Nicotine promotes the progression and metastasis of non-small cell lung cancer by modulating the OTUB1-c-Myc-EZH2 axis

Abstract

Smoking has been identified as a major risk factor for the development and progression of non-small cell lung cancer (NSCLC). As a key component of tobacco smoke, nicotine is believed to play a significant role in promoting NSCLC growth and progression. EZH2 is an epigenetic regulator highly expressed in the tumor tissues of smokers. However, whether and how nicotine regulates the expression of EZH2 and the underlying mechanisms remain unclear. Bioinformatics analysis and immunohistochemistry were used to compare the expression of EZH2 in NSCLC samples between smokers and nonsmokers. Western blotting, real-time quantitative PCR, and immunofluorescence were employed to confirm the effects of nicotine on EZH2 expression. Cell Counting Kit-8 assays, colony formation assays, 5-ethynyl-2-deoxyuridine staining, and Transwell assays were conducted to analyze the proliferation and metastasis of A549 and H1650 cells treated with siRNA or EZH2 inhibitors. Real-time quantitative PCR and chromatin immunoprecipitation assays were performed to assess the regulatory effect of nicotine on EZH2 transcript levels via c-Myc. Coimmunoprecipitation and ubiquitination assays were used to assess the deubiquitination of c-Myc by OTUB1. Finally, a nude mouse model was used to evaluate the impact of combined c-Myc and EZH2 inhibitors on tumor proliferation and metastasis in vivo. EZH2 is expressed at relatively high levels in NSCLC patients, as determined by both bioinformatic and IHC analyses. Nicotine upregulates EZH2 expression and promotes the proliferation and metastatic ability of lung cancer cells. Inhibition of EZH2 with either DZNep or EPZ6438, EZH2 inhibitors, or siRNA significantly decreased the proliferative and metastatic capacity of NSCLC cells induced by nicotine treatment. Moreover, the study revealed that nicotine induces OTUB1 expression, stabilizes the c-Myc protein via deubiquitination, and enables c-Myc-mediated transcriptional activation of EZH2. Furthermore, the c-Myc inhibitor 10058-F4 exhibited synergistic effects with the EZH2 inhibitor DZNep in suppressing NSCLC cell proliferation and metastasis both in vitro and in vivo.Nicotine regulates the c-Myc/EZH2 signaling pathway via OTUB1-mediated deubiquitination, thereby promoting the proliferation and metastasis of NSCLC cells. This research reveals novel molecular mechanisms of nicotine in the development of NSCLC, providing a theoretical foundation for future therapeutic strategies.

Introduction

According to statistics from the World Health Organization, lung cancer ranks second in incidence and first in mortality rate among malignancies [1]. Lung cancer is primarily divided into non-small cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), with NSCLC accounting for approximately 85%–88% of cases and SCLC accounting for approximately 12%–15% of cases [2]. In the past decade, there have been significant advances in the treatment of advanced NSCLC, with improved survival rates and improved quality of life for patients owing to targeted therapies and immunotherapies [3]. However, the overall survival (OS) rate for patients with lung cancer remains low; thus, a deeper exploration of the mechanisms underlying its development is needed.

Smoking is a severe global health issue and is directly or indirectly associated with the occurrence of various diseases, with lung cancer being the most common [4–6]. Long-term exposure to cigarette smoke has a profound effect on the immune system and overall health, making individuals more susceptible to diseases and less capable of recovering [7, 8]. Nicotine, a primary addictive component of cigarettes, leads to nicotine dependence with prolonged smoking, making smoking cessation challenging [9]. The inhalation of nicotine in cigarette smoke stimulates the release of dopamine in the brain, resulting in a sense of pleasure. The chemical structure of nicotine includes one pyridine and one pyrrolidine ring, both of which contain a tertiary amine [10]. Nicotine selectively binds to multiple positions on nicotinic acetylcholine receptors (nAChRs), which are distributed in the brain, lungs, neuromuscular junctions, adrenal medulla, and ganglia [11]. α9-nAChR plays a critical role in nicotine-induced angiogenesis, and this bispecific antibody (α9 BsAb) may serve as a potential therapeutic candidate for the treatment of α9-positive cancers [12]. Research indicates that nicotine can promote tumor progression through various pathways. Studies have shown that nicotine induces the proliferation and growth of tumor cells while reducing apoptosis [13]. This may be attributed to the ability of nicotine to increase DNA damage repair within cells, thereby increasing cell survival [14]. Recent studies have shown that the OTUD3/ZFP36/VEGF-C axis plays a vital role in nicotine addiction-induced lymphatic metastasis in human esophageal cancer [15]. Nicotine stimulates the invasion and metastasis of colon cancer cells in vitro via activation of nAChRs and the p38 MAPK downstream signaling pathway [16]. Although numerous studies suggest that nicotine may play a role in promoting tumor development, the underlying molecular mechanisms remain unclear. Therefore, further research is needed to elucidate the specific pathways and potential impacts of nicotine on tumor initiation and progression.

The enhancer of zeste homolog (EZH2) gene, located on chromosome 7q35, comprises 20 exons encoding 746 amino acid residues [17]. EZH2 is a key member of the polycomb group of proteins and one of the core subunits of the polycomb repressive complex 2 (PRC2), which possesses histone methyltransferase activity and plays a crucial role in epigenetic modifications [18]. The role of EZH2 in cancer has been extensively studied. Research indicates that EZH2 is highly expressed in various types of tumors, especially malignant tumors such as breast cancer, prostate cancer, lung cancer, lymphoma, melanoma, and pancreatic cancer [19, 20]. Elevated expression of EZH2 is associated with tumor differentiation, infiltration, and metastasis [21]. Research has shown that nicotine can increase EZH2 expression and promote breast cancer progression [22]. Previous studies have revealed that EZH2 expression is significantly correlated with male sex and smoking history. However, in NSCLC, there is currently no reported evidence on the relationship between nicotine and EZH2, highlighting the need for further investigations to elucidate the impact of nicotine on EZH2 and its potential role in the development and progression of NSCLC.

In our present study, we investigated the correlation between EZH2 expression and nicotine-induced NSCLC progression; this report provides substantial evidence that nicotine regulates proliferation and metastasis in NSCLC through the OTUB1/c-Myc/EZH2 axis, suggesting that EZH2 is a critical downstream target in the response to nicotine and regulates the malignant phenotype of NSCLC cells. More broadly, our current study highlights the potential therapeutic value of targeting EZH2 in NSCLC.

Materials and methods

Cells and reagents

The NSCLC cell lines A549 and H1650 were obtained from the American Type Culture Collection and cultured in DMEM (Biological Industries, USA) supplemented with 10% FBS (Gibco, USA). All culture media contained 100 U/ml penicillin and 100 mg/ml streptomycin and were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. DMSO, MG132 (S2619), and cycloheximide (CHX, 508739) were purchased from Sigma‒Aldrich. 3-Deazaneplanocin A hydrochloride (DZNep, S7120) and 10058-F4 (S7153) were purchased from Selleck. Nicotine (AB0644) was purchased from Chengdu Alfa Biotechnology Co., Ltd.

Plasmids and vectors

The OTUB1 and c-Myc coding sequences were purchased from the Public Protein/Plasmid Library. siRNAs targeting EZH2, c-Myc and OTUB1 were purchased from Guangzhou RiboBio Co., Ltd. The cells were transfected with plasmids or siRNA duplexes using Lipofectamine 2000 (Thermo Fisher, USA) according to the manufacturer’s instructions.

Cell proliferation assay

For Cell Counting Kit-8 (CCK-8; K1018, APExBIO, USA) assays, the cells were seeded in a 96-well culture plate at a density of 3 × 10³ cells per well. Subsequently, 10 μL of CCK-8 solution was added to each well, followed by a 2-h incubation. The absorbance was measured at 450 nm using a microplate reader for a total of 4 days. For the colony formation assays, the cells were seeded at a density of 500 cells per well in a 6-well plate. After 14 days, the cells were fixed with 4% paraformaldehyde at room temperature for 20 min and then stained with crystal violet for 20 min. For 5-ethynyl-2’-deoxyuridine (EdU) incorporation assays, 1 × 10⁵ cells per group were seeded onto slides and cultured for 24 h. EdU incorporation was detected using the Click-iT EdU Imaging Kit (C10310-1; RiboBio, China) according to the manufacturer’s instructions. The stained samples were observed under an inverted fluorescence microscope (IX73P1F; Olympus, Japan). The results were analyzed and interpreted accordingly.

Transwell assay

Migration and invasion assays were conducted using 24-well cell culture inserts (3422; Corning, USA) with transparent PET membranes. The cells (2 × 10⁵) in 200 µL of serum-free DMEM were added to the upper chamber, with or without Matrigel. In the lower chamber, 600 µL of DMEM supplemented with 10% FBS was added. After 24 h, the migrated cells were fixed with 4% paraformaldehyde for 20 min and stained with crystal violet.

Protein extraction and Western blot analysis

Total protein was extracted, quantified with BCA protein assay reagent, separated via SDS‒PAGE, and detected by immunoblotting with specific antibodies. Antibodies against EZH2 (CST, 5246 s), c-Myc (CST, 5605 s), OTUB1 (SANTA, sc-130458), GAPDH (ABclonal, Ac002), β-ACTIN (CST, 4967), and ubiquitin (CST, 3933 s) were purchased from the designated manufacturers. GAPDH or β-actin was used as the loading control.

RNA isolation and quantitative real-time PCR

TRIzol reagent (Invitrogen) was used to extract total RNA. Reverse transcription of RNA was performed using the Takara PrimeScript RT Kit. The expression of candidate genes was measured via quantitative real-time PCR using an ABI 7900HT real-time PCR system (Applied Biosystems). The primer sequences are listed in Supplementary Table 1.

Clinical samples

NSCLC and paired noncancerous tissues were obtained from 60 patients who underwent surgical resection without preoperative chemotherapy or radiotherapy at TJMUGH from 2012 to 2013. Prior patient consent and approval from the Institutional Research Ethics Committee were obtained. The patients were followed up every 3 months after surgery. All the tissues were frozen at −80 °C until use.

Immunohistochemistry (IHC) and Immunofluorescence (IF)

IHC staining of paraffin-embedded tissues with antibodies was performed, and the results were scored according to standard procedures. The staining score was determined by two independent pathologists at our center. Immunofluorescence images were acquired via a confocal microscope. The antibodies used for IHC or IF were as follows: anti-EZH2 (CST, 5246 s, 1:100), anti-c-Myc (CST, 5605 s, 1:200), anti-OTUB1 (SANTA, sc-130458, 1:200), and anti-Ki-67 (ab205921, Abcam, 1:200).

Coimmunoprecipitation (co-IP) assay

Co-IP assays were performed to determine the interaction between OTUB1 and c-Myc. The complexes were precipitated using protein A/agarose beads and then subjected to Western blotting.

ChIP assay

After A549 cells were transfected with c-Myc, ChIP experiments were performed according to the manufacturer’s instructions. A549 cells were crosslinked with formaldehyde, lysed with SDS buffer and sonicated. Sheared DNA was precleared with a salmon sperm DNA/protein A agarose slurry and immunoprecipitated with c-Myc and IgG. The agarose beads were incubated with the antibody/protein/DNA complex and washed with low-salt buffer, high-salt buffer, and LiCl wash buffer. DNA was eluted in 1% SDS/0.1 M NaHCO₃ and decrosslinked with 0.2 M NaCl.

PDO tumor models

To prepare lung adenocarcinoma organoids, sterile scissors were used to mechanically dissect fresh lung adenocarcinoma tissue obtained from the patient. Then, 3 ml of digestion solution was added, and the mixture was incubated horizontally on a shaker (70 rpm, 37 °C) for 20 min. Next, the cell suspension was filtered through a 100 μm mesh sieve to remove larger cell clumps, and the filtrate was centrifuged (1200 rpm, 5 min). The filtered cell suspension was subsequently mixed with an organoid culture matrix (Corning, 356255), and the mixture was transferred evenly to the bottom of a 24-well low-attachment culture plate. The mixture was inoculated into preheated 12/24-well plates at 50 μl/well and placed in a 37 °C incubator. After 5 min, the culture plate was carefully inverted. After 30 min, the gel was allowed to solidify, and preheated culture medium was carefully added along the well walls. The growth and morphology of the organoids were observed every 2–3 days, and the culture medium was changed as needed to maintain the healthy growth state of the organoids.

Animal models

BALB/c mice (female, 4–6 weeks of age, 18–20 g) were housed in a specific pathogen-free environment. A total of 2 × 10⁴ A549 cells were injected subcutaneously into the right flank of each mouse. Once the tumors reached an average volume of 100 mm³, the mice were randomly divided into different groups and subjected to the following treatments: vehicle (saline), nicotine (0.25 mg/kg), DZNep (3 mg/kg), and 10058-F4 (30 mg/kg) for 2 consecutive weeks. All drugs were intraperitoneally injected into the mice every other day. Tumor size was measured using digital calipers. After 24 days, the tumors were surgically dissected. To assess metastasis, A549 cells (3 × 10⁶) were injected via the tail vein into nude mice. After injection, the health status of the mice was monitored over a period of 4 weeks. The mice were randomly divided into different groups and treated with vehicle (saline), nicotine (0.25 mg/kg), DZNep (3 mg/kg), or 10058-F4 (30 mg/kg) for 4 consecutive weeks. At the end of the experiment, the mice were humanely euthanized, and their lung tissues were immediately harvested. The tumor nodules visible on the surface of the lungs were photographed and counted. The lung tissues were then fixed in formalin and subjected to hematoxylin and eosin (H&E) staining. Continuous lung tissue sections were examined and analyzed under a microscope. All animal experiments were performed according to procedures approved by the institutional animal care and use committee of Tianjin Medical University General Hospital.

Statistical analysis

Statistical analyses were performed via GraphPad Prism 8. Differences between the means of two groups were analyzed via Student’s t test. The threshold for statistical significance was set at P < 0.05.

Results

Nicotine promotes the proliferation and metastasis of NSCLC cells

To investigate the impact of nicotine on the proliferation of NSCLC cells, CCK-8, colony formation, and EdU incorporation assays were performed. The results of the CCK-8 assay indicated that nicotine treatment significantly promoted the proliferation of NSCLC cells (Fig. 1a, b). According to the EdU immunofluorescence staining assay, the proportion of EdU-positive cells in the nicotine treatment group was greater than that in the control group (Fig. 1c, d). The colony formation assay also revealed that, compared with the control group, the nicotine treatment group had more colonies (Fig. 1e, f). To further investigate the impact of nicotine on cell invasion and migration, we employed Transwell assays. The results revealed that nicotine treatment significantly enhanced the invasion and migration of A549 and H1650 cells (Fig. 1g–i). Our research results indicate that nicotine promotes the proliferation and metastasis of NSCLC cells.

Fig. 1. Nicotine promotes the proliferation and metastasis of NSCLC cells.

a, b Nicotine promoted A549 and H1650 cell proliferation, as measured by a CCK-8 assay. c, d Cell proliferation was also evaluated via EdU staining. e, f Nicotine promoted colony-forming capacity, as measured by a colony formation assay. g–i Nicotine promoted NSCLC cell migration and invasion, as measured by a Transwell assay. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, with P values calculated via unpaired t tests.

EZH2 is upregulated by nicotine and its expression level is correlate with the prognosis of NSCLC

To further explore the potential mechanism of the effect of nicotine on NSCLC cells, NSCLC cells were treated with nicotine, and the results revealed a gradual increase in EZH2 protein expression with increasing concentrations of nicotine. In subsequent experiments, after the addition of 10 μM nicotine and treatment for 24 h, 48 h, or 72 h, the protein expression of EZH2 and H3K27me3 increased with increasing duration of nicotine treatment (Fig. 2a). RT‒qPCR analysis was performed to detect changes in EZH2 mRNA levels following nicotine treatment, yielding consistent results (Fig. 2b). Additionally, immunofluorescence experiments revealed a significant increase in EZH2 expression in lung adenocarcinoma cells after nicotine treatment, further confirming that nicotine promotes EZH2 expression in lung adenocarcinoma cells (Fig. 2c).

Fig. 2. EZH2 is upregulated by nicotine and its expression correlates with the prognosis of NSCLC.

a Western blot experiments revealed time- and concentration-dependent increases in EZH2 and H3K27me3 protein levels following nicotine treatment. b RT‒qPCR experiments revealed increased EZH2 mRNA levels in response to various durations and concentrations of nicotine. c Immunofluorescence staining experiments revealed an increase in EZH2 expression after nicotine treatment. d, e Immunohistochemistry of tissues from 60 lung cancer patients revealed increased expression levels of EZH2 in smokers; smokers, n = 20; nonsmokers, n = 40. f, g Immunohistochemistry of tissues from lung cancer patients revealed increased expression levels of EZH2 in smokers; smokers, n = 10; nonsmokers, n = 10. h, i Analysis of LUAD and LUSC cohorts from the TCGA database revealed higher expression levels of EZH2 in smokers than in nonsmokers. j Analysis of the GSE31210 dataset revealed higher expression levels of EZH2 in smokers than in nonsmokers. k, l Immunohistochemistry revealed significantly higher expression levels of EZH2 in lung cancer tissues than in adjacent noncancer tissues. m Kaplan‒Meier plotter analysis indicating that high EZH2 expression is correlated with poor LUAD prognosis. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, with P values calculated via unpaired t tests.

For the analysis of clinical samples, tumor and tumor-adjacent tissues were collected from 60 postoperative lung cancer patients at the Department of Lung Cancer Surgery, Tianjin Medical University General Hospital. Immunohistochemical staining experiments confirmed a substantial increase in EZH2 expression in smokers (Fig. 2d, e). Moreover, the experiments confirmed that H3K27me3 expression was significantly increased in smokers (Fig. 2f, g, smokers = 10, nonsmokers = 10). Data on the expression of EZH2 in LUAD and LUSC patients were extracted from The Cancer Genome Atlas (TCGA) database. Analysis of these data revealed significantly greater EZH2 expression in smokers with lung adenocarcinoma and squamous cell carcinoma than in nonsmokers with these tumor types (Fig. 2h, i); similar results were obtained in the GSE31210 cohort (Fig. 2j). IHC data revealed greater EZH2 expression in lung cancer tissues than in adjacent tissues (Fig. 2k, l). The Kaplan‒Meier plotter database revealed that the higher the expression of EZH2 was, the worse the prognosis of LUAD was (Fig. 2m). In summary, nicotine significantly promotes the expression of EZH2 in NSCLC cells, and this effect is dependent on both time and drug concentration. EZH2 may be an important downstream molecule in the nicotine-induced progression of NSCLC.

EZH2 is an important downstream target of nicotine in NSCLC

Research indicates that EZH2 can promote the proliferation and metastasis of various tumors, including lung cancer. To validate whether EZH2 is a crucial downstream target of nicotine in NSCLC, we investigated whether the promoting effect of nicotine on the malignant phenotype of NSCLC cells was blocked by EZH2 inhibition. CCK-8, EdU, and colony formation assays revealed that the proliferative capacity of NSCLC cells was significantly increased with nicotine treatment alone. However, after we treated A549 cells with an EZH2 inhibitor (DZNep or EPZ6438), the proliferative capacity of NSCLC cells was significantly decreased. Similar results were obtained when EZH2 expression was knocked down with siRNA. When EZH2 was inhibited and then nicotine was administered, the results revealed that inhibiting EZH2 expression significantly attenuated nicotine-induced proliferation in NSCLC (Fig. 3a–j, Supplementary Fig. S1a–e). Furthermore, when NSCLC cells were treated with nicotine alone, their migration and invasion capabilities were significantly increased. However, inhibiting EZH2 expression with DZNep or EPZ6438 markedly suppressed NSCLC migration and invasion. Similar results were observed when EZH2 expression was knocked down. Analysis of cells treated with EZH2 inhibitor and nicotine indicated that inhibiting EZH2 expression significantly weakened nicotine-induced migration and invasion in NSCLC (Fig. 3k–p, Supplementary Fig. S1f–h). To further confirm our in vitro observations, we subcutaneously injected A549 cells into BALB/c mice. The promoting effect of nicotine on NSCLC was reversed by DZNep (Fig. 3q, r). The same conclusion was also obtained by IHC staining analysis of tumors in mice (Supplementary Fig. S1i). In conclusion, these results suggest that EZH2 is a crucial downstream target of nicotine that promotes the proliferation and metastasis of NSCLC cells.

Fig. 3. DZNep and EPZ6438 reversed the nicotine-induced inhibition of A549 cell proliferation and metastasis.

a–j Effects of nicotine. The effects of DZNep, EPZ6438 or their combinations with nicotine on A549 cell proliferation were evaluated via CCK-8 (a, b), EdU (c–f) and colony formation (g–j) assays. k‒p A549 cell migration and invasion under the four conditions were also evaluated via a Transwell assay. q, r Photograph of dissected tumors (first line: vehicle; second line: nicotine; third line: DZNep; fourth line: nicotine + DZNep; n = 5). Nicotine promoted tumor growth in mice. DZNep inhibited tumor growth in mice. Nicotine combined with DZNep did not inhibit tumor growth in the mice. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, with P values calculated via unpaired t tests.

c-Myc serves as a downstream effector of nicotine to increase EZH2 transcription

The transcription factor c-Myc plays a pivotal role in regulating cellular differentiation and proliferation, including the amplification of target gene transcription. In NSCLC, the nicotine derivative NNK has been shown to induce the expression of c-Myc, a multifaceted regulator implicated in various cellular processes [23]. Immunohistochemical staining confirmed a substantial increase in c-Myc expression in smokers (Fig. 4a, b). Furthermore, we investigated whether c-Myc mediates nicotine-induced EZH2 expression in NSCLC, and the results revealed that nicotine upregulates c-Myc protein expression without affecting its mRNA level (Fig. 4c, d). Furthermore, a direct association between c-Myc and EZH2 was observed, with EZH2 protein levels decreasing upon c-Myc knockdown and increasing with c-Myc overexpression (Fig. 4e, f). Meanwhile, the results showed that the mRNA level of EZH2 was increased when c-Myc was overexpressed (Fig. 4g). The same results were obtained by immunofluorescence (Fig. 4h). Notably, inhibiting c-Myc expression reversed the nicotine-induced increase in EZH2 protein expression (Fig. 4i, j). IHC analysis of consecutive tumor sections from 10 individuals revealed a distinct colocalization pattern of EZH2 and c-Myc (Fig. 4k). Additionally, analysis of TCGA data revealed a modest positive correlation between EZH2 and c-Myc expression levels, with R values of 0.23 for LUSC and 0.20 for LUAD (Fig. 4l). These results suggest a weak, yet consistent, association between the expression of these two genes in both LUSC and LUAD, although the correlation is relatively low. Using Jaspar, an open-access database storing transcription factor binding profiles, potential c-Myc binding sites in the EZH2 promoter were predicted, with two putative binding sites identified in the genomic region (Fig. 4m). ChIP assays confirmed that c-Myc was recruited to the promoter region containing one of the potential binding sites (region 862-873) exclusively in cells overexpressing c-Myc (Fig. 4n, o).

Fig. 4. c-Myc serves as a downstream effector of nicotine to increase EZH2 transcription.

a, b Immunohistochemistry of tissues from patients with lung cancer revealed increased expression levels of c-Myc in smokers; smokers, n = 10; nonsmokers, n = 10. c Western blot experiments revealed a time- and concentration-dependent increase in c-Myc protein levels following nicotine treatment. d RT‒qPCR confirmed that there was no change in the mRNA level of c-Myc after nicotine treatment. e, f c-Myc knockdown decreased the protein level of EZH2, whereas c-Myc overexpression increased the protein level of EZH2 in A549 and H1650 cells. g c-Myc overexpression increased the level of the EZH2 mRNA. h c-Myc knockdown decreased the protein level of EZH2 according to immunofluorescence (IF) analysis. i, j Inhibiting the expression of c-Myc attenuated the promoting effect of nicotine on EZH2 protein expression. k Representative IHC images of c-Myc and EZH2 in NSCLC. c-Myc and EZH2 colocalization was indicated by overlapping staining. l c-Myc expression was positively correlated with EZH2 expression in the LUAD and LUSC cohorts in the TCGA analysis. m Putative EZH2-binding sites within the genomic sequence adjacent to the transcription start site of the c-Myc gene. n, o ChIP with IgG and c-Myc antibodies was performed in A549 cells after c-Myc was overexpressed. ****P < 0.0001, **P < 0.01, with P values calculated via unpaired t tests.

To investigate the role of the c-Myc-EZH2 axis in NSCLC, we conducted experiments in which c-Myc was knocked down and EZH2 was overexpressed. Through CCK-8, EdU incorporation, and colony formation assays, we found that EZH2 overexpression partially counteracted the inhibition of NSCLC proliferation induced by c-Myc knockdown (Supplementary Fig. S2a–e). Additionally, Transwell assays revealed that, compared with c-Myc knockdown alone, the combination of c-Myc knockdown and EZH2 overexpression significantly restored NSCLC cell migration and invasion (Supplementary Fig. S2f–h). These results suggest that EZH2 plays a crucial role in reversing the inhibitory effects of c-Myc knockdown, further implicating the c-Myc-EZH2 axis as a key regulator of NSCLC progression.

Inhibition of c-Myc expression attenuates the promoting effect of nicotine on NSCLC cell behaviors

We further investigated whether the inhibition of c-Myc could block the promoting effect of nicotine on the malignant phenotype of NSCLC cells. 10058-F4, a specific inhibitor of c-Myc expression, was used to suppress c-Myc expression, resulting in a significant decrease in the proliferative capacity of NSCLC cells. Similar results were obtained when c-Myc expression was knocked down. Our findings demonstrated that inhibiting c-Myc expression markedly attenuated nicotine-induced NSCLC proliferation (Fig. 5a–e, Supplementary Fig. S3a–e). Additionally, inhibiting c-Myc expression with 10058-F4 significantly suppressed the migration and invasion of NSCLC cells. Similar results were observed when c-Myc expression was knocked down. These results indicate that inhibiting c-Myc expression significantly attenuates nicotine-induced migration and invasion of NSCLC cells (Fig. 5f–h, Supplementary Fig. S3f–h). To further confirm our in vitro observations, A549 cells were subcutaneously injected into BALB/c mice. The promoting effect of nicotine on lung cancer was reversed by 10058-F4, with IHC yielding similar conclusions (Fig. 5i–k). Collectively, our findings suggest that c-Myc acts downstream of nicotine, increasing EZH2 transcription.

Fig. 5. 10058-F4 combined with nicotine reversed the inhibitory effect of nicotine on H1650 cell proliferation and metastasis.

a–e The effects of 10058-F4, nicotine and 10058-F4 combined with nicotine on H1650 cell proliferation were evaluated via CCK-8, EdU and colony formation assays. f–h H1650 cell migration and invasion under the four conditions were also evaluated via Transwell assays. i, j Photograph of dissected tumors (first line: vehicle; second line: nicotine; third line: 10058-F4; fourth line: nicotine +10058-F4; n = 5). Nicotine promoted tumor growth in mice. 10058-F4 inhibited tumor growth in mice. Nicotine combined with 10058-F4 did not inhibit tumor growth in the mice. k The protein expression of c-Myc and Ki-67 in dissected tumor samples was evaluated by IHC. ****P < 0.0001, ***P < 0.001, *P < 0.05, with P values calculated via unpaired t tests.

OTUB1 is required for nicotine-mediated c-Myc stabilization

Under nicotine stimulation, although there were differences in c-Myc protein levels, c-Myc mRNA levels remained unchanged (Fig. 4c, d), suggesting that nicotine-induced c-Myc may be posttranscriptional rather than transcriptional. To validate this, NSCLC cells were coincubated with MG132 and the protein synthesis inhibitor CHX. MG132 treatment significantly increased c-Myc protein levels (Fig. 6a). Concurrently, a CHX assay revealed that nicotine markedly attenuated c-Myc degradation (Fig. 6b, c). Further ubiquitination experiments revealed that nicotine reduced c-Myc ubiquitination (Fig. 6d). Previous studies have shown that OTUB1 can induce c-Myc deubiquitination [24, 25]. We hypothesized that OTUB1 might be involved in nicotine-induced c-Myc expression. The results revealed that nicotine promoted OTUB1 expression (Fig. 6e). Immunohistochemical staining confirmed a substantial increase in OTUB1 expression in smokers (Fig. 6f, g). OTUB1 overexpression increased c-Myc protein levels, whereas OTUB1 silencing decreased these levels (Fig. 6h, i). The CHX assay results indicated that OTUB1 significantly attenuated c-Myc degradation, whereas OTUB1 knockdown increased c-Myc degradation (Fig. 6j–m). Co-IP assays were subsequently conducted to verify the interaction between OTUB1 and c-Myc. OTUB1 was immunoprecipitated using an anti-c-Myc antibody, and vice versa (Fig. 7a). IF analysis also revealed the colocalization of OTUB1 and c-Myc (Fig. 7b).

Fig. 6. OTUB1 is required for nicotine-mediated c-Myc stability.

a Immunoblot (IB) analysis of whole-cell lysates (WCLs) derived from nicotine-treated A549 cells treated with 10 µM MG132. b, c IB analysis of WCLs from nicotine-treated A549 cells following treatment with 100 µg/ml cycloheximide (CHX) at the indicated time points. d IB analysis of WCLs and anti-c-Myc immunoprecipitates derived from lysates of A549 cells treated with 10 µM nicotine and incubated with the indicated antibodies. e Nicotine (10 µM) promoted the protein expression of OTUB1 in A549 cells. f, g Immunohistochemistry of tissues from lung cancer patients revealed increased expression levels of OTUB1 in smokers; smokers, n = 10, nonsmokers, n = 10. h, i OTUB1 knockdown decreased the protein level of c-Myc, whereas OTUB1 overexpression increased the protein level of c-Myc in A549 cells. j, k IB analysis of WCLs derived from oe-control- or oe-OTUB1-treated A549 cells treated with 100 µg/ml CHX at the indicated time points. l, m IB analysis of WCLs derived from si-control- or si-OTUB1-treated A549 cells treated with 100 µg/ml CHX at the indicated time points. ****P < 0.0001, with P values calculated via unpaired t tests.

Fig. 7. OTUB1-mediated deubiquitination promotes c-Myc stability.

a IB analysis of anti-OTUB1 and anti-c-Myc immunoprecipitates derived from A549 cells. b Confocal microscopy image showing the colocalization of OTUB1 with c-Myc. c, d c-Myc was pulled down and subsequently subjected to IB using an anti-ubiquitin antibody after the indicated transfection treatment and 10 µM MG132 for 6 h in A549 cells.

We analyzed c-Myc ubiquitination in NSCLC cells and found that, compared with that in control cells or cells with OTUB1 knockdown, the ubiquitination level of c-Myc was significantly increased in cells with OTUB1 knockdown. OTUB1 knockdown significantly increased the K48-linked ubiquitination of c-Myc in cells, whereas the K63-linked ubiquitination level remained unchanged (Fig. 7c). We utilized HA-tagged Ub mutants (K48R, K63R) to identify the lysine residues required for polyubiquitin chain formation, and we found that the K48R mutation disrupted the OTUB1-mediated deubiquitination of c-Myc (Fig. 7d). In conclusion, this series of experiments revealed that OTUB1 can directly mediate the removal of the K48-linked ubiquitination of c-Myc, providing crucial evidence for understanding the molecular mechanism by which OTUB1 regulates c-Myc protein stability.

10058-F4 increases the efficacy of DZNep therapy

We further assessed whether inhibiting c-Myc expression could enhance the therapeutic efficacy of NSCLC treatment when combined with DZNep or EPZ6438. Using CCK-8, EdU incorporation, and colony formation assays, we found that 10058-F4 treatment significantly suppressed NSCLC cell proliferation. Moreover, the combination of 10058-F4 with DZNep or EPZ6438 had a more potent inhibitory effect on NSCLC proliferation than either treatment alone (Fig. 8a–e, Supplementary Fig. S4a–e). Additionally, Transwell assays demonstrated that the combination of 10058-F4 with DZNep or EPZ6438 had the strongest inhibitory effect on lung cancer cell migration and invasion (Fig. 8f–h, Supplementary Fig. S4f–h). We subsequently conducted experiments using si-Myc combined with DZNep. The results revealed that the knockdown of c-Myc expression with siRNA combined with DZNep significantly enhanced the inhibitory effects on NSCLC cell proliferation, migration, and invasion (Supplementary Fig. S5). These findings further support the synergistic effect of c-Myc inhibition in enhancing the therapeutic potential of DZNep. Patient-derived organoid (PDO) models have become crucial preclinical models for assessing drug responses and efficacy. Initially, we obtained tumor tissue from a patient with LUAD to establish a PDO model. We found that the combination of 10058-F4 and DZNep significantly suppressed cell viability (Fig. 8i). To further confirm our in vitro observations, we subcutaneously injected A549 cells into BALB/c mice. The combination of 10058-F4 and DZNep significantly inhibited tumor growth (Fig. 8j, k), which was also supported by the IHC results (Fig. 8l). These findings collectively demonstrate that the c-Myc inhibitor 10058-F4 significantly increases the therapeutic efficacy of DZNep in treating NSCLC.

Fig. 8. 10058-F4 combined with DZNep increases the efficacy of DZNep therapy in H1650 cells.

a–e The effects of 10058-F4, DZNep and 10058-F4 combined with DZNep on H1650 cell proliferation were evaluated via CCK-8, EdU staining and colony formation assays. f–h H1650 cell migration and invasion under the four conditions were also evaluated via Transwell assays. i The effects of 10058-F4 and DZNep on organoid morphology were assessed. j, k Photograph of dissected tumors (first line: vehicle; second line: DZNep; third line: 10058-F4; fourth line: DZNep +10058-F4; n = 5); DZNep inhibited tumor growth in mice; 10058-F4 inhibited tumor growth in mice; DZNep combined with 10058-F4 increased the efficacy of DZNep therapy. l The protein expression of EZH2, c-Myc and Ki-67 in dissected tumor samples was evaluated by IHC. m Bioluminescence images of representative mice showing the progression of NSCLC metastasis. The top row shows whole-body images of the mice, while the bottom row shows the lung tissue. n Representative images of lung metastasis. The top row shows bright-field images of the lungs, with visible metastatic nodules. The bottom row presents corresponding H&E staining of the lung tissue, confirming the presence and extent of metastasis. o Statistical analysis of the lung nodule counts. p Schematic diagram showing that nicotine promotes the progression and metastasis of non-small cell lung cancer by modulating the OTUB1-c-Myc-EZH2 axis. ****P < 0.0001, ***P < 0.001, **P < 0.01, with P values calculated via unpaired t tests.

To further investigate the impact of nicotine on NSCLC metastasis mediated by the c-Myc-EZH2 axis, we injected A549 tumor cells into mice via the tail vein and monitored tumor metastasis and progression by small animal imaging. These results indicated that nicotine significantly promoted the metastasis of NSCLC cells. Both DZNep and 10058-F4, when administered alone, effectively inhibited nicotine-induced metastasis in the mice. Notably, the combination of DZNep and 10058-F4 further significantly suppressed the prometastatic effects of nicotine on tumor spread (Fig. 8m). All the mice were euthanized 28 days after tumor injection, and their brains, livers, and lungs were subjected to histological analysis. The results demonstrated that metastasis predominantly occurred in the lungs. H&E staining of the lung tissues and nodule counting confirmed these findings, which were consistent with the imaging results (Fig. 8n, o). These results suggest that the c-Myc-EZH2 axis plays a crucial role in nicotine-induced NSCLC metastasis.

Discussion

The use of tobacco products leads a deadly combination of nicotine addiction and carcinogen exposure and contributes to millions of cancer-related deaths per year worldwide [26]. Consistent with a number of clinically relevant studies, nicotine was found to significantly promote NSCLC cell proliferation and metastasis in this study. A deeper understanding of the relationship between tobacco smoke carcinogens and NSCLC, as well as elucidation of the specific mechanisms by which nicotine induces NSCLC (Fig. 8p), will aid in devising protective strategies to reduce NSCLC risk.

Nicotine is one of the main substances responsible for smoking addiction and affects various cellular signaling pathways, including those involved in cell cycle regulation, apoptosis, proliferation, and differentiation [27]. Nicotine exerts its carcinogenic effects by promoting tumor cell survival, growth, metastasis, and resistance to chemotherapy or radiotherapy through its genotoxic effects and creation of a tumor-supportive microenvironment. Previous research has indicated that the effects of nicotine on tumors are primarily mediated by nAChRs, which, upon stimulation, lead to the sustained activation of intracellular pathways such as the PI3K/Akt/mTOR, RAS/RAF/MEK/ERK, and JAK/STAT pathways, inducing NF-κB activity, enhancing the transcription of promitotic factors, suppressing mitochondrial apoptotic pathways, or stimulating angiogenic factors [28]. Studies have demonstrated that nicotine disrupts the intracellular redox state in lung epithelial cells, fostering an environment conducive to lung cancer growth [29].

Here, we present compelling evidence suggesting that nicotine modulates NSCLC cell proliferation and metastasis via EZH2. In NSCLC patients, EZH2 promotes carcinogenesis by epigenetically silencing tumor suppressor genes [30]. Previous studies have shown that EZH2 expression is significantly correlated with male sex and smoking history [31] and that cigarette smoke induces the carcinogenesis of human bronchial epithelial cells by epigenetically silencing miR-218 through EZH2-mediated H3K27 trimethylation, thereby initiating a carcinogenic response [32]. Therefore, EZH2 plays a central role in smoking-related lung carcinogenesis and may serve as a target for lung cancer prevention and treatment. In this study, we demonstrated that a high tumor grade is associated with greater EZH2 expression and that high EZH2 expression is correlated with greater tumor invasion depth. Our research also revealed that increased EZH2 expression is mediated primarily through c-Myc upregulation. c-Myc, a transcription factor, plays crucial roles in processes such as cell cycle regulation, proliferation and apoptosis, cell differentiation, and DNA repair [33]. Aberrant expression of c-Myc is closely associated with the occurrence and development of various tumors. Notably, these tumor cells depend on high levels of c-Myc for survival [33, 34]. Our study revealed that nicotine regulates the ubiquitination level of c-Myc and that this process is mediated by OTUB1. OTUB1 is the most important element of the OTU deubiquitinase superfamily and has been identified as an essential regulator of diverse physiological processes. OTUB1 regulates GPX4 protein stability, inhibits ferroptosis and promotes the metastasis of gastric cancer; thus, it may be a new target for the treatment of gastric cancer [35]. OTUB1 mediates ferroptosis by stabilizing SLC7A11, identifying OTUB1 as a key regulator of ferroptosis and implicating it as a potential target for cancer therapy [36]. This study provides a theoretical basis for the clinical management of nicotine-induced lung cancer by clarifying the detailed regulatory mechanism by which nicotine regulates the c-Myc/EZH2 signaling pathway, which leads to the proliferation and metastasis of NSCLC.

DZNep, an inhibitor that suppresses DNA methyltransferase, inhibits cancer cell proliferation and metastasis [20]. It was initially used to treat leukemia and later found to have anticancer effects on various tumors, including lung cancer. DZNep can inhibit lung cancer cell proliferation and metastasis, induce apoptosis, and enhance the efficacy of chemotherapy and radiotherapy, improving the treatment outcomes of patients with lung cancer [37, 38]. The mechanism of action of DZNep involves the inhibition of DNA methyltransferase; thus, it reduces DNA methylation levels in cancer cells and suppresses cancer cell proliferation and metastasis. DZNep is a highly promising novel treatment for lung cancer, but further research is needed. Treating nicotine-stimulated tumors with DZNep leads to suppression of tumor growth, indicating its effectiveness as a therapeutic agent for NSCLC treatment. Although several EZH2 inhibitors have entered clinical trials, they are associated with significant clinical side effects, and progress remains slow, with the issue of EZH2 inhibitors’ ineffectiveness against certain solid tumors still unresolved. In previous studies, EZH2 was found to directly interact with MYC family oncogenes, MYC and MYCN, promoting their stability through a methyltransferase-independent mechanism, thereby driving tumor cell growth in neuroblastoma and small cell lung cancer [39]. Additionally, EZH2 performs noncanonical functions in acute leukemia by binding to c-Myc at non-PRC2 target sites, further contributing to tumorigenesis [40]. On the basis of these findings, EZH2 and c-Myc inhibitors can be applied to simultaneously block two major pathways essential for tumor cell proliferation and survival. By reducing c-Myc stability and activity, EZH2 inhibitors may further weaken c-Myc-driven tumor growth and inhibit tumor progression. Moreover, combination therapy may attenuate tumor resistance to single-agent inhibitors and increase therapeutic efficacy. In this study, we found that the combination of a specific c-Myc inhibitor with DZNep has a synergistic effect on reducing NSCLC cell proliferation and metastasis, indicating the potential clinical application prospects of applying c-Myc inhibitors in combination with DZNep for treating NSCLC, which is highly important for accelerating clinical trials on targeting EZH2. In summary, the results of this study provide new insights into the regulatory mechanisms of nicotine-related NSCLC progression.

In conclusion, our study indicates that nicotine promotes cell proliferation and metastasis in NSCLC through the OTUB1/c-Myc/EZH2 signaling pathway. Combined treatment with 10058-F4, a c-Myc inhibitor, and DZNep enhances the therapeutic effects against NSCLC. This research reveals novel molecular mechanisms of nicotine in the development of NSCLC, providing a theoretical foundation for future therapeutic strategies.

Author contributions

This study was designed by JC, HYL, and XBL and conducted by HH, CD, and WHZ. Data analysis was performed by HBZ, ZXZ, YJW, BSL, and YWL, and the manuscript was written by HH, PJC, and XGL. Funding was provided by JC, and the study was supervised by JC, HYL, and YWL. All the authors have read and approved the final version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant Nos. 82072595 and 82473191), the Natural Science Foundation of Tianjin (Grant No. 23JCZDJC00710), the Tianjin Key Medical Discipline (Specialty) Construction Project (Grant No. TJYXZDXK-061B), and the Tianjin Health Science and Technology Project (Grant No. TJWJ2022XK005). Beijing Science and Technology Innovation Medical Development Fund (Grant No. KC2023-JX-0288-PZ78).

Data availability

All the data in our study are available upon request.

Competing interests

The authors declare no competing interests.

Ethics approval

All experiments were performed in compliance with the relevant regulations, and all patients provided written informed consent. In addition, all animal experiments were performed according to procedures approved by the institutional animal care and use committee of Tianjin Medical University General Hospital. The experiments followed the Guidelines for the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research. The maximum tumor size allowed by the ethics committee is not more than 2000 mm³, and this requirement was met for all the described experiments.

Consent for publication

All the authors agree with the content of the paper.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-025-01527-5.