pipette and grown in serum-free media for 48 hours once the cell density in the 6-well plate exceeded 90%. Using a microscope, pictures of the scratch region were taken at 0 hours, 24 hours, and 48 hours. To assess the percentage decrease in the scratch area at 48 hours compared to the original measurement at 0 hours, the scratch area was examined using FIJI software (version 1.0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Xenograft animal model\u003c/h2\u003e \u003cp\u003eThe Institution of Medical Biology of the Chinese Academy of Medical Sciences, Peking Union Medical College, provided the BALB/c nude mice, four-week-old male. For in vivo research, mice were given subcutaneous injections of 5\u0026times;106 HT29 or SW620 cells in 100 \u0026micro;L of serum-free growth media under their armpits. Every day, body weight and tumor volume (length and breadth) were noted. The following formula was used to determine tumor volumes: tumor volume\u0026thinsp;=\u0026thinsp;length\u0026times;width\u0026times;width / 2. The mice were killed two weeks later, and the tumors were removed. The xenografts were separated into three sections for qRT-PCR, Western blot, HE staining, and immunohistochemical staining in order to further examine the stimulating impact of MUC1 in vivo.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe data was analyzed by GraphPad Prism (Version 10.2.0), which included One-Way ANOVA, correlation analysis, and t-tests. The threshold for statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003e \u003cb\u003e3.1 MUC1 and β-catenin are abnormally highly expressed in colorectal cancer, and patients with both high expression have a worse prognosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe used the TCGA database to examine the mRNA levels of MUC1 and β-catenin in tumors and their corresponding normal tissues in order to compare these proteins expression between tumor and normal tissues in colorectal cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). Higher expression of either MUC1 or β-catenin was linked to patient age, tumor stage, and TNM stage, according to our study of the association between these proteins and patient clinical features (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). The survival curves for both markers did not, however, differ significantly between the groups with high and low expression. The survival curves of the HMUC1_HCTNNB1 group and the LMUC1_LCTNNB1 group differed significantly when we classified patients according to expression levels, designating the top three-quarters of MUC1 expression as the HMUC1 group and the remaining one-quarter as the LMUC1 group, and the top one-quarter of β-catenin expression as the HCTNNB1 group and the remaining three-quarters as the LCTNNB1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Interestingly, there was no discernible difference in the survival curves between the HMUC1_HCTNNB1 and LMUC1_LCTNNB1 groups when we set the median as the threshold for high and low expression of MUC1 and β-catenin. We suggest that whereas MUC1 elevation is less common and weaker in colorectal cancer, β-catenin elevation is more common and much greater than baseline levels. MUC1 expression may have an impact on survival outcomes only once it surpasses a certain threshold. Additionally, the HMUC1_HCTNNB1 group is linked to microsatellite instability (MSI) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), but not to tumor mutational burden (TMB) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) or neoantigen levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Furthermore, qPCR and Western blot analysis were used to confirm these findings in our patient cohort using four typical matched colorectal cancer samples and nearby normal frozen tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-Q). These findings lead us to hypothesize that MUC1 and β-catenin overexpression may be important in influencing colorectal cancer behavior.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 In APC mutant colorectal cell lines HT29 and SW620, knockdown of MUC1 inhibits the proliferation, migration, and invasion of tumor cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe used siRNA to knock down MUC1 in APC mutant colorectal cell lines HT29 and SW620 in order to ascertain the function of MUC1 in colorectal cancer. Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C), qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E), and immunofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-H) were used to evaluate the effectiveness of knockdown. The Cell Counting Kit-8 (CCK-8) test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B) and colony formation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-F) showed that MUC1 knockdown in HT29 and SW620 cells led to decreased proliferation in comparison to control cells. Furthermore, it was shown by transwell assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-K) and wound healing assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM-P) that MUC1 knockdown markedly reduced the HT29 and SW620 cells' capacity for migration, invasion, and wound healing. In conclusion, the si-MUC1 group markedly reduced the capacity of APC mutant HT29 and SW620 cells to proliferate, migrate, and invade.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 In APC mutant colorectal cell lines HT29 and SW620, overexpression of MUC1 promotes the proliferation, migration, and invasion of tumor cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn contrast, we used lentivirus or control lentivirus to infect APC mutant colorectal cell lines HT29 and SW620 in order to stably overexpress MUC1 and examine its function in colorectal cancer. Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D), qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F), fluorescence and bright field imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and immunofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-I) were used to demonstrate the overexpression efficiency. The Cell Counting Kit-8 (CCK-8) test (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B) and colony formation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-F) demonstrated that HT29 and SW620 cells overexpressing MUC1 proliferated more than control cells. Additionally, MUC1 overexpression markedly enhanced the migration, invasion, and wound healing capacities of HT29 and SW620 cells, as shown by transwell assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-K) and wound healing tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM-P). In summary, the oe-MUC1 group significantly improved the APC mutant HT29 and SW620 cells' capacities for invasion, migration, and proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 In APC mutant colorectal cell lines HT29 and SW620, β-catenin regulates the expression of MUC1 mRNA and protein.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs is well known, mutations in the APC gene cause aberrant accumulation of β-catenin in the nucleus and cytoplasm and alter complex function in individuals with Familial Adenomatous Polyposis (FAP). This leads to a number of molecular alterations that encourage the growth of tumors and impair regular cellular function. We looked at five cell lines: the normal intestinal epithelial cell line (NCM460), APC mutant cell lines (HT29 and SW620), and APC non-mutant cell lines (DLD-1 and RKO) in order to determine the precise function of MUC1 in the formation of FAP. Western blot examination revealed that APC mutant cells had much greater levels of MUC1 and β-catenin expression than normal cells, while APC non-mutant cells had significantly lower levels of these proteins than normal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). This implies that, in the setting of APC mutations, MUC1 could play a role in the development of FAP. We hypothesize that MUC1 may contribute to the transition from FAP adenoma to adenocarcinoma because of its elevated expression levels, which may impact patients' clinical prognoses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur earlier investigation showed a substantial link between MUC1 and β-catenin. By suppressing, knocking down, or overexpressing β-catenin, we were able to identify changes in MUC1 at both the mRNA and protein levels, which helped us elucidate the precise link between them. First, the transcription and translation levels of MUC1 were significantly downregulated when we treated HT29 and SW620 cells with the β-catenin inhibitor MSAB at a dose of 10 \u0026micro;M for 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C, H). Second, we noticed the similar downregulation of MUC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-E, H) as with MSAB therapy when β-catenin was knocked down using siRNA. On the other hand, transfection with a plasmid to overexpress β-catenin seems to increase MUC1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5 In APC mutant colorectal cell lines HT29 and SW620, MUC1 and β-catenin exhibit a phenomenon of protein interaction.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe used the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.stringdb.org/\u003c/span\u003e\u003cspan address=\"https://cn.stringdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to identify possible proteins that may interact with MUC1 in order to further investigate the link between MUC1 and β-catenin proteins. CTNNB1, MUC4, MUC6, MUC5AC, EGFR, ERBB2, SRC, LGALS3, ICAM1, and SIGLEC1 were among the noteworthy possibilities (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Furthermore, we used PyMOL (Version 3.1.3) and the Alphafold website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk/\u003c/span\u003e\u003cspan address=\"https://alphafold.ebi.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to perform molecular docking experiments between MUC1 and β-catenin. A 2D molecular docking picture (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), a 3D molecular docking diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), and an examination of the development of salt bridges, hydrogen bonds, and hydrophobic interactions between the AB chains were among the findings. The binding energy of the AB chains was \u0026minus;\u0026thinsp;13.7 kcal/mol (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). The tail of MUC1's intracellular portion, known as MUC1-C, can wrap around the 7\u0026ndash;12 repeat sequences of the stable region of β-catenin to create a U-shaped domain. This implies that their stability could be aided by the development of the MUC1-β-catenin complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, MUC1 and β-catenin protein binding was shown by immunofluorescence tests in APC mutant cells (HT29 and SW620). MUC1 is shown by red fluorescence, β-catenin by green fluorescence, and DAPI indicating nuclear localization by blue fluorescence. When MUC1 and β-catenin are co-localized, their binding is shown by yellow or orange fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B). Notably, we saw possible binding events in the nucleus or regions near the nuclear membrane in addition to the binding of these two proteins in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e Ae-Be). TIn APC mutant colorectal cancer cells, the overabundance of β-catenin may trigger downstream signaling pathways by generating MUC1-β-catenin complexes that reach the nucleus through nuclear pores, a behavior that has never been seen before. Furthermore, MUC1 and β-catenin co-immunoprecipitation tests were conducted on five cell lines: APC non-mutant cell lines (DLD-1 and RKO), APC mutant cell lines (HT29 and SW620), and normal intestinal epithelial cell lines (NCM460). MUC1 and β-catenin interacted in the APC mutant cell lines (HT29 and SW620) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC), but not in the APC wild-type cell lines (DLD-1, RKO) or normal intestinal epithelial cell line (NCM460) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.6 In APC mutant colorectal cell lines HT29 and SW620, the regulation of MUC1 or β-catenin expression can promote or inhibit the binding or dissociation of MUC1-β-catenin complexes, leading to alterations in key molecules within downstream signaling pathways.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe evaluated the complex's expression levels by immunoprecipitation after MUC1 knockdown or overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA), treatment with the β-catenin inhibitor MSAB, or β-catenin knockdown in order to better understand how MUC1 and β-catenin affect the MUC1-β-catenin complex. According to the experimental findings, downregulating the expression of either MUC1 or β-catenin prevents the MUC1-β-catenin complex from forming and inhibits the production of important molecules in the downstream Wnt signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). On the other hand, the opposite outcome occurs when MUC1 expression is increased.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.7 In vivo, overexpression of MUC1 promotes the growth of xenograft tumors and increase the expression of MUC1 and β-catenin, facilitating the formation of MUC1-β-catenin complexes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMUC1 overexpression (oe-MUC1) dramatically increases the tumor formation capacity of APC mutant cell lines HT29 and SW620, as shown by xenograft tumors removed from BALB/c nude mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA, F). The tumors in the oe-MUC1 groups are noticeably bigger than those in the oe-NC groups, according to the examination of tumor development curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB, G), tumor volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC, H), and tumor weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eD, I). Interestingly, the mice's body weights (D, I) are identical. The xenograft tumors' overexpression of MUC1 and β-catenin is confirmed by Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eK, N-Q), qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eR-U), immunohistochemical tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA-E), and immunofluorometric assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eF-G). It's interesting to note that whereas MUC1 overexpression in vivo dramatically raises β-catenin expression, this impact is less noticeable in vitro. On the other hand, we also saw that MUC1 and β-catenin interacted in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eMutations in the APC gene cause abnormal β-catenin breakdown, which causes it to accumulate in intestinal epithelial cells and is the hallmark of Familial Adenomatous Polyposis (FAP). The development of adenomas from these cells is largely dependent on this accumulation. Genes that support the development of adenocarcinoma are altered when β-catenin levels fluctuate quantitatively, causing qualitative changes in downstream signaling pathways.\u003c/p\u003e \u003cp\u003eAccording to our research, colorectal cancer (CRC) tissues overexpress both MUC1 and β-catenin, which is associated with a worse prognosis. MUC1 knockdown dramatically reduced tumor cell migration, invasion, and proliferation in APC mutant cell lines including HT29 and SW620, but overexpression had the reverse impact. These findings highlight β-catenin's regulatory function in MUC1 expression and imply that their connection is essential for WNT/β-catenin signaling pathway activation.\u003c/p\u003e \u003cp\u003eThe bidirectional control of β-catenin degradation is crucial to the traditional WNT signaling pathway. Disrupting aberrant WNT/β-catenin signaling and stopping tumor growth can be achieved by blocking β-catenin's transfer from the cytoplasm to the nucleus\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. We explicitly highlight how MUC1 and β-catenin interact, precisely how the MUC1 protein (amino acids 1187\u0026ndash;1245) binds to the β-catenin armadillo repeat region. The complexes co-localize in the cytoplasm, which is where this interaction mostly takes place. Furthermore, immunofluorescence research has shown that these complexes also bind to nuclear regions and the nuclear envelope. It is noteworthy that MUC1 overexpression facilitates the nuclear translocation of β-catenin by improving the synthesis of the MUC1-β-catenin complex, extending its half-life, and increasing its stability\u003csup\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. These mechanisms contribute to the conversion of adenomas into adenocarcinomas in FAP by activating the WNT/β-catenin signaling pathways.\u003c/p\u003e \u003cp\u003eAll things considered, our study emphasizes how important the MUC1-β-catenin complex is to the development of adenocarcinoma in FAP from adenoma. A possible therapeutic approach for the prevention and treatment of FAP and related colorectal malignancies may be to target this connection. In order to prevent FAP from progressing to more advanced stages of cancer, additional research is necessary to investigate the potential of MUC1 and β-catenin as therapeutic targets and to devise ways that could interfere with their interaction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST STATEMENT\u003c/h2\u003e \u003cp\u003eThe authors have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003e \u003cb\u003eETHICS STATEMENT\u003c/b\u003e \u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cp\u003ewas granted by the Ethics Committee of the Third Affiliated Hospital of Kunming Medical University.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING INFORMATION\u003c/h2\u003e \u003cp\u003eKey Laboratory of Cell Therapy Technology Transformation Medicine of Yunnan Province, Grant/Award Number: 2015DG034. Graduate Education Innovation Fund of Kunming Medical University in 2024, Grant/Award Number: 2024S330.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYinan Li: conceptualization (equal), data curation (equal), formal analysis (equal), methodology (equal), writing original draft (equal). Wenjun Shi: conceptualization (equal), writing review and editing (equal). Pei Luo: conceptualization (equal), writing review and editing (equal). Xianshuo Cheng: conceptualization (equal), writing review and editing (equal). Jun Yang: methodology (equal). Yunfeng Li: methodology (equal). Linghan Tian: writing review and editing (equal), Nanlu Ren: clinical sample collection (equal), Jian Dong: conceptualization (equal), funding acquisition (equal), project administration (equal), supervision (equal), writing review and editing (equal).\u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e \u003cp\u003eThe authors acknowledge Xin Cai, Gen Pei, Jie Du, Lan Wang for excellent technical support.\u003c/p\u003e\u003ch2\u003eDATA AVAILABILITY STATEMENT\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eR.L. Siegel, N.S. Wagle, A. Cercek, R.A. Smith, A. Jemal, Colorectal cancer statistics, 2023. CA Cancer J. 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Rep. \u003cb\u003e39\u003c/b\u003e (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1042/BSR20182193\u003c/span\u003e\u003cspan address=\"10.1042/BSR20182193\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Familial Adenomatous Polyposis (FAP), adenomas, adenocarcinoma, MUC1, β-catenin, protein interactions","lastPublishedDoi":"10.21203/rs.3.rs-5722397/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5722397/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThis study aims to elucidate the mechanisms of interaction between MUC1 and β-catenin in the transformation from adenoma to adenocarcinoma in FAP, providing insights for potential interventions in this progression and establishing a scientific basis for the prevention and treatment of FAP.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e(1)Bioinformatics and Clinical Specimen Analysis to investigate the differential expression of MUC1 and β-catenin and their association with clinical characteristics, including tumor staging, grading, metastasis, and survival outcomes in CRC. (2)Confirmation of MUC1-β-catenin Complexes using Alphafold, PyMol, immunofluorescence, and co-immunoprecipitation techniques. (3)Regulation of β-catenin through inhibitors, siRNA, and lentivirus to modulate β-catenin levels and assess MUC1 expression. (4)Biological behavior observation to evaluate changes in the biological behavior of APC mutant cells following MUC1 knockdown or overexpression.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e(1)Both MUC1 and β-catenin are overexpressed in colorectal cancer, with high expression correlating with poor prognosis. (2)In APC mutant colorectal cell lines, MUC1 knockdown inhibits cell proliferation, migration, and invasion, while MUC1 overexpression enhances these behaviors. (3)Both in vitro and in vivo models, β-catenin regulates MUC1 expression and interacts with it. Modulating MUC1 or β-catenin influences the binding or dissociation of MUC1-β-catenin complexes, impacting downstream signaling pathways.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn FAP patients, APC gene mutations impair β-catenin degradation within the WNT signaling pathway. Consequently, MUC1 is recruited around free β-catenin, forming a stable complex that promotes the progression from adenoma to adenocarcinoma by activating the WNT/β-catenin pathways. Inhibiting the interaction between MUC1 and β-catenin may offer significant potential for the prevention and treatment of FAP.\u003c/p\u003e","manuscriptTitle":"Interaction between MUC1 and β-catenin promotes transformation of adenomas to adenocarcinomas in Familial Adenomatous Polyposis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-08 19:54:58","doi":"10.21203/rs.3.rs-5722397/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c6da14d9-abae-4212-9614-b93b6382d59a","owner":[],"postedDate":"January 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-12T04:53:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-08 19:54:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5722397","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5722397","identity":"rs-5722397","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}