The cells (BEAS-2B, A549, H1299 and PC-9) were cultured in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. For stem cell growth, cells were seeded in ultra-low attachment plates (Costar, Corning, NY, USA) and cultured in serum-free DMEM (Cat. No. 11320033, Gibco, Waltham, MA, USA)/F-12 medium supplemented with 0.4% BSA (Cat. No. 15260037, Gibco, Waltham, MA, USA), 50 µg/mL EGF (Cat. No. P02307, Solaria, Beijing, China), 20 µg/mL bFGF (Cat. No. P00032, Gibco, Beijing, China), 5 µg/mL ITS (Cat. No. YA0811, Gibco, Beijing, China) PREMIX, 2% B-27, 2 µM L-glutamine, and 200 µg/mL penicillin/streptomycin. The medium was changed every 3 days, and spheroid formation was assessed using a microscope on days 0, 7, and 14.

All cell lines used in this study were obtained from National Infrastructure of Cell Line Resource (NICR; Beijing, China) who authenticated the cell lines through Short Tandem Repeat profiling. Mycoplasma tests were routinely performed by the suppliers to ensure the cell lines were free from contamination.

The University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) [20] was utilized to analyze the difference in FBXO33 expression levels between tumor and non-tumor tissues. Through the ‘TCGA Analysis’ module of UALCAN, differential expression of FBXO33 mRNA was obtained. UALCAN accesses TCGA level 3 RNA-seq and clinical data from 31 cancer types, offering comprehensive insights into gene expression and survival correlations. The analysis was performed using default settings, comparing normal and tumor tissue samples to identify significant differences in FBXO33 expression.

The Kaplan-Meier (KM) plotter, a bioinformatics tool [21], was employed to evaluate the potential prognostic value of FBXO33 expression in NSCLC patients. The ‘automatic selection of the best cutoff’ model was utilized in this analysis. All possible cutoffs were calculated to determine the best-performing threshold, ensuring the most statistically significant division between high and low FBXO33 expression groups. The KM plotter integrates gene expression data and survival information from various databases, including Gene Expression Omnibus (GEO), European Genome-phenome Archive (EGA), and TCGA, to provide robust survival analysis results.

RNA extraction was performed using the RNA extraction kit (Labled, Beijing, China), and reverse transcription was conducted using the reverse transcription kit (Labled, Beijing, China) on the NSCLC tissue samples used in this study were sourced from patients at the Affiliated Hospital 2 of Nantong University, with all procedures approved by the Ethics Committee (Approval No. 2023KT053), and informed consent was obtained from all participants. RT-qPCR was carried out using SYBR Green Master Mix (Labled, Beijing, China). The expression of the internal control gene, $\beta$-actin, was compared to assess gene expression levels using 2^{-Δ⁢Δ⁢Ct} Method. The primer sequences utilized are listed as follows: FBXO33: Forward: 5${}^{\prime }$-TCATGGTTACACGGTGTGGG-3${}^{\prime }$ Reverse: 5${}^{\prime }$-TGACTGCATGCCAAGGTTGA-3${}^{\prime }$; $\beta$-actin: Forward: 5${}^{\prime }$-TCCCTGGAGAAGAGCTACG-3${}^{\prime }$ Reverse: 5${}^{\prime }$-GTAGTTTCGTGGATGCCACA-3${}^{\prime }$.

FBXO33 shRNA lentiviral particles (Cat. No. sc-92223-V, Santa Cruz, CA, USA), control lentiviral particles (Cat. No. sc-108080, Santa Cruz, CA, USA), and the pGEX vector containing the cDNA sequence of FBXO33 -6P-3 (Cat. No. K240020, Addgene, MA, USA) overexpression vector (sequenced) were used to transfect A549 and H1299 cells. Briefly, according to the manufacturer’s protocol, the cells were seeded onto a 12-well plate and cultured with Opti-MEM (Cat. No. 31985062, Thermo Fisher, MA, USA) for 24 hours until reaching approximately 50% confluence. Then, the complete medium was mixed with polybrene (Cat. No. sc-134220, Santa Cruz, CA, USA) to a final concentration of 5 µg/mL. The original medium was then replaced with 1 mL of the polybrene mixture. After 24 hours, the original medium was replaced with 1 mL of complete medium devoid of polybrene. After overnight incubation, stable clones expressing the shRNA were selected at a 1:4 split cell ratio and incubated for an additional 48 hours. Subsequently, stable clones were screened for 72 hours using puromycin dihydrochloride to assess shRNA efficiency.

The cells were transfected or overexpressed with FBXO33 for 72 hours before conducting cell viability assays using the cell counting kit-8 (CCK-8) (Cat.No. CA1210, Solarbio, Beijing, China). Briefly, cells were seeded into 96-well plates at a density of 5 $\times$ 10³ cells per well. After 24 hours of cell culture, the cells adhered to the plate. Prior to the assay, each well was replaced with fresh medium containing 10% CCK-8 reagent and incubated for 2 hours. The optical density (OD) was measured daily at 450 nm using an Epoch microplate reader (BioTek, Winooski, VT, USA).

Cell proliferation was assessed using EdU staining. After transfection or FBXO33 overexpression, the cells were seeded into 96-well plates at a density of 5 $\times$ 10³ cells per well and cultured overnight. Before staining, EdU (20 mmol/L) was added to each well and incubated for 2 hours, the cells were then fixed with 4% paraformaldehyde, and EdU-positive cells were analyzed in the different experimental groups.

A transwell system with 8 µm pore size membranes (Costar, Corning, NY, USA) was utilized. Briefly, the upper chamber was seeded with 6 $\times$ 10⁴ cells in medium containing 0.02% FBS, while the lower chamber was filled with 650 µL of medium supplemented with 20% FBS. The migration period lasted for 18 hours. Subsequently, the cells on the lower surface of the chamber were fixed with 4% paraformaldehyde and stained with crystal violet for 10 minutes. The cells were assessed using a microscope (Stereo Discovery.V20, Zeiss, Germany) at 1$\times$ magnification, capturing the entire field of view in each case. The crystal violet-stained area was quantified using ImageJ (1.54, National Institutes of Health, Bethesda, MD, USA).

Tumorspheres were generated by trypsinizing cells using 0.05% trypsin-EDTA and seeding them into ultra-low attachment plates (Costar, Corning, NY, USA). The culture medium consisted of 0.4% BSA, 50 µg/mL EGF, 20 µg/mL bFGF, 5 µg/mL ITS PREMIX, 2% B-27, and serum-free RPMI-1640 medium supplemented with 2 $\times$ 10² µg/mL penicillin/streptomycin and 2 µM L-glutamine. Cells were then incubated in DMEM/F-12 for 14 days. Microscopic images were captured on days 0, 7, and 14 to monitor spheroid formation. To quantify spheroid formation, the percentage of cells forming spheroids was calculated using the formula: Y (n)/X (n) $\times$ 100%, where X (n) represents the number of wells with single cells and Y (n) represents the number of wells with formed tumorspheres. The number of formed tumorspheres was also determined. Subsequently, cells were collected for Western Blot (WB) analysis to examine protein expression.

FBXO33 and Myc proteins were extracted using RIPA buffer (Thermo Fisher, Waltham, MA, USA). Protein A/G agarose beads were incubated with anti-FBXO33 (Cat. No. NBP1-91890, 1:50, Novus Biologicals, Co., Centennial, CO, USA), c-Myc (Cat. No. NB600-335, 1–4 µg/mg lysate, Novus Biologicals, Co., USA) or IgG (Cat. No. NBP3-21815, 1:50, Novus Biologicals, Co., USA) antibodies for 30 minutes on a rotating wheel at 4 °C, followed by two washes. The beads were then mixed with the protein lysate and incubated on a rotating wheel overnight at 4 °C. After three washes with extraction buffer, the immunoprecipitates were collected by centrifugation at 3000 g and analyzed by WB. Cell extracts were immunoprecipitated with the specified antibodies for 4 hours at 4 °C to determine ubiquitination levels. The immune complexes were washed four times with RIPA buffer and then separated by WB for analysis.

The cells were fixed with 4% paraformaldehyde for 15 minutes at 37 °C, followed by permeabilization with 0.2% Triton X-100 (Cat. No. T8787, Sigma-Aldrich, Waltham, MA, USA) at room temperature for 15 minutes. After blocking with 5% BSA (Cat. No. 15260037, Gibco, Waltham, MA, USA) for 1 hour, cells were incubated with primary antibodies against FBXO33 (Cat. No. NBP1-91890, 0.25–2 µg/mL, Novus Biologicals, Co., USA) and c-Myc (Cat. No. NB600-335, 1:100, Novus Biologicals, Co., USA) at 4 °C overnight. The following day, the cells were incubated with fluorescently labeled secondary antibodies for 1 hour at room temperature.

For colocalization analysis, the cells were mounted with a DAPI-containing mounting medium and examined under an Olympus FV1000 (Hamburg, Germany) laser scanning confocal microscope. Images were captured at higher magnification to clearly demonstrate the colocalization of FBXO33 and Myc. The intensity profiles of both proteins were analyzed to confirm their colocalization within the cells.

Transfected cells were treated with CHX at a final concentration of 50 µg/mL to inhibit protein synthesis. After CHX treatment, cells were harvested at various time points (0, 1, and 2 hours). Protein extraction was performed using RIPA buffer supplemented with protease inhibitors and deubiquitination inhibitors. The samples were then subjected to WB analysis to detect Myc protein levels. The degradation rate of Myc was calculated by comparing the protein levels at different time points.

An animal model was established to assess the effect of FBXO33. Briefly, 12 BALB/c nude mice (female, 6–8 weeks old, weighing 18–22 g) were obtained from the Laboratory Animal Center of Nantong University and divided into two groups: a control group and an FBXO33 overexpression group, each containing 6 mice. The FBXO33 overexpression group received intratumoral injections of 50 µL adenovirus expressing FBXO33 (1 $\times$ 10⁹ PFU/mL) every 3 days for a total of 5 injections. The control group received an equivalent volume of adenovirus expressing an empty vector following the same schedule. A total of 3 $\times$ 10⁶ A549 cells were injected into the right flank of nude mice to establish a nude mouse xenograft model of NSCLC. Once the NSCLC tumors were established, tumor shape, weight, and volume were recorded or measured every four days. Tumor volume was measured using volumetric imaging techniques, such as Magnetic Resonance Imaging (MRI) or Computed Tomography (CT) scans, to ensure accurate assessment of the irregularly shaped tumors in the intraperitoneal cavity. Tumor measurements were performed using digital calipers. At the end of the experiment (21 days after tumor cell inoculation), mice were euthanized by CO₂ inhalation followed by cervical dislocation to ensure death. Tumors were immediately excised, weighed, and divided into two portions. One portion was fixed in 10% neutral buffered formalin for 24 hours, then processed for paraffin embedding and sectioning (5 µm thickness) for immunohistochemistry (IHC). The other portion was flash-frozen in liquid nitrogen and stored at –80 °C for future molecular analyses. IHC was performed on the formalin-fixed, paraffin-embedded sections to assess the number of positive cells stained for KI67, FBXO33 and Myc Stained sections were evaluated by two independent pathologists blinded to the experimental groups. All animal experiments conducted in this study were approved by the Animal Care and Use Committee of Affiliated Hospital 2 of Nantong University (Nantong First People’s Hospital) (Approval No. 2023KT053).

NSCLC tissues or cells were lysed using RIPA buffer, and protein quantification was conducted with the BCA protein assay kit (PC0020, Solarbio, China). Subsequently, equal amounts (30 µg) of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. The membranes were incubated separately with specific primary antibodies, including FBXO33 (Cat. No. NBP1-91890, 1:20, Novus Biologicals, Co., USA), CD133 (Cat. No. NB120-16518, 1:1000, Novus Biologicals, Co., USA), ALDH1A1 (Cat. No. NBP2-15339, 1:1000, Novus Biologicals, Co., USA), OCT4 (Cat. No. NB100-2379, 1:500, Novus Biologicals, Co., USA), SOX2 (Cat. No. NB110-37235, 1:2000, Novus Biologicals, Co., USA), Myc (Cat. No. NB600-335, 1:1000, Novus Biologicals, Co., USA), and $\beta$-actin (Cat. No. 14395-1-AP, 1:1000, San Diego, CA, USA), followed by incubation with HRP-conjugated secondary antibodies. Enhanced chemiluminescent reagents were used to detect target proteins. The gray value of each target protein band was normalized to that of $\beta$-actin in the same sample. The relative protein expression was calculated as the ratio of the normalized gray value of the target protein to that of the control group, which was set as 1.

Data analyses were conducted using SPSS 22.0 (IBM Corp., Chicago, IL, USA). Prior to comparisons, normality and variance homogeneity tests were conducted. If the data met the criteria of normality and homogeneity of variances, t-tests or one-way ANOVA were applied. Conversely, if the data did not meet these criteria, the Wilcoxon signed-rank test was utilized. A significance level of p 0.05 was considered statistically significant. Each experiment was repeated three times to ensure reliability.

The analysis conducted using UALCAN comprised 59 normal lung tissues and 515 primary Lung Adenocarcinoma (LUAD) tissues, as well as 52 normal lung tissues and 503 primary Lung Squamous Cell Carcinoma (LUSC) tissues. The findings revealed a significant reduction in the expression level of FBXO33 in primary tumors (LUAD/LUSC) compared to normal tissues (Fig. 1A,B). Subsequent survival analysis indicated a correlation between low FBXO3 expression and poor overall survival (OS) in NSCLC (Fig. 1C).

Fig. 1.

Prediction of F-box Only Protein 33 (FBXO33) expression in non-small cell lung cancer (NSCLC) tissues. (A) Comparison of FBXO33 expression between normal lung tissues and Lung Adenocarcinoma (LUAD) tumor tissues. (B) Comparison of FBXO33 expression between normal lung tissues and Lung Squamous Cell Carcinoma (LUSC) tumor tissues. (C) Kaplan-Meier survival analysis depicting the correlation between FBXO33 expression and overall survival (OS) in NSCLC. (D) Relative expression of FBXO33 mRNA in NSCLC tissues assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). (E) Protein expression of FBXO33 in NSCLC tissues analyzed by Western blot (WB). (F) Relative expression of FBXO33 mRNA in human normal lung epithelial cells and NSCLC cell lines. (G) Protein expression levels of FBXO33 in human normal lung epithelial cell line and NSCLC cell lines. (H) Immunofluorescence localization of FBXO33 in cells. N = 3, ***p 0.001 vs. normal group or BEAS-2B group. Scale bar 30 µm.

To validate these bioinformatics predictions, FBXO33 mRNA and protein expression levels were evaluated in clinical NSCLC samples and cell lines. Both RT-qPCR and WB analyses demonstrated a significant reduction in FBXO33 mRNA expression in tumor tissue relative to the control group (Fig. 1D,E). Additionally, mRNA and protein expression levels of FBXO33 were examined in human normal lung epithelial cells (BEAS-2B) and NSCLC cell lines (A549, H1299, PC-9). The results revealed significantly lower expression of FBXO33 in all NSCLC cell lines compared to normal lung epithelial cells (Fig. 1F,G). Furthermore, IF staining illustrated relatively higher FBXO33 expression in BEAS-2B cells compared to A549, H1299, and PC-9 cells (Fig. 1H). Collectively, these findings underscore the diminished expression of FBXO33 in NSCLC.

The effects of FBXO33 overexpression or knockdown on NSCLC cell behavior were examined in A549 and H1299 cell lines. Initially, WB analysis demonstrated a significant increase in FBXO33 expression relative to the control group following overexpression. Conversely, compared to the shNC group, FBXO33 expression notably decreased after knockdown, indicating the effectiveness of overexpression and knockdown (Fig. 2A). Subsequently, the influence of FBXO33 expression on NSCLC cell growth was investigated. As shown in Fig. 2B, at 24 h, 48 h, and 72 h post-transfection, inhibiting FBXO33 expression led to significantly higher cell viability in the shFBXO33 group of both cell lines compared to the shNC group. Comparatively, FBXO33 overexpression significantly reduced cell viability in both cell lines compared to the control group. Furthermore, EdU staining (Fig. 2C) revealed that overexpression of FBXO33 significantly reduced cell proliferation compared to the control group. FBXO33 knockdown significantly increased cell proliferation relative to the shNC group. Moreover, cell migration and invasion were assessed (Fig. 2D,E). Overexpression of FBXO33 significantly reduced the number of migrating and invading cells compared to the control group, while knockdown of FBXO33 led to a significant increase in the number of migrating and invading cells compared to the shNC group. Overall, these findings indicate that FBXO33 suppresses the growth, migration, and invasion of NSCLC cells.

Fig. 2.

Effects of FBXO33 on the growth, migration, and invasion of NSCLC cells. (A) Western blot analysis confirming the efficiency of FBXO33 overexpression or knockdown. (B) Impact of FBXO33 overexpression or knockdown on cell growth. (C) Influence of FBXO33 overexpression or knockdown on cell proliferation. Scale bar 100 µm. (D) Effects of FBXO33 knockdown on cell migration. Scale bar 50 µm. (E) Effects of FBXO33 overexpression or knockdown on cell invasion. Scale bar 50 µm. N = 3, *p 0.05 vs. control or shNC group, **p 0.01 vs. control or shNC group, ***p 0.001 vs. control or shNC group.

To assess the impact of FBXO33 on the stemness characteristics of NSCLC cells, the protein expressions of stemness markers, including CD133, OCT4 and SOX2, were evaluated via WB. As shown in Fig. 3A, in the A549 and H1299 cell lines, the protein levels of CD133, OCT4 and SOX2 were significantly reduced compared to the control group after FBXO33 overexpression, while the expression levels of these proteins were elevated compared to the shNC group. Additionally, the number of tumor spheres formed per 500 cells was determined through tumor sphere formation and MSFE experiments. As shown in Fig. 3B, the control group exhibited a certain number of cells with a spherical morphology. However, after FBXO33 overexpression, the observed field of view was less populated with tumor spheres compared to the shNC group, and knocking down FBXO33 resulted in an increased number of tumor spheres observed in the field of view. These findings collectively suggest that FBXO33 suppresses the stemness characteristics of NSCLC cells.

Fig. 3.

Effects of FBXO33 on the stemness characteristics of NSCLC cells. (A) Impact of FBXO33 overexpression or knockdown on the protein expression levels of CD133, OCT4, and SOX2. (B) Influence of FBXO33 overexpression or knockdown on the formation of tumor spheres. N = 3, *p 0.05 vs. control or shNC group, **p 0.01 vs. control or shNC group, ***p 0.001 vs. control or shNC group. Scale bar 400 µm.

Firstly, the expression of Myc was assessed in A549 cells. As shown in Fig. 4A, the protein expression level of Myc significantly decreased following FBXO33 overexpression, whereas it markedly increased after FBXO33 knockdown compared to the shNC group. Subsequently, the interaction between FBXO33 and Myc was investigated using Co-IP. The results shown in Fig. 4B revealed the expression of both FBXO33 and Myc, indicating their binding to each other. Notably, IgG expression was absent. Furthermore, Myc ubiquitination levels were evaluated via Immunoprecipitation (IP). The findings indicated an increase in Myc ubiquitination in the presence of FBXO33. Additionally, CHX degradation experiments were conducted to assess the degradation rate of Myc. Within 2 hours of CHX treatment, the degradation rate of Myc was significantly lower in cells with FBXO33 knockdown compared to the shNC group (Fig. 4C–E). These results collectively suggest that FBXO33 promotes the ubiquitination and degradation of Myc.

Fig. 4.

Effects of FBXO33 on the ubiquitination and degradation of Myelocytomatosis (Myc). (A) Impact of FBXO33 overexpression or knockdown on the protein expression level of Myc. (B) Co-immunoprecipitation assay illustrating the protein interaction between FBXO33 and Myc. (C) Immunofluorescence localization of FBXO33 and Myc in cells. Scale bar 30 µm. (D) Cycloheximide (CHX) assay demonstrating the ubiquitination degradation of Myc following FBXO33 overexpression. (E) Western blot detection of the effect of FBXO33 knockdown on the degradation rate of Myc within 2 hours. N = 3, **p 0.01, *** p 0.001 vs. control or shNC group.

A xenograft NSCLC tumor model was established in BALB/c nude mice, and tumor weight and volume were monitored over 21 days. The results showed that compared to the control group, which exhibited intact tumor morphology, tumors overexpressing FBXO33 had reduced volume (Fig. 5A). Furthermore, within the 21-day timeframe, both tumor volume and mass were lower in the FBXO33 overexpression group compared to the control group. Subsequent analysis to detect KI67, FBXO33, and Myc staining in tumor tissues demonstrated a significant reduction in the number of KI67 and Myc-positive cells following FBXO33 overexpression compared to the control group (Fig. 5B), while the number of FBXO33-positive cells was significantly increased. These findings collectively indicate that FBXO33 inhibits NSCLC tumor growth in vivo.

Fig. 5.

Effects of FBXO33 on NSCLC tumor growth in vivo. (A) Morphological changes, tumor volume, and weight over 21 days. (B) Immunohistochemical staining showing KI67 and Myc positive cells following FBXO33 intervention. N = 6, ***p 0.001 vs. control group. Scale bar 200 µm