In vitro and in vivo studies on the impact of the familial adenomatous polyposis heterogeneous mutation MUC20-S671C on colorectal carcinogenesis and progression

Abstract

Background

Familial adenomatous polyposis (FAP) is a hereditary colorectal cancer (CRC). We performed genetic testing on nine FAP patients and identified a recurrent mutation at the 671st site of the MUC20 gene—MUC20-S671C. This mutation has a detection frequency of zero in the 1000 Genomes Project database. Previous studies have demonstrated that MUC20 can promote CRC progression through epithelial-mesenchymal transition (EMT). We conducted a series of experiments to analyze the impact of this mutation on CRC cells, aiming to infer its potential role and significance in CRC patients.

Methods

We introduced the MUC20-S671C mutation into the CRC SW480 cell line using the CRISPR-Cas9 technique and established a stable cell line carrying this mutation. We then conducted various experiments to assess the effects of this mutation. The Transwell assay was used to evaluate cell invasion and migration. We also examined cell proliferation, cell cycle progression, and apoptosis rate. Furthermore, we tested the tumorigenic ability of these cells in NOD-scid IL2Rγ[null] (NSG) mice. Additionally, transcriptome sequencing was performed on both cell lines and mouse tumor tissues to obtain molecular regulatory network data, and key molecules were further validated.

Results

The results of Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and colony formation assays indicated that the proliferation ability of mutant cells was significantly reduced. The Transwell assay demonstrated a marked decline in the invasion and migration capabilities of mutant cells. Flow cytometry analysis revealed that the mutation increased the apoptosis rate of CRC cells and might have caused S-phase arrest. The tumor formation assay in nude mice showed that the tumorigenic ability of mutant cells was weakened. Transcriptome sequencing of both the cells and tumor tissues suggested that the mutation altered the expression of apoptosis- and cell cycle-related molecules and also affected EMT. Further experiments confirmed that key molecules involved in the EMT process, such as E-cadherin, were upregulated, while Vimentin, MMP9, and MMP14 were significantly downregulated, indicating that the mutation weakened the EMT capability of CRC cells.

Conclusions

We have identified a novel mutation, MUC20-S671C, in patients with FAP. Our study demonstrates that this mutation exerts its tumor-suppressive effect by reversing the EMT process.

Highlight box.

Key findings

• The MUC20-S671C mutation, identified in familial adenomatous polyposis (FAP) patients, suppresses colorectal cancer (CRC) cell proliferation, migration, invasion, and tumorigenicity while inducing apoptosis and S-phase arrest. This mutation reverses epithelial-mesenchymal transition (EMT) by upregulating E-cadherin and downregulating Vimentin, MMP9, and MMP14.

What is known and what is new?

• MUC20 is known to promote CRC progression via EMT, but the functional impact of its germline mutations in FAP remains unexplored.

• This study provides the first functional evidence that the MUC20-S671C mutation acts as a protective factor in CRC by reversing EMT, challenging the conventional pro-tumorigenic view of MUC20.

What is the implication, and what should change now?

• MUC20-S671C may serve as a novel prognostic biomarker for FAP patients and a potential therapeutic target for mimicking its protective effect. Larger cohort validation and mechanistic exploration of the mutation’s impact on EMT regulation are warranted.

Introduction

Familial adenomatous polyposis (FAP) is a hereditary colorectal cancer (CRC) (1). Due to its characteristic “polyp-adenoma-adenocarcinoma” progression, FAP serves as an ideal model for studying the mechanisms of CRC development (2).

To investigate the pathogenic genes and mutation sites in FAP patients, we performed whole-genome sequencing on nine randomly selected FAP patients from different families. In addition to identifying conventional pathogenic adenomatous polyposis coli (APC) gene mutations (3), we unexpectedly discovered 13 shared germline mutations among these patients. These 13 mutations had a detection frequency of less than 0.01% in the 1000 Genomes database, suggesting a strong correlation between these newly identified mutations and FAP, and indicating their potential role as genetic susceptibility factors for FAP.

Bioinformatics analysis revealed that, except for the MUC20 (Mucin 20) missense mutation MUC20-S671C, which was classified as a variant of “uncertain significance”, all other mutations were deemed “benign”. Additionally, the detection frequency of the MUC20-S671C mutation in the 1000 Genomes database was zero (4), indicating that MUC20-S671C is a newly identified disease-associated mutation site. However, since bioinformatics analysis did not clarify the functional impact of the MUC20-S671C mutation—meaning it could be protective, deleterious, or even neutral—we sought to determine its specific role in CRC progression.

In this study, we introduced the MUC20-S671C mutation into the CRC cell line SW480 and conducted both in vitro and in vivo experiments to assess its effects. Furthermore, we performed transcriptome sequencing to analyze key molecular alterations and regulatory networks associated with this mutation. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0904/rc).

Methods

Whole genome sequencing

We conducted whole-genome sequencing analysis on nine probands from different FAP families (5). The nine FAP probands were recruited from the Department of Oncology, The First Affiliated Hospital of Kunming Medical University between 2005 and 2018. The diagnosis of FAP was based on the presence of ≥100 colorectal adenomas and/or a confirmed APC germline mutation. We performed whole-genome sequencing analysis on these nine probands and identified shared mutations present in these patients, further analyzing their relevance using bioinformatics tools such as SIFT and Polyphen2 (6,7). The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The First Affiliated Hospital of Kunming Medical University (No. 2023-186). Informed consent was obtained from all individual participants included in the study.

Construction and identification of MUC20-S671C mutant SW480 cell line

The MUC20 gene point mutation was introduced into CRC SW480 cells using CRISPR/Cas9 gene-editing technology mediated by electroporation (8). The specific mutation was introduced in Exon 3, p.S671 (TCC to TGC). After electroporation, single clones were selected, and the homozygous mutant cell line was successfully obtained through polymerase chain reaction (PCR) and sequencing validation.

Cell culture and reagents

SW480 cells were purchased from Cyagen Biosciences. Cells were authenticated and tested for mycoplasma contamination. Both SW480-WT and SW480-MUT cell lines were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin. The cells were maintained at 37 ℃ in a humidified incubator with 5% CO₂. Cell culture was performed in 25 cm² culture flasks.

Transcriptome sequencing

Total RNA was extracted and purified from the two cell lines and corresponding tumor tissues from nude mice using TRIzol (9). The quantity and purity of total RNA were assessed using a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA), and RNA integrity was evaluated using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Sequencing was performed using the Illumina NovaSeq™ 6000 platform (LC-Bio Technology CO., Ltd., Hangzhou, China).

Cell proliferation assays

Cell Counting Kit-8 (CCK-8) assay

Both cell lines were seeded in 96-well plates at a density of 2,000 cells per well in 100 µL of culture medium. Three replicate wells were set for each cell line and time point (24, 48, 72, 96, 120 h). After the designated incubation time, 10 µL of CCK-8 reagent was added to each well and incubated at 37 ℃ in the dark for 2 hours before measuring absorbance.

Colony formation assay

Cells were seeded in 6-well plates at a density of 1,000 cells per well (three replicates per cell line). Cells were cultured in 2 mL of complete medium for 7–14 days, with the medium changed every 3–4 days. When most colonies contained more than 50 cells or had been cultured for 14 days, the cells were fixed with 4% paraformaldehyde and stained with crystal violet before imaging.

Transwell assay

Invasion assay

A 24-well plate with Matrigel-coated inserts (pore size 8 µm) was used. Matrigel (ECM550, Chemicon, Temecula, CA, USA) was diluted at a ratio of 1:8 in serum-free culture medium, and 90 µL was added to each insert. The inserts were incubated at 37 ℃ for 3 hours. Then, 50,000 cells were seeded in the upper chamber in 200 µL of serum-free RPMI 1640 medium, while the lower chamber contained 1.5 mL of complete medium with 10% FBS. After 48 hours, non-invading cells on the upper surface were removed with a cotton swab. The invaded cells on the lower surface were fixed with formaldehyde and stained with 0.1% crystal violet. Five random fields per insert were counted under an inverted microscope.

Migration assay

The migration assay was conducted using the same procedure as the invasion assay, except that Matrigel coating was omitted.

Flow cytometry

Concurrent analysis of cell cycle distribution and apoptosis was performed on SW480-WT and SW480-MUT cells using flow cytometry.

For cell cycle analysis, cells were fixed in 70% ice-cold ethanol at 4 ℃ overnight. After washing with phosphate-buffered saline (PBS), the fixed cells were stained with FxCycle™ PI/RNase Staining Solution (Thermo Fisher Scientific, Waltham, MA, USA; Catalog #: F10797) following the manufacturer’s instructions and incubated in the dark at room temperature for 30 minutes.

For apoptosis analysis, unstained single-cell suspensions were stained using the Annexin V-APC/7-AAD Apoptosis Kit (Liankebio, Hangzhou, China; Catalog #: AP105-100) according to the manufacturer’s protocol. Briefly, cells were resuspended in Annexin V Binding Buffer and incubated with Annexin V-APC and 7-AAD for 15 minutes at room temperature in the dark.

Data acquisition for both assays was performed using a (BD FACSCanto II) flow cytometer. A minimum of 10,000 events were recorded for each sample. The cell cycle distribution (G0/G1, S, and G2/M phases) was analyzed based on propidium iodide (PI) fluorescence. The percentages of apoptotic cells (Annexin V-APC+/7-AAD− for early apoptosis and Annexin V-APC+/7-AAD+ for late apoptosis) were determined from the unstained samples. All data were analyzed using FlowJo software (version 10.8).

Nude mouse xenograft model

All animal experiments were conducted at the experimental animal facility of Yunnan Zeen Biomedicine Technology Co., Ltd. (Kunming, China) under a service agreement with Kunming Medical University. All animal experiments were performed under a project license (No. kmmu20240954) granted by the Animal Ethics Committee of Kunming Medical University, in compliance with national guidelines for the care and use of animals. NOD-scid IL2Rγ[null] (NSG) mice were purchased from SPF Biotechnology (10). Male NSG mice, 6–8 weeks old, weighing 18–22 g. Mice were housed in groups of five per cage in individually ventilated cages (IVCs) under specific pathogen-free (SPF) conditions. Autoclaved corncob bedding was used, and environmental enrichment included nesting material (cotton squares) and a polycarbonate tunnel. The animal room maintained a 12-hour light/dark cycle (lights on at 7:00 AM), temperature of 22±2 ℃, and relative humidity of 50%±10%. Standard rodent chow and autoclaved water were provided ad libitum. Mice were randomly assigned to two groups using a random number generator: WT and MUT (n=10 mice per group) (all animals included in analysis; no exclusions). Sample size was determined based on power analysis of preliminary data. To establish subcutaneous xenografts, 5×10⁶ cells in the logarithmic growth phase, suspended in 100 µL of PBS, were injected into the right axilla of each mouse. No blinding was performed due to the exploratory nature of the study. Mice were monitored regularly for tumor growth. A predefined humane endpoint was set at a tumor volume of 1,000 mm³. A fixed experimental endpoint was defined at 3 weeks post-injection. Animals that reached the humane endpoint before 3 weeks were euthanized immediately. In the wild-type group, tumor growth was faster, and some mice reached the humane endpoint earlier than 3 weeks; in the mutant group, no mice reached the humane endpoint before 3 weeks. All remaining mice were sacrificed at the 3-week time point. Consequently, tumor tissues were collected at the time of euthanasia for each animal (11). The tumorigenesis was confirmed by histopathological examination using hematoxylin and eosin (H&E) staining. The collected tissues were subsequently subjected to RNA and protein extraction, as well as transcriptome sequencing.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from both cell lines and tumor tissues using the Total RNA Extraction Kit (SevenFast, Beijing, China) according to the manufacturer’s instructions. Genomic DNA was removed and first-strand cDNA was synthesized from 1 µg of total RNA using the All-in-one 1st Strand cDNA Synthesis SuperMix (gDNA Purge) (NovoProtein, Suzhou, China) on a conventional PCR instrument (Heal Force, Shanghai, China). qRT-PCR was performed using the SYBR qPCR SuperMix Plus (NovoProtein) on an Applied Biosystems (Foster City, CA, USA) quantitative PCR instrument. The reaction mixture was subjected to the following cycling protocol: initial denaturation at 95 ℃ for 1 minute, followed by 40 cycles of 95 ℃ for 30 seconds and 60 ℃ for 1 minute. The specificity of the PCR amplification was confirmed by the presence of a single peak in the melting curve analysis. The sequences of all primers, synthesized by Sangon Biotech (Shanghai, China), are listed: CDH1: forward: 5'-GCCATCGCTTACACCATCCTCAG-3', reverse: 5'-CTCTCTCGGTCCAGCCCAGTG-3'; FN1: forward: 5'-GGCGACAGGACGGACATCTTTG-3', reverse: 5'-GGCACAAGGCACCATTGGAATTTC-3'; VIM: forward: 5'-TGAATGACCGCTTCGCCAACTAC-3', reverse: 5'-CTCCCGCATCTCCTCCTCGTAG-3'; MMP9: forward: 5'-CCCTGGTCCTGGTGCTCCTG-3', reverse: 5'-CTGCCTGTCGGTGAGATTGGTTC-3'; MMP14: forward: 5'-TGCCTGCGTCCATCAACA-3', reverse: 5'-ATCACCTCCGTCTCCTCCT-3'; GAPDH (internal control): forward: 5'-GAAAGTCCGGAAGTCTCTGG-3', reverse: 5'-TAGAGACTTGGGCAGTGTGG-3'.

The relative gene expression levels were calculated using the comparative 2^−ΔΔCt method (12). Each experiment included three technical replicates and was independently repeated three times.

Western blotting analysis

Total protein was extracted from both cell lines and xenograft tumor tissues using radioimmunoprecipitation assay (RIPA) lysis buffer, and concentrations were determined by bicinchoninic acid (BCA) assay. Proteins (20 µg per lane) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies (see Table 1 for details) overnight at 4 ℃, followed by incubation with HRP-conjugated secondary antibodies. Specific protein bands were visualized using an enhanced chemiluminescence (ECL) substrate. The antibodies used are listed in Table 1.

Table 1. Antibodies used for Western blot analysis.

Target	Host species	Clonality	Vendor	Catalog number	Dilution
MMP9	Rabbit	Monoclonal	Proteintech	10375-2-AP	1:5,000
MMP14	Rabbit	Polyclonal	Proteintech	14552-1-AP	1:500
E-cadherin	Mouse	Monoclonal	Proteintech	60902-1-Ig	1:5,000
Vimentin	Mouse	Monoclonal	Proteintech	60330-1-Ig	1:20,000
GAPDH	Mouse	Monoclonal	Proteintech	60004-1-Ig	1:50,000
HRP-conjugated Goat Anti-Rabbit IgG	Goat	–	Proteintech	SA00001-2	1:1,000
HRP-conjugated Goat Anti-Mouse IgG	Goat	–	Proteintech	SA00001-1	1:1,000

Statistical analysis

Transcriptome data analysis

Differentially expressed genes (DEGs) were identified from the transcriptome sequencing data. The analysis of read count data was performed using a negative binomial distribution model. Differential expression analysis was performed using the DESeq2 package in R (version 4.2.1) (13,14). P values were adjusted for multiple testing using the Benjamini-Hochberg method to control the false discovery rate (FDR) (15). Genes with an absolute log₂ fold change of ≥1 and an FDR-adjusted P value (q-value) of < 0.05 were defined as statistically significant DEGs.

Other quantitative data

Data from qRT-PCR and functional assays are presented as the mean ± standard error of the mean (SEM) from at least three biologically independent experiments. Statistical comparisons between two groups were performed using a two-tailed Student’s t-test, data were tested for normality before applying parametric tests. A P value <0.05 was considered statistically significant.

Phosphorylation site prediction

Phosphorylation site prediction for the MUC20 protein (UniProt: Q8N307) was performed using PhosphoSitePlus (https://www.phosphosite.org), GPS 5.0 (Group-based Prediction System), and PhosphoNET. All predictions were based on the full-length human MUC20 protein sequence (709 amino acids). Kinase prediction scores were generated using default parameters; higher scores indicate greater prediction confidence.

Results

Bioinformatics analysis suggests MUC20-S671C as a novel mutation in FAP

We performed whole-genome sequencing on nine randomly selected probands from different FAP families. The clinical characteristics of the 9 sporadic FAP patients are shown in Table 2. The results revealed pathogenic germline mutations in the APC gene (16) in the majority of the samples (8/9), suggesting that APC mutations remain the primary cause of FAP in most cases (Tables S1,S2). To further investigate whether other shared mutations associated with FAP exist in these nine samples, we organized and classified the sequencing data, identifying a total of 67 shared mutations. Among them, 13 mutations had a detection frequency of less than 0.01% in the 1,000 Genomes database, indicating a high potential correlation with FAP (Table S3). Further screening using bioinformatics tools such as SIFT, Polyphen2, and MutationTaster for disease association analysis showed that, except for the missense mutation MUC20 671S→C (rs3762739), which was classified as a “variant of uncertain significance (VUS)” (17,18), all other mutations were classified as “benign” (Table S4). This suggests that MUC20-S671C may be a newly discovered FAP-associated mutation site, though its specific impact remains unknown.

Table 2. Clinical characteristics of 9 patients with FAP.

Characteristic	Number [%]
Sex
Male	6 [67]
Female	3 [33]
Age
<40 years	8 [88]
≥40 years	1 [12]
Family structure
Unknown	9 [100]
Population
China-Yunan	9 [100]
Phenotype
Nonpolyposis	0 [0]
Polyposis	9 [100]
Cancer	9 [100]
Metastasis	0 [0]

FAP, familial adenomatous polyposis.

Successful construction of MUC20-S671C mutant SW480 cells and establishment of a stable cell line

To explore the potential effects or alterations caused by this mutation in CRC cells, we introduced the point mutation Exon 3, p.S671C (TCC→TGC) into the MUC20 gene in SW480 cells using CRISPR/Cas9 gene-editing technology mediated by electroporation (Table S5).

After electroporation, single clones were selected, and successful homozygous mutant cells of the human MUC20 gene were obtained through PCR and sequencing validation (Figure S1).

The MUC20 point mutation suppresses oncogenic phenotypes in CRC cells

After establishing SW480 cells stably expressing the MUC20-S671C mutation, we conducted a series of functional assays to evaluate its impact on the malignant phenotypes of CRC cells. CCK-8, colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) assays consistently revealed that the mutation significantly suppressed cell proliferation compared to wild-type controls (Figure 1A-1C). Transwell migration and invasion assays further demonstrated that the mutant cells exhibited markedly reduced migratory and invasive capabilities (Figure 1D). Flow cytometry analysis indicated a significant increase in the total apoptosis rate in the mutant group, characterized by a slight decrease in early apoptosis and a notable increase in late apoptosis (Figure 1E). Cell cycle analysis showed a significant accumulation of mutant cells in the S phase, along with a reduced proportion in the G2 phase, suggesting S-phase arrest (Figure 1F). Taken together, these findings demonstrate that the MUC20-S671C point mutation impairs multiple oncogenic properties of CRC cells, including proliferation, migration, invasion, apoptosis evasion, and cell cycle progression.

Figure 1.

Functional characterization of SW480 cells expressing wild-type or MUC20-S671C mutant constructs. (A) Representative images and quantification of colony formation assay. After seeding, cells were cultured for 10–14 days, and visible colonies were stained with crystal violet. (B) Cell viability measured by CCK-8 assay at 24, 48, and 72 hours. (C) EdU incorporation assay. EdU-positive cells (red); nuclei counterstained with Hoechst (blue). Scale bar =100 μm. (D) Representative images and quantification of Transwell migration and invasion assays (0.1% crystal violet; scale bar: 100 µm). (E,F) Representative images and quantification of flow cytometry apoptosis and cell cycle assays. Data are presented as mean ± standard deviation. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 vs. WT (Student’s t-test). CCK-8, Cell Counting Kit-8; EdU, 5-ethynyl-2'-deoxyuridine; MUT, mutation type; WT, wild-type.

MUC20-S671C mutation attenuates tumorigenicity in CRC cells

Xenograft tumor formation in nude mice is one of the most widely used experiments to assess the tumorigenic potential of cancer cells. We conducted tumor-bearing experiments in NSG mice using both wild-type and mutant CRC cells. The results showed that mutant-type cells exhibited significantly lower tumorigenicity, with smaller average tumor sizes compared to the wild type cell group (Figure 2A-2D).

Figure 2.

Effect of mutation on the tumorigenic ability of colorectal cancer cells in vivo. Subcutaneous inoculation of WT and MUC20-S671C MUT SW480 cells (5×10⁶ cells/mouse) was performed in NSG mice (n=10/group). (A) Representative external views of tumor-bearing mice. Tumor formation in the mutant group was delayed by 1–2 days. (B) Changes in tumor volume growth after inoculation in mice. (C) Comparison of gross tumor specimens (arranged in order of decreasing volume from left to right). (D) Statistical analysis of tumor tissue volume. (E) H&E staining of WT group tumors (×100; ×200), showing features of poorly differentiated adenocarcinoma (increased nuclear-to-cytoplasmic ratio, increased pathological mitotic figures). (F) H&E staining of MUT group tumors (×100; ×200), with more regular cell arrangement and reduced mitotic figures. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. H&E, hematoxylin and eosin; NSG, NOD-scid IL2Rγ[null]; MUT, mutation type; WT, wild-type.

After obtaining tumor tissues, H&E staining was performed for histological analysis. Microscopic examination revealed significant atypia in both groups, consistent with the pathological features of colonic adenoma. However, the wild-type tumor tissues displayed more pronounced atypia, with poor differentiation, large hyperchromatic nuclei, disorganized cellular arrangement, and fewer glandular structures. In contrast, the mutant cell-derived tumors exhibited slightly better differentiation, more orderly cell arrangement, and an increased number of glandular structures (Figure 2E,2F).

These findings indicate that the introduction of the MUC20-S671C mutation significantly reduces tumorigenicity in CRC cells, further suggesting a protective role for this mutation in CRC progression.

Transcriptome sequencing analysis

Gene clustering and enrichment profiles

Transcriptome sequencing of wild-type and mutant CRC cells and their corresponding tumors revealed significant molecular differences between the two groups, as demonstrated by Pearson correlation analysis (Figure 3). This indicates that the MUC20-S671C mutation induces extensive transcriptional alterations.

Figure 3.

Impact of MUC20-S671C mutation on the transcriptome of colorectal cancer cells. (A) Pearson correlation analysis was used to assess expression consistency between cell and tumor tissue samples. Each cell line and its corresponding tumor tissue had three biological replicates, with darker colors indicating stronger correlations. (B) The number of DEGs in cells and tumor tissues was quantified, showing upregulated and downregulated genes separately. (C) Volcano plots display the distribution of DEGs in cells and tumor tissues. Red dots represent significantly upregulated genes, blue dots indicate significantly downregulated genes, and gray dots denote genes with nonsignificant expression changes. (D) GO enrichment analysis illustrates the distribution of upregulated genes in biological processes, cellular components, and molecular functions for both cells and tumor tissues. (E) Enrichment analysis was performed on the top 100 most significantly upregulated and downregulated genes in cells and tumor tissues. Red indicates higher enrichment, while blue indicates lower enrichment. The left three columns show enrichment results for the mutant group, and the right three columns display those for the wild-type group. DEG, differentially expressed gene; GO, Gene Ontology.

In the mutant group, 3,871 genes were upregulated in cells and 2,622 in tumor tissues (Figure 3B,3C). Heatmaps of the top 100 DEGs highlight the most significant changes (Figure 3E). Notably, among the upregulated genes were SEMA3A, which has been reported to suppress tumor invasion and metastasis by modulating the tumor microenvironment (19), and ROBO1, a known regulator of cell adhesion and migration that can inhibit tumor spread in certain cancers (20).

Gene Ontology (GO) analysis indicated that the upregulated genes in the mutant group were significantly enriched in pathways related to cell differentiation and transcriptional regulation (Figure 3D). These findings suggest that the MUC20-S671C mutation may enhance tumor-suppressive pathways by modulating transcriptional activity, signal transduction, and microenvironment interactions.

Differential expression of partial genes

Through analysis of overall gene expression levels, we found that after introducing the MUC20-S671C mutation, SW480 cells showed significant changes in the expression of EMT-related genes (21), as well as apoptosis and cell cycle-related genes (Tables 3,4). The analysis revealed that among cell cycle-related genes, the expression levels of genes regulating S-phase progression and affecting DNA replication and checkpoint control, such as CDK2 (22) and CDC6 (23), were decreased, which may be one of the reasons for S-phase arrest in mutant cells. Additionally, the expression levels of pro-apoptotic molecules like ATF3 (24), BAX (25) and CYCS (26) increased, while anti-apoptotic molecules like HSPB1 (27) and PIK3CB (28) decreased, overall consistent with the observed trend of increased apoptosis rates in mutant cells. Regarding the decreased early apoptosis rate in mutant cells, we speculate this may be caused by the high expression of another anti-apoptotic gene—BCL2 (29). BCL2 is a core member of the BCL-2 family and a classical anti-apoptotic gene that can inhibit the mitochondrial apoptosis pathway, primarily functioning in the early stages of apoptosis. Furthermore, the S-phase arrest occurring in mutant cells may activate internal repair mechanisms during this period, preventing apoptosis (30), which could also contribute to the decreased early apoptosis rate. However, since the cells are generally in a pro-apoptotic state, over time more cells will still irreversibly enter late apoptosis, leading to a significant increase in the overall apoptosis rate.

Table 3. The effect of introducing MUC20-S671C into SW480 on signaling molecules such as the c-MET pathway (cells).

Gene ID	Gene name	FPKM-MUT-SW480	FPKM-WT-SW480	Regulation
ENSG00000039068	CDH1	7.00	4.31	Up
ENSG00000146648	EGFR	0.38	8.34	Down
ENSG00000115414	FN1	8.21	13.69	Down
ENSG00000157227	MMP14	4.03	11.38	Down
ENSG00000123342	MMP19	0.21	0.62	Down
ENSG00000125966	MMP24	0.54	5.65	Down
ENSG00000026025	VIM	5.87	102.90	Down
ENSG00000162772	ATF3	69.77	8.97	Up
ENSG00000087088	BAX	13.72	11.40	Up
ENSG00000106211	HSPB1	18.06	112.40	Down
ENSG00000171791	BCL2	0.47	0.2	Up
ENSG00000123374	CDK2	9.2	24.9	Down
ENSG00000145386	CCNA2	22.4	31.8	Down
ENSG00000094804	CDC6	10.8	30.2	Down
ENSG00000073111	MCM2	39.6	62.4	Down

Table 4. The effect of introducing MUC20-S671C into SW480 on signaling molecules such as the c-MET pathway (subcutaneous tumors in nude mice).

Gene ID	Gene name	FPKM-MUT-SW480	FPKM-WT-SW480	Regulation
ENSG00000146648	EGFR	0.46	9.50	Down
ENSG00000039068	CDH1	18.30	7.93	Up
ENSG00000115414	FN1	0.81	7.28	Down
ENSG00000026025	VIM	25.68	413.36	Down
ENSG00000157227	MMP14	18.22	27.82	Down
ENSG00000100985	MMP9	0.04	3.59	Down
ENSG00000172115	CYCS	61.86	50.25	Up
ENSG00000106211	HSPB1	230.99	439.47	Down
ENSG00000051382	PIK3CB	4.10	6.59	Down
ENSG00000171791	BCL2	0.53	0.22	Up
ENSG00000123374	CDK2	20.60	43.60	Down
ENSG00000094804	CDC6	12.90	23.9	Down
ENSG00000101412	E2F1	21.40	32	Down

Protein-protein interaction (PPI) network analysis

To further identify potential regulatory hub genes associated with the MUC20-S671C mutation, PPI networks were constructed using the DEGs identified from both the SW480 cellular and xenograft tumor transcriptomic datasets [Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, confidence score threshold ≥0.4 (31)]. In SW480 cells, the PPI network comprised 228 interaction pairs, with CCND1, HSP90AA1, CD44, CXCR4, and EPAS1 exhibiting the highest connectivity (Figure 4A). In xenograft tumors, the PPI network comprised 267 interaction pairs, with EGFR, FN1, ITGAV, WNT5A, and SERPINE1 emerging as the principal hub genes (Figure 4B). These hub genes are mainly involved in cell-cycle regulation, cell adhesion, extracellular matrix remodeling, growth factor signaling, and EMT-related biological processes. Notably, several of these hub genes—including EGFR, FN1, WNT5A, CD44, and CXCR4—have established roles in epithelial-mesenchymal transition (EMT) and CRC progression (32). These hub genes may represent candidate regulatory molecules associated with the biological effects of the MUC20-S671C mutation and warrant further mechanistic investigation.

Figure 4.

Protein-protein interaction networks associated with MUC20-S671C. (A) PPI network constructed from differentially expressed genes in SW480 MUC20-S671C versus SW480 WT cells. (B) PPI network constructed from differentially expressed genes in MUC20-S671C versus WT xenograft tumors. Hub genes with the highest connectivity are highlighted in both panels. Edge thickness corresponds to interaction confidence score; node size reflects connectivity degree. PPI, protein-protein interaction; WT, wild-type.

In addition, gene set enrichment analysis (GSEA) was performed on the ranked transcriptome datasets using GO, KEGG gene sets. Phosphorylation site prediction details are provided in Tables S6,S7. Summarized GSEA results are provided in Table S8.

The mutation reduces the EMT transformation ability of CRC cells

Based on transcriptome sequencing results, we found significant differences in the expression of EMT-related genes between the two types of cells, suggesting that after the mutation was introduced, the EMT ability of SW480 cells was weakened, leading to a decrease in the motility and migration ability of CRC cells (33,34). We observed changes in the expression of genes related to EMT, such as MMPs, E-cadherin, Vimentin, and Fibronectin, and thus, we validated the expression of these genes (35-38).

The results showed that after the introduction of the MUC20-S671C mutation, the expression of molecules promoting metastasis, including MMP9, MMP14, Vimentin, and Fibronectin, were downregulated, while the expression of the typical molecule inhibiting metastasis, E-cadherin, was upregulated (Figure 5). This suggests that the mutation can suppress CRC cell metastasis by inhibiting the EMT process.

Figure 5.

MUC20-S671C mutation downregulates the expression of pro-invasive EMT-related genes in SW480 cells and xenograft tumors. (A,B) qRT-PCR analysis shows that compared with the WT group, the mRNA expression of pro-invasive EMT genes (FN1, VIM, MMP9, MMP14) was significantly downregulated, while the expression of the epithelial marker CDH1 was upregulated in MUT SW480 cells (A) and their derived xenograft tissues (B). (C-F) Western blot results verified these changes at the protein level. The expression of Vimentin, MMP9, and MMP14 proteins was decreased, while E-cadherin protein expression was increased in MUT cells (C,E) and xenograft tissues (D,F). β-actin was used as a loading control. All data are presented as mean ± standard deviation (n=3). *, P<0.05; ***, P<0.001; ****, P<0.0001; ns, not significant vs. the WT group (Student’s t-test). EMT, epithelial-mesenchymal transition; MUT, mutation type; qRT-PCR, quantitative real-time polymerase chain reaction; WT, wild type.

Discussion

Mucins (MUCs) are high-molecular-weight epithelial glycoproteins consisting of 24 members (MUC1 to MUC24) (39). Mucins constitute a large part of the mucus covering the epithelial surfaces of organ cavities, serving as a selective physical barrier between the extracellular environment and the cell membrane and interior, providing protection and lubrication for the ducts and cavities in the human body (40). Mucins are also involved in cell or cell-matrix interactions, signal transduction, and mediating various processes such as wound healing, immune response regulation, and metastasis (41). Additionally, some mucins have been found to be associated with cell proliferation, differentiation, and tumorigenesis (42).

FAP is a special type of hereditary CRC that has a typical “polyp-adenoma-adenocarcinoma” progression, making it an ideal model for studying the development of CRC (43). In our recent research on FAP, we discovered a phosphorylation site missense mutation SNP directly related to the progression of FAP, namely MUC20-S671C. The MUC20-S671C mutation is a mutation at the 671st position of the MUC20 gene, where a serine codon carrying a phosphate group is replaced by cysteine (S→C), resulting in the loss of the phosphate group. We found that MUC20-S671C is present in all the samples tested, and its mutation frequency is zero in the 1000 Genomes Project population, suggesting that MUC20-S671C may be a shared genetic susceptibility factor in some cases of FAP.

MUC20 is a mucin, and as a recently discovered mucin, MUC20 is expressed in normal tissues such as the colon, esophagus, gallbladder, kidneys, and bladder (44). To date, research on MUC20 remains limited, mainly focusing on the relationship between MUC20 and various human organ tumors. It has been found that changes in MUC20 expression can affect the malignant phenotype of tumor cells, thereby influencing patient prognosis. For instance, Xue et al. found that high expression of MUC20 is associated with the occurrence of clear cell renal carcinoma and poor prognosis in patients (40). High expression of MUC20 also promotes the occurrence and development of endometrial cancer (45,46) and ovarian cancer (47), and is similarly associated with poor prognosis in patients. Furthermore, Xiao et al. found that overexpression of MUC20 enhances the invasiveness of CRC cells, which was the first evidence that high MUC20 expression predicts poor clinical outcomes in CRC patients (48).

Due to the significant challenges in establishing FAP cell lines, most fundamental research in the field currently utilizes cell lines or mouse models carrying APC mutations to simulate FAP conditions. Since FAP lesions originate from colorectal epithelial cells, using closely related CRC epithelial cells to study the MUC20-S671C mutation is a reasonably appropriate approach. By querying the Catalogue Of Somatic Mutations In Cancer (COSMIC) database (49), we found that the SW480 cell line carries an APC mutation. Furthermore, as this cell line was derived from transformed colorectal epithelial carcinoma, it provides a suitable basis for modeling FAP. We introduced the MUC20-S671C mutation into SW480 cells to observe the potential effects of this mutation. We first used the CCK-8 assay to test the cell proliferation ability and found that the proliferation rate of mutant cells was significantly lower than that of wild-type SW480 cells. Moreover, as time progressed, the gap between the two groups gradually widened. The EdU experiment also confirmed that the proliferation of mutant cells was significantly affected. Additionally, the cell cloning experiment, which reflects the proliferative ability of single-cell colonies, further indicated that the mutation had a significant impact on CRC cells. Next, we used flow cytometry to assess cell apoptosis and the cell cycle, finding that the overall apoptosis rate of mutant cells was higher and statistically significant. The cell cycle results showed an increase in the S phase and a decrease in the G2/M phase, suggesting a possible S-phase block. Combined with the EdU results, we speculated that the mutation might have affected DNA synthesis efficiency, thus impacting cell proliferation. Additionally, we tested the invasion and migration abilities of both cell types using the Transwell assay and found that the migration and invasion abilities of the mutant cells were significantly decreased. These phenomena suggest that MUC20-S671C mutation reduces the malignancy of CRC cells in vitro. Based on these findings, we conducted animal experiments. We performed xenograft experiments in nude mice to evaluate the tumorigenic ability of both cell types. Since SW480 cells have low tumorigenic potential, we chose the immunodeficient NSG mice for the experiment. We injected cell suspensions into the axilla of NSG mice, and tumor formation began 4–7 days later. Tumors in the wild-type group appeared 1–2 days earlier than those in the mutant group. Overall, the mutant tumors grew more slowly, with more frequent tumor regression during the implantation period. At day 21, we harvested the tumors and found that the tumor size in the mutant group was significantly smaller than in the wild-type group, further confirming our findings at the cellular level.

We subsequently performed transcriptome sequencing on both cell types and their corresponding tumor tissues to obtain molecular-level regulatory information. The sequencing results revealed that, overall, the mutant cells exhibited gene enrichment patterns skewed toward cellular differentiation and transcriptional regulation, suggesting that the introduced mutation may enhance the differentiation capacity of intestinal cancer cells while reducing their malignant potential—a finding consistent with the H&E staining results from xenograft tumor tissues. Furthermore, when screening and analyzing apoptosis- and cell cycle-related genes, we observed that pro-apoptotic genes were generally upregulated in mutant cells, while anti-apoptotic genes were downregulated. Additionally, significant alterations were detected in cell cycle-related genes, particularly those associated with S-phase arrest (e.g., CDK2, CDC6). These findings collectively demonstrate that the mutation exerts extensive and profound effects on cellular behavior.

EMT is a biological process in which epithelial cells lose their polarity and cell-cell junction characteristics, transforming into mesenchymal-like cells with enhanced migration and invasion capabilities (50). EMT plays a key role in embryonic development, wound healing, and cancer invasion and metastasis. Through the analysis of transcriptome sequencing results, we found significant changes in the expression levels of genes involved in EMT, such as E-cadherin, MMP9, MMP14, and Vimentin. E-cadherin and Vimentin, key markers for epithelial and mesenchymal cells in EMT, were significantly upregulated and downregulated in the mutant cells, respectively, weakening the EMT process. MMPs, including MMP9 and MMP14, were significantly downregulated, providing space for tumor cell invasion and metastasis. Protein results also confirmed that the mutation weakened the EMT process in CRC cells, which may be one of the fundamental reasons for the reduction in the various abilities of the mutant cells.

Previous studies by Xiao et al. showed that MUC20 can promote the EMT process through its overexpression, thus increasing the invasiveness and migration of CRC cells in vitro (48). Our study suggests that the expression trends of MUC20 in both cells and tumor tissues are opposite, but the commonality is that the EMT process is inhibited both in vitro and in mouse tumors. This suggests that the MUC20-S671C mutation may be a key molecule in altering the role of MUC20 in CRC. This change may not be solely mediated through attenuation of the EMT process, and other pathways likely exist, requiring further exploration and validation.

In addition, PPI network analysis identified several hub genes closely associated with EMT and CRC progression, including EGFR, FN1, WNT5A, CD44, and CXCR4. These findings provide additional network-level support for the molecular pathways potentially regulated by the MUC20-S671C mutation.

An important question arising from our findings is: if the MUC20-S671C mutation exerts tumor-suppressive effects, why do FAP patients carrying this mutation still develop polyps and cancer? This apparent paradox can be explained by considering the hierarchical roles of APC and MUC20 mutations in FAP pathogenesis. FAP is primarily driven by biallelic inactivation of the APC gene, which leads to constitutive activation of the Wnt/β-catenin signaling pathway and serves as the initiating event in colorectal carcinogenesis (1,2). The APC mutation-driven tumorigenic signal is remarkably potent, involving not only Wnt pathway activation but also chromosomal instability and alterations in cell adhesion and apoptosis (3). In contrast, the MUC20-S671C mutation identified in this study appears to function as a disease modifier that partially counteracts the oncogenic effects through the reversal of EMT. Notably, our experimental model inherently carries an APC mutation (SW480 cell line) (49), providing a relevant genetic context in which we observed that MUC20-S671C significantly suppressed, but did not completely abolish, malignant phenotypes. This is consistent with an antagonistic model in which APC loss drives Wnt-dependent EMT and proliferation while MUC20-S671C partially reverses EMT, thereby moderating—but not eliminating—the malignant phenotype. However, the current study was not specifically designed to evaluate genetic interactions between APC and MUC20. Because all functional experiments were performed in SW480 cells, which already harbor APC mutations, our findings support a modulatory effect of MUC20-S671C in an APC-deficient background but do not allow a direct assessment of epistatic interactions between the two genes. Future studies employing APC-controlled organoid models or APCmin/+ mouse models with controlled MUC20-S671C status will be required to determine whether the relationship between these alterations is truly antagonistic at the genetic level.

Furthermore, whether the S671C substitution alters the biochemical properties of MUC20 warrants further investigation. Ser671 is located within the C-terminal MET-interacting domain (residues 657–709) of MUC20 (Figure S2). Computational analyses using PhosphoSitePlus, GPS, and PhosphoNET consistently identified Ser671 as a potential phosphorylation site. The substitution of serine with cysteine may abolish phosphorylation at this residue and replace the hydroxyl group of serine with the reactive sulfhydryl group of cysteine, thereby altering the regulatory properties and PPIs of the MET-interacting domain. While these computational predictions provide a plausible mechanistic explanation for the functional changes observed in the present study, direct experimental evidence regarding the effects of the S671C substitution on MUC20 phosphorylation, protein stability, glycosylation, or subcellular localization is still lacking.

The FAP patient cohort included in this study was derived from the long-term clinical accumulation of our research group. As a rare autosomal dominant genetic disorder, the population incidence of FAP is approximately 1/10,000 to 1/30,000. Consequently, the collection of a large-scale, homogeneous cohort itself poses a significant challenge. Our research aims to explore novel genetic modifiers in FAP, which requires that enrolled patients must undergo strict clinical and genetic diagnosis and that high-quality genomic DNA samples can be obtained for whole-genome sequencing. Within a limited timeframe, we successfully collected and completed the analysis of samples from nine sporadic FAP probands from different families. This sample size is already considerable for in-depth molecular mechanism exploration research targeting such a rare disease. Nevertheless, we fully recognize that the sample size remains a limitation of this study. Future multi-center collaboration is needed to expand the validation cohort to further confirm the generalizability of our findings. Additionally, the retrospective nature of this cohort and the extended data collection period have resulted in a lack of standardized longitudinal clinical data, including serial endoscopic records and uniform histopathological assessments. This precludes meaningful genotype-phenotype correlation analysis between MUC20-S671C and clinical outcomes such as polyp progression and cancer risk, which should be addressed in future prospective studies.

Furthermore, since we used CRC cell lines to investigate the effects of the MUC20-S671C mutation, the results partially reflect the mutation’s effects. However, there are limitations: (I) the effects observed in CRC cells may not fully represent the situation in FAP cells; (II) the cell line used for validation was limited, and results obtained from SW480 may not be identical in other CRC cell lines. Therefore, establishing FAP cell lines represents an important future direction. The effects of the S671C substitution on MUC20 phosphorylation, glycosylation, protein stability, and subcellular localization were not directly investigated and therefore remain to be experimentally validated. Furthermore, the expression of MUC20 and key EMT-related proteins was not validated in polyp or colorectal tumor tissues from FAP patients in this retrospective cohort. Therefore, the clinical relevance of the molecular findings identified in the present study requires further confirmation in well-characterized patient tissue specimens.

Conclusions

In conclusion, we have discovered a new mutation in the MUC20 gene, MUC20-S671C. Our research provides initial evidence that this mutation may act as a protective factor in CRC, but more precise and large-scale studies are needed to confirm these findings.

Supplementary

The article’s supplementary files as

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The First Affiliated Hospital of Kunming Medical University (Approval No. 2023-186). Informed consent was obtained from all individual participants included in the study. All animal experiments were performed under a project license (No. kmmu20240954) granted by the Animal Ethics Committee of Kunming Medical University, in compliance with national guidelines for the care and use of animals.

Data Sharing Statement

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DOI: 10.21037/tcr-2026-0904