2025 年 50 巻 9 号 p. 459-469
Hazardous environmental factors contribute to various irreversible threats to human health worldwide. Accumulating evidence suggests that exposure to particulate matter with an aerodynamic diameter of <2.5 µm (PM2.5) plays a critical role in lung carcinogenesis. Previously, we reported that 1,2-naphthoquinone (1,2-NQ), a component of atmospheric PM2.5 and diesel exhaust particles, forms a covalent bond with the epidermal growth factor receptor (EGFR) via protein N-arylation, thereby activating the downstream protein kinase B (Akt) signaling pathway. Here, we elucidate a regulatory mechanism by which 1,2-NQ modulates the migratory activity of human lung adenocarcinoma A549 cells. Specifically, exposure of A549 cells to 1,2-NQ induces phosphorylation of EGFR, leading to the activation of extracellular signal-regulated kinase 1/2 (ERK1/2). This activation is significantly suppressed by anti-EGFR antibodies (cetuximab and panitumumab) and inhibitors targeting rapidly accelerated fibrosarcoma (Raf; LY3009120) and mitogen-activated protein kinase kinase (MEK; U0126). These findings suggest that 1,2-NQ induces ERK1/2 phosphorylation by activating the Raf-MEK pathway. Notably, suppression of EGFR-ERK1/2 signaling resulted in a decrease in migratory activity. Our findings provide new insights into lung cancer carcinogenesis and may contribute to the development of novel therapeutic strategies.
Hazardous environmental factors, such as air pollution, pose both short- and long-term threats to human health (Mazzarella et al., 2007). Among air pollutants, particulate matter with a diameter of less than 2.5 µm (PM2.5) is a major contaminant in urban areas (Thangavel et al., 2022). Naphthoquinones (NQs) have been identified in ambient PM2.5, and 1,2-NQ has attracted significant attention due to its chemical properties and diverse biological effects (Kumagai et al., 2012; Iwamoto et al., 2007; Nakahara et al., 2021; Beei et al., 2013). For instance, 1,2-NQ exhibits strong electrophilic properties, allowing it to regulate cellular protein functions through covalent bond formation (Kumagai et al., 2012). Previously, we demonstrated that 1,2-NQ triggers epidermal growth factor receptor (EGFR) activation by modulating protein tyrosine phosphatase 1B (PTP1B) (Cho et al., 2004; Iwamoto et al., 2007). Furthermore, 1,2-NQ induces protein N-arylation of EGFR, a unique mechanism that enhances the downstream protein kinase B (Akt) signaling pathway and promotes anti-apoptotic activity in human non-small cell lung cancer (NSCLC) cells (Nakahara et al., 2021).
Recent epidemiological studies have revealed significant associations between PM2.5 exposure and the pathogenesis of respiratory diseases, particularly lung cancer development (Lee et al., 2020; Nakhjirgan et al., 2023; Li et al., 2018). NSCLC accounts for approximately 85% of lung cancer cases and is characterized by frequent driver mutations in oncogenes and tumor suppressor genes, such as EGFR, rat sarcoma viral oncogene homolog (Ras), and phosphatase and tensin homolog (PTEN) (Nicholson et al., 2022; Herbst et al., 2018). Emerging evidence suggests that PM2.5 acts as a mutagen, disrupting gene expression, including EGFR, through mechanisms such as DNA methylation, histone modification, and microRNA regulation (Holme et al., 2023; Sun et al., 2007; Li et al., 2017). EGFR intracellular signaling is primarily mediated by two key pathways: the Ras–rapidly accelerated fibrosarcoma (Raf)–mitogen-activated protein kinase kinase (MEK)–extracellular signal-regulated kinase (ERK) pathway and the phosphatidylinositol-3 kinase (PI3K)–PTEN–Akt pathway (Santos et al., 2010).
Cell migration is a highly coordinated and dynamic process essential for various physiological functions, such as wound healing, and is also closely associated with cancer metastasis (Tanimura and Takeda, 2017; Lawson and Ridley, 2018). Studies have shown that biphasic activation of ERK signaling pathway regulates cancer cell migration and invasion (Choi and Helfman, 2014; Meng et al., 2009). However, the role of 1,2-NQ, a component of PM2.5, in modulating cell migration remains unclear. In this study, we demonstrate that 1,2-NQ promotes the migration of human lung adenocarcinoma-derived A549 cells via the EGFR–ERK signaling pathway. Our findings provide new insights into lung carcinogenesis and may contribute to the development of strategies to mitigate lung cancer pathogenesis.
1,2-NQ was purchased from Sigma Aldrich (St. Louis, MO, USA). Cetuximab was purchased from Merck KGaA (Darmstadt, Germany). Panitumumab was purchased from Takeda Pharmaceuticals (Osaka, Japan). LY3009120 and U0126 were purchased from FUJIFILM Wako (Osaka, Japan). Recombinant epidermal growth factor (EGF) (236-EG), Anti-Akt (9272S), anti-phospho-Akt (Ser473) (D9E), anti-EGFR (D38B1) (#4267), anti-phospho-EGFR (Tyr1068) (D7A5) (#3777) anti-p44/42 MAPK (ERK1/2) (137F5), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
Cell culture and inhibitors treatmentA549 cells were cultured in Dulbecco’s modified Eagle’s medium (D-MEM) (FUJIFILM Wako, Osaka, Japan) supplemented with 10% [v/v] 56°C heat inactivated fetal bovine serum (FBS; Sigma-Aldrich or Biosera, Cholet, France) and 1% [v/v] penicillin-streptomycin solution (FUJIFILM Wako, Osaka, Japan) in a 5% CO2 humidified incubator at 37°C.
Cells were seeded and incubated overnight, washed twice with serum-free DMEM (SFM), and cultured in SFM. 24 hr later, the cells were treated with 1,2-NQ and the supernatant was replaced. In some experiments, the cells were pretreated with inhibitors, including cetuximab and panitumumab, LY3009120, and U0126.
Cell viability assayCell viability assay (WST-8 assay) was performed using Cell Counting Kit-8 (CCK-8, Dojindo, Tokyo, Japan) according to the manufacturer’s instructions. Briefly, cells were pre-seeded in 6-well plates at a density of 1 × 105 cells/well for 72 hr. After serum starvation for 24 hr, the cells were treated with the inhibitor for 1 hr prior to exposure to 1, 2-NQ or EGF. Cell treatments were repeated after 24 hr under equivalent conditions. 24 hr later, 20 µL of CCK-8 solution was added and incubated in the dark for 2 hr. The supernatant was transferred to 1.5 mL centrifuge tubes, followed by centrifugation at 15,000 g at 4°C for 3 min. Subsequently, 200 µL of supernatant was added in 96-well plate. The absorbance at 450 nm (OD450) was measured using iMark microplate reader (Bio-Rad Inc. Hercules, CA, USA).
Western blottingCells were washed with PBS and lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1% [w/v] sodium deoxycholate, 0.1% [w/v] SDS, 1% [v/v] Triton X-100, and a protease inhibitor cocktail) with PhosSTOP (4906845001, phosphatase inhibitor cocktail, Roche Diagnostics, Switzerland). After quantification of protein concentration by the BCA assay kit (T9300A, Takara, Shiga, Japan), protein samples were boiled in 1×Laemmli SDS sample buffer (62.5 mM Tris–HCl [pH 6.8], 5% [v/v] 2-mercaptoethanol, 2% [w/v] SDS, and 10% [v/v] glycerol) for 5 min. The samples were subjected to SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and electrophoresed in running buffer at 40 mA for 120 min. Separated proteins were transferred onto a PVDF membrane (Merck KGaA, Darmstadt, Germany) in the transfer buffer at a constant voltage of 100 V at 4°C. After 1 hr, the membranes were blocked in 5% BSA at room temperature for 1 hr. The membranes were gently shaken to allow reacting with primary antibodies overnight. On the next day, the membranes were washed 3 times by TBS-T for 5 min, and further incubated with HRP-conjugated secondary antibodies for 1 hr at room temperature. After 3 times washing with TBS-T for 5 min, membranes were reacted with the 100 µL of Immunostar LD or Zeta series (FUJIFILM Wako, Osaka, Japan) for 3 min at room temperature, the band signals were detected by Chemi DocTM MP system (Bio-Rad Inc. Hercules, CA, USA).
qPCRTotal RNA was extracted using TRI reagent (TR118, Molecular Research Center, Inc., USA), according to the manufacturer’s instructions. A ReverTra Ace qPCR RT kit (FSQ-201, TOYOBO, Osaka, Japan) was used to synthesize cDNAs according to the manufacturer’s instructions. qPCR was performed using the KOD SYBR qPCR Mix (QKD-201, TOYOBO, Osaka, Japan) under the following conditions: 98 °C for 2 min, followed by 40 cycles of 98°C for 10 sec, 60°C for 10 sec, and 68°C for 30 sec. The following primer sets were used: human c-fos 5′- TGG CGT TGT GAA GAC CAT GA -3′ and 5′- AGT TGG TCT GTC TCC GCT TG′; human ACTB 5′-TCA CCC ACA CTG TGC CCA TCT ACG A-3′ and 5′-CAG CGG AAC CGC TCA TTG CCA ATG G-3′. The 2-∆∆Ct method, using ACTB gene expression to normalize the target gene expression.
Wound healing assayCells were pre-seeded in 6-well plate at a density of 1× 105 cells/well for 72 hr until they reached 80% confluence as a monolayer. The scratch was performed with 20 µL pipette tips from one direction, followed by washing with 1× PBS (-) for three times to remove all detached cells and cell debris. Subsequently, the cells were treated by inhibitors in prior to 1,2-NQ exposure. DMSO-treated and EGF-treated A549 cells were used as negative control and positive control, respectively. Phase contrast images were photographed by phase contrast microscope BZ-X810 (Keyence, Osaka, Japan) and quantified using the ImageJ software (Schneider et al., 2012).
Statistical analysesAll experiments were independently performed at least 3 times. All data are expressed as the mean ± standard error of the mean (SEM) values. The experiments were analyzed using one-way ANOVA with Bonferroni’s multiple comparison test using GraphPad Prism 10.2.2 (GraphPad Software, San Diego, CA, USA). Statistical significance was set at p < 0.05.
To investigate the impact of 1,2-NQ on EGFR downstream signaling, we examined ERK1/2 phosphorylation in serum-starved A549 cells. As shown in Fig. 1, endogenous ERK1/2 phosphorylation was detected under certain conditions. Exposure to 1,2-NQ significantly increased ERK1/2 phosphorylation in a concentration-dependent manner (Fig. 1A, B). Notably, the levels of ERK1/2 phosphorylation in EGF-treated cells were comparable to those observed in A549 cells treated with 50 µM 1,2-NQ (Fig. 1A, B). Consistent with our previous study, baseline phosphorylation of EGFR and Akt was detected in serum-starved A549 cells, and 1,2-NQ modestly enhanced their phosphorylation in a dose-dependent manner (Fig. 1C–F) (Nakahara et al., 2021). We also monitored ERK1/2 phosphorylation induced by 1,2-NQ at various time points. As shown in Fig. 2A and B, ERK1/2 phosphorylation occurred rapidly upon 1,2-NQ exposure, with a significant increase within 5 min, peaking at 10 min, and then gradually declining over the next 60 min in a time-dependent manner. These findings suggest that 1,2-NQ induces activation of both the Akt and ERK1/2 pathways downstream of EGFR.
1,2-NQ treatment induces ERK1/2 phosphorylation. (A, B) A549 cells were serum-starved in SFM for 24 hr, followed by treatment with the indicated concentrations of 1,2-NQ or 100 pg/mL EGF for 15 min. ERK1/2 phosphorylation was analyzed by Western blotting. Quantitative data corresponding to (A) is shown in (B). (C–F) 1,2-NQ induces EGFR-Akt signaling. A549 cells were serum-starved for 24 hr, followed by treatment with the indicated concentrations of 1,2-NQ or 100 pg/mL EGF for 15 min. The phosphorylation of EGFR (C) and Akt (E) was examined by Western blotting. Quantitative analyses are presented in (D) and (F), respectively. Data represent means ± SEM (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001.
Regulation of ERK1/2 phosphorylation and effect of EGFR-targeting antibodies. (A, B) Time-course analysis of 1,2-NQ-induced ERK1/2 phosphorylation. A549 cells were pre-cultured in SFM for 24 hr and subsequently incubated with 20 µM 1,2-NQ. The time-dependent phosphorylation of ERK1/2 was analyzed by Western blotting. Quantitative analysis is presented in (B). (C–F) Dose-dependent effects of EGFR antibodies on ERK1/2 phosphorylation. After serum starvation, A549 cells were treated with cetuximab (C) or panitumumab (E) at the indicated concentrations for 3 hr, followed by treatment with 20 µM 1,2-NQ for 15 min. The phosphorylation of ERK1/2 (C, E) was examined by Western blotting. Quantitative analyses are presented in (D) and (F), respectively. Data represent means ± SEM (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001.
To determine whether 1,2-NQ-induced ERK1/2 phosphorylation is mediated via EGFR, we treated serum-starved A549 cells with cetuximab and panitumumab, monoclonal antibodies that competitively bind to EGFR and prevent ligand binding (Li et al., 2005; Giusti et al., 2007). We examined that a concentration of 20 µg/mL of cetuximab or panitumumab was sufficient to completely block 1,2-NQ-induced ERK1/2 phosphorylation (Fig. 2C–F). Furthermore, Co-treatment with either EGFR antibody significantly suppressed 1,2-NQ-induced ERK1/2 phosphorylation compared to treatment with 1,2-NQ alone (Fig. 3A–D). These results confirm that 1,2-NQ activates the ERK1/2 signaling pathway through EGFR.
Regulation of ERK1/2 phosphorylation by EGFR-targeting antibodies. (A–D) A549 cells were serum-starved in SFM for 24 hr, followed by incubation in the absence or presence of 20 µg/mL cetuximab or panitumumab for 3 hr. The cells were then exposed to 20 µM 1,2-NQ for 15 min. Negative and positive controls were cells treated with 0.2% DMSO and 100 ng/mL EGF, respectively. ERK1/2 phosphorylation was analyzed by Western blotting (A, C), with quantitative analysis shown in (B, D). Data represent means ± SEM (n = 3). ***p < 0.001. Fig. 4. Effects of LY3009120 and U0126 on ERK1/2 phosphorylation.A549 cells were serum-starved in SFM for 24 hr, followed by incubation with or without 5 µM Raf inhibitor (LY3009120) or 20 µM MEK inhibitor (U0126) for 30 min. Cells were then treated with 20 µM 1,2-NQ for 15 min. ERK1/2 phosphorylation was analyzed by Western blotting (A, C), with quantitative data shown in (B, D). Negative and positive controls were cells treated with 0.2% DMSO and 100 ng/mL EGF, respectively. Data represent means ± SEM (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001.
To further elucidate the mechanism of 1,2-NQ-induced ERK1/2 phosphorylation, we investigated the role of Raf, a key downstream effector of EGFR, using LY3009120, a pan-Raf inhibitor (Henry et al., 2015). Treatment with LY3009120 strongly suppressed ERK1/2 phosphorylation induced by either 1,2-NQ or EGF, without affecting total ERK1/2 expression (Fig. 4A, B). These findings suggest that Raf is involved in 1,2-NQ-induced ERK1/2 phosphorylation. Next, we examined the role of MEK, a downstream mediator of Raf, using U0126, a selective MEK inhibitor (Favata et al., 1998). In A549 cells treated with U0126, ERK1/2 phosphorylation induced by both 1,2-NQ and EGF was completely inhibited (Fig. 4C, D). These data indicate that 1,2-NQ activates the ERK1/2 signaling cascade via the EGFR–Raf–MEK pathway.
Effects of LY3009120 and U0126 on ERK1/2 phosphorylation. A549 cells were serum-starved in SFM for 24 hr, followed by incubation with or without 5 µM Raf inhibitor (LY3009120) or 20 µM MEK inhibitor (U0126) for 30 min. Cells were then treated with 20 µM 1,2-NQ for 15 min. ERK1/2 phosphorylation was analyzed by Western blotting (A, C), with quantitative data shown in (B, D). Negative and positive controls were cells treated with 0.2% DMSO and 100 ng/mL EGF, respectively. Data represent means ± SEM (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001.
As a key transcription factor downstream of ERK1/2, c-Fos regulates various cellular functions (Deng and Karin, 1994; Nakakuki et al., 2010). To assess whether 1,2-NQ modulates c-Fos expression, we analyzed c-fos mRNA levels following 1,2-NQ exposure. 1,2-NQ significantly upregulated c-fos expression, peaking at 30 min post-exposure before returning to baseline within the next 30 min (Fig. 5A). Notably, U0126 treatment significantly reduced c-fos mRNA levels in A549 cells exposed to either 1,2-NQ or EGF (Fig. 5B), confirming that ERK1/2 signaling via MEK regulates c-fos expression. These findings further support the notion that 1,2-NQ activates the EGFR-mediated ERK1/2 pathway through the Raf–MEK signaling axis.
1,2-NQ induces upregulation of c-fos mRNA levels. (A) A549 cells were serum-starved in SFM for 24 hr, followed by treatment with 20 µM 1,2-NQ for 15 min and incubation in SFM for the indicated times. c-Fos mRNA levels were analyzed by real-time qPCR. (B) After serum starvation, A549 cells were incubated in the absence or presence of 20 µM U0126 for 1 hr, followed by treatment with 0.2% DMSO, 20 µM 1,2-NQ, or 100 pg/mL EGF for 15 min and incubation in SFM for 30 min. c-Fos gene expression was quantified by real-time qPCR and normalized to ACTB. Data represent means ± SEM (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001.
Given the well-established role of ERK signaling in tumor progression (Aoki et al., 2017; Grandal and Madshus, 2008), we investigated the effect of 1,2-NQ on A549 cell migration, a critical process in cancer metastasis. Using a wound healing assay, we observed that 1,2-NQ significantly enhanced A549 cell migration after 24 hr of exposure (Fig. 6A, B). Similarly, EGF-treated cells displayed a slight but significant increase in migration compared to untreated controls (Fig. 6A, B). After 48 hr, both 1,2-NQ- and EGF-treated cells exhibited a marked increase in migration relative to controls (Fig. 6A, B). These results suggest that 1,2-NQ promotes cell migration via EGFR-mediated ERK1/2 signaling.
Regulation of A549 cell migration by 1,2-NQ-mediated EGFR-ERK signaling. (A, B) A549 cells were pre-cultured for 3 days and serum-starved in SFM. Wounds were introduced and photographed at 0 hr. Cells were then exposed to 20 µM 1,2-NQ or 1 ng/mL EGF for 10 min, and images were taken every 24 hr. The percentage of cell migration is shown in (B).(C, D) Effects of ERK signaling inhibitors on 1,2-NQ-enhanced cell migration. A549 cells were pre-cultured for 3 days, serum-starved for 24 hr, and wounded. Cells were then incubated in the absence or presence of 20 µg/mL cetuximab for 3 hr, 5 µM LY3009120 for 1 hr, or 20 µM U0126 for 1 hr before exposure to 20 µM 1,2-NQ or 1 ng/mL EGF for 10 min every 24 hr. Images were taken at 0 hr (before inhibitor treatment) and 48 hr (after 1,2-NQ treatment). The percentage of cell migration is shown in (D). (E) A549 cells were pre-cultured for 72 hr until reaching 80% confluence, followed by serum starvation in SFM. After 24 hr, cells were incubated with or without 20 µM U0126 for 1 hr and then exposed to 20 µM 1,2-NQ or 1 ng/mL EGF for 10 min every 24 hr. Cell viability was assessed using the WST-8 assay. Data represent means ± SEM (n = 3). **p < 0.01, and ***p < 0.001. Scale bar: 100 µm. ns, not significant.
To further investigate the relationship between 1,2-NQ-induced cell migration and ERK signaling, we pretreated A549 cells with inhibitors targeting EGFR, Raf, and MEK before 1,2-NQ exposure. As shown in Fig. 6C, D, both 1,2-NQ and EGF significantly increased A549 cell migration compared to control cells. However, cetuximab markedly suppressed migration induced by both 1,2-NQ and EGF (Fig. 6C, D). Similarly, inhibition of Raf with LY3009120 and MEK with U0126 nearly abolished 1,2-NQ-induced migration (Fig. 6C, D). Importantly, none of the inhibitors, including cetuximab, LY3009120, and U0126, exhibited cytotoxic effects in A549 cells (Fig. 6E). These results suggest that 1,2-NQ promotes cell migration through the EGFR-mediated ERK1/2 signaling pathway.
EGFR plays a central role in the tumorigenesis of various malignant carcinomas, particularly in NSCLC, where its aberrant activation drives tumor progression (da Cunha Santos et al., 2011; Imyanitov et al., 2021; Normanno et al., 2006). It has been reported that 1,2-NQ activates EGFR by reducing the activity of PTP1B, a negative regulator of EGFR, through the covalent binding of 1,2-NQ to cysteine residue (Cys) 121 within PTP1B (Iwamoto et al., 2007). More recently, our group demonstrated that 1,2-NQ specifically activates EGFR, but not other EGFR family members, through direct covalent N-arylation of lysine residue (Lys) 80 within EGFR (Nakahara et al., 2021). These findings collectively suggest that multiple molecular targets are involved in 1,2-NQ-mediated EGFR signaling. Furthermore, we observed that 1,2-NQ treatment also promotes ERK1/2 phosphorylation, and anti-EGFR antibodies suppressed this 1,2-NQ-induced phosphorylation, suggesting that EGFR is a key mediator of ERK1/2 signal transduction in response to 1,2-NQ exposure (Figs. 2 and 3). Although we did not directly assess the effect of an EGFR kinase inhibitor on 1,2-NQ-induced ERK1/2 activation in this study, our previous work (Nakahara et al., 2021) demonstrated that treatment with an EGFR kinase inhibitor markedly suppressed 1,2-NQ-induced Akt phosphorylation in A549 cells. This finding suggests that the EGFR kinase activity is functionally required for the activation of downstream signaling pathways following 1,2-NQ exposure. Given that both Akt and ERK1/2 are well-known downstream effectors of EGFR, it is reasonable to infer that ERK activation also depends on EGFR kinase activity.
ERK is a downstream component of a conserved signaling module that is activated by Raf, which, in turn, phosphorylates MEK, leading to the activation of ERK1/2. Therefore, we examined the activation of these molecules using their respective inhibitors. Although we did not directly assess the activation of Raf and MEK, treatment of A549 cells with these inhibitors significantly suppressed ERK1/2 phosphorylation upon 1,2-NQ exposure, indicating that 1,2-NQ induces ERK1/2 phosphorylation via the EGFR-Raf-MEK cascade (Fig. 4).
Multiple studies have demonstrated that EGFR-mediated ERK signaling plays a critical role in cell proliferation, survival, and metastasis during tumorigenesis (Roberts and Der, 2007; McCubrey et al., 2007). Consistently, our observations revealed enhanced A549 cell migration when cells were cultured in the presence of 1,2-NQ (Fig. 6). Although we didn’t examine the effects of other downstream effectors of EGFR, such as Akt, JNK and p38, 1,2-NQ-enhanced cell migration was abolished by treatment with inhibitors targeting the EGFR-Raf-MEK-ERK axis, suggesting that this pathway is a major driver of 1,2-NQ-induced cell migration (Fig. 6). Our findings provide insight into the mechanisms by which environmental electrophiles contribute to lung cancer pathogenesis.
Additionally, c-Fos is a key downstream transcription factor of the EGFR signaling pathway, regulated by ERK1/2 activation, and is known to promote invasive growth and metastasis in human cancers (Muhammad et al., 2017; Sachdev et al., 2008). For example, c-Fos overexpression has been shown to regulate the inhibitor of differentiation/DNA binding 1 (ID1), disrupting the balance of cell proliferation. This imbalance may contribute to cell transformation and tumor formation (Zhao et al., 2016). Furthermore, c-Fos-mediated upregulation of activator protein-1 (AP-1) has been implicated in the development of osteosarcoma (Xiao et al., 2016). Notably, we observed that exposure to 1,2-NQ enhances c-fos mRNA expression, and the addition of a MEK inhibitor suppressed this upregulation by inhibiting ERK signaling, as expected (Fig. 5). Based on these observations, the enhancement of A549 cell migration by 1,2-NQ may be attributed to ERK-mediated c-fos upregulation, warranting further investigation in future studies.
Taken together, our findings provide evidence that 1,2-NQ promotes A549 cell migration through the EGFR-Raf-MEK-ERK signaling pathway. Pharmacological inhibition of EGFR-mediated signaling, including EGFR, Raf, and MEK, significantly suppressed downstream ERK phosphorylation. Interestingly, inhibition of this axis also suppressed A549 cell migration. These results offer insights into the molecular mechanisms underlying 1,2-NQ-induced carcinogenesis and may lead to potential therapeutic targets for lung cancer progression.
This research was funded by the Grants-in-Aid for Challenging Exploratory Research (22K19380) (to T.U.), Scientific Research (A) (24H00678) (to T.U.), and Scientific Research (C) (24K09795) (to S.K.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and the Smoking Research Foundation (to T.U.).
Conflict of interestThe authors declare that there is no conflict of interest.
