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. 2026 Feb 21;12(1):2629346. doi: 10.1080/20565623.2026.2629346

Targeting FMO3 suppresses the malignant phenotype of colorectal cancer: evidence from bioinformatics to functional experiments

Rong Su a,b,#, Cuncun Huang a,c,#, Zhe Jing a, Shuping Qiu a, Lihua Shao a, Tinghong Jiao a,d, Hongwei Wang e,✉, Hailong Li a,f,✉
PMCID: PMC12928650  PMID: 41721713

Abstract

Aims

Flavin-containing monooxygenase 3 (FMO3) is a key metabolic enzyme involved in endogenous metabolism; however, its role in colorectal cancer (CRC) remains poorly understood. This study aimed to investigate the expression pattern, biological function, and potential clinical relevance of FMO3 in CRC.

Materials and methods

Publicly available transcriptomic datasets were analyzed to assess FMO3 expression and its association with clinical characteristics. Immunohistochemistry and in vitro functional assays were performed to evaluate the effects of FMO3 on CRC cell proliferation, cell-cycle progression, migration, and invasion. Bioinformatics analyses were used to explore potential signaling pathways and immune-related associations.

Results

FMO3 was significantly upregulated in CRC tissues and cell lines and was associated with unfavorable clinicopathological features and poor prognosis. Silencing of FMO3 inhibited CRC cell proliferation by inducing G0/G1 cell-cycle arrest and suppressed migration and invasion by reversing epithelial–mesenchymal transition–related phenotypes. Mechanistically, FMO3 depletion attenuated PI3K/AKT signaling activity. In addition, FMO3 expression was correlated with immune cell infiltration patterns and immune checkpoint-related molecules.

Conclusions

FMO3 promotes malignant progression of colorectal cancer and may serve as a potential biomarker and therapeutic target. Further in vivo and mechanistic studies are warranted to validate these findings.

Keywords: FMO3, TMAO, colorectal cancer, bioinformatics analysis, PI3K/AKT signaling

PLAIN LANGUAGE SUMMARY

Colorectal cancer (CRC) is one of the most common cancers worldwide and remains difficult to treat, especially when it becomes aggressive or spreads. To improve diagnosis and treatment, it is important to better understand the biological factors that help cancer cells grow and spread.

In this study, we focused on a protein called flavin monooxygenase 3 (FMO3). Although FMO3 is known to play a role in normal metabolism, its involvement in colorectal cancer has not been well studied. By analyzing large public databases and performing experiments in cancer cells, we found that FMO3 levels were higher in colorectal cancer tissues than in normal tissues. Higher levels of FMO3 were also associated with more aggressive cancer features and poorer patient outcomes.

When we reduced the amount of FMO3 in colorectal cancer cells, the cells grew more slowly and were less able to move and invade, which are key steps in cancer progression. We also found that FMO3 may be linked to changes in the tumor environment that affect how cancer cells interact with the immune system. However, these immune-related findings are based on data analysis and need further confirmation in future studies.

Overall, our results suggest that FMO3 may play an important role in promoting colorectal cancer progression. Although more research is needed, especially in animal models and clinical studies, FMO3 could become a useful marker for cancer progression and a potential target for future treatments.

ARTICLE HIGHLIGHTS

  1. Flavin monooxygenase 3 (FMO3) expression is significantly upregulated in colorectal cancer tissues and cell lines and is associated with unfavorable clinicopathological features and poor prognosis.

  2. Functional in vitro experiments demonstrate that flavin monooxygenase 3 (FMO3) promotes colorectal cancer (CRC) cell proliferation by facilitating cell-cycle progression, particularly the G1/S transition.

  3. Silencing of flavin monooxygenase 3 (FMO3) markedly suppresses colorectal cancer cell migration and invasion by reversing epithelial–mesenchymal transition (EMT)–related phenotypes.

  4. Mechanistically, flavin monooxygenase 3 (FMO3) depletion attenuates PI3K/AKT signaling activity, suggesting a potential role for FMO3 in regulating oncogenic signaling pathways associated with tumor aggressiveness.

  5. Bioinformatics analyses reveal that flavin monooxygenase 3 (FMO3) expression is closely associated with immune cell infiltration patterns and immune checkpoint–related molecules, indicating a possible link between FMO3 and the tumor immune microenvironment.

  6. Although further in vivo and mechanistic validation is required, this study identifies flavin monooxygenase 3 (FMO3) as a previously underappreciated metabolic regulator in colorectal cancer (CRC) and highlights its potential value as a biomarker and therapeutic target.

1. Introduction

Colorectal cancer (CRC) is a major global health burden, ranking as the third most commonly diagnosed malignancy and the second leading cause of cancer-related mortality worldwide [1]. According to GLOBOCAN 2022, CRC accounts for more than 900,000 deaths annually, with a markedly higher incidence in developed than in developing countries [2,3]. Beyond traditional risk factors, accumulating evidence highlights obesity, high intake of red and processed meat, high-fat dietary patterns and gut microbiota dysbiosis as key contributors to CRC initiation and progression [4,5]. Trimethylamine N-oxide (TMAO), a gut microbiota–derived metabolite, has emerged as a critical mediator linking diet, intestinal flora and colorectal carcinogenesis [6]. Prospective studies in postmenopausal women have shown that higher circulating TMAO levels, a choline-derived oxidation product generated by gut bacteria, are positively associated with CRC risk, especially among individuals with lower plasma vitamin B12 concentrations [7]. Clinical and epidemiological data further indicate that TMAO concentrations are elevated in patients with CRC, and are even higher in obese individuals with CRC, suggesting a potential interaction between metabolic status, gut microbial metabolism and tumorigenesis [6,8].

Mechanistic studies have further support a direct oncogenic role of TMAO in the colorectum. In human fetal colon cells, TMAO has been shown to inhibit autophagy-related proteins, activate the NLRP3 inflammasome and promote a pro-inflammatory phenotype, thereby exacerbating intestinal inflammation and barrier dysfunction [9,10]. In CRC models, TMAO promotes colorectal cancer cell proliferation and enhances angiogenesis by upregulating vascular endothelial growth factor A (VEGFA) expression and secretion in HCT116 cells, leading to increased tumor volume, neovascularization and CD31 expression in vivo [11,12]. More broadly, preclinical and translational studies suggest that TMAO may facilitate CRC development through chronic inflammation, oxidative stress, endothelial dysfunction and modulation of oncogenic pathways [6,13,14].

In this context, flavin-containing monooxygenase 3 (FMO3) is a hepatic microsomal enzyme that catalyzes the oxidation of trimethylamine (TMA) to TMAO and is regarded as the major endogenous source of circulating TMAO in humans [15,16]. As one of the principal phase I xenobiotic-metabolizing enzymes after cytochromes P450, FMO3 participates in the oxidative metabolism of a broad range of endogenous and exogenous substrates and exhibits marked inter-individual variation in activity [17]. Genetic studies in Koreans have suggested that single-nucleotide polymorphisms in FMO3 may influence susceptibility to CRC, implicating this enzyme in colorectal carcinogenesis at the host-genetic level [18]. In addition, pan-cancer and tumor-specific analyses have shown that members of the FMO family are dysregulated in multiple malignancies and can be associated with adverse prognosis, including in CRC where FMO5 overexpression predicts poor outcome [19]. Recent work in non-small-cell lung cancer has further demonstrated that FMO3 overexpression promotes tumor cell proliferation and survival, supporting its potential oncogenic properties in solid tumors [19,20].

Despite these observations, the specific role of FMO3 in CRC remains largely unexplored. In particular, its expression pattern in colorectal tumors, prognostic relevance, downstream signaling networks and impact on the tumor immune microenvironment have not been systematically characterized. Given that FMO3 is the key enzymatic driver of TMAO production, and that TMAO has been implicated in CRC development via inflammatory and angiogenic mechanisms, it is reasonable to hypothesize that FMO3 may function as a pro-tumorigenic factor in CRC by integrating metabolic, inflammatory and immune regulatory signals.

In this study, we combined multi-cohort bioinformatics analyses with experimental validation to comprehensively investigate the role of FMO3 in CRC. First, we assessed FMO3 expression and prognostic significance in pan-cancer datasets and colon adenocarcinoma (COAD) cohorts from TCGA and GEO, and examined its protein expression in colorectal cancer tissue microarrays and cell lines. Second, we constructed FMO3-related co-expression networks, identified hub genes and enriched pathways, and evaluated the association between FMO3 and immune cell infiltration as well as immune checkpoint molecules. Finally, we established stable FMO3-knockdown CRC cell models to examine the effects of FMO3 depletion on cell proliferation, cell-cycle progression, migration, invasion, epithelial–mesenchymal transition (EMT) and oncogenic signaling. This study identifies the oncogenic and immunomodulatory functions of FMO3 in CRC and to provide a theoretical basis for considering FMO3 as a potential biomarker and therapeutic target.

2. Materials and methods

2.1. Bioinformatics analysis

Gene expression and clinical data for pan-cancer and colon adenocarcinoma (COAD) were obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database (GSE39582). FMO3 expression levels and their prognostic significance were evaluated. Differential expression between tumor and normal tissues was analyzed statistically. For Kaplan-Meier survival analysis, optimal cutoff values were determined using the surv_cutpoint function (survival package, R), stratifying patients into high- and low-expression groups.

2.2. Cell culture maintenance and reagent preparation

Human colorectal cancer cell lines HCT116 and HT29 were purchased from Servicebio (Wuhan, China), and the normal colon epithelial cell line NCM460 from Zhongqiao Xinzhou Biotechnology (Shanghai, China). Cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 incubator. The cell cycle analysis kit was obtained from Multi Sciences Biotechnology (Hangzhou, China).

2.3. Tissue microarray and immunohistochemistry

A colorectal cancer tissue microarray (HColA160Bc01) containing paired tumor and adjacent normal tissues was purchased from Shanghai Xinchao Biotechnology. FMO3 protein expression was assessed by immunohistochemistry (IHC). All samples were commercially obtained and anonymized; therefore, ethical approval was not required.

2.4. Lentiviral transfection

Cells were prepared at a density of 3,000–5,000 cells/mL and seeded into six-well plates. Viral volume was calculated using the formula: Virus Volume = MOI × Cell Number/Viral Titer. Lentiviruses (sh-FMO3#1-3) and negative controls, along with transfection enhancer GP, were added to each well. Cells were selected with puromycin (2 µg/mL; Beyotime Biotechnology, Lot No. 44240816020) for at least two weeks to establish stable knockdown.

2.5. Colony formation assay

Stable FMO3-knockdown and control cells were digested, counted, and verified to have viability >95%. A total of 500 cells were seeded per well into six-well plates (triplicates). After 2–3 weeks of incubation, colonies were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 20 minutes, washed, air-dried, and counted.

2.6. Cell cycle analysis

Stable transfected and control cells were cultured to 70–80% confluence, harvested using 0.25% trypsin-EDTA, and fixed overnight in 75% ethanol at 4 °C. Cells were washed and stained according to the Cell Cycle Staining Kit instructions (Multi Sciences), followed by analysis using a CytoFlex-LX flow cytometer (Beckman Coulter, USA).

2.7. Wound healing assay

Viable cells (>95%) were seeded into six-well plates and allowed to reach full confluence. Subsequently, a straight wound was introduced in the cell monolayer using a sterile 200 µL pipette tip. Following this, the wells were gently washed with PBS to clear dislodged cells, and fresh serum-free medium was added. Images of the wound area were captured at predefined intervals to monitor closure.

2.8. Transwell migration and invasion assays

Transwell chambers (8.0 µm pore size; Corning, USA) were used. For invasion assays, the upper chamber was coated with Matrigel (Yeasen Bio-Tech, Shanghai, China). The lower chamber was filled with medium containing 20% FBS. A total of 5 × 104 cells in 100 µL serum-free medium were added to the upper chamber. After incubation, non-migrating cells were removed, and remaining cells were fixed with 4% paraformaldehyde for 25 minutes and stained with 0.1% crystal violet for 25 minutes. Images were captured under a light microscope for quantification.

2.9. Western blot analysis

Stably transfected colorectal adenocarcinoma (COAD) cells and corresponding control cells were seeded into six‑well plates and cultured under standard conditions until reaching the logarithmic growth phase (approximately 70–80% confluence). After removal of the culture medium, cells were washed once with PBS and digested with 0.25% trypsin. Digestion was terminated using serum‑containing medium, followed by centrifugation and resuspension in PBS for cell counting. Only cell suspensions with >95% viability were used for subsequent protein extraction. Cells were lysed using RIPA buffer (P0013B, Beyotime), and total proteins were extracted. Protein samples were separated on 10–15% SDS‑PAGE gels and transferred onto PVDF membranes (0.45 μm). Membranes were incubated overnight at 4 °C with the following primary antibodies: GAPDH (1:500, YM3029, Immunoway), FMO3 (1:1000, GTX113698, GeneTex), E‑Cadherin (1:1000, #9139, GeneTex), N‑Cadherin (1:1000, YT2988, Immunoway), CDK4 (1:1000, #3230, Immunoway), CDK6 (1:1000, #3771, Immunoway), Vimentin (1:2000, 60330‑1‑IG, Immunoway), MMP2 (1:1000, 10373‑2‑AP, Immunoway), MMP9 (1:1000, YT1892, Immunoway), P53 (1:500, T55546, Immunoway), Bcl‑2 (1:1000, T40056, Immunoway), Bax (1:1000, YM3619, Immunoway), and P21 (1:1000, YT3497, Immunoway). After washing, membranes were incubated for 1 h at room temperature with horseradish peroxidase‑conjugated secondary antibodies (1:10000, RS0002, Immunoway). Protein bands were visualized using SuperKine™ chemiluminescent substrate (femto‑level sensitivity, Beyotime).

3. Results

3.1. FMO3 is upregulated in COAD and predicts poor prognosis via oncogenic and immune pathways

Pan-cancer analysis revealed marked heterogeneity in FMO3 expression across malignancies, with significant upregulation in GBM, LGG, UCEC, COAD, READ, BRCA, and LUAD, whereas no significant differences were observed in THCA or PAAD compared with normal tissues (Figure 1A). Among these, COAD displayed one of the most notable increases, with tumor tissues (n = 287) expressing substantially higher FMO3 levels than normal tissues (n = 41). Consistently, analysis of TCGA colorectal cancer data confirmed a significant elevation of FMO3 expression in COAD (Figure 1B). Moreover, high FMO3 levels were associated with worse overall survival in both TCGA and GSE39582 cohorts (Figure 1E,F). Validation experiments demonstrated markedly increased FMO3 protein expression in COAD cell lines compared to normal colon epithelial cells (Figure 1D,G). GSEA further indicated that elevated FMO3 expression was enriched in pathways related to Toll-like receptor signaling, T-cell receptor signaling, JAK-STAT and MAPK signaling, as well as colorectal cancer–associated pathways (Figure 1C).

Figure 1.

Figure 1.

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Comprehensive analysis of FMO3 expression, functional enrichment, and clinical relevance in human cancers. (A) Differential expression of FMO3 in tumor versus normal tissues across TCGA datasets. (B) FMO3 overexpression in tumor tissues. (C) Functional enrichment analysis of FMO3. (D, G) Validation of FMO3 expression in human cancer cell lines at the protein levels. (E, F) Kaplan–Meier survival analysis demonstrating that high FMO3 expression is associated with significantly worse overall survival in cancer patients.

3.2. Expression pattern and clinical relevance of FMO3 in colorectal cancer

Tissue microarray analysis revealed that FMO3 expression was markedly elevated in colorectal cancer tissues compared with adjacent non-tumorous tissues (Figure 2A). Quantitative assessment further demonstrated that high FMO3 expression was significantly associated with advanced pathological grading (P < 0.05; Figure 2B). In contrast, FMO3 levels showed no significant correlation with patients’ baseline characteristics (gender, age; Figure 2C,D), tumor burden (tumor size, TNM stage; Figure 2E,F), immune microenvironment indicators (PD-L1 expression, CD8+ T-cell infiltration; Figure 2G,H), or common oncogenic mutations including KRAS/NRAS status (Figure 2I–O). Consistently, immunohistochemical validation confirmed robust upregulation of FMO3 protein in colorectal cancer tissues, further corroborating the microarray findings (Figure 2P,Q).

Figure 2.

Figure 2.

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Clinical relevance and tissue validation of FMO3 expression in colorectal cancer. (A) FMO3 expression is significantly upregulated in colorectal cancer tissues compared with adjacent normal tissues. (B) Elevated FMO3 levels are positively associated with advanced pathological stage (P < 0.05). (C–O) FMO3 expression shows no significant association with other clinicopathological or molecular features. (P–Q) Tissue microarray analysis further confirms that FMO3 is highly expressed in colorectal cancer tissues.

3.3. Functional enrichment of genes associated with FMO3 interactions and expression

Through a comprehensive analysis utilizing the cBioPortal online database (https://www.cbioportal.org/), the Sangerbox academic platform (http://www.sangerbox.com/), and the Xiantao academy (https://www.xiantao.love/). It was identified that there are 2,088 co-expressed genes associated with the FMO3 gene, which were significantly enriched in immune and cancer-related pathways. Among the 31 highly enriched pathways (enrichment factor > 2), 20 were tumor-associated, including PI3K-AKT, MAPK, cytokine-receptor interaction, CAMs, and PD-1/PD-L1 checkpoint signaling (Figure 3A). Integration of tumor-related genes yielded 166 candidates, and STRING-CytoNCA analysis further identified ten hub genes—PIK3R1, ITGB1, TLR4, HRAS, CD4, CXCL10, CD8A, ITGAL, CCL2, ITK (Figure 3B). Venn analysis revealed 39 overlapping genes between FMO3-interacting and FMO3-related expression modules (Figure 3C). The top correlated genes showed strong positive associations with FMO3 expression, and the ten hub genes exhibited high inter-correlation in colorectal cancer (Figure 3D–G). Functional enrichment of hub genes demonstrated involvement in immune activation pathways (cytokine/TCR signaling) and pro-invasive programs (adhesion/MAPK activation) (Figure 3H–I). GSCALite analysis showed that key hubs (HRAS, PIK3R1, ITGB1) activate PI3K-AKT and RAS-MAPK pathways while suppressing apoptosis and DNA-repair signaling. Immune-related hubs (CD8A, CXCL10) were also linked to TSC-mTOR metabolic regulation, suggesting interplay between immune evasion and metabolic reprogramming in colorectal cancer (Figure 3J).

Figure 3.

Figure 3.

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Functional enrichment and interaction networks of FMO3 in colorectal cancer. (A) KEGG analysis of FMO3 co-expressed genes shows enrichment in cancer-related pathways. (B) PPI analysis identifies 10 hub genes. (C) Overlap between FMO3-interacting and co-expressed genes. (D–F) FMO3 positively correlates with key associated genes. (G) Co-expression network of hub genes. (H, I) GO/KEGG analyses indicate immune and metastasis-related processes. (J) Hub genes are involved in PI3K-AKT, RAS-MAPK, and immune-metabolic pathways.

3.4. Correlation between FMO3 expression and immune cell infiltration as well as immune checkpoints

Comprehensive immune profiling revealed that FMO3 expression was significantly associated with 24 immune cell subsets (Figure S1A). FMO3 showed positive correlations with macrophages, iDCs, DCs, and Th1 cells, but negative correlations with Th17 and NK CD56bright cells (Figure S1B–I). Comparison of high-versus low-FMO3 groups demonstrated that FMO3 significantly altered the infiltration of 23 immune cell types, with high expression favoring Th1/cytotoxic immune signatures and low expression indicating an immunosuppressive phenotype (Figure S1J). FMO3 expression also correlated positively with the Immune Score, Stromal Score, and ESTIMATE Score (Figure S1K–M). Additionally, FMO3 showed strong positive correlations with multiple immune checkpoint genes, including PD-L1, LAG3, TNF, and IFN-γ (Figure S1N).

3.5. Lentiviral transfection for knockdown of FMO3 gene

Lentiviral shRNA transduction was used to generate stable FMO3-knockdown models in HCT116 and HT29 colorectal cancer cells (Figure S2A,B). Western blotting confirmed efficient suppression of FMO3, with sh#2–sh#3 effective in HCT116 and HT29 (Figure S2C,D). Robust GFP fluorescence in all groups verified high transduction efficiency and successful establishment of a reliable FMO3-deficient model for subsequent functional investigations.

3.6. Knockdown of FMO3 inhibits cell migration and invasion in COAD cells

The metastatic phenotype of colorectal cancer cells following FMO3 silencing was assessed using wound-healing and Transwell assays. FMO3 knockdown markedly reduced the migratory capacity of HCT116 and HT29 cells at both 24 and 48 h (Figure 4A–F), and significantly suppressed cell invasion over 72 h (Figure 4G–L). To elucidate the underlying mechanisms, EMT-related protein expression was examined. FMO3 silencing led to a decrease in mesenchymal markers (Vimentin, N-cadherin) and MMP2/MMP9, accompanied by a significant increase in E-cadherin expression in both cell lines (Figure 4M–P). Furthermore, FMO3 knockdown attenuated PI3K-AKT pathway activity, as evidenced by the reduction of p-PI3K and p-AKT, while total PI3K and AKT levels remained stable (Figure 4Q–T). The signaling changes were consistent with the observed inhibition of EMT.

Figure 4.

Figure 4.

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FMO3 knockdown suppresses migration, invasion, EMT, and PI3K/Akt signaling in CRC cells. (A–F) Reduced migration of HCT116 and HT29 cells after FMO3 knockdown. (G–L) Transwell assays show decreased migration and invasion. (M–P) EMT inhibition indicated by increased E-cadherin and reduced N-cadherin, Vimentin, and MMP2/9. (Q–T) Decreased PI3K and AKT phosphorylation following FMO3 silencing.

3.7. FMO3 knockdown induces G0/G1 cell-cycle arrest and suppresses clonogenic growth in COAD cells

Flow cytometry analysis revealed that FMO3 knockdown induced a pronounced G0/G1 phase arrest in both HCT116 and HT29 cells, accompanied by a significant reduction in the S-phase population (Figure 5A–C, E–G). Consistently, the expression of key G1/S transition regulators CDK4 and CDK6 was markedly down-regulated following FMO3 silencing (Figure 5D,H). Functionally, colony formation assays demonstrated that FMO3 depletion substantially impaired clonogenic growth, reducing colony numbers by 52.5% (sh#2) and 78.1% (sh#3) in HCT116 cells, and by 68.34% (sh#2) and 84.70% (sh#3) in HT29 cells (Figure 5I,J). Colonies formed by knockdown cells were also visibly smaller, indicating diminished proliferative potential. At the molecular level, FMO3 knockdown induced upregulation of P21 and P53, together with increased Bax and decreased Bcl-2 expression (Figure 5K,L).

Figure 5.

Figure 5.

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FMO3 knockdown induces G0/G1 arrest, inhibits colony formation, and alters cell-cycle and apoptosis-related proteins in CRC cells. (A–C, E–G) FMO3 knockdown increases G0/G1-phase and decreases S-phase cell populations. (D, H) CDK4 and CDK6 protein levels are reduced after FMO3 silencing. (I, J) Colony formation is significantly suppressed in HCT116 and HT29 cells. (K, L) Bax, P21, and P53 are upregulated, while Bcl-2 is decreased following FMO3 knockdown.

4. Discussion

In this study, we systematically investigated the expression patterns, biological functions, and immunological relevance of FMO3 in colorectal cancer (CRC). Our results demonstrated that FMO3 was significantly upregulated in CRC tissues and cell lines, and that elevated FMO3 expression was closely associated with poor prognosis and aggressive tumor phenotypes, suggesting a potential oncogenic and immunomodulatory role in CRC progression. As a key hepatic flavin-containing monooxygenase, FMO3 catalyzes the conversion of gut microbiota-derived TMA into TMAO, thereby constituting a critical metabolic node linking microbial metabolism to host tumor-promoting signaling pathways [20–22]. Notably, FMO3 exhibits pronounced sex-dependent expression patterns, with higher hepatic expression observed in female mice in certain strains, indicating that its regulation may be influenced by endocrine and metabolic status [23].

Accumulating evidence suggests that TMAO, the principal enzymatic product of FMO3, participates in multiple pro-tumorigenic processes, including activation of the NLRP3 inflammasome, enhancement of NF-κB signaling, induction of oxidative stress, and promotion of angiogenesis [24,25]. In CRC models, TMAO has been reported to upregulate VEGFA and various pro-inflammatory cytokines, thereby amplifying tumor-associated inflammatory responses [11]. As the rate-limiting enzyme in TMAO biosynthesis, aberrant FMO3 expression may serve as an important upstream driver that potentiates TMAO-mediated oncogenic effects [15]. However, the regulatory mechanisms governing FMO3 expression in CRC remain incompletely understood. Potential contributors include bile acid-FXR signaling, sex hormone-dependent regulation, and the availability of microbial substrates, yet their coordinated roles have not been systematically elucidated [26]. In particular, impaired FXR signaling may simultaneously enhance FMO3-mediated TMAO production and compromise the FXR-FGF15 axis, thereby facilitating aberrant activation of Wnt/β-catenin signaling [11,13,27]. At the downstream level, activation of the FMO3-TMAO axis may promote angiogenesis and sustain inflammatory signaling through induction of VEGFA, ultimately fostering a pro-tumorigenic tumor microenvironment.

Within this mechanistic framework, we further validated the functional role of FMO3 in CRC malignant phenotypes through a series of functional assays. Silencing of FMO3 markedly suppressed CRC cell proliferation, induced G0/G1 phase cell-cycle arrest, and reduced clonogenic capacity, while significantly inhibiting cell migration and invasion. Notably, downregulation of FMO3 was accompanied by reversal of EMT, as evidenced by increased E-cadherin expression and decreased levels of N-cadherin, vimentin, MMP-2, and MMP-9. These findings suggest that FMO3 may facilitate CRC metastasis by sustaining EMT-associated migratory programs. Beyond its direct oncogenic effects, high FMO3 expression was closely associated with remodeling of the CRC immune microenvironment, including increased immune cell infiltration, elevated immune and stromal scores, and positive correlations with multiple immune checkpoint molecules. Collectively, these observations indicate that FMO3 may contribute to immune evasion through metabolic-immune crosstalk, thereby promoting the establishment of an immunosuppressive tumor microenvironment [28–30].

Several limitations of the present study should be acknowledged, including the relatively limited sample size, lack of treatment response and long-term follow-up data, and the absence of circulating FMO3 level assessment, which constrain the evaluation of its clinical predictive value. Importantly, however, these limitations do not affect the principal conclusions supported by the consistent multi-level experimental evidence presented herein. The regulatory pathway of the FMO3-TMAO axis has been well characterized in the fields of cardiovascular and metabolic research, while direct evidence for its role in colorectal cancer (CRC) remains limited. Future studies integrating in vivo models with multi-omics approaches will be required to further delineate the functional roles and molecular mechanisms of the FMO3-TMAO axis in CRC progression.

5. Conclusion

In summary, FMO3 is significantly overexpressed in colorectal cancer and drives malignant progression by promoting epithelial–mesenchymal transition, activating PI3K/AKT signaling, and disrupting cell-cycle regulation. Silencing FMO3 suppresses proliferation, migration, invasion, and clonogenic growth while inducing G0/G1 arrest and pro-apoptotic pathways. FMO3 also shapes an immune-regulatory tumor microenvironment, suggesting its potential role in immune escape. These findings identify FMO3 as a promising biomarker and therapeutic target for colorectal cancer.

Supplementary Material

Supplemental Material
IFSO_A_2629346_SM0149.docx (48.6MB, docx)

Acknowledgments

The authors acknowledge the contributions and support of all authors involved in this study. We also thank the project administrators for their support in project coordination and funding managemen. It has been confirmed that this research has not been published before.

Funding Statement

This study was supported by the Joint Research Fund of Provincial Science and Technology Program of Gansu Province [23JRRA1527], Entrepreneurship Project of Science and Technology bereau of Chengguan District of Lanzhou city [2023-2-6]. Lanzhou City Science and Technology Project [2023-ZD-199], Planning Project of the Health Industry in Gansu Province. [GSWSKY2022-73]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contributions

Rong Su: Conceptualization, Data curation, Writing-original draft (equal contribution). Cuncun Huang: Data curation (equal contribution). Zhe Jing: Data curation (equal contribution). Shuping Qiu: Data curation (equal contribution). Lihua Shao: Data curation (equal contribution). Tinghong Jiao: Project administration (equal contribution). Hongwei Wang: Writing – review & editing (equal contribution). Hailong Li: Project administration, Writing – review & editing (equal contribution).

Declaration of generative AI and AI-assisted technologies in the writing process

We hereby declare that during the preparation of this manuscript, we utilized [deepseek,quillbot] solely for [language polishing/grammar checking, or syntax refinement]. The authors employed artificial intelligence tools solely for language refinement purposes. All AI-generated content underwent rigorous review and modification to ensure (1) accuracy through manual verification of factual claims and data interpretations, (2) originality with core arguments and conclusions remaining entirely author-created, and (3) integrity by excluding any AI-influenced substantive content. After using this tool/service, the authors reviewed and edited the content as needed and takes full responsibility for the content of the publication.

Ethical disclosure statement

This study did not involve human participants, human tissue samples, animals, or clinical interventions. All data used in this research were obtained from publicly available databases and established commercial cell lines. Therefore, ethical approval from an institutional or national ethics committee was not required. The study was conducted in accordance with relevant guidelines and regulations and complies with the principles of the Declaration of Helsinki.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Data availability statement

All data generated or analyzed in this study are included in this published article and its supplementary materials. Publicly available datasets used in this work were obtained from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/) and the Gene Expression Omnibus (GEO) under accession number GSE39582 (https://www.ncbi.nlm.nih.gov/geo/). Experimental data supporting the findings of this study, including raw Western blot images, flow cytometry files, and original microscopy images, are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Material
IFSO_A_2629346_SM0149.docx (48.6MB, docx)

Data Availability Statement

All data generated or analyzed in this study are included in this published article and its supplementary materials. Publicly available datasets used in this work were obtained from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/) and the Gene Expression Omnibus (GEO) under accession number GSE39582 (https://www.ncbi.nlm.nih.gov/geo/). Experimental data supporting the findings of this study, including raw Western blot images, flow cytometry files, and original microscopy images, are available from the corresponding author upon reasonable request.

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