Abstract
Background/Aim: Particulate matter 2.5 (PM2.5) is known to adversely affect human health. While its involvement in lung cancer pathogenesis is recognized, its specific impact on metastasis-related behaviors of lung cancer cells remains largely unexplored.
Materials and Methods: In this study, we employed cell culture models, proteomic analysis, and bioinformatic analysis. Target proteins and signaling pathways were validated using western blotting and immunofluorescence assay. Wound healing, transwell migration and phalloidin-rhodamine assays were used to determine the migratory activity.
Results: Proteomic analysis identified 3,795 proteins in both control and PM2.5-treated groups. Among these, proteins associated with metastasis, particularly those related to “cell migration” (GO: 0016477), were highlighted, identifying six key proteins involved in cancer metastasis. Protein-protein interaction analysis pinpointed fibroblast growth factor receptor 1 (FGFR1) as a central target influenced by PM2.5, which promoted cell migration via the Rap1 signaling pathway through integrin signaling. The enhanced migratory behavior of PM2.5-treated cells aligned with proteomic findings, demonstrating that PM2.5 exposure increases the motility of lung cancer cells. Western blotting and immunofluorescence confirmed that PM2.5 exposure led to up-regulation of FGFR1, integrin αV, β1, and activated p-Akt. Notably, PM2.5-treated cells exhibited significantly increased motility and a higher number of filopodia per cell.
Conclusion: These results indicate that FGFR1 is a crucial target in PM2.5-induced metastasis in lung cancer cells, operating through an FGFR1/integrin/Akt signaling axis. This study advances our understanding of the role of PM2.5 in lung cancer metastasis and suggests potential therapeutic strategies to mitigate cancer progression.
Introduction
Particulate matter 2.5 (PM2.5), characterized as airborne particles with a diameter of less than 2.5 μm, has emerged as a significant air pollution with serious consequences for human health. PM2.5 can adversely affect various physiological systems, including the cardiovascular, neurological, immunological, and predominantly the respiratory system (1, 2). PM2.5 has the potential to be readily absorbed into the respiratory system and subsequently deposited inside the lung alveoli. This can lead to potential structural damage to the lungs and impairments in their functionality. The International Agency for Research on Cancer (IARC) has classified PM from outdoor air pollution as a Group 1 carcinogen for humans, which is implicated in the etiology of lung cancer (3). Numerous studies have explored the correlation between PM2.5 exposure and the heightened risk of both lung cancer incidence and mortality (4, 5). Cytokine production, inflammatory reactions, and angiogenesis have been recognized to be associated with tumor cell proliferation and metastasis (6). Although metastasis constitutes the primary etiology of mortality in individuals afflicted with cancer, the specific molecular target proteins of PM2.5 that facilitate the promotion of cancer metastasis remain predominantly unidentified.
Fibroblast growth factors (FGFs) are key signaling molecules that bind to FGF receptors (FGFRs), playing a vital role in numerous physiological processes. FGFRs directly engage with extracellular matrix (ECM) and cell adhesion molecules, enhancing cancer cells’ invasive and motile characteristics (7). Dysregulation of FGFR signaling has been considered in various tumor forms, particularly non-small cell lung cancer (NSCLC), prostate, breast, glioblastoma, and gastrointestinal cancers (7). The fibroblast growth factor receptor 1 (FGFR1) is a receptor presenting on the cell membrane and is classified as a member of the FGFR family. FGFR1 plays crucial roles in cancer development (8). Upon the binding of FGF, the FGFR1 is involved in dimerization and autophosphorylation. The downstream signaling pathways associated with FGFR1 further influence cellular activities by regulating gene expression, in addition to the direct alteration of cellular processes through the phosphorylation of proteins. This process serves to initiate phosphorylation and the activation of downstream signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt), rat sarcoma virus (Ras)/rapidly accelerated fibrosarcoma (Raf)/Mitogen-activated protein kinase kinase (MEK)-mitogen-activated protein kinase (MAPKs), phospholipase C gamma (PLCγ)/protein kinase C (PKC), and signal transducer and activator of transcription (STAT) (9-11). These downstream pathways enable FGFR1 to execute its various roles in regulating processes such as cell proliferation, cell differentiation, metabolic homeostasis, and the mechanisms that contribute to disease (9-11). FGFR1 plays a crucial role in the epithelial-mesenchymal transition (EMT), enhancing cancer cells’ invasiveness and motility. Therefore, targeting FGFR1 could potentially reduce the metastatic dissemination of cancer. Although it is established that FGFR1 signaling can induce many genes, the molecular mechanisms and the key genes that mediate FGFR1-promoted lung cancer cell growth remain unclear.
Integrins are a category of cell-adhesion molecules consisting of two interconnected heterodimeric subunits, namely the α and β subunits, facilitating both cell-cell and cell-ECM adhesions (12). The expression levels of various integrin molecules have been documented to be associated with cancer invasion, metastasis, and prognostic outcomes in specific malignant tumors (13). Integrin β1 serves as the principal subunit and has been recognized as a vital mediator in oncogenesis, playing a pivotal role in various aspects of cancer progression, including cellular motility, adhesion, migration, proliferation, differentiation, and resistance to chemotherapy (14). The integrin αV family plays a particularly significant role in the development and progression of malignant tumors (15). Integrins have the capacity to engage directly with growth factor receptors, thereby modulating the functionality of integrin/growth factor receptor complexes in the transmission of downstream signaling pathways (16). This intricate coordination is pivotal for regulating various dimensions of cellular behavior, encompassing survival, proliferation, differentiation, and migration. The interaction between integrins and growth factor receptors forms a complex network that is crucial for understanding cell behavior and the development of cancer (17).
Previous research has demonstrated the relationship between PM2.5 exposure and the metastatic progression of lung cancer cells (18, 19). The metastatic process involves a series of complex biological events, including cellular proliferation and migration through the extracellular matrix (ECM), invasion of vascular structures, evasion of anoikis during circulatory transit, extravasation into target metastatic tissues, and the initiation of a secondary neoplasm (20, 21). Cell migration is a crucial prerequisite process in the detachment of cells from the ECM and the disruption of cellular polarity. This process promotes the invasion and metastasis of cancer cells (22). However, the comprehensive understanding of PM2.5’s influence on cell migration remains unclear. The current study aimed to elucidate the critical proteins and signaling cascades modulated by PM2.5, as these alterations are intrinsically linked to the metastatic capability of cancer. We conducted a proteomic analysis on lung cancer cells treated with PM2.5 and utilized bioinformatics approaches to explore the affected signaling pathways. Our findings might be useful for the development of diagnostic and therapeutic strategies.
Materials and Methods
Particulate matter 2.5 (PM2.5) sampling and extraction. Under weather conditions containing the PM2.5 concentration of 55 μg/m3, the temperature of 32°C and relative humidity of 55%, air samples were collected into the filter membrane Whatman No.5 using air-sampler System MAS-100 NT with 100 liters per min sampling flow rate. Sampling was performed at 12, 24, 48 and 72 hr. According to the previous report by Roper et al. (23), the sonication method was employed to extract particulate matter from the filter. The 1.5×1.5 cm of the filter was cut and put into a 15 ml tube containing 6 ml of water. Filters were sonicated for 1 h. Then the filters were removed and washed with water to eliminate any remaining particles on the filter. The suspension was concentrated by freeze-drying. The morphology of PM2.5 is shown in Figure 1A.
Effect of PM2.5 on cell viability and apoptosis of non-small cell lung cancer H460 and A549 cells. (A) The morphology of PM2.5 was characterized using a scanning electron microscope at an electron energy of 15 keV with 2.00 kx. (B) H460 and (C) A549 cells were treated with PM2.5 at 0-200 μg/ml for 24 h. The effect of PM2.5 on cell viability was examined by the MTT assay, and the percentage of cell viability was calculated. (D-E) H460 cells and (F-G) A549 cells were treated with PM2.5 of 0-100 μg/ml for 24 h. The morphology of apoptotic nuclei was examined by Hoechst 33342/PI staining and visualized by fluorescence microscopy. The percentage of apoptotic cells in PM2.5 was calculated. Data are presented as mean±standard deviation (n=3). Multiple comparisons were conducted using one-way analysis of variance (ANOVA) followed by Turkey’s post hoc test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 versus untreated control cells. PM2.5: Particulate matter 2.5.
Chemicals and reagents. Human non-small cell lung cancer (NSCLC) cell lines, including H460, and A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Roswell Park Memorial Institute 1640 Medium (RPMI) and Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA), respectively. Media were added with 2 mM L-glutamine (Gibco, Gaithersburg, MA, USA) and 10% FBS (Gibco, Gaithersburg, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Hoechst 33342, and propidium iodide (PI) were obtained from Invitrogen (Thermo Fisher Scientific, Carlsbad, CA, USA). The primary antibodies against FGFR1 (#9740), integrin αV (#4711), integrin β1 (#4706), Akt (#9272), p-Akt (#4060), and β-actin (#4970), and the secondary antibody anti-rabbit IgG (#7074) were all acquired from Cell Signaling Technology (Danvers, MA, USA).
Cell viability assay. The cell viability was determined by MTT colorimetric assay. Human NSCLC cells were seeded with a density of 1×104 cells/well in 96-well plates overnight for cell attachment. After that, NSCLC cells were treated with varying concentrations of PM2.5 (0-200 μg/ml) for 24 h at 37°C. The culture media was subsequently substituted with 100 μl/well of MTT solution (4 mg/ml in PBS) and incubated at 37°C for 4 h. Subsequently, 100 μl of DMSO was added to solubilize the formazan crystals. The absorbance was measured at wavelength 570 nm with a microplate reader (Anthros, Durham, NC, USA). The percentage of cell viability was calculated as absorbance of PM2.5-treated cells relative to the untreated cells.
Nuclear staining assay. NSCLC cells were seeded at a density of 1×104 cells/well in 96-well plates and incubated overnight. After that, cells were incubated with a 0-100 μg/ml concentration of PM2.5 for 24 h. Then, cells were co-stained with 10 μg/ml of Hoechst 33342 and 0.5 μg/ml of propidium iodide (PI) for 30 min in the darkness. The cells were analyzed for fluorescence using a fluorescence microscope (Nikon ECLIPSE Ts2, Tokyo, Japan). The percentage of apoptotic cells was calculated by counting the number of condensed nuclear and DNA-fragmented cells.
Wound healing assay. NSCLC cells were seeded with a density of 4×104 cells/well in 96-well plates for 24 h at 37°C. The cell monolayer was subjected to a scratch using a 10 mm micropipette tip, creating a wound space. Subsequently, the cells were permitted to migrate and occupy the wound area. The detached cells were removed by PBS and the cell monolayer was treated with PM2.5 (0-200 μg/ml) in medium with 1% FBS. The wound spaces were captured with phase contrast images at different time points at 0, 24, 48 and 72 h using a phase-contrast microscope (Nikon ECLIPSE Ts2). The wound space was measured by image J software (version 1.52a, National Institutes of Health, Bethesda, MD, USA). The change in the wound space percentage was calculated as following: % change=(average space at time 0 h) – (average space at time 24 h, 48 h, 72 h)/(average space at time 0 h) ×100. The wound area at each time point was measured by using Image J software. The results are reported as relative cell migration.
Transwell migration assay. NSCLC cells were seeded at a density of 1×105 cells/well in the upper chamber. The medium with various doses of PM2.5 (0-200 μg/ml) was added to the lower chamber. The cells were allowed to migrate to the lower chamber for 24 h at 37°C. The cells underneath the membrane were fixed with 3.7% formaldehyde and incubated for 15 min at RT. After that, formaldehyde was removed, and cells were washed twice with PBS. The cells were then stained by adding 0.5% w/v crystal violet in 25% v/v methanol for 10 min at room temperature in the dark. After that, the crystal violet solution was removed, and cells were washed twice with PBS. The migrated cells were counted and captured using a fluorescence microscope (Nikon ECLIPSE Ts2). The number of migrated cells was counted using image J software. The results are reported as relative cell migration.
Cell morphology and filopodia characterization. NSCLC cells were treated with PM2.5 at a concentration 0-200 μg/ml for 24 h. The treated cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 10 min at 37°C. Then, the cells were permeabilized with 0.1% Triton-×100 for 5 min and blocked with 0.2% bovine serum albumin (BSA) for 30 min. Subsequently, cells were stained with phalloidin-rhodamine solution (1:100 ratio) in PBS for 15 min and mounted with 50% glycerol. Cell morphology and filopodia were visualized and imaged under a fluorescence microscope (Nikon ECLIPSE Ts2). The relative numbers of filopodia were determined by comparing the filopodia per cell in PM2.5-treated cells to that in control cells.
Proteomics sample preparation. H460 cells were seeded at a density of 5×105 cells/well in 6-well plates overnight before being treated with 200 μg/ml PM2.5 for 24 h. Cells were dissolved in 0.5% SDS and were then centrifuged at 10,000 g for 15 min. The supernatant was transferred to a new tube, mixed well with 2 volumes of cold acetone, and incubated overnight at −20°C. The mixture was centrifuged at 12,000 g for 15 min and the supernatant was removed. Subsequently, the pellet was dried and stored at −80°C for further proteomic analysis.
LC-MS/MS. An equal amount of pooled protein (5 μg) from control and PM2.5-treated cells were subjected to in-solution digestion. Samples were subjected to reduction of disulfide bonds in 10 mM ammonium bicarbonate (NH4HCO3) containing 10 mM dithiothreitol (DTT) for 1 h at 60°C and alkylated with 30 mM iodoacetamide (IAA) in 10 mM NH4HCO3 at room temperature for 45 min in the dark. The protein samples were digested with trypsin (1:20 ratio) (Sequencing Grade, Promega, Germany) for 16 h at 37°C. The trypsinized peptides were dried and protonated with 0.1% formic acid and followed by nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) analysis. The tryptic peptide samples were analyzed using an Ultimate 3000 Nano/Capillary LC System (Thermo Scientific, UK) coupled to a HCTUltra LC-MS system (Bruker Daltonics Ltd, Hamburg, Germany) equipped with a Nano-captive spray ion source as described in our previous report (24). LC-MS analysis of each sample was performed in triplicate.
Bioinformatics and data analysis. LC-MS data were operated using DecyderMS (GE Healthcare, Chicago, IL, USA) using the Homo sapiens protein database from Uniprot, as previously described (24). Identified proteins were filtered using one-way ANOVA and p<0.05. Only proteins with at least two peptides and at least one unique peptide was considered and used for data analysis. The protocol of bioinformatic analysis was conducted as described in our previous report (24). Briefly, a Venn diagram (http://jvenn.toulouse.inra.fr/app/index.html) was used to illustrate the identified proteins in control and PM2.5-treated cells group. Multi Experiment Viewer (MeV) (MeV Version 4.9, https://webmev.tm4.org)) software was used to determine the expression pattern of differentially expressed proteins. Protein Analysis through Evolutionary Relationships (PANTHER) (http://www.pantherdb.org) was used for the identification of gene ontology underlying cell migration (GO: 0016477). The Search Tool for the Retrieval of Interacting Genes (STRING) software (https://string-db.org/cgi/input.pl; version: 11.0) was used to construct the protein-protein interaction (PPI) networks. Cytoscape software 3.7.2 (https://cytoscape.org) was used to construct the hub genes. This software computes a comprehensive set of topological parameters for directed and undirected networks, as well as degree, betweenness centrality and closeness centrality, in order to identify hub genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) mapper analysis (https://www.genome.jp/kegg/mapper.html) was used to confirm the signaling pathways related to cell migration.
Western blot analysis. NSCLC cells were seeded at a density of 4×105 cells/well into 6-well plates for 24 h. H460 cells were treated with PM2.5 concentrations of 0-200 μg/ml, and A549 cells with concentrations of 0-100 μg/ml, for 24 h 37°C. The cells were then lysed with RIPA buffer for 30 min at 4°C. The protein concentrations were quantified using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Cell lysates were analyzed for protein content and an equivalent volume of the sample was loaded onto a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk in TBST (25 mm Tris-HCl, pH 7.4, 125 mm NaCl, and 0.05% Tween 20) for 1 h. They were then incubated with the specific primary antibodies against FGFR1, integrin αV, integrin β1, Akt, p-Akt and β-actin (Cell Signaling, Danvers, MA, USA). Then, membranes were washed three times with TBS-T and incubated with appropriate horseradish peroxidase (HRP)-labeled secondary antibodies (Cell Signaling) for 1 h at room temperature. The protein bands were detected by an enhanced chemiluminescent detection system (Supersignal West Pico, Pierce). The protein intensity was quantitatively analyzed using image J software to determine the level of protein expression in the sample.
Immunofluorescence. NSCLC cells were seeded at a density of 1×104 cells/well into 96-well plates for 24 h. The cells were treated with various PM2.5 concentrations (0-200 μg/ml) for 24 h and then removed from the medium. Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton-X in PBS for 5 min. Cells were subjected to blocking and incubated with primary antibodies against FGFR1, integrin αV, integrin β1, p-Akt at 4°C overnight. Then, cells were then incubated with secondary antibody for 1 h, stained with Hoechst 33342 for 30 min at room temperature in darkness, and mounted using 50% glycerol (Merck, Darmstadt, Germany). The images were captured by a fluorescence microscope (Nikon ECLIPSE Ts2), and the fluorescence intensity was quantitatively analyzed using Image J software and created profile plots based on randomized selections of single cells, with a focus on the cytoplasmic parts of cells. The program will generate a plot of intensity values along the cells, showing how fluorescence intensity varies across that line. The relative of fluorescence intensity was calculated.
Statistical analysis. All experiments were repeated at least three times, and results were expressed as the mean±standard deviation (SD). Multiple comparisons for statistically significant differences between multiple groups were performed by one-way analysis of variance (ANOVA) followed by Turkey’s post hoc test. The statistical difference between the two groups was compared using t-test. Statistical significance was considered at p<0.05. GraphPad Prism software version 8.0 (GraphPad Software, La Jolla, CA, USA) was used to create graphs in this experiment.
Results
Effect of PM2.5 on cell viability in human lung cancer cells. The appropriate concentrations of PM2.5 were assessed in NSCLC cells (H460 and A549). Cells were treated with PM2.5 at various concentrations (0-200μg/ml) for 24 h. Subsequently, the viability of the cells was assessed as described. According to the results, PM2.5 at concentrations of 50 to 200 μg/ml had a significant effect on cell variability in H460 cells (Figure 1B); however, PM2.5 at concentrations of 200 μg/ml had effects in A549 cells (Figure 1C). Hoechst 33342 staining was utilized to evaluate the nuclear morphology of cells and propidium iodide (PI) fluorescent dye was utilized to detect necrosis. The results indicated that a PM2.5 concentration of less than 100 μg/ml significantly affected apoptosis in H460 cells (Figure 1D, E), whereas PM2.5 concentrations at 100 μg/ml had a significant effect on apoptosis in A549 cells (Figure 1F, G). In addition, the results indicated that necrosis was not detected in all treatment conditions in H460 and A549 cells (Figure 1D-G).
PM2.5 induces human lung cancer cell migration. Migration is a crucial process in cancer metastasis. The migratory properties of H460 and A549 cell lines were examined using wound healing and transwell migration assays. The cells were treated with PM2.5 at 0-200 μg/ml for 24, 48, and 72 h, compared with the non-treated controls. The results indicated that PM2.5 significantly increased the migration of H460 (Figure 2A, B) at concentrations of 50 to 200 μg/ml at 48 and 72 h. Meanwhile, PM2.5 at 50 μg/ml had significant effects on cell migration at 24 h (Figure 2A, B). For A549 cells, PM2.5 at concentrations of 50 to 200 μg/ml significantly induced the cell migration at 24,48 and 72 h (Figure 2C, D). Consistently, transwell migration assay indicated that PM2.5 significantly enhanced the migratory capacity of H460 cells at a concentration of 50 to 200 μg/ml, whereas A549 cell migration was induced by PM2.5 at a concentration of 100 to 200 μg/ml (Figure 2E, F). Filopodia formation, characterized by cellular protrusions, plays a critical role in numerous essential biological processes, particularly facilitating the migration of cancer cells. As shown in Figure 2G-J, filopodia formation in H460 and H292 cells indicated an increase in the number of filopodia per cell after treatment with PM2.5 in a dose-dependent manner. According to these findings, PM2.5 facilitated the migration of lung cancer cells, which constitutes a vital step in the metastatic cascade.
PM2.5 induces cell migration and filopodia formation in non-small cell lung cancer H460 and A549 cells. (A-B) H460 cells and (C-D) A549 cells were treated with PM2.5 (0-200 μg/ml) for 24, 48, and 72 h, and the migration activity was determined by the wound healing assay. The results are presented as relative cell migration. (E-F) Transwell migration assay was performed to evaluate cell migration after treatment with various concentrations of PM2.5 (0-200 μg/ml) for 24 h. The migrated cells that were underneath the membrane were stained with crystal violet and the results are presented as relative cell migration. (G-H) H460 cells and (I-J) A549 cells were treated with PM2.5 (0-200 μg/ml) for 24 h before staining with phalloidin-rhodamine and visualization by fluorescence microscopy. Features representing filopodia are indicated by arrowheads. The results are presented as a relative number of filipodia/cell. Data are presented as mean±standard deviation (n=3). Multiple comparisons were conducted using one-way analysis of variance (ANOVA) followed by Turkey’s post hoc test. **p<0.01, ***p<0.001 and ****p<0.0001 versus untreated control cells. PM2.5: Particulate matter 2.5.
Proteomic analysis of differentially expressed proteins in PM2.5-treated cells. From proteomic analysis, the differentially expressed proteins were compared between groups; 428 and 329 proteins were uniquely expressed within the control and PM2.5-exposed groups, respectively. The utilization of a Venn diagram showed that 3,038 proteins were common to both groups (Figure 3A). Subsequently, a total of 3,795 proteins, which were obtained from the proteomic data for both groups, were categorized according to the Gene Ontology (GO) terms related to “cell migration” (GO: 0016477), crucial to cancer metastasis. The MultiExperiment Viewer program (version 4.9, https://webmev.tm4.org) was employed to quantify the differentially expressed proteins among groups, as illustrated in Figure 3A. Among the identified proteins, 61 were associated with cell migration. Of these proteins, 9 proteins including SORL1, STRIP2, PTK2, PBXIP1, SCYL3, PTPRF, AVL9, LIMA1 and PSPIP2 that were expressed in the control group but were absent in the PM2.5 treatment group, indicating down-regulation. Conversely, seven proteins, namely FGFR1, ARF4, CTNNA2, TYRO3, FSCN1, SH3KBP1, and LIMD1 were up-regulated in the PM2.5 treatment group and were absent in control group. Additionally, 45 proteins related to cell migration were found to be common to both groups.
Proteins affected by PM2.5-treated lung cancer cells. (A) Venn diagram showing the number of proteins expressed in the control and PM2.5-treated cells. Heatmap representing the level of expression of 61 proteins associated with “cell migration” (GO:0016477), with 9 down-regulated proteins, 7 up-regulated proteins, and 45 proteins present in both groups. (B) The protein-protein interaction (PPI) network of the down- and up-regulated proteins related to the GO term “cell migration” (GO: 0016477) in response to exposure to PM2.5. The blue circle represents the down-regulated protein, and the red circles represent the up-regulated proteins involved in cell migration. PM2.5: Particulate matter 2.5; GO: gene ontology; PTPRF: receptor-type tyrosine-protein phosphatase F; FGFR1: fibroblast growth factor receptor 1; SH3KBP1: SH3 domain-containing kinase-binding protein 1; FSCN1: fascin; ARF4: ADP-ribosylation factor 4; TYRO3: tyrosine-protein kinase receptor TYRO3.
Consequently, we carried out an investigation to assess the impact of the down-regulated and up-regulated proteins on the facilitation of cancer cell migration (Table I). The findings revealed that the proteins showing down-regulation as PTPRF, while those exhibiting up-regulation comprised FGFR1, ARF4, TYRO3, FSCN1 and SH3KBP1, which were significantly linked to the enhancement of cancer cell migration. The potential correlations of these six proteins associated with cell migration were further established through a protein-protein interaction (PPI) network utilizing the Search Tool for Interacting Chemicals 5.0 (http://stitch.embl.de/) (Figure 3B).
List of up-regulated and down-regulated proteins associated with cancer cell migration.
FGFR1 has a key role in the mechanism of action of PM2.5. Subsequently, our investigation aimed to identify the most critical hub gene affected by PM2.5. Cytoscape software 3.7.2 (https://cytoscape.org) was used to analyze the protein nodes derived from the PPI networks in order to visualize the protein interaction network and identify hub proteins. The hub genes were identified and further investigated using degree, betweenness centrality, and closeness centrality metrics. Degree measures the number of direct connections a node has in a network (25). In the context of protein-protein interaction (PPI) networks, nodes with a high degree are known as hub proteins, indicating they have many interactions with other proteins (25). Betweenness centrality of a node is a measure that quantifies how often a node acts as a bridge along the shortest paths between two other nodes in a network (26). Closeness centrality is a measure of the shortest paths distance from a node to all other nodes in the network (27). In the PPI network, the nodes with high degree are defined as hub proteins, indicating their significant number of connections to other proteins. (28). Therefore, according to its large degree as well as closeness centrality value, we identified FGFR1 as a pivotal protein target of PM2.5 in facilitating cell migration (Table II).
Hub genes associated with cell migration in PM2.5-treated cells.
We utilized the KEGG mapper (https://www.genome.jp/kegg/mapper.html) to construct the signaling pathway affected by PM2.5 in order to elucidate the specific signaling pathway related to PM2.5 exposure. The six hub genes associated with cell migration were mapped in the KEGG mapper to generate the pathways in Table III. The KEGG mapper indicated that FGFR1 [as members of the receptor tyrosine kinase (RTK) family] was a key player in the mechanism of action of PM2.5 in the promotion of cell migration through the Rap1 signaling pathway involving integrin signaling (Figure 4).
KEGG pathway enrichment analysis of the hub genes associated with cell migration in PM2.5-treated cells.
Effect of PM2.5 on proteins related to cell migration through the Rap1 signaling pathway obtained from the KEGG pathway database. Several downstream effectors are modulated by FGFR1 through integrin signaling. The red box represents proteins affected by PM2.5 treatment. PM2.5: Particulate matter 2.5; Rap1: Ras-associated protein-1; FGFR1: fibroblast growth factor receptor 1 (as members of the receptor tyrosine kinase (RTK) family); KEGG: Kyoto Encyclopedia of Genes and Genomes.
PM2.5 induces expression of FGFR1 in lung cancer cells. Having established that FGFR1 was suggested to be a key player in the PM2.5 effect and that integrin signaling is implicated in this process, we sought to validate the influence of PM2.5 on the protein levels and activation of these signaling pathways through western blot analysis. H460 cells were treated with PM2.5 concentrations of 0-200 μg/ml, and A549 cells with concentrations of 0-100 μg/ml, for 24 h and the expression levels of FGFR1 were measured. Our results indicated that exposure to PM2.5 significantly increased FGFR1 expression levels at 200 μg/ml in H460 cells (Figure 5A, B) and concentration of 50 to 100 μg/ml in A549 cells (Figure 5C, D), compared with untreated control cells. FGFR1 levels were also detected by immunofluorescence assay. These findings were consistent with the results obtained from the western blot analysis. We found a significantly increased fluorescence intensity of the FGFR1 protein at a concentration 200 μg/ml in H460 and A549 cells (Figure 5E-H). These results indicated that PM2.5 increased the levels of FGFR1, confirming the results of the previous experiment.
The FGFR1-dependent pathway is a target of PM2.5 in non-small cell lung cancer H460 and A549 cells. (A-B) H460 cells were treated with PM2.5 of 0-200 μg/ml and (C-D) A549 cells were treated with PM2.5 of 0-100 μg/ml for 24 h. Then, the expression level of FGFR1 was determined by western blot analysis. β-actin was used as a loading control. The band intensities of the PM2.5 treatment groups were quantified by densitometry using ImageJ, and the results are presented as a relative protein level. (E-F) H460 cells and (G-H) A549 cells were treated with PM2.5 of 0-200 μg/ml for 24 h. The expression of FGFR1 was determined by immunofluorescence. The fluorescence intensity was analyzed by ImageJ, and the results are presented as relative protein intensity. Data are presented as mean±standard deviation (n=3). Multiple comparisons were conducted using one-way analysis of variance (ANOVA) followed by Turkey’s post hoc test and the statistical difference between the two groups was compared using t-test.*p<0.05 and **p<0.01 versus untreated control cells. PM2.5: Particulate matter 2.5.
PM2.5 induced integrin signaling and the migratory proteins involved in cell migration. FGFR1 has been recognized as a target of PM2.5 and the downstream target integrin suggesting a possible mechanism of action. Our analysis using KEGG mapping revealed that the FGFR1/integrin pathway is a crucial mechanism by which PM2.5 affects cell migration. To elucidate the downstream signaling of FGFR1, we performed western blot analysis for protein determination. H460 cells were treated with PM2.5 concentrations of 0-200 μg/ml, and A549 cells with concentrations of 0-100 μg/ml, for 24 h. The expression levels of integrin αV and β1, Akt and p-Akt were determined. Our results show that integrin αV and β1, and p-Akt/Akt significantly increased expression levels at PM2.5 in a dose-dependent manner of H460 cells (Figure 6A, B). In A549 cells, treatment with 50 to 100 μg/ml PM2.5 significantly induced integrin β1 expression (Figure 6C, D). However, a significant increase in p-Akt/Akt protein was observed only at 100 μg/ml, while integrin αV expression levels increased at 50 μg/ml (Figure 6C, D).
PM2.5 induces the expression of proteins involved cell migration in non-small cell lung cancer H460 and A549 cells. (A-B) H460 cells were treated with PM2.5 of 0-200 μg/ml and (C-D) A549 cells were treated with PM2.5 of 0-100 μg/ml for 24 h. Then, the expression level of integrin αV, β1, Akt, and p-Akt determined by western blot analysis. β-actin was used as a loading control. The band intensities of the PM2.5 treatment groups were quantified by densitometry using ImageJ, and the results are presented as relative protein level. Data are presented as mean ± standard deviation (n=3). Multiple comparisons were conducted using one-way analysis of variance (ANOVA) followed by Turkey’s post hoc test.*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 versus untreated control cells. PM2.5: Particulate matter 2.5.
We used an immunofluorescence staining assay to confirm how PM2.5 affects the integrin signaling pathway. Our result found that integrin αV and β1 and p-Akt fluorescence intensity significantly increased in H460 (Figure 7A-F) and A549 cells (Figure 7G-L). Overall, these findings suggest that PM2.5 enhances the metastasis regulation mechanism of lung cancer cells through FGFR1/integrin/Akt signaling.
PM2.5 induces the expression of proteins associated with cell migration in non-small cell lung cancer H460 and A549 cells. (A-F) H460 cells and (G-H) A549 cells were treated with PM2.5 (0-200 μg/ml) for 24 h. The expression of integrin αV, β1, Akt, and p-Akt were determined by immunofluorescence assay. The fluorescence intensity was analyzed by ImageJ, and the results are presented as relative protein intensity. Data are presented as mean±standard deviation (n=3). The statistical difference between the two groups was compared using t-test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 versus untreated control cells. PM2.5: Particulate matter 2.5.
Discussion
Lung cancer is among the most common diseases related to PM2.5 (37). EMT and migratory activity are crucial phenotypic activities that generally get activated during tumor invasion and metastasis (38, 39). PM2.5 has been linked to various health issues and cancer risks. Prolonged exposure to PM2.5 is associated with higher cancer mortality rates, with specific risks noted for different types of cancer. The inflammation caused by PM2.5 leads to the production of chemokines and cytokines, which support angiogenesis. This process helps metastatic tumor cells invade epithelial cells, aiding the survival of malignant cells in distant organs (40). In this study, we demonstrated that PM2.5 treatment impacts many proteins related to cell migration (GO: 0016477) using proteomic and bioinformatics analyses. Additionally, we identified the crucial protein target and its downstream regulatory mechanism through which PM2.5 affects the migration of lung cancer cells.
EMT, which is associated with the process of cancer metastasis, facilitates the spread of neoplastic cells by reducing the adhesion between cells as well as between cells and the ECM, thus enhancing cell migration. The latter involves the movement of cells by morphological alterations, dissociation from one adhesion surface, and subsequent adherence to another (41). The present study demonstrated that PM2.5 treated cells had migration capacity. The wound healing assay and the transwell migration assay showed that PM2.5 induced the migration rate of lung cancer cells (Figure 2). A previous study has demonstrated that PM2.5 enhanced cell migration wounding and transwell assay revealed that cell migration was significantly higher following PM2.5 treatment (42). These data imply that PM2.5 enhanced the migratory behavior of NSCLC cells.
PPI network helps in identifying proteins that play essential roles in cancer. Nodes with high degrees are known as network hubs, indicating their significant number of connections to other proteins. Hubs play crucial roles in information flow and are important in the network. Hub proteins may serve as potential drug targets or biomarkers for cancer diagnosis and prognosis (43). In the context of protein-protein interaction networks, hub proteins often serve as key regulators of cellular processes. FGFR1 was identified as such, i.e., a key protein target of PM2.5. The results highlight FGFR1 as a central protein with the highest degree of interaction, indicating its key role in the mechanism of action of PM2.5 (Table II, Figure 4). FGFR1 represents a class of receptor tyrosine kinases (RTKs) that are linked to growth factors, which possess the ability to initiate a diverse array of tumor-promoting mechanisms, such as cellular proliferation, angiogenesis, suppression of apoptosis, and cellular migration (44). The activation of FGFR1 signaling possesses the capacity to commence oncogenic processes and promote both invasion and metastasis (45, 46). Increased FGFR1 expression is related to EMT in cancer cells derived from diverse tissues. FGFR1 activation is also recognized as a significant driver of EMT in breast (47) and prostate cancer (45, 46). In prostate cancer, abnormal FGFR1 expression is closely linked to the development of poorly differentiated tumors. Additionally, ongoing FGFR1 signaling plays a crucial role in the metastasis of prostate cancer, particularly in neuroendocrine tumor types (32). FGFR1 was shown to activate the cellular signaling of PI3K/Akt and RAS/MAPK pathways, thereby contributing to tumor development (48). In addition, a previous study (49) showed that Sry-related HMG box 2 (SOX2) is essential for FGFR1-induced EMT and highlighted that the FGFR1-ERK1/2-SOX2 axis facilitates cell proliferation, EMT, migration, invasion, and tumor metastasis in FGFR1-amplified lung cancer cells, both in vitro and in vivo. In this study, we considered FGFR1 as a potential target for the promotion of cell migration mediated by PM2.5. The expression levels of FGFR1 were assessed in lung cancer cells, with validation achieved through western blot analysis and immunofluorescence assays. These analyses revealed a significant induction of FGFR1 levels in cells treated with PM2.5 (Figure 5).
Our study focused primarily on the enrichment pathway associated with the increase of cell migration. Consequently, the enrichment pathway demonstrated that FGFR1 plays a pivotal role in the mechanism by which PM2.5 promotes cell migration via the Ras-associated protein-1 (Rap1) signaling pathway. Rap1 signaling pathway is crucial for regulating several cellular processes such tumor cell migration, invasion, and metastasis. Rap1 activation contributes to the acquisition of cancer hallmarks through the extracellular signal-regulated kinase (ERK), MAPK, and steroid receptor coactivator (Src)/focal adhesion kinase (FAK) (50). Rap1 signaling may play a role in the aggressive behavior of certain breast cancer types. Rap1 signaling affects Rho GTPase activating protein 29 (ARHGAP29), which is linked to increased invasiveness of tamoxifen-resistant breast cancer cells (51). Moreover, activation of Rap1 enhances the adhesion of lymphoma cells to endothelial cells, facilitating their transmigration into the hematopoietic system, which allows the lymphoma cells to spread to distant organs (52). A previous study showed that Rap1 supports the maintenance of cell-cell contacts within the border cell cluster during migration (53). As a multifunctional protein, Rap1 participates in regulating adhesion that depends on integrins and cadherins (54). Additionally, Rap1 is involved in the recycling process of integrins and modulates their affinity (55). The activation of Rap1 can trigger tumor initiation and EMT via Notch signaling. Furthermore, the epidermal growth factor receptor (EGFR) and Src/FAK may be stimulated by the activated Rap1, leading to integrin-mediated cell adhesion in cancer (56). Rap1 specifically regulates integrin activation and integrin-mediated adhesion by forming a complex with talin and the Rap1-GTP-interacting adaptor molecule (RIAM), which facilitates talin recruitment to integrin (57). Integrins are receptor proteins that play a role in cell adhesion and tumor cell migration and invasion by interacting with the ECM (58). Integrin-mediated cell adhesion to the ECM proteins is influenced by several growth factors (59-61). Integrin-mediated processes can be activated by growth factor receptors, leading to significant alterations in cellular behavior. Integrin-regulated FGFR signaling is intimately associated with cancer, particularly in angiogenesis, a vital phase in the development of metastases. Increasing evidence shows that integrins modify FGF/FGFR signaling (62). Here, our study demonstrated that FGFR1 is a target of PM2.5, with integrins serving as downstream targets. PM2.5-mediated induction of integrins was specific to integrin αV and β1 (Figure 6 and Figure 7).
Akt, also known as protein kinase B (PKB) (63), is a critical downstream mediator in integrin signaling (64). Integrins not only provide structural support through adhesion but also initiate signaling cascades that activate Akt, thereby regulating cytoskeletal dynamics and promoting cell movement (65). Elevated Akt phosphorylation corresponds to metastatic activity in lung cancer (66). The expression levels of p-Akt were consistently elevated in PM2.5 treated lung cancer cells (Figure 6 and Figure 7). Several reports have indicated that Akt activation is essential for EMT in cancer cells (67, 68). According to this study, lung cancer cells treated with PM2.5 increased their migration capacity and were linked to a notable rise in the number of filopodia per cell (Figure 2).
Conclusion
The current study used proteomics and bioinformatics analyses to clarify the molecular processes by which PM2.5 influences the migration of lung cancer cells. PM2.5 enhanced the metastatic regulatory mechanism of lung cancer cells via FGFR1/integrin/Akt signaling (Figure 8). These findings offer significant insights into the processes of cell migration, potentially guiding the creation of innovative therapeutic techniques to regulate cancer cell metastasis.
Schematic diagram that summarizes the underlying mechanism of PM2.5 that promotes lung cancer metastasis through FGFR1/integrin/Akt signaling. NSCLC: non-small cell lung cancer. FGFR1: Fibroblast growth factor receptor 1; PI3K: phosphatidylinositol 3-kinases; Akt: protein kinase B (PKB); FAK: focal adhesion kinase. Figure created with BioRender.com.
Acknowledgements
This work (Grant No. RGNS 64-128) was supported by Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (OPS MHESI), Thailand Science Research and Innovation (TSRI) and Navamindradhiraj University.
Footnotes
Authors’ Contributions
Conceptualization: P.C.; Research design: P.C. and N.A.; Experiments: N.A., Z.Z.E., K.P., N.P. and S.T.; PM2.5 collection: C.S.; Data analysis: P.C., S.R. and N.A.; Writing, review, and/or revision of the manuscript: P.C. and N.A. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
The Authors declare that they have no competing interests.
- Received December 16, 2024.
- Revision received March 5, 2025.
- Accepted March 10, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.