FMO3 as a novel prognostic biomarker and therapeutic target in non-small cell lung cancer by attenuating endoplasmic reticulum stress

FMO3 as a novel prognostic biomarker and therapeutic target in non-small cell lung cancer by attenuating endoplasmic reticulum stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article FMO3 as a novel prognostic biomarker and therapeutic target in non-small cell lung cancer by attenuating endoplasmic reticulum stress Hainaer Haisaer, Wolong Zhou, Xizhe Li, Yuanda Cheng, Ruimin Chang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7984231/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 18 You are reading this latest preprint version Abstract Non-small cell lung cancer (NSCLC) accounts for > 85% of lung malignancies and is characterized by late presentation and limited therapeutic options. While members of the flavin-containing monooxygenase (FMO) family have been implicated in various solid tumors, the expression pattern and biological significance of FMO3 in NSCLC remain undefined. In this study, immunohistochemistry was performed on 10 paired fresh NSCLC-adjacent specimens and a tissue microarray of 115 additional pairs for FMO1-5 and Ki67, with clinicopathological data and 5-year follow-up analyzed. Stable FMO3-overexpressing A549 cells and FMO3-knockdown H1703 cells were generated. Cell proliferation was evaluated by CCK-8, EdU and colony formation assays; apoptosis by Annexin V-FITC/PI flow cytometry; and ER-stress signaling by western blotting of the IRE1α/caspase-12/caspase-3/PARP axis. Results showed FMO3 was selectively upregulated in NSCLC, positively correlated with tumor size and T stage, predicted poorer 5-year survival as an independent prognostic factor. In vitro, FMO3 overexpression promoted proliferation and suppressed apoptosis, while knockdown had the opposite effects, via negative regulation of the IRE1α pathway to attenuate ER-stress. Thus, targeting FMO3 could be a novel NSCLC therapy. Biological sciences/Cancer Biological sciences/Cell biology Health sciences/Oncology cell death endoplasmic reticulum stress FMO3 IRE1α pathway non-small cell lung cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Lung cancer is ranked as the second most commonly diagnosed malignancy worldwide (following breast cancer) and remains the leading cause of cancer-related mortality[ 1 ]. The prognosis for lung cancer remains grim, with a five-year survival rate spanning a mere 7% to 22%, markedly inferior to that of other prevalent cancers[ 2 ]. Non-small cell lung cancer (NSCLC), which constitutes over 85% of all lung cancer cases, encompasses major histological subtypes including squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and adenosquamous carcinoma[ 3 ]. Over the past decade, the therapeutic paradigm for NSCLC has undergone substantial transformation. Precision-targeted agents and immune checkpoint blockade, in concert with state-of-the-art radiotherapeutic modalities, have been integrated alongside conventional chemotherapy, radiotherapy, and surgical resection as cornerstone interventions[ 4 , 5 ]. Consequently, therapeutic algorithms are now predicated on a multidimensional assessment that encompasses tumor-specific molecular profiles, actionable biomarkers, disease stage, and host physiological status[ 6 , 7 ]. Therefore, there is an urgent and compelling need to identify novel tumor biomarkers for early diagnosis, guiding treatment strategies, evaluating therapeutic efficacy, and predicting prognosis in NSCLC. The flavin-containing monooxygenase (FMOs) family comprises six members, namely FMO1-6, among which only FMO1-5 exhibit biological functions in the human body[ 8 ]. As a family of enzymes reliant on flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide phosphate (NADPH), and molecular oxygen (O₂), FMOs primarily participate in the oxidative metabolism of both exogenous and endogenous substrates, playing a pivotal role in drug metabolism and detoxification processes[ 9 ]. The FMOs family is expressed in numerous cancers and is closely associated with tumorigenesis and progression. Aberrant expression of FMOs has been observed in oral squamous cell carcinoma [ 10 ], hepatocellular carcinoma [ 11 ], colorectal cancer [ 12 ], and ovarian cancer [ 13 ]. Furthermore, FMOs have been identified as potential biomarkers for gastric cancer [ 14 ]. Additionally, FMO2⁺ cancer-associated fibroblasts (CAFs) enhance the efficacy of anti-PD-1 therapy by modulating the immune properties of the tumor microenvironment [ 15 ]. Nevertheless, the precise mechanisms and functional significance of the FMO family in NSCLC pathogenesis and progression are yet to be determined. Members of the FMOs exhibit diverse cytological functions across various diseases. Specifically, FMO5 significantly inhibits cell apoptosis by suppressing the NF-κB signaling pathway and reducing oxidative stress[ 16 ]. FMO1 is involved in the regulation of light-triggered cell death and directly induces caspase 3 activation due to its inhibited activity in Parkinson's disease models[ 17 , 18 ]. FMO2 reduces cardiomyocyte apoptosis in cardiovascular diseases by inhibiting endoplasmic reticulum (ER) stress[ 19 ]. Conversely, FMO3 induces ER stress and cell apoptosis through the CREB3/P4HB axis in drug-induced liver injury[ 20 ]. However, the specific roles and mechanisms of the FMO family in NSCLC remain unclear. To clarify the unclear roles and mechanisms of the FMO family in NSCLC, we investigated FMOs' expression, functions, and mechanisms in NSCLC. Immunohistochemical analysis revealed significant FMO3 upregulation in NSCLC tissues, positively linked to larger tumor size and advanced T stage, as well as poor prognosis, serving as an independent risk factor. In vitro, FMO3 promoted cell proliferation and inhibited apoptosis in NSCLC cell lines. Mechanistically, FMO3 mediated ER stress-induced apoptosis via negative regulation of the IRE1α/caspase12/caspase3/PARP pathway, suggesting FMO3 as a potential prognostic biomarker and therapeutic target for NSCLC. 2 Material and Methods 2.1 Selection of clinical samples To assess FMOs expression in NSCLC, we first collected ten fresh tumor–adjacent tissue pairs (adenocarcinoma and squamous cell carcinoma) from surgically resected specimens. Subsequently, a tissue microarray (TMA) was constructed containing 115 independent tumor–adjacent pairs derived from patients who underwent curative lung resection at the Department of Thoracic Surgery, Xiangya Hospital, Central South University. Inclusion criteria were: (1) complete clinical data available (age, sex, histologic subtype, TNM stage, tumor size, smoking status, differentiation grade); (2) definitive pathologic report; (3) absence of any preoperative chemotherapy, radiotherapy, targeted therapy, or radiofrequency ablation; and (4) postoperative histopathologic confirmation of NSCLC. Postoperative follow-up was conducted at 3-month intervals for the first year and every 6 months thereafter until month 60 or death. Only deaths attributable to lung cancer were considered events; deaths from other causes were censored. Among the 115 patients, 89 had complete follow-up data and were included in survival analyses. The study adhered to the principles of the Declaration of Helsinki and received approval from the Ethics Committee of Xiangya Hospital, Central South University (Ethics Approval No. 2022020671). 2.2 Immunohistochemistry FFPE sections (4 µm) were baked at 60°C for 30–60 min, dewaxed, rehydrated, and subjected to antigen retrieval in 0.01 M citrate buffer (pH 6.0) via microwave for 20 min. After blocking endogenous peroxidase with 3% H₂O₂ (10 min) and serum (30 min), sections were incubated overnight at 4°C with primary antibody, followed by HRP-conjugated secondary antibody for 1 h at RT. Visualization employed DAB (≤ 10 min), counterstaining with Mayer’s hematoxylin. Slides were dehydrated, cleared, and mounted in neutral balsam. 2.3 Cell culture NSCLC cell lines, including the A549 (CL-0016), NCI-H1299 (CL-0165), and NCI-H1703 (CL-0390) were provided from Procell (Wuhan, China), while the BEAS-2B (C6106) was obtained from Beyotime Biotechnology. All NSCLC cell lines, except A549 cells, were cultured in RPMI-1640 medium (PM150110, Procell), while A549 cells were maintained in Ham's F-12K cell medium (PM150910, Procell). BEAS-2B cells were cultured in DMEM (PM150210, Procell). All of these cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37°C in a humidified atmosphere containing 5% CO 2 . 2.4 Cell-line construction Stable FMO3-over-expressing A549 cells and FMO3-knockdown H1703 cells were generated by lentiviral transduction. Recombinant lentiviruses carrying human FMO3 cDNA (GeneChem) or three independent shRNAs targeting FMO3 (sh1–sh3, GeneChem) were added at a multiplicity of infection (MOI) pre-determined by pilot infections. After 16 h exposure, the medium was replaced and 48 h later cells were selected with puromycin (2 µg mL⁻¹) or neomycin (800 µg mL⁻¹), respectively. Surviving colonies were expanded for two weeks, validated by western blot, and stocks were cryopreserved in 90% FBS plus 10% DMSO. 2.5 CCK-8 proliferation assay Cells were seeded in 96-well plates (3 × 10³ cells/well) and incubated for 0–72 h. At each time-point, 10 µL CCK-8 reagent (CK04, Dojindo) was added and plates were returned to 37°C for 2 h. Absorbance at 450 nm was measured in a microplate reader (BioTek). All conditions were run in quintuplicate, and viability was expressed as the percentage of untreated controls after background subtraction. 2.6 EdU incorporation assay Exponentially growing cells were exposed to 50 µM EdU for 2 h, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and labelled with Apollo-488 (KGA1108, keygenbio) according to the manufacturer’s protocol. Nuclei were counterstained with Hoechst 33342. EdU-positive cells were quantified by fluorescence microscopy (×20 objective, five random fields/well) and expressed as the percentage of total nuclei. 2.7 Colony formation assay Single-cell suspensions (500 cells/well) were plated in 6-well plates and cultured for 14 days. Colonies were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet for 20 min, washed, air-dried, and counted manually under a stereomicroscope (≥ 50 cells/colony). Results were normalized to plating efficiency and presented as fold change relative to controls. 2.8 Apoptosis assessment Apoptosis was quantified using Annexin V-FITC/PI double staining (KGA1102, keygenbio). Briefly, 1 × 10⁵ cells were harvested, washed twice with cold PBS, resuspended in 100 µL binding buffer and incubated with 5 µL Annexin V-FITC plus 5 µL PI for 15 min at room temperature in the dark. Samples were analyzed within 1 h on a FACSCanto II flow cytometer (BD). Early and late apoptotic populations were combined to calculate total apoptosis. 2.9 Western blotting Total protein was extracted with RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts (30 µg) were separated on 10% SDS-PAGE gels, transferred to PVDF membranes. The PVDF membranes were cropped prior to hybridization with the antibodies. Then, the membranes were blocked with 5% non-fat milk, and probed overnight at 4°C with primary antibodies against FMO3 (ab126711, Abcam), IRE1α (27528-1-AP, Proteintech), p-IRE1α-S724 (ab48187, Abcam), caspase-12 (55238-1-AP, Proteintech), caspase-3 (19677-1-AP, Proteintech), cleaved PARP (13371-1-AP, Proteintech), α-tubulin (11224-1-AP, Proteintech) and GAPDH (60004-1-Ig, Proteintech). After incubation with HRP-conjugated secondary antibodies, bands were visualized using ECL (Millipore) and quantified by densitometry (ImageJ). 2.10 Statistical analysis All statistical analyses were performed with SPSS 24.0 and illustrated using GraphPad Prism 8. Continuous variables are presented as mean ± SD and were compared by independent-sample t tests or one-way ANOVA; categorical data are expressed as counts and analyzed by χ² tests. Five-year overall survival was estimated by Kaplan–Meier analysis and visualized with survival curves. Covariates showing significance in univariate analysis were entered into a Cox proportional-hazards regression model for multivariate assessment. P < 0.05 (two-tailed) was considered statistically significant. 3 Results 3.1 FMO3 exhibits overexpression in non-small cell lung cancer tissues To delineate the expression profiles of flavin-containing monooxygenase family (FMOs 1–5) in non-small cell lung cancer (NSCLC), we conducted immunohistochemical (IHC) staining on ten paired NSCLC and adjacent non-tumor tissues (n = 20 total). All five FMO family genes exhibited detectable protein expression in both tumor and para-tumor tissue. Notably, quantitative analysis demonstrated significantly upregulated FMO3 expression in NSCLC tissues compared to matched adjacent non-tumor controls (P = 0.0003) (Fig. 1 A-B). In contrast, no statistically significant differences were observed for FMO1, FMO2, FMO4, or FMO5 (all P > 0.05) (Fig. 1 B). These findings prompted us to select FMO3 as the primary target for subsequent functional investigations. To further validate the differential expression of FMO3 in NSCLC, we constructed a tissue microarray (TMA) comprising of 115 paired NSCLC and adjacent non-tumor tissues. IHC staining was performed on the TMA sections, followed by whole-slide scanning to generate high-resolution digital images. Quantitative analysis using ImageJ software revealed significantly upregulated FMO3 expression in NSCLC tissues compared to matched controls, as determined by average optical density (AOD) measurements (P = 0.0003) (Fig. 2 A upper). Further analysis of tumor proliferation via Ki67 immunohistochemistry demonstrated significantly elevated labeling indices in NSCLC tissues versus para-tumor controls (P = 0.0002) (Fig. 2 A lower). Notably, FMO3 expression levels exhibited a significant positive correlation with Ki67 (Fig. 2 B). These findings collectively suggest that FMO3 is overexpressed in NSCLC tissues and its expression is closely associated with enhanced tumor cell proliferation. 3.2 FMO3 serves as an independent prognostic factor in non-small cell lung cancer To evaluate the clinical significance of FMO3 in NSCLC progression, patients were stratified into FMO3-high and FMO3-low subgroups using median IHC scores as the cutoff. Comparative analysis of clinicopathological parameters revealed significant associations between elevated FMO3 expression and larger tumor size (P = 0.0021) as well as T-stage classification (P = 0.016) (Table 1 ). Notably, Kaplan-Meier survival analysis demonstrated a significant inverse correlation between FMO3 expression levels and 5-year overall survival (P < 0.001) (Fig. 2 C). Table 1 Association between FMO3 expression and clinicopathological characteristics in NSCLC patients Variable Subgroup FMO3-Low (%) FMO3-High (%) P-value Age ≥ 60 years 26 (53.06) 23 (46.94) 0.3460 0.9999 Female 18 (60.00) 12 (40.00) Histological Type Adenocarcinoma 37 (62.71) 22 (37.29) 0.3491 Squamous 30 (53.57) 26 (46.43) TNM Stage I-II 43 (58.11) 31 (41.89) > 0.9999 III-IV 24 (58.54) 17 (41.46) Lymph Node Metastasis Present 23 (60.53) 15 (39.47) 0.8412 Absent 44 (57.14) 33 (42.86) Smoking Status Non-smoker 33 (67.35) 16 (32.65) 0.1258 Smoker 34 (51.52) 32 (48.48) Tumor Differentiation Moderate/High 45 (61.64) 28 (38.36) 0.4324 Poor 22 (52.38) 20 (47.62) Tumor Size ≤ 5 cm 54 (65.06) 29 (34.94) 0.0035 * > 5 cm 11 (34.38) 21 (65.63) T Stage T1-T2 53 (65.43) 28 (34.57) 0.0224 * T3-T4 14 (41.18) 20 (58.82) Univariate Cox regression analysis identified FMO3 overexpression (IHC score ≥ median) as a significant predictor of reduced 5-year overall survival (OS) (HR = 3.32, 95% CI: 1.59–6.93, P = 0.01) (Table 2 ). After adjusting for potential confounders in multivariate analysis, FMO3 overexpression retained independent prognostic significance (HR = 2.95, 95% CI: 1.38–6.32, P = 0.005) (Table 2 ). These results collectively indicate that FMO3 overexpression is closely linked to poor prognosis in NSCLC. Importantly, the prognostic impact of FMO3 even surpasses that of traditional clinicopathological factors such as TNM stage in the final model, highlighting its potential as a novel and robust biomarker for predicting survival outcomes and guiding therapeutic strategies in NSCLC patients. Table 2 Univariate and multivariate Cox regression analyses of prognostic factors in NSCLC Variable Univariate Analysis Multivariate Analysis HR (95% CI) P-value HR (95% CI) P-value Age (≥ 60 vs < 60) 1.55 (0.77–3.10) 0.22 - - Gender (Male vs Female) 1.44 (0.62–3.33) 0.38 - - Histological Type (Adeno vs Squamous) 0.87 (0.43–1.73) 0.68 - - T Stage (T3-T4 vs T1-T2) 1.47 (0.71–3.05) 0.02 * 1.52 (0.66–3.72) 0.16 Lymph Node Metastasis (Yes vs No) 1.69 (0.83–3.43) 0.15 - - Tumor Size (> 5 cm vs ≤ 5 cm) 2.54 (1.26–5.13) 0.01 * 2.03 (0.99–4.17) 0.05 TNM Stage (III-IV vs I-II) 1.78 (0.88–3.58) 0.02 * 1.67 (0.99–3.77) 0.03 * Differentiation (Poor vs Moderate/High) 1.95 (0.97–3.91) 0.07 - - Smoking (Yes vs No) 2.47 (1.11–5.51) 0.02 * 1.90 (0.84–4.31) 0.13 FMO3 Expression (High vs Low) 3.32 (1.59–6.93) 0.01 * 2.84 (1.34–6.01) 0.01 * Notes: *Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001 Adeno: Adenocarcinoma; Squamous: Squamous cell carcinoma Multivariate model adjusted for T stage, tumor size, TNM stage, smoking status, and FMO3 expression HR: Hazard Ratio; CI: Confidence Interval "-" indicates variables not included in multivariate analysis 3.3 FMO3 promotes non - small cell lung cancer cell proliferation To investigate the functional role of FMO3 in NSCLC, we first screened basal FMO3 protein levels in A549, H1299, H1703 (NSCLC), and HBEAS-2B (normal) cells by Western blot. The results demonstrated that FMO3 exhibited relatively higher expression in HBEAS-2B, H1299, and H1703 cells, while showing relatively lower expression in A549 cells (figure S1 ). A549 (lowest FMO3) was selected for stable overexpression via lentiviral transduction, while H1703 (highest FMO3) underwent FMO3 knockdown by shRNA (figure S1 ). Overexpression of FMO3 was found to significantly bolster cell viability in A549 cells. In stark contrast, when FMO3 was knocked down in H1703 cells, a marked suppression of cell viability was observed (Fig. 3 A). Regarding DNA synthesis, FMO3 overexpression in A549 cells led to a notable enhancement of this process, while in H1703 cells, FMO3 knockdown resulted in a significant inhibition of DNA synthesis activity (Fig. 3 B). Furthermore, our results indicated that overexpression of FMO3 in A549 cells enhanced their clonogenic potential. However, when FMO3 was knocked down in H1703 cells, there was no discernible impact on their clonogenic potential (Fig. 3 C). These results collectively suggest that FMO3 plays a crucial role in promoting NSCLC cell growth and survival. 3.4 FMO3 overexpression alleviates endoplasmic reticulum stress and inhibits apoptosis in NSCLC Cells Flow cytometry analysis demonstrated that FMO3 overexpression in A549 cells significantly reduced early and late populations compared to controls, whereas FMO3 knockdown in H1703 cells selectively elevated early apoptosis without affecting late apoptotic or necrotic rates (Fig. 4 A). These findings suggest that FMO3 differentially suppresses apoptosis: inhibiting both early and late stages in A549 cells but primarily modulating early signaling in H1703 cells. Previous studies have demonstrated that FMO3 exerts dual regulatory effects on endoplasmic reticulum (ER) stress-mediated apoptosis. Mechanistically, FMO3 attenuates ER stress-induced apoptosis via JNK dephosphorylation and reduced cleaved caspase-3/XBP1s levels (IRE1 pathway)[ 21 , 22 ]. Concurrently, FMO3 modulates PERK pathway activity by interacting with CREB3 and regulating PERK/eIF2α phosphorylation, thereby contributing to ER stress homeostasis[ 20 – 22 ]. Building on these findings, we conducted mechanistic investigations using Western blot analysis to evaluate the expression profile of ER stress-associated proteins (IRE1α, caspase-12, caspase-3, and PARP) in FMO3-overexpressing A549 cells. The results demonstrated significant downregulation of total and phosphorylated IRE1α (p-IRE1α), along with reduced cleavage of caspase-12, caspase-3 and PARP (Fig. 4 B). To elucidate these in vitro observations in clinical specimens, we conducted IHC analysis on paired NSCLC tissues and adjacent non-malignant counterparts. Quantitative assessment demonstrated significantly elevated p-IRE1α immunoreactivity in tumor specimens compared to matched normal controls (P < 0.0001) (Fig. 5 A). Correlation analysis revealed a strong inverse relationship between p-IRE1α accumulation and FMO3 expression levels (P < 0.001) (Fig. 5 B), suggesting a potential reciprocal regulatory mechanism between FMO3 and ER stress signaling in NSCLC pathogenesis. 4 Discussion In China, non-small cell lung cancer (NSCLC) incidence and mortality continue to rise, with over 820,000 new cases annually [ 23 ]. Globally, NSCLC 5-year survival remains < 20% due to frequent late-stage diagnosis[ 24 , 25 ]. While molecular targeted therapies have improved outcomes for patients with actionable mutations[ 26 ], acquired resistance limits long-term efficacy[ 27 ]. This underscores the need to identify novel regulatory proteins modulating tumor proliferation and apoptosis as alternative therapeutic targets. The FMO (Flavin-Containing Monooxygenase) family constitutes a group of enzymes pivotal in the oxidative metabolism of both endogenous and exogenous substrates, encompassing drugs and xenobiotics[ 28 ]. Among the mammalian isoforms, FMO1, FMO2, and FMO3 represent the predominant subtypes implicated in drug and xenobiotic biotransformation across various species. Notably, transcriptional expression and enzymatic activity of FMO1, FMO2, and FMO3 have been detected in murine pulmonary tissues[ 29 ]. Specifically, FMO2 emerges as a critical tumor suppressor in lung adenocarcinoma, wherein its diminished expression correlates significantly with augmented resistance to paclitaxel and heightened malignant phenotypic progression[ 30 ]. This underscores the importance of FMO family members in modulating lung cancer behavior. In this study, we observed significantly elevated FMO3 expression in NSCLC tissues compared to adjacent non-tumor regions. Clinically, FMO3 overexpression correlated positively with larger tumor size, advanced T-stage, and served as an independent prognostic factor for reduced overall survival. IHC correlation analysis demonstrated a strong positive association between FMO3 and Ki67 expression, suggesting that FMO3 promotes NSCLC malignancy by enhancing cellular proliferation. This finding positions FMO3 as a potential prognostic biomarker for postoperative survival prediction in NSCLC patients. The development and progression of cancer are intricately governed by the delicate balance between cell proliferation and apoptosis[ 31 , 32 ]. Previous studies have predominantly focused on the prognostic significance of FMOs in various malignancies, including colorectal, gastric, breast, and ovarian cancers[ 12 , 13 , 33 ]. Notably, FMO2 has been identified as a biomarker for predicting immunotherapy response in hepatocellular carcinoma[ 15 ], and its knockdown has been shown to promote proliferation and suppress apoptosis in lung cancer cells[ 30 ]. However, the functional role of FMO3 in cancer remains largely unexplored. Our study reveals, overexpression of FMO3 promoted cell viability and enhanced proliferation in A549 cells, whereas FMO3 knockdown suppressed cell viability and inhibited DNA synthesis activity in H1703 cells without impacting their clonogenic potential. Flow cytometry showed FMO3 overexpression in A549 cells reduced early and late apoptotic populations, while FMO3 knockdown in H1703 cells selectively increased early apoptosis. These findings collectively suggest that FMO3 serves as a potential biomarker for the treatment of NSCLC. The intricate interplay between cellular proliferation and apoptosis in lung cancer remains a central focus in oncology research, with endoplasmic reticulum (ER) stress emerging as a pivotal regulatory mechanism[ 34 , 35 ]. Persistent ER stress induced by chronic ischemia, hypoxia, and nutrient deprivation in lung cancer cells drives apoptotic cascades through coordinated signaling networks[ 36 ]. Previous studies indicate FMO3 regulates ER stress-mediated apoptosis by modulating the activity of IRE1α and PERK pathways [ 20 – 22 ]. In our investigation, Western blot analysis revealed that overexpression of FMO3 in NSCLC cells resulted in the downregulation of both total and phosphorylated IRE1α (p-IRE1α). Concurrently, it led to a reduction in the cleavage of caspase-12, caspase-3, and poly(ADP-ribose) polymerase (PARP). IHC analysis of paired NSCLC tissues and adjacent non-malignant tissues revealed elevated p-IRE1α immunoreactivity in tumors, inversely correlated with FMO3 expression levels. The IRE1/Caspase-12/Caspase-3 signaling pathway mediates ER stress[ 37 , 38 ]. In the context of lung cancer and cognitive dysfunction, the IRE1α/caspase 12 apoptotic pathway is implicated in ER stress-induced cell apoptosis[ 39 , 40 ]. Concurrently, the IRE1α/JNK/caspase-3 pathway also contributes to endothelial cell apoptosis by inducing ER stress[ 41 ]. Under sustained ER stress stimulation, caspases are activated, leading to the cleavage of poly(ADP-ribose) polymerase (PARP). The cleaved PARP loses its poly(ADP-ribose) polymerase (PARylation) activity and becomes incapable of repairing DNA damage, thereby accelerating cell apoptosis [ 42 , 43 ]. These findings suggest that FMO3 plays a crucial role in inhibiting key components of the ER stress-induced IRE1/Caspase-12/Caspase-3/PARP apoptotic cascade in NSCLC cells. Our study, by demonstrating the inhibitory effect of FMO3 on p-IRE1α and downstream apoptotic molecules, provides new insights into the regulatory network of ER stress-mediated apoptosis in NSCLC. It suggests that FMO3 could be a potential therapeutic target for modulating ER stress and preventing apoptosis in NSCLC. In conclusion,this study redefines FMO3, traditionally seen as a metabolic enzyme, as a context-dependent oncoprotein, thereby opening up new avenues for molecularly stratified therapy in non-small cell lung cancer (NSCLC). We propose that FMO3 is a promising therapeutic target for precision oncology in NSCLC. Looking ahead, in-depth research into the distinct functions and regulatory mechanisms of different FMO3 subtypes is warranted. For patients exhibiting high FMO3 expression, molecular profiling of predominant FMO3 isoforms could guide the development of isoform-specific inhibitors, potentially improving postoperative survival and quality of life through targeted interventions. Abbreviations NSCLC, Non-small cell lung cancer; FMOs, Flavin-containing monooxygenase; FAD, Flavin adenine dinucleotide; NADPH, Nicotinamide adenine dinucleotide phosphate; AOD, Average optical density; ER, Endoplasmic reticulum Declarations Funding Information This research was supported by National Natural Science Foundation of China (grant number 82203833). Competing interests The authors declare no competing interests. Ethics Statement Approval of the research protocol by an Institutional Reviewer Board:The study involving clinical sample collection was approved by the Ethics Committee of Xiangya Hospital, Central South University (Ethics Approval No. 2022020671, 5 March 2022). Informed Consent Informed consent was obtained from all subjects involved in the study. Author Contributions Hainaer Haisaer: Conceptualization, Investigation, Formal analysis, Writing - original draft. Wolong Zhou: Conceptualization. Xizhe Li: Funding acquisition, Resources. Chunfang Zhang: Funding acquisition, Resources. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. References Jawed, R., Bhatti, H. & Khan, A. Genetic profile of ferroptosis in non-small cell lung carcinoma and pharmaceutical options for ferroptosis induction . Clin Transl Oncol. 27, 1867-1886(2025) Siegel, R.L., Miller, K.D., Wagle, N.S. & Jemal, A. Cancer statistics, 2023 . CA Cancer J Clin. 73, 17-48(2023) Xi, Z. et al. Traditional Chinese medicine in lung cancer treatment . Mol Cancer. 24, 57(2025) Popat, S. et al. Navigating Diagnostic and Treatment Decisions in Non-Small Cell Lung Cancer: Expert Commentary on the Multidisciplinary Team Approach . Oncologist. 26, e306-e315(2021) Cohen, D. et al. Optimizing Mutation and Fusion Detection in NSCLC by Sequential DNA and RNA Sequencing . J Thorac Oncol. 15, 1000-1014(2020) Riely, G.J. et al. Non-Small Cell Lung Cancer, Version 4.2024, NCCN Clinical Practice Guidelines in Oncology . J Natl Compr Canc Netw. 22, 249-274(2024) Duma, N., Santana-Davila, R. & Molina, J.R. Non-Small Cell Lung Cancer: Epidemiology, Screening, Diagnosis, and Treatment . Mayo Clin Proc. 94, 1623-1640(2019) Bhat, A., Carranza, F.R., Tuckowski, A.M. & Leiser, S.F. Flavin-containing monooxygenase (FMO): Beyond xenobiotics . Bioessays. 46, e2400029(2024) Sun, B.Y. et al. Structure-guided engineering of a flavin-containing monooxygenase for the efficient production of indirubin . Bioresour Bioprocess. 9, 70(2022) Fialka, F. et al. CPA6, FMO2, LGI1, SIAT1 and TNC are differentially expressed in early- and late-stage oral squamous cell carcinoma--a pilot study . Oral Oncol. 44, 941-948(2008) Luo, Y. et al. FMO4 shapes immuno-metabolic reconfiguration in hepatocellular carcinoma . Clin Transl Med. 12, e740(2022) Zhang, T. et al. Overexpression of flavin-containing monooxygenase 5 predicts poor prognosis in patients with colorectal cancer . Oncol Lett. 15, 3923-3927(2018) Yu, S. et al. Cancer-associated fibroblasts-derived FMO2 as a biomarker of macrophage infiltration and prognosis in epithelial ovarian cancer . Gynecol Oncol. 167, 342-353(2022) Gong, X. et al. FMO family may serve as novel marker and potential therapeutic target for the peritoneal metastasis in gastric cancer . Front Oncol. 13, 1144775(2023) Xu, W. et al. FMO2(+) cancer-associated fibroblasts sensitize anti-PD-1 therapy in patients with hepatocellular carcinoma . J Immunother Cancer. 13, (2025) Kong, L. et al. Alcoholic fatty liver disease inhibited the co-expression of Fmo5 and PPARα to activate the NF-κB signaling pathway, thereby reducing liver injury via inducing gut microbiota disturbance . J Exp Clin Cancer Res. 40, 18(2021) Czarnocka, W. et al. FMO1 Is Involved in Excess Light Stress-Induced Signal Transduction and Cell Death Signaling . Cells. 9, (2020) Li, B. et al. Flavin-containing monooxygenase, a new clue of pathological proteins in the rotenone model of parkinsonism . Neurosci Lett. 566, 11-16(2014) Liu, Q. et al. Flavin-containing monooxygenase 2 confers cardioprotection in ischemia models through its disulfide bond catalytic activity . J Clin Invest. 134, (2024) Zhang, H. et al. FMO3 exacerbates hepatic endoplasmic reticulum stress in drug-induced liver injury by inhibiting CREB3/P4HB axis and activating TMAO-mediated PERK pathway . Life Sci. 374, 123699(2025) Liao, B.M., McManus, S.A., Hughes, W.E. & Schmitz-Peiffer, C. Flavin-Containing Monooxygenase 3 Reduces Endoplasmic Reticulum Stress in Lipid-Treated Hepatocytes . Mol Endocrinol. 30, 417-428(2016) Wang, J. et al. Deficiency of flavin-containing monooxygenase 3 protects kidney function after ischemia-reperfusion in mice . Commun Biol. 7, 1054(2024) Chen, P., Liu, Y., Wen, Y. & Zhou, C. Non-small cell lung cancer in China . Cancer Commun (Lond). 42, 937-970(2022) Huang, Q. et al. Advances in molecular pathology and therapy of non-small cell lung cancer . Signal Transduct Target Ther. 10, 186(2025) Odintsov, I. & Sholl, L.M. Prognostic and predictive biomarkers in non-small cell lung carcinoma . Pathology. 56, 192-204(2024) Tan, A.C. & Tan, D.S.W. Targeted Therapies for Lung Cancer Patients With Oncogenic Driver Molecular Alterations . J Clin Oncol. 40, 611-625(2022) Wu, J. & Lin, Z. Non-Small Cell Lung Cancer Targeted Therapy: Drugs and Mechanisms of Drug Resistance . Int J Mol Sci. 23, (2022) Rossner, R., Kaeberlein, M. & Leiser, S.F. Flavin-containing monooxygenases in aging and disease: Emerging roles for ancient enzymes . J Biol Chem. 292, 11138-11146(2017) Siddens, L.K., Henderson, M.C., Vandyke, J.E., Williams, D.E. & Krueger, S.K. Characterization of mouse flavin-containing monooxygenase transcript levels in lung and liver, and activity of expressed isoforms . Biochem Pharmacol. 75, 570-579(2008) Qian, X., Chen, C., Tong, S. & Zhang, J. Circ_MACF1 targets miR-421 to upregulate FMO2 to suppress paclitaxel resistance and malignant cellular behaviors in lung adenocarcinoma . Thorac Cancer. 14, 3348-3357(2023) He, R. et al. Mechanisms and cross-talk of regulated cell death and their epigenetic modifications in tumor progression . Mol Cancer. 23, 267(2024) Jenča, A. et al. Herbal Therapies for Cancer Treatment: A Review of Phytotherapeutic Efficacy . Biologics. 18, 229-255(2024) Wu, L., Chu, J., Shangguan, L., Cao, M. & Lu, F. Discovery and identification of the prognostic significance and potential mechanism of FMO2 in breast cancer . Aging (Albany NY). 15, 12651-12673(2023) Xu, Z. et al. Novel SERCA2 inhibitor Diphyllin displays anti-tumor effect in non-small cell lung cancer by promoting endoplasmic reticulum stress and mitochondrial dysfunction . Cancer Lett. 598, 217075(2024) Chen, J. et al. RPL11 promotes non-small cell lung cancer cell proliferation by regulating endoplasmic reticulum stress and cell autophagy . BMC Mol Cell Biol. 24, 7(2023) Li, X. et al. Endoplasmic reticulum stress in non-small cell lung cancer . Am J Cancer Res. 15, 1829-1851(2025) Wang, Z. et al. Doxycycline-Induced Apoptosis in Brucella suis S2-Infected HMC3 Cells via Calreticulin Suppression and Activation of the IRE1/Caspase-3 Signaling Pathway . Infect Drug Resist. 18, 2005-2020(2025) Wang, Z., Wang, Y., Yang, S., Wang, Z. & Yang, Q. Brucella suis S2 strain inhibits IRE1/caspase-12/caspase-3 pathway-mediated apoptosis of microglia HMC3 by affecting the ubiquitination of CALR . mSphere. 10, e0094124(2025) Lou, Z.H. et al. [Anti-lung cancer mechanisms of diterpenoid tanshinone via endoplasmic reticulum stress-mediated apoptosis signal pathway] . Zhongguo Zhong Yao Za Zhi. 43, 4900-4907(2018) Hu, X. et al. Sevoflurane postconditioning improves the spatial learning and memory impairments induced by hemorrhagic shock and resuscitation through suppressing IRE1α-caspase-12-mediated endoplasmic reticulum stress pathway . Neurosci Lett. 685, 160-166(2018) Wu, L. et al. Exendin-4 protects HUVECs from tunicamycin-induced apoptosis via inhibiting the IRE1a/JNK/caspase-3 pathway . Endocrine. 55, 764-772(2017) Los, M. et al. Activation and caspase-mediated inhibition of PARP: a molecular switch between fibroblast necrosis and apoptosis in death receptor signaling . Mol Biol Cell. 13, 978-988(2002) Aikin, R., Rosenberg, L., Paraskevas, S. & Maysinger, D. Inhibition of caspase-mediated PARP-1 cleavage results in increased necrosis in isolated islets of Langerhans . J Mol Med (Berl). 82, 389-397(2004) Additional Declarations No competing interests reported. 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09:09:41","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116422,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7984231/v1/426a16fe0a25fe7905144dc4.html"},{"id":96065356,"identity":"ce55d93e-0c53-40b0-9083-c1ec28f86fac","added_by":"auto","created_at":"2025-11-17 09:09:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":469879,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential expression of FMO family genes in NSCLC tissues and adjacent non-tumor counterparts. (A) Representative immunohistochemical staining of FMO1-5.Scale bar = 50 µm. (B) Quantitative analysis demonstrating significantly elevated FMO family genes expression in NSCLC tissues compared with matched controls. Data is expressed as mean ± SEM, n = 10. ****p \u0026lt; 0.0001, ns = no significance.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7984231/v1/cd7143dce12dd82b148123a3.png"},{"id":96065292,"identity":"427fffd9-f217-4687-8c0c-3c9206468577","added_by":"auto","created_at":"2025-11-17 09:09:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":506049,"visible":true,"origin":"","legend":"\u003cp\u003eCo-upregulation of Ki67 and FMO3 in non-small cell lung cancer. (A) Comparative immunohistochemical analysis of Ki67 and FMO3 expression in lung cancer and paired para-tumor tissues (left). Scale bar = 50 µm. Quantitative analysis demonstrating significantly elevated FMO3 and Ki67 expression (right). (B) Scatter plot demonstrating significant positive correlation between FMO3 and Ki67 expression levels. (C) Kaplan-Meier survival curves demonstrating significantly reduced 5-year overall survival in the FMO3-high expression group compared to the FMO3-low cohort. Data is expressed as mean ± SEM, n = 115. ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7984231/v1/8377a3b9c01c3ec019d8f2dd.png"},{"id":96065283,"identity":"2a88b54c-bf4d-415b-ab3b-064bc6e669e5","added_by":"auto","created_at":"2025-11-17 09:09:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":361276,"visible":true,"origin":"","legend":"\u003cp\u003eFMO3 inhibits NSCLC cells proliferation and colony formation. (A) CCK-8 assay reveals FMO3-dependent proliferation dynamics in NSCLC cells. (B) EdU assay reveals FMO3-dependent proliferation in NSCLC cells. Scale bar = 25 µm. (C) Colony formation assay reveals FMO3-dependent clonogenic potential in NSCLC cells. Data is expressed as mean ± SEM, n = 3. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, ns: no significance.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7984231/v1/0eca92152954b9f3e8dce90f.png"},{"id":96065334,"identity":"9dee4c39-3a08-4f8d-ad39-4ffcda4a0b21","added_by":"auto","created_at":"2025-11-17 09:09:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":374387,"visible":true,"origin":"","legend":"\u003cp\u003eFMO3 suppresses apoptosis via endoplasmic reticulum stress modulation in NSCLC Cells. (A) Annexin V-FITC/PI dot plots of apoptosis in FMO3-engineered NSCLC cells. (B) Representative western blot images of IRE1α pathway components (left). The PVDF membranes were cropped at the molecular weight range corresponding to the target protein regions before antibody incubation. The original blots are presented in Supplementary Figure S2. Quantitative analysis of protein expression and phosphorylation levels normalized to α-tubulin (right). Data is expressed as mean ± SEM, n = 3. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ns = no significance.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7984231/v1/4f1f972ff800a0bcfc4bb6c3.png"},{"id":96065280,"identity":"90bc1045-113c-4d50-b4c9-4cb811db51d5","added_by":"auto","created_at":"2025-11-17 09:09:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":184094,"visible":true,"origin":"","legend":"\u003cp\u003eNegative association of p-IRE1α and FMO3 in non-small cell lung cancer. (A) Comparative immunohistochemical analysis of p-IRE1α expression in lung cancer and paired para-tumor tissues (left). Scale bar = 50 µm. Quantitative analysis demonstrating significantly elevated p-IRE1α expression (right). (B) Scatter plot demonstrating significant positive correlation between FMO3 and p-IRE1α expression levels. Data is expressed as mean ± SEM, n = 115. ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7984231/v1/fcbbe319edb51918792e2810.png"},{"id":96065360,"identity":"ddd4c71a-18c6-4f48-8506-d71d384ec001","added_by":"auto","created_at":"2025-11-17 09:09:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2976519,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984231/v1/9353bbd7-b753-4766-9c65-d3c2a6506992.pdf"},{"id":96065294,"identity":"23259ab6-345d-4915-ae2e-f6759adacac1","added_by":"auto","created_at":"2025-11-17 09:09:29","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4196458,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7984231/v1/c51a3c9af8716ae223c56606.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"FMO3 as a novel prognostic biomarker and therapeutic target in non-small cell lung cancer by attenuating endoplasmic reticulum stress","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eLung cancer is ranked as the second most commonly diagnosed malignancy worldwide (following breast cancer) and remains the leading cause of cancer-related mortality[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The prognosis for lung cancer remains grim, with a five-year survival rate spanning a mere 7% to 22%, markedly inferior to that of other prevalent cancers[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Non-small cell lung cancer (NSCLC), which constitutes over 85% of all lung cancer cases, encompasses major histological subtypes including squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and adenosquamous carcinoma[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Over the past decade, the therapeutic paradigm for NSCLC has undergone substantial transformation. Precision-targeted agents and immune checkpoint blockade, in concert with state-of-the-art radiotherapeutic modalities, have been integrated alongside conventional chemotherapy, radiotherapy, and surgical resection as cornerstone interventions[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Consequently, therapeutic algorithms are now predicated on a multidimensional assessment that encompasses tumor-specific molecular profiles, actionable biomarkers, disease stage, and host physiological status[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, there is an urgent and compelling need to identify novel tumor biomarkers for early diagnosis, guiding treatment strategies, evaluating therapeutic efficacy, and predicting prognosis in NSCLC.\u003c/p\u003e\u003cp\u003eThe flavin-containing monooxygenase (FMOs) family comprises six members, namely FMO1-6, among which only FMO1-5 exhibit biological functions in the human body[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As a family of enzymes reliant on flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide phosphate (NADPH), and molecular oxygen (O₂), FMOs primarily participate in the oxidative metabolism of both exogenous and endogenous substrates, playing a pivotal role in drug metabolism and detoxification processes[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The FMOs family is expressed in numerous cancers and is closely associated with tumorigenesis and progression. Aberrant expression of FMOs has been observed in oral squamous cell carcinoma [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], hepatocellular carcinoma [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], colorectal cancer [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and ovarian cancer [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, FMOs have been identified as potential biomarkers for gastric cancer [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additionally, FMO2⁺ cancer-associated fibroblasts (CAFs) enhance the efficacy of anti-PD-1 therapy by modulating the immune properties of the tumor microenvironment [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Nevertheless, the precise mechanisms and functional significance of the FMO family in NSCLC pathogenesis and progression are yet to be determined.\u003c/p\u003e\u003cp\u003eMembers of the FMOs exhibit diverse cytological functions across various diseases. Specifically, FMO5 significantly inhibits cell apoptosis by suppressing the NF-κB signaling pathway and reducing oxidative stress[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. FMO1 is involved in the regulation of light-triggered cell death and directly induces caspase 3 activation due to its inhibited activity in Parkinson's disease models[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. FMO2 reduces cardiomyocyte apoptosis in cardiovascular diseases by inhibiting endoplasmic reticulum (ER) stress[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Conversely, FMO3 induces ER stress and cell apoptosis through the CREB3/P4HB axis in drug-induced liver injury[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the specific roles and mechanisms of the FMO family in NSCLC remain unclear.\u003c/p\u003e\u003cp\u003eTo clarify the unclear roles and mechanisms of the FMO family in NSCLC, we investigated FMOs' expression, functions, and mechanisms in NSCLC. Immunohistochemical analysis revealed significant FMO3 upregulation in NSCLC tissues, positively linked to larger tumor size and advanced T stage, as well as poor prognosis, serving as an independent risk factor. In vitro, FMO3 promoted cell proliferation and inhibited apoptosis in NSCLC cell lines. Mechanistically, FMO3 mediated ER stress-induced apoptosis via negative regulation of the IRE1α/caspase12/caspase3/PARP pathway, suggesting FMO3 as a potential prognostic biomarker and therapeutic target for NSCLC.\u003c/p\u003e"},{"header":"2 Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Selection of clinical samples\u003c/h2\u003e\u003cp\u003eTo assess FMOs expression in NSCLC, we first collected ten fresh tumor\u0026ndash;adjacent tissue pairs (adenocarcinoma and squamous cell carcinoma) from surgically resected specimens. Subsequently, a tissue microarray (TMA) was constructed containing 115 independent tumor\u0026ndash;adjacent pairs derived from patients who underwent curative lung resection at the Department of Thoracic Surgery, Xiangya Hospital, Central South University. Inclusion criteria were: (1) complete clinical data available (age, sex, histologic subtype, TNM stage, tumor size, smoking status, differentiation grade); (2) definitive pathologic report; (3) absence of any preoperative chemotherapy, radiotherapy, targeted therapy, or radiofrequency ablation; and (4) postoperative histopathologic confirmation of NSCLC.\u003c/p\u003e\u003cp\u003ePostoperative follow-up was conducted at 3-month intervals for the first year and every 6 months thereafter until month 60 or death. Only deaths attributable to lung cancer were considered events; deaths from other causes were censored. Among the 115 patients, 89 had complete follow-up data and were included in survival analyses. The study adhered to the principles of the Declaration of Helsinki and received approval from the Ethics Committee of Xiangya Hospital, Central South University (Ethics Approval No. 2022020671).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Immunohistochemistry\u003c/h2\u003e\u003cp\u003eFFPE sections (4 \u0026micro;m) were baked at 60\u0026deg;C for 30\u0026ndash;60 min, dewaxed, rehydrated, and subjected to antigen retrieval in 0.01 M citrate buffer (pH 6.0) via microwave for 20 min. After blocking endogenous peroxidase with 3% H₂O₂ (10 min) and serum (30 min), sections were incubated overnight at 4\u0026deg;C with primary antibody, followed by HRP-conjugated secondary antibody for 1 h at RT. Visualization employed DAB (\u0026le;\u0026thinsp;10 min), counterstaining with Mayer\u0026rsquo;s hematoxylin. Slides were dehydrated, cleared, and mounted in neutral balsam.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Cell culture\u003c/h2\u003e\u003cp\u003eNSCLC cell lines, including the A549 (CL-0016), NCI-H1299 (CL-0165), and NCI-H1703 (CL-0390) were provided from Procell (Wuhan, China), while the BEAS-2B (C6106) was obtained from Beyotime Biotechnology. All NSCLC cell lines, except A549 cells, were cultured in RPMI-1640 medium (PM150110, Procell), while A549 cells were maintained in Ham's F-12K cell medium (PM150910, Procell). BEAS-2B cells were cultured in DMEM (PM150210, Procell). All of these cell lines were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Cell-line construction\u003c/h2\u003e\u003cp\u003eStable FMO3-over-expressing A549 cells and FMO3-knockdown H1703 cells were generated by lentiviral transduction. Recombinant lentiviruses carrying human FMO3 cDNA (GeneChem) or three independent shRNAs targeting FMO3 (sh1\u0026ndash;sh3, GeneChem) were added at a multiplicity of infection (MOI) pre-determined by pilot infections. After 16 h exposure, the medium was replaced and 48 h later cells were selected with puromycin (2 \u0026micro;g mL⁻\u0026sup1;) or neomycin (800 \u0026micro;g mL⁻\u0026sup1;), respectively. Surviving colonies were expanded for two weeks, validated by western blot, and stocks were cryopreserved in 90% FBS plus 10% DMSO.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 CCK-8 proliferation assay\u003c/h2\u003e\u003cp\u003eCells were seeded in 96-well plates (3 \u0026times; 10\u0026sup3; cells/well) and incubated for 0\u0026ndash;72 h. At each time-point, 10 \u0026micro;L CCK-8 reagent (CK04, Dojindo) was added and plates were returned to 37\u0026deg;C for 2 h. Absorbance at 450 nm was measured in a microplate reader (BioTek). All conditions were run in quintuplicate, and viability was expressed as the percentage of untreated controls after background subtraction.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 EdU incorporation assay\u003c/h2\u003e\u003cp\u003eExponentially growing cells were exposed to 50 \u0026micro;M EdU for 2 h, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and labelled with Apollo-488 (KGA1108, keygenbio) according to the manufacturer\u0026rsquo;s protocol. Nuclei were counterstained with Hoechst 33342. EdU-positive cells were quantified by fluorescence microscopy (\u0026times;20 objective, five random fields/well) and expressed as the percentage of total nuclei.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Colony formation assay\u003c/h2\u003e\u003cp\u003eSingle-cell suspensions (500 cells/well) were plated in 6-well plates and cultured for 14 days. Colonies were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet for 20 min, washed, air-dried, and counted manually under a stereomicroscope (\u0026ge;\u0026thinsp;50 cells/colony). Results were normalized to plating efficiency and presented as fold change relative to controls.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Apoptosis assessment\u003c/h2\u003e\u003cp\u003eApoptosis was quantified using Annexin V-FITC/PI double staining (KGA1102, keygenbio). Briefly, 1 \u0026times; 10⁵ cells were harvested, washed twice with cold PBS, resuspended in 100 \u0026micro;L binding buffer and incubated with 5 \u0026micro;L Annexin V-FITC plus 5 \u0026micro;L PI for 15 min at room temperature in the dark. Samples were analyzed within 1 h on a FACSCanto II flow cytometer (BD). Early and late apoptotic populations were combined to calculate total apoptosis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Western blotting\u003c/h2\u003e\u003cp\u003eTotal protein was extracted with RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts (30 \u0026micro;g) were separated on 10% SDS-PAGE gels, transferred to PVDF membranes. The PVDF membranes were cropped prior to hybridization with the antibodies. Then, the membranes were blocked with 5% non-fat milk, and probed overnight at 4\u0026deg;C with primary antibodies against FMO3 (ab126711, Abcam), IRE1α (27528-1-AP, Proteintech), p-IRE1α-S724 (ab48187, Abcam), caspase-12 (55238-1-AP, Proteintech), caspase-3 (19677-1-AP, Proteintech), cleaved PARP (13371-1-AP, Proteintech), α-tubulin (11224-1-AP, Proteintech) and GAPDH (60004-1-Ig, Proteintech). After incubation with HRP-conjugated secondary antibodies, bands were visualized using ECL (Millipore) and quantified by densitometry (ImageJ).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed with SPSS 24.0 and illustrated using GraphPad Prism 8. Continuous variables are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and were compared by independent-sample t tests or one-way ANOVA; categorical data are expressed as counts and analyzed by χ\u0026sup2; tests. Five-year overall survival was estimated by Kaplan\u0026ndash;Meier analysis and visualized with survival curves. Covariates showing significance in univariate analysis were entered into a Cox proportional-hazards regression model for multivariate assessment. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (two-tailed) was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 FMO3 exhibits overexpression in non-small cell lung cancer tissues\u003c/h2\u003e\u003cp\u003eTo delineate the expression profiles of flavin-containing monooxygenase family (FMOs 1\u0026ndash;5) in non-small cell lung cancer (NSCLC), we conducted immunohistochemical (IHC) staining on ten paired NSCLC and adjacent non-tumor tissues (n\u0026thinsp;=\u0026thinsp;20 total). All five FMO family genes exhibited detectable protein expression in both tumor and para-tumor tissue. Notably, quantitative analysis demonstrated significantly upregulated FMO3 expression in NSCLC tissues compared to matched adjacent non-tumor controls (P\u0026thinsp;=\u0026thinsp;0.0003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). In contrast, no statistically significant differences were observed for FMO1, FMO2, FMO4, or FMO5 (all P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These findings prompted us to select FMO3 as the primary target for subsequent functional investigations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further validate the differential expression of FMO3 in NSCLC, we constructed a tissue microarray (TMA) comprising of 115 paired NSCLC and adjacent non-tumor tissues. IHC staining was performed on the TMA sections, followed by whole-slide scanning to generate high-resolution digital images. Quantitative analysis using ImageJ software revealed significantly upregulated FMO3 expression in NSCLC tissues compared to matched controls, as determined by average optical density (AOD) measurements (P\u0026thinsp;=\u0026thinsp;0.0003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA upper).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurther analysis of tumor proliferation via Ki67 immunohistochemistry demonstrated significantly elevated labeling indices in NSCLC tissues versus para-tumor controls (P\u0026thinsp;=\u0026thinsp;0.0002) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA lower). Notably, FMO3 expression levels exhibited a significant positive correlation with Ki67 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These findings collectively suggest that FMO3 is overexpressed in NSCLC tissues and its expression is closely associated with enhanced tumor cell proliferation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2 FMO3 serves as an independent prognostic factor in non-small cell lung cancer\u003c/h2\u003e\u003cp\u003eTo evaluate the clinical significance of FMO3 in NSCLC progression, patients were stratified into FMO3-high and FMO3-low subgroups using median IHC scores as the cutoff. Comparative analysis of clinicopathological parameters revealed significant associations between elevated FMO3 expression and larger tumor size (P\u0026thinsp;=\u0026thinsp;0.0021) as well as T-stage classification (P\u0026thinsp;=\u0026thinsp;0.016) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, Kaplan-Meier survival analysis demonstrated a significant inverse correlation between FMO3 expression levels and 5-year overall survival (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAssociation between FMO3 expression and clinicopathological characteristics in NSCLC patients\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSubgroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFMO3-Low (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFMO3-High (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;60 years\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e26 (53.06)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23 (46.94)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.3460\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;60 years\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e41 (62.12)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e25 (37.88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e49 (57.65)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e36 (42.35)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e18 (60.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12 (40.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHistological Type\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAdenocarcinoma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37 (62.71)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e22 (37.29)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.3491\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSquamous\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30 (53.57)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e26 (46.43)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTNM Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eI-II\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e43 (58.11)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e31 (41.89)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIII-IV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24 (58.54)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e17 (41.46)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLymph Node Metastasis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23 (60.53)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e15 (39.47)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.8412\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e44 (57.14)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e33 (42.86)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSmoking Status\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNon-smoker\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e33 (67.35)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16 (32.65)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.1258\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSmoker\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e34 (51.52)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e32 (48.48)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTumor Differentiation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eModerate/High\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e45 (61.64)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e28 (38.36)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.4324\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e22 (52.38)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20 (47.62)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTumor Size\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026le;\u0026thinsp;5 cm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e54 (65.06)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e29 (34.94)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.0035\u003c/b\u003e*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;5 cm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11 (34.38)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e21 (65.63)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT1-T2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e53 (65.43)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e28 (34.57)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.0224\u003c/b\u003e*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT3-T4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14 (41.18)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20 (58.82)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eUnivariate Cox regression analysis identified FMO3 overexpression (IHC score\u0026thinsp;\u0026ge;\u0026thinsp;median) as a significant predictor of reduced 5-year overall survival (OS) (HR\u0026thinsp;=\u0026thinsp;3.32, 95% CI: 1.59\u0026ndash;6.93, P\u0026thinsp;=\u0026thinsp;0.01) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). After adjusting for potential confounders in multivariate analysis, FMO3 overexpression retained independent prognostic significance (HR\u0026thinsp;=\u0026thinsp;2.95, 95% CI: 1.38\u0026ndash;6.32, P\u0026thinsp;=\u0026thinsp;0.005) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results collectively indicate that FMO3 overexpression is closely linked to poor prognosis in NSCLC. Importantly, the prognostic impact of FMO3 even surpasses that of traditional clinicopathological factors such as TNM stage in the final model, highlighting its potential as a novel and robust biomarker for predicting survival outcomes and guiding therapeutic strategies in NSCLC patients.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eUnivariate and multivariate Cox regression analyses of prognostic factors in NSCLC\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnivariate Analysis\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMultivariate Analysis\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHR (95% CI)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHR (95% CI)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAge (\u0026ge;\u0026thinsp;60 vs\u0026thinsp;\u0026lt;\u0026thinsp;60)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.55 (0.77\u0026ndash;3.10)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGender (Male vs Female)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.44 (0.62\u0026ndash;3.33)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHistological Type (Adeno vs Squamous)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.87 (0.43\u0026ndash;1.73)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eT Stage (T3-T4 vs T1-T2)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e1.47 (0.71\u0026ndash;3.05)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.02\u003c/b\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.52 (0.66\u0026ndash;3.72)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLymph Node Metastasis (Yes vs No)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.69 (0.83\u0026ndash;3.43)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTumor Size (\u0026gt;\u0026thinsp;5 cm vs\u0026thinsp;\u0026le;\u0026thinsp;5 cm)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e2.54 (1.26\u0026ndash;5.13)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.01\u003c/b\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.03 (0.99\u0026ndash;4.17)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTNM Stage (III-IV vs I-II)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e1.78 (0.88\u0026ndash;3.58)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.02\u003c/b\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e1.67 (0.99\u0026ndash;3.77)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.03\u003c/b\u003e*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDifferentiation (Poor vs Moderate/High)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.95 (0.97\u0026ndash;3.91)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSmoking (Yes vs No)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e2.47 (1.11\u0026ndash;5.51)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.02\u003c/b\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.90 (0.84\u0026ndash;4.31)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFMO3 Expression (High vs Low)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e3.32 (1.59\u0026ndash;6.93)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.01\u003c/b\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e2.84 (1.34\u0026ndash;6.01)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.01\u003c/b\u003e*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eNotes:\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e*Statistical significance: *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eAdeno: Adenocarcinoma; Squamous: Squamous cell carcinoma\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eMultivariate model adjusted for T stage, tumor size, TNM stage, smoking status, and FMO3 expression\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eHR: Hazard Ratio; CI: Confidence Interval\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\"-\" indicates variables not included in multivariate analysis\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3 FMO3 promotes non - small cell lung cancer cell proliferation\u003c/h2\u003e\u003cp\u003eTo investigate the functional role of FMO3 in NSCLC, we first screened basal FMO3 protein levels in A549, H1299, H1703 (NSCLC), and HBEAS-2B (normal) cells by Western blot. The results demonstrated that FMO3 exhibited relatively higher expression in HBEAS-2B, H1299, and H1703 cells, while showing relatively lower expression in A549 cells (figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A549 (lowest FMO3) was selected for stable overexpression via lentiviral transduction, while H1703 (highest FMO3) underwent FMO3 knockdown by shRNA (figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Overexpression of FMO3 was found to significantly bolster cell viability in A549 cells. In stark contrast, when FMO3 was knocked down in H1703 cells, a marked suppression of cell viability was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Regarding DNA synthesis, FMO3 overexpression in A549 cells led to a notable enhancement of this process, while in H1703 cells, FMO3 knockdown resulted in a significant inhibition of DNA synthesis activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, our results indicated that overexpression of FMO3 in A549 cells enhanced their clonogenic potential. However, when FMO3 was knocked down in H1703 cells, there was no discernible impact on their clonogenic potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These results collectively suggest that FMO3 plays a crucial role in promoting NSCLC cell growth and survival.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4 FMO3 overexpression alleviates endoplasmic reticulum stress and inhibits apoptosis in NSCLC Cells\u003c/h2\u003e\u003cp\u003eFlow cytometry analysis demonstrated that FMO3 overexpression in A549 cells significantly reduced early and late populations compared to controls, whereas FMO3 knockdown in H1703 cells selectively elevated early apoptosis without affecting late apoptotic or necrotic rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These findings suggest that FMO3 differentially suppresses apoptosis: inhibiting both early and late stages in A549 cells but primarily modulating early signaling in H1703 cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePrevious studies have demonstrated that FMO3 exerts dual regulatory effects on endoplasmic reticulum (ER) stress-mediated apoptosis. Mechanistically, FMO3 attenuates ER stress-induced apoptosis via JNK dephosphorylation and reduced cleaved caspase-3/XBP1s levels (IRE1 pathway)[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Concurrently, FMO3 modulates PERK pathway activity by interacting with CREB3 and regulating PERK/eIF2α phosphorylation, thereby contributing to ER stress homeostasis[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Building on these findings, we conducted mechanistic investigations using Western blot analysis to evaluate the expression profile of ER stress-associated proteins (IRE1α, caspase-12, caspase-3, and PARP) in FMO3-overexpressing A549 cells. The results demonstrated significant downregulation of total and phosphorylated IRE1α (p-IRE1α), along with reduced cleavage of caspase-12, caspase-3 and PARP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eTo elucidate these in vitro observations in clinical specimens, we conducted IHC analysis on paired NSCLC tissues and adjacent non-malignant counterparts. Quantitative assessment demonstrated significantly elevated p-IRE1α immunoreactivity in tumor specimens compared to matched normal controls (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Correlation analysis revealed a strong inverse relationship between p-IRE1α accumulation and FMO3 expression levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), suggesting a potential reciprocal regulatory mechanism between FMO3 and ER stress signaling in NSCLC pathogenesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn China, non-small cell lung cancer (NSCLC) incidence and mortality continue to rise, with over 820,000 new cases annually [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Globally, NSCLC 5-year survival remains\u0026thinsp;\u0026lt;\u0026thinsp;20% due to frequent late-stage diagnosis[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. While molecular targeted therapies have improved outcomes for patients with actionable mutations[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], acquired resistance limits long-term efficacy[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This underscores the need to identify novel regulatory proteins modulating tumor proliferation and apoptosis as alternative therapeutic targets.\u003c/p\u003e\u003cp\u003eThe FMO (Flavin-Containing Monooxygenase) family constitutes a group of enzymes pivotal in the oxidative metabolism of both endogenous and exogenous substrates, encompassing drugs and xenobiotics[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Among the mammalian isoforms, FMO1, FMO2, and FMO3 represent the predominant subtypes implicated in drug and xenobiotic biotransformation across various species. Notably, transcriptional expression and enzymatic activity of FMO1, FMO2, and FMO3 have been detected in murine pulmonary tissues[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Specifically, FMO2 emerges as a critical tumor suppressor in lung adenocarcinoma, wherein its diminished expression correlates significantly with augmented resistance to paclitaxel and heightened malignant phenotypic progression[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This underscores the importance of FMO family members in modulating lung cancer behavior. In this study, we observed significantly elevated FMO3 expression in NSCLC tissues compared to adjacent non-tumor regions. Clinically, FMO3 overexpression correlated positively with larger tumor size, advanced T-stage, and served as an independent prognostic factor for reduced overall survival. IHC correlation analysis demonstrated a strong positive association between FMO3 and Ki67 expression, suggesting that FMO3 promotes NSCLC malignancy by enhancing cellular proliferation. This finding positions FMO3 as a potential prognostic biomarker for postoperative survival prediction in NSCLC patients.\u003c/p\u003e\u003cp\u003eThe development and progression of cancer are intricately governed by the delicate balance between cell proliferation and apoptosis[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Previous studies have predominantly focused on the prognostic significance of FMOs in various malignancies, including colorectal, gastric, breast, and ovarian cancers[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Notably, FMO2 has been identified as a biomarker for predicting immunotherapy response in hepatocellular carcinoma[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and its knockdown has been shown to promote proliferation and suppress apoptosis in lung cancer cells[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, the functional role of FMO3 in cancer remains largely unexplored. Our study reveals, overexpression of FMO3 promoted cell viability and enhanced proliferation in A549 cells, whereas FMO3 knockdown suppressed cell viability and inhibited DNA synthesis activity in H1703 cells without impacting their clonogenic potential. Flow cytometry showed FMO3 overexpression in A549 cells reduced early and late apoptotic populations, while FMO3 knockdown in H1703 cells selectively increased early apoptosis. These findings collectively suggest that FMO3 serves as a potential biomarker for the treatment of NSCLC.\u003c/p\u003e\u003cp\u003eThe intricate interplay between cellular proliferation and apoptosis in lung cancer remains a central focus in oncology research, with endoplasmic reticulum (ER) stress emerging as a pivotal regulatory mechanism[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Persistent ER stress induced by chronic ischemia, hypoxia, and nutrient deprivation in lung cancer cells drives apoptotic cascades through coordinated signaling networks[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Previous studies indicate FMO3 regulates ER stress-mediated apoptosis by modulating the activity of IRE1α and PERK pathways [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In our investigation, Western blot analysis revealed that overexpression of FMO3 in NSCLC cells resulted in the downregulation of both total and phosphorylated IRE1α (p-IRE1α). Concurrently, it led to a reduction in the cleavage of caspase-12, caspase-3, and poly(ADP-ribose) polymerase (PARP). IHC analysis of paired NSCLC tissues and adjacent non-malignant tissues revealed elevated p-IRE1α immunoreactivity in tumors, inversely correlated with FMO3 expression levels. The IRE1/Caspase-12/Caspase-3 signaling pathway mediates ER stress[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In the context of lung cancer and cognitive dysfunction, the IRE1α/caspase 12 apoptotic pathway is implicated in ER stress-induced cell apoptosis[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Concurrently, the IRE1α/JNK/caspase-3 pathway also contributes to endothelial cell apoptosis by inducing ER stress[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Under sustained ER stress stimulation, caspases are activated, leading to the cleavage of poly(ADP-ribose) polymerase (PARP). The cleaved PARP loses its poly(ADP-ribose) polymerase (PARylation) activity and becomes incapable of repairing DNA damage, thereby accelerating cell apoptosis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. These findings suggest that FMO3 plays a crucial role in inhibiting key components of the ER stress-induced IRE1/Caspase-12/Caspase-3/PARP apoptotic cascade in NSCLC cells.\u003c/p\u003e\u003cp\u003eOur study, by demonstrating the inhibitory effect of FMO3 on p-IRE1α and downstream apoptotic molecules, provides new insights into the regulatory network of ER stress-mediated apoptosis in NSCLC. It suggests that FMO3 could be a potential therapeutic target for modulating ER stress and preventing apoptosis in NSCLC.\u003c/p\u003e\u003cp\u003eIn conclusion,this study redefines FMO3, traditionally seen as a metabolic enzyme, as a context-dependent oncoprotein, thereby opening up new avenues for molecularly stratified therapy in non-small cell lung cancer (NSCLC). We propose that FMO3 is a promising therapeutic target for precision oncology in NSCLC. Looking ahead, in-depth research into the distinct functions and regulatory mechanisms of different FMO3 subtypes is warranted. For patients exhibiting high FMO3 expression, molecular profiling of predominant FMO3 isoforms could guide the development of isoform-specific inhibitors, potentially improving postoperative survival and quality of life through targeted interventions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNSCLC, Non-small cell lung cancer; FMOs, Flavin-containing monooxygenase; FAD, Flavin adenine dinucleotide; NADPH, Nicotinamide adenine dinucleotide phosphate; AOD, Average optical density; ER, Endoplasmic reticulum\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by National Natural Science Foundation of China (grant number 82203833).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproval of the research protocol by an Institutional Reviewer Board:The study involving clinical sample collection was approved by the Ethics Committee of Xiangya Hospital, Central South University (Ethics Approval No. 2022020671, 5 March 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHainaer Haisaer:\u003c/strong\u003e Conceptualization, Investigation, Formal analysis, Writing - original draft. \u003cstrong\u003eWolong Zhou:\u0026nbsp;\u003c/strong\u003eConceptualization. \u003cstrong\u003eXizhe Li:\u0026nbsp;\u003c/strong\u003eFunding acquisition, Resources.\u003cstrong\u003e\u0026nbsp;Chunfang Zhang:\u0026nbsp;\u003c/strong\u003eFunding acquisition, Resources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJawed, R., Bhatti, H. \u0026amp; Khan, A. Genetic profile of ferroptosis in non-small cell lung carcinoma and pharmaceutical options for ferroptosis induction\u003cem\u003e.\u003c/em\u003e \u003cem\u003eClin Transl Oncol.\u003c/em\u003e 27, 1867-1886(2025)\u003c/li\u003e\n\u003cli\u003eSiegel, R.L., Miller, K.D., Wagle, N.S. \u0026amp; Jemal, A. Cancer statistics, 2023\u003cem\u003e.\u003c/em\u003e \u003cem\u003eCA Cancer J Clin.\u003c/em\u003e 73, 17-48(2023)\u003c/li\u003e\n\u003cli\u003eXi, Z. et al. Traditional Chinese medicine in lung cancer treatment\u003cem\u003e.\u003c/em\u003e \u003cem\u003eMol Cancer.\u003c/em\u003e 24, 57(2025)\u003c/li\u003e\n\u003cli\u003ePopat, S. et al. Navigating Diagnostic and Treatment Decisions in Non-Small Cell Lung Cancer: Expert Commentary on the Multidisciplinary Team Approach\u003cem\u003e.\u003c/em\u003e \u003cem\u003eOncologist.\u003c/em\u003e 26, e306-e315(2021)\u003c/li\u003e\n\u003cli\u003eCohen, D. et al. Optimizing Mutation and Fusion Detection in NSCLC by Sequential DNA and RNA Sequencing\u003cem\u003e.\u003c/em\u003e \u003cem\u003eJ Thorac Oncol.\u003c/em\u003e 15, 1000-1014(2020)\u003c/li\u003e\n\u003cli\u003eRiely, G.J. et al. Non-Small Cell Lung Cancer, Version 4.2024, NCCN Clinical Practice Guidelines in Oncology\u003cem\u003e.\u003c/em\u003e \u003cem\u003eJ Natl Compr Canc Netw.\u003c/em\u003e 22, 249-274(2024)\u003c/li\u003e\n\u003cli\u003eDuma, N., Santana-Davila, R. \u0026amp; Molina, J.R. Non-Small Cell Lung Cancer: Epidemiology, Screening, Diagnosis, and Treatment\u003cem\u003e.\u003c/em\u003e \u003cem\u003eMayo Clin Proc.\u003c/em\u003e 94, 1623-1640(2019)\u003c/li\u003e\n\u003cli\u003eBhat, A., Carranza, F.R., Tuckowski, A.M. \u0026amp; Leiser, S.F. Flavin-containing monooxygenase (FMO): Beyond xenobiotics\u003cem\u003e.\u003c/em\u003e \u003cem\u003eBioessays.\u003c/em\u003e 46, e2400029(2024)\u003c/li\u003e\n\u003cli\u003eSun, B.Y. et al. Structure-guided engineering of a flavin-containing monooxygenase for the efficient production of indirubin\u003cem\u003e.\u003c/em\u003e \u003cem\u003eBioresour Bioprocess.\u003c/em\u003e 9, 70(2022)\u003c/li\u003e\n\u003cli\u003eFialka, F. et al. CPA6, FMO2, LGI1, SIAT1 and TNC are differentially expressed in early- and late-stage oral squamous cell carcinoma--a pilot study\u003cem\u003e.\u003c/em\u003e \u003cem\u003eOral Oncol.\u003c/em\u003e 44, 941-948(2008)\u003c/li\u003e\n\u003cli\u003eLuo, Y. et al. FMO4 shapes immuno-metabolic reconfiguration in hepatocellular carcinoma\u003cem\u003e.\u003c/em\u003e \u003cem\u003eClin Transl Med.\u003c/em\u003e 12, e740(2022)\u003c/li\u003e\n\u003cli\u003eZhang, T. et al. Overexpression of flavin-containing monooxygenase 5 predicts poor prognosis in patients with colorectal cancer\u003cem\u003e.\u003c/em\u003e \u003cem\u003eOncol Lett.\u003c/em\u003e 15, 3923-3927(2018)\u003c/li\u003e\n\u003cli\u003eYu, S. et al. Cancer-associated fibroblasts-derived FMO2 as a biomarker of macrophage infiltration and prognosis in epithelial ovarian cancer\u003cem\u003e.\u003c/em\u003e \u003cem\u003eGynecol Oncol.\u003c/em\u003e 167, 342-353(2022)\u003c/li\u003e\n\u003cli\u003eGong, X. et al. FMO family may serve as novel marker and potential therapeutic target for the peritoneal metastasis in gastric cancer\u003cem\u003e.\u003c/em\u003e \u003cem\u003eFront Oncol.\u003c/em\u003e 13, 1144775(2023)\u003c/li\u003e\n\u003cli\u003eXu, W. et al. FMO2(+) cancer-associated fibroblasts sensitize anti-PD-1 therapy in patients with hepatocellular carcinoma\u003cem\u003e.\u003c/em\u003e \u003cem\u003eJ Immunother Cancer.\u003c/em\u003e 13, (2025)\u003c/li\u003e\n\u003cli\u003eKong, L. et al. Alcoholic fatty liver disease inhibited the co-expression of Fmo5 and PPAR\u0026alpha; to activate the NF-\u0026kappa;B signaling pathway, thereby reducing liver injury via inducing gut microbiota disturbance\u003cem\u003e.\u003c/em\u003e \u003cem\u003eJ Exp Clin Cancer Res.\u003c/em\u003e 40, 18(2021)\u003c/li\u003e\n\u003cli\u003eCzarnocka, W. et al. FMO1 Is Involved in Excess Light Stress-Induced Signal Transduction and Cell Death Signaling\u003cem\u003e.\u003c/em\u003e \u003cem\u003eCells.\u003c/em\u003e 9, (2020)\u003c/li\u003e\n\u003cli\u003eLi, B. et al. Flavin-containing monooxygenase, a new clue of pathological proteins in the rotenone model of parkinsonism\u003cem\u003e.\u003c/em\u003e \u003cem\u003eNeurosci Lett.\u003c/em\u003e 566, 11-16(2014)\u003c/li\u003e\n\u003cli\u003eLiu, Q. et al. Flavin-containing monooxygenase 2 confers cardioprotection in ischemia models through its disulfide bond catalytic activity\u003cem\u003e.\u003c/em\u003e \u003cem\u003eJ Clin Invest.\u003c/em\u003e 134, (2024)\u003c/li\u003e\n\u003cli\u003eZhang, H. et al. FMO3 exacerbates hepatic endoplasmic reticulum stress in drug-induced liver injury by inhibiting CREB3/P4HB axis and activating TMAO-mediated PERK pathway\u003cem\u003e.\u003c/em\u003e \u003cem\u003eLife Sci.\u003c/em\u003e 374, 123699(2025)\u003c/li\u003e\n\u003cli\u003eLiao, B.M., McManus, S.A., Hughes, W.E. \u0026amp; Schmitz-Peiffer, C. Flavin-Containing Monooxygenase 3 Reduces Endoplasmic Reticulum Stress in Lipid-Treated Hepatocytes\u003cem\u003e.\u003c/em\u003e \u003cem\u003eMol Endocrinol.\u003c/em\u003e 30, 417-428(2016)\u003c/li\u003e\n\u003cli\u003eWang, J. et al. Deficiency of flavin-containing monooxygenase 3 protects kidney function after ischemia-reperfusion in mice\u003cem\u003e.\u003c/em\u003e \u003cem\u003eCommun Biol.\u003c/em\u003e 7, 1054(2024)\u003c/li\u003e\n\u003cli\u003eChen, P., Liu, Y., Wen, Y. \u0026amp; Zhou, C. Non-small cell lung cancer in China\u003cem\u003e.\u003c/em\u003e \u003cem\u003eCancer Commun (Lond).\u003c/em\u003e 42, 937-970(2022)\u003c/li\u003e\n\u003cli\u003eHuang, Q. et al. Advances in molecular pathology and therapy of non-small cell lung cancer\u003cem\u003e.\u003c/em\u003e \u003cem\u003eSignal Transduct Target Ther.\u003c/em\u003e 10, 186(2025)\u003c/li\u003e\n\u003cli\u003eOdintsov, I. \u0026amp; Sholl, L.M. Prognostic and predictive biomarkers in non-small cell lung carcinoma\u003cem\u003e.\u003c/em\u003e \u003cem\u003ePathology.\u003c/em\u003e 56, 192-204(2024)\u003c/li\u003e\n\u003cli\u003eTan, A.C. \u0026amp; Tan, D.S.W. Targeted Therapies for Lung Cancer Patients With Oncogenic Driver Molecular Alterations\u003cem\u003e.\u003c/em\u003e \u003cem\u003eJ Clin Oncol.\u003c/em\u003e 40, 611-625(2022)\u003c/li\u003e\n\u003cli\u003eWu, J. \u0026amp; Lin, Z. Non-Small Cell Lung Cancer Targeted Therapy: Drugs and Mechanisms of Drug Resistance\u003cem\u003e.\u003c/em\u003e \u003cem\u003eInt J Mol Sci.\u003c/em\u003e 23, (2022)\u003c/li\u003e\n\u003cli\u003eRossner, R., Kaeberlein, M. \u0026amp; Leiser, S.F. Flavin-containing monooxygenases in aging and disease: Emerging roles for ancient enzymes\u003cem\u003e.\u003c/em\u003e \u003cem\u003eJ Biol Chem.\u003c/em\u003e 292, 11138-11146(2017)\u003c/li\u003e\n\u003cli\u003eSiddens, L.K., Henderson, M.C., Vandyke, J.E., Williams, D.E. \u0026amp; Krueger, S.K. Characterization of mouse flavin-containing monooxygenase transcript levels in lung and liver, and activity of expressed isoforms\u003cem\u003e.\u003c/em\u003e \u003cem\u003eBiochem Pharmacol.\u003c/em\u003e 75, 570-579(2008)\u003c/li\u003e\n\u003cli\u003eQian, X., Chen, C., Tong, S. \u0026amp; Zhang, J. Circ_MACF1 targets miR-421 to upregulate FMO2 to suppress paclitaxel resistance and malignant cellular behaviors in lung adenocarcinoma\u003cem\u003e.\u003c/em\u003e \u003cem\u003eThorac Cancer.\u003c/em\u003e 14, 3348-3357(2023)\u003c/li\u003e\n\u003cli\u003eHe, R. et al. Mechanisms and cross-talk of regulated cell death and their epigenetic modifications in tumor progression\u003cem\u003e.\u003c/em\u003e \u003cem\u003eMol Cancer.\u003c/em\u003e 23, 267(2024)\u003c/li\u003e\n\u003cli\u003eJenča, A. et al. Herbal Therapies for Cancer Treatment: A Review of Phytotherapeutic Efficacy\u003cem\u003e.\u003c/em\u003e \u003cem\u003eBiologics.\u003c/em\u003e 18, 229-255(2024)\u003c/li\u003e\n\u003cli\u003eWu, L., Chu, J., Shangguan, L., Cao, M. \u0026amp; Lu, F. Discovery and identification of the prognostic significance and potential mechanism of FMO2 in breast cancer\u003cem\u003e.\u003c/em\u003e \u003cem\u003eAging (Albany NY).\u003c/em\u003e 15, 12651-12673(2023)\u003c/li\u003e\n\u003cli\u003eXu, Z. et al. Novel SERCA2 inhibitor Diphyllin displays anti-tumor effect in non-small cell lung cancer by promoting endoplasmic reticulum stress and mitochondrial dysfunction\u003cem\u003e.\u003c/em\u003e \u003cem\u003eCancer Lett.\u003c/em\u003e 598, 217075(2024)\u003c/li\u003e\n\u003cli\u003eChen, J. et al. RPL11 promotes non-small cell lung cancer cell proliferation by regulating endoplasmic reticulum stress and cell autophagy\u003cem\u003e.\u003c/em\u003e \u003cem\u003eBMC Mol Cell Biol.\u003c/em\u003e 24, 7(2023)\u003c/li\u003e\n\u003cli\u003eLi, X. et al. Endoplasmic reticulum stress in non-small cell lung cancer\u003cem\u003e.\u003c/em\u003e \u003cem\u003eAm J Cancer Res.\u003c/em\u003e 15, 1829-1851(2025)\u003c/li\u003e\n\u003cli\u003eWang, Z. et al. Doxycycline-Induced Apoptosis in Brucella suis S2-Infected HMC3 Cells via Calreticulin Suppression and Activation of the IRE1/Caspase-3 Signaling Pathway\u003cem\u003e.\u003c/em\u003e \u003cem\u003eInfect Drug Resist.\u003c/em\u003e 18, 2005-2020(2025)\u003c/li\u003e\n\u003cli\u003eWang, Z., Wang, Y., Yang, S., Wang, Z. \u0026amp; Yang, Q. Brucella suis S2 strain inhibits IRE1/caspase-12/caspase-3 pathway-mediated apoptosis of microglia HMC3 by affecting the ubiquitination of CALR\u003cem\u003e.\u003c/em\u003e \u003cem\u003emSphere.\u003c/em\u003e 10, e0094124(2025)\u003c/li\u003e\n\u003cli\u003eLou, Z.H. et al. [Anti-lung cancer mechanisms of diterpenoid tanshinone via endoplasmic reticulum stress-mediated apoptosis signal pathway]\u003cem\u003e.\u003c/em\u003e \u003cem\u003eZhongguo Zhong Yao Za Zhi.\u003c/em\u003e 43, 4900-4907(2018)\u003c/li\u003e\n\u003cli\u003eHu, X. et al. Sevoflurane postconditioning improves the spatial learning and memory impairments induced by hemorrhagic shock and resuscitation through suppressing IRE1\u0026alpha;-caspase-12-mediated endoplasmic reticulum stress pathway\u003cem\u003e.\u003c/em\u003e \u003cem\u003eNeurosci Lett.\u003c/em\u003e 685, 160-166(2018)\u003c/li\u003e\n\u003cli\u003eWu, L. et al. Exendin-4 protects HUVECs from tunicamycin-induced apoptosis via inhibiting the IRE1a/JNK/caspase-3 pathway\u003cem\u003e.\u003c/em\u003e \u003cem\u003eEndocrine.\u003c/em\u003e 55, 764-772(2017)\u003c/li\u003e\n\u003cli\u003eLos, M. et al. Activation and caspase-mediated inhibition of PARP: a molecular switch between fibroblast necrosis and apoptosis in death receptor signaling\u003cem\u003e.\u003c/em\u003e \u003cem\u003eMol Biol Cell.\u003c/em\u003e 13, 978-988(2002)\u003c/li\u003e\n\u003cli\u003eAikin, R., Rosenberg, L., Paraskevas, S. \u0026amp; Maysinger, D. Inhibition of caspase-mediated PARP-1 cleavage results in increased necrosis in isolated islets of Langerhans\u003cem\u003e.\u003c/em\u003e \u003cem\u003eJ Mol Med (Berl).\u003c/em\u003e 82, 389-397(2004)\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cell death, endoplasmic reticulum stress, FMO3, IRE1α pathway, non-small cell lung cancer","lastPublishedDoi":"10.21203/rs.3.rs-7984231/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7984231/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNon-small cell lung cancer (NSCLC) accounts for \u0026gt;\u0026thinsp;85% of lung malignancies and is characterized by late presentation and limited therapeutic options. While members of the flavin-containing monooxygenase (FMO) family have been implicated in various solid tumors, the expression pattern and biological significance of FMO3 in NSCLC remain undefined. In this study, immunohistochemistry was performed on 10 paired fresh NSCLC-adjacent specimens and a tissue microarray of 115 additional pairs for FMO1-5 and Ki67, with clinicopathological data and 5-year follow-up analyzed. Stable FMO3-overexpressing A549 cells and FMO3-knockdown H1703 cells were generated. Cell proliferation was evaluated by CCK-8, EdU and colony formation assays; apoptosis by Annexin V-FITC/PI flow cytometry; and ER-stress signaling by western blotting of the IRE1α/caspase-12/caspase-3/PARP axis. Results showed FMO3 was selectively upregulated in NSCLC, positively correlated with tumor size and T stage, predicted poorer 5-year survival as an independent prognostic factor. In vitro, FMO3 overexpression promoted proliferation and suppressed apoptosis, while knockdown had the opposite effects, via negative regulation of the IRE1α pathway to attenuate ER-stress. Thus, targeting FMO3 could be a novel NSCLC therapy.\u003c/p\u003e","manuscriptTitle":"FMO3 as a novel prognostic biomarker and therapeutic target in non-small cell lung cancer by attenuating endoplasmic reticulum stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 09:08:45","doi":"10.21203/rs.3.rs-7984231/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-27T07:38:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-25T13:37:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"103339840069131233786375523751875190620","date":"2025-11-23T06:21:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-18T08:00:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-18T07:13:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305045825522349139154377977767990410581","date":"2025-11-18T04:43:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-17T13:35:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28071530514249780949775476754232958275","date":"2025-11-17T10:02:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"134541960907447329863390930976728937858","date":"2025-11-17T05:02:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"112785929826641354386480912388036924999","date":"2025-11-17T02:15:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"122028600691437686718034204433588545841","date":"2025-11-17T02:06:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326238231396397427244545518799057893559","date":"2025-11-16T13:26:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38846686372266271195732956858472924218","date":"2025-11-16T10:47:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-06T09:46:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-06T09:40:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-06T09:22:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-04T06:29:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-04T06:25:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"16553b55-db36-41fa-b83b-590fd35d1034","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58074433,"name":"Biological sciences/Cancer"},{"id":58074434,"name":"Biological sciences/Cell biology"},{"id":58074435,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-05-22T03:38:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-17 09:08:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7984231","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7984231","identity":"rs-7984231","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}