NPM1 Drives ERK1/2-dependent Tumor Progression in Lung Cancer.

Introduction
Lung cancer remains the leading cause of cancer-related mortality worldwide, accounting for approximately 1.8 million deaths annually, and its incidence continues to rise despite advances in early detection and treatment (1). Although recent developments in targeted therapies and immunotherapies have improved outcomes for selected patients, the overall five-year survival rate for advanced lung cancer remains below 20% (2, 3). Therefore, identifying actionable molecular drivers and reliable biomarkers is essential to enhance early detection, improve prognostic accuracy, and develop more effective therapeutic strategies.
Nucleophosmin 1 (NPM1) is a multifunctional nucleolar phosphoprotein that shuttles between the nucleolus and cytoplasm and participates in ribosomal RNA processing, centrosome duplication, chromatin remodeling, and transcriptional regulation (4, 5). Mutations in the NPM1 gene are well recognized as defining events in acute myeloid leukemia (AML), where they serve as diagnostic and prognostic markers (6). Beyond hematologic malignancies, aberrant NPM1 expression has been reported in several solid tumors, including gastric, colorectal, and breast cancers, where its overexpression correlates with enhanced tumor aggressiveness and poor patient survival (7-9). Recent studies have further linked NPM1 to the regulation of the tumor immune microenvironment, m6A RNA modification, and metabolic reprogramming, highlighting its pleiotropic oncogenic potential in solid tumors and particularly in lung adenocarcinoma (7, 9-11). The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway represents one of the most critical oncogenic axes in lung cancer, regulating cell proliferation, migration, invasion, and survival (12, 13). Persistent ERK1/2 activation is strongly associated with therapeutic resistance and poor clinical outcomes in non-small cell lung cancer (12, 13). Crosstalk between ERK and parallel signaling pathways, including PI3K/AKT, further reinforces malignant phenotypes and contributes to adaptive signaling under therapeutic pressure (14). Additionally, emerging evidence suggests that nucleolar homeostasis influences MAPK activity, implicating nucleolar proteins as upstream modulators of kinase-driven oncogenic signaling (11, 15). ERK-driven transcription factors such as MYC and the AP-1 complex (c-Fos/c-Jun) play crucial roles in lung cancer proliferation, invasion, and survival, acting as key integrators of MAPK output (12, 13, 16).
Despite emerging evidence implicating nucleolar proteins in tumorigenic signaling, the mechanistic contribution of NPM1 to lung cancer pathophysiology remains insufficiently defined, and its potential diagnostic or prognostic utility has not been systematically evaluated. Therefore, in this study, we aimed to comprehensively characterize the expression landscape of NPM1 in lung cancer and investigate its functional role in regulating malignant phenotypes using patient-derived datasets and experimental lung cancer models. In particular, we focused on elucidating whether NPM1 is associated with key oncogenic signaling pathways relevant to lung cancer progression and assessing its potential clinical relevance as a biomarker. This work is intended to address the current gaps in understanding NPM1-driven molecular mechanisms in lung cancer and to clarify its possible implications in disease diagnosis and biological behavior.

Materials and Methods
Analysis of TCGA datasets. Transcriptomic data of lung cancer patients were obtained from the TCGA portal (https://portal.gdc.cancer.gov). RNA-seq expression data [log2(TPM+1)] of NPM1 were extracted from the lung adenocarcinoma (TCGA-LUAD) and lung squamous cell carcinoma (TCGA-LUSC) cohorts, together with matched normal lung tissues. Boxplots were generated to compare NPM1 expression between tumor and normal tissues. Statistical significance was evaluated using Student’s t-test.
Cell culture and siRNA transfection.  Human lung cancer cell lines (A549, H460, Calu-1, H1299, H358, Calu-3, SK-MES-1) and the normal lung fibroblast cell line (MRC-5) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) at 37°C in a humidified atmosphere containing 5% CO₂, unless otherwise specified by the supplier. Cells were transfected with 20 nM siRNA targeting NPM1 or non-targeting control siRNA (Santa Cruz Biotechnology, Dallas, TX, USA) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.
Cell proliferation assay (MTT assay). Cell proliferation was evaluated using the MTT assay. Briefly, cells were seeded in 96-well plates (3×10³ cells/well). At the indicated time points (0, 24, 48, 72 h), 10 μl of MTT solution (1 mg/ml in PBS) was added to each well and incubated for 4 h. After removing the medium, 100 μl dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) was added, and absorbance was measured at 570 nm using a microplate reader (Tecan Trading AG, Männedorf, Switzerland). Experiments were performed using three biological replicates (n=3) and three technical replicates per condition.
Migration assay. Cell migration was assessed using transwell chambers (8-μm pore size, Corning Inc., Kennebunk, ME, USA). A total of 5×10⁴ cells in serum-free medium were seeded into the upper chamber, and medium containing 10% FBS was placed in the lower chamber as a chemoattractant. After incubation for 24 h, migrated cells were fixed with methanol, stained with 0.005% crystal violet, and counted under a light microscope in five random fields. Transwell migration assays were performed using three independent experiments (n=3), with each experiment measured in duplicate.
Soft agar colony formation assay. Anchorage-independent growth was analyzed using soft agar assays. A base layer of 0.6% low-melting-point agarose (Duchefa, Haarlem, the Netherlands) in complete medium was prepared in 6-well plates. Cells (5×10⁴) were suspended in 0.3% agarose in medium and overlaid onto the base layer. After 14 days of incubation, colonies were stained with crystal violet and counted in five random fields under a microscope. Each condition included three biological replicates, and colonies were quantified from five random fields per replicate.
Immunoblotting. Cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk and incubated overnight at 4°C with primary antibodies against NPM1 (ab15440, Abcam, Cambridge, UK), β-actin (ab6276, Abcam), ERK1/2 (#9102, Cell Signaling Technology, Danvers, MA, USA), phospho-ERK1/2 (Thr202/Tyr204) (#9101, Cell Signaling Technology), c-MYC (#9402, Cell Signaling Technology), c-FOS (#4384, Cell Signaling Technology) and c-Jun (#9165, Cell Signaling Technology). HRP-conjugated secondary antibodies were applied, and protein bands were visualized using an ECL kit (iNtRON Biotechnology, Seongnam, Republic of Korea). β-Actin was used as a loading control. Western blotting was repeated three times using independent biological samples.
Human phospho-kinase array. To evaluate changes in kinase activation, a Proteome Profiler Human Phospho-Kinase Array Kit (ARY003C, R&D Systems, Minneapolis, MN, USA) was used according to the manufacturer’s instructions. Lysates from H1299 cells transfected with si-control or si-NPM1 were incubated with the array membranes. Signals were detected by chemiluminescence, and relative spot intensities were quantified using the ImageJ software (https://imagej.net/ij/). Phospho-kinase array was performed once per manufacturer’s protocol, but spot intensity validation was repeated using three independent biological replicates by western blot.
In vivo tumorigenicity assay. Six-week-old male BALB/c nude mice (Orient Bio, Seongnam, Korea) were maintained under pathogen-free conditions. H1299 cells (1×10⁶) transfected with si-control or si-NPM1 were suspended in serum-free medium and injected subcutaneously into the left flank of nude mice (1×10⁶ cells per mouse; n=3 per group). Tumor volumes were measured every other day using calipers and calculated as: [length × (width)2]/2. Mice were euthanized on day 40, and tumors were excised and weighed. Xenograft experiments were conducted using three mice per group (n=3).
Statistical analysis. Statistical analysis. All experiments were independently repeated at least three times. Data are presented as the mean±standard deviation (SD). Statistical comparisons between two groups were performed using Student’s t-test, and multiple group comparisons were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Normality was assessed before applying parametric tests. Differences were considered statistically significant at p<0.05. GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses. Levels of statistical significance are indicated as *p<0.05, **p<0.01, and ***p<0.001.

Results
NPM1 is highly expressed in lung cancer tissues and cell lines. To evaluate NPM1 expression in lung cancer, TCGA transcriptomic data were analyzed. NPM1 expression was significantly elevated in tumor tissues compared with adjacent normal lung tissues (***p<0.001, Figure 1A). Western blotting further confirmed that NPM1 protein levels were markedly increased in multiple lung cancer cell lines, including A549, H460, Calu-1, H1299, H358, Calu-3, and SK-MES-1, compared with the normal lung fibroblast cell line MRC-5 (Figure 1B).
NPM1 silencing suppresses lung cancer cell proliferation. MTT assays demonstrated that siRNA-mediated NPM1 knockdown significantly reduced cell proliferation in both A549 and H1299 cells compared to si-control groups. Growth curves revealed a time-dependent divergence, with NPM1-depleted cells exhibiting markedly lower OD values at 48 and 72 h (**p<0.01, Figure 2A and B).
NPM1 depletion reduces migration capacity of lung cancer cells. Transwell migration assays revealed that NPM1 knockdown substantially impaired the migratory potential of lung cancer cells. Both A549 and H1299 cells transfected with si-NPM1 showed a significant decrease in the number of migrated cells compared to si-control groups (***p<0.001, Figure 3A and B), suggesting that NPM1 contributes to a pro-migratory phenotype.
Anchorage-independent growth is impaired by NPM1 knockdown. Soft agar colony formation assays showed that silencing NPM1 markedly decreased the number and size of colonies in A549 and H1299 cells compared to controls (***p<0.001, Figure 4A and B). These results indicate that NPM1 is required for anchorage-independent growth and tumorigenic potential.
NPM1 regulates ERK1/2 signaling in lung cancer cells. To explore the molecular mechanism underlying NPM1-driven oncogenesis, phospho-kinase array profiling was performed. NPM1 knockdown significantly reduced phosphorylation of ERK1/2 (Thr202/Tyr204) (Figure 5A). Western blot analysis validated these findings, showing that NPM1 depletion decreased phosphorylated ERK1/2, as well as the downstream effectors MYC, AP-1, and c-FOS, while total ERK1/2 levels remained unchanged (Figure 5B). These results demonstrate that NPM1 promotes lung cancer progression, at least in part, through activation of ERK1/2 signaling and downstream transcriptional networks.
NPM1 silencing inhibits tumor growth in vivo. In xenograft models, mice injected with H1299 cells transfected with si-NPM1 developed significantly smaller tumors compared to those injected with si-control cells (**p<0.01, Figure 6A and B). Tumor growth curves revealed a sustained reduction in tumor volume throughout the experimental period, confirming the essential role of NPM1 in promoting lung cancer tumorigenesis in vivo.

Discussion
This study demonstrates that NPM1 is up-regulated in lung cancer and promotes tumor progression through ERK1/2 signaling. Analysis of TCGA datasets revealed significantly elevated NPM1 expression in tumor tissues compared with normal lung tissues, and siRNA-mediated knockdown experiments confirmed that NPM1 depletion suppresses proliferation, migration, anchorage-independent growth, and in vivo tumor formation. Mechanistically, NPM1 silencing attenuated ERK1/2 phosphorylation and down-regulated key oncogenic transcription factors, including MYC, AP-1, and c-FOS, suggesting that NPM1 contributes to lung cancer progression by enhancing MAPK signaling activity. These findings are consistent with previous reports showing that aberrant NPM1 expression promotes aggressive phenotypes and poor prognosis in a variety of solid tumors, particularly breast and lung cancers (7-9).
To our knowledge, direct functional evidence linking NPM1 to ERK1/2 regulation in lung cancer remains limited, although NPM1 has been implicated in modulating EGFR/MAPK signaling and immune evasion mechanisms in other malignancies (7, 9-11). Our data expand these observations by demonstrating that NPM1 enhances oncogenic signaling through ERK1/2 activation and transcriptional up-regulation of downstream effectors. The MAPK/ERK pathway is a central signaling cascade frequently activated in lung cancer, driving cell proliferation, invasion, and survival (12, 13).  Persistent ERK1/2 activation is often associated with resistance to targeted therapies and poor clinical outcomes, and crosstalk with parallel pathways such as PI3K/AKT further stabilizes malignant signaling networks (12-14). In addition, recent evidence indicates that nucleolar stress and disruption of nucleolar homeostasis can modulate MAPK output and reprogram transcriptional responses, suggesting that nucleolar proteins such as NPM1 may serve as upstream regulators that integrate ribosomal function with oncogenic kinase activation (11, 15).
Our results extend these findings by demonstrating that NPM1 silencing reduces ERK1/2 phosphorylation and down-regulates key downstream effectors, including MYC, c-Fos, and c-Jun, suggesting that NPM1 sustains oncogenic MAPK activity. Functionally, NPM1 depletion suppressed proliferation, migration, anchorage-independent growth, and in vivo tumorigenicity, highlighting its essential role in driving malignant phenotypes. These observations support the hypothesis that NPM1 not only functions as a nucleolar structural and stress-response regulator but also acts as an upstream enhancer of ERK1/2 signaling in lung cancer. Notably, ERK-driven transcription factors such as MYC and AP-1 are recognized drivers of lung cancer proliferation, invasiveness, and survival (12, 13, 16), which is consistent with the observed impact of NPM1 depletion on these downstream programs.
Clinically, elevated NPM1 expression could serve as a biomarker of poor prognosis and may help predict responsiveness to therapies targeting ERK1/2 or upstream receptor tyrosine kinases (7-9). Similar to previous findings in breast and other solid cancers (7, 8), NPM1 overexpression appears to reinforce oncogenic signaling networks, supporting tumor survival and progression. Moreover, recent studies indicate that NPM1 may modulate the tumor immune microenvironment, m6A RNA modification, and metabolic reprogramming, further contributing to immune evasion and therapeutic resistance (9-11). Given that nucleolar stress regulators have been increasingly recognized as modulators of MAPK signaling (11, 15), therapeutic inhibition of NPM1 may simultaneously disrupt multiple oncogenic axes, including ERK1/2-driven transcriptional programs. Therefore, targeting NPM1 or its downstream ERK1/2 signaling axis may represent a promising strategy to suppress tumor progression and overcome drug resistance in lung cancer.
Nevertheless, this study has several limitations. Although our in vitro and in vivo data support an oncogenic role of NPM1 via ERK1/2 activation, the precise molecular intermediates linking NPM1 to ERK1/2 phosphorylation remain to be elucidated. Future research should focus on identifying direct protein–protein interactions or post-translational modifications that mediate this signaling relationship. Additionally, validation in larger patient cohorts and functional studies using NPM1 inhibitors will be essential to confirm its therapeutic potential and to determine whether NPM1 expression can serve as a predictive biomarker for response to ERK-targeted therapies.

Conclusion
Collectively, NPM1 acts as an oncogenic driver in lung cancer by activating ERK1/2 signaling and promoting cell proliferation, migration, and tumorigenicity. NPM1 silencing reduced ERK1/2 phosphorylation and down-regulated MYC, AP-1, and c-FOS, indicating its role in sustaining oncogenic transcriptional activity. Elevated NPM1 expression may serve as a diagnostic and prognostic biomarker, and targeting NPM1 or its downstream ERK1/2 pathway could provide a promising therapeutic strategy for lung cancer.

Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.

Authors’ Contributions
KHB was involved in the experiments, data organization, and writing of the manuscript. PSG and LHJ contributed to writing, editing, and reviewing the manuscript. All Authors read and approved the final version of the manuscript.

Funding
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education [NRF-2017R1D1A1B04031741, NRF-2018R1D1A1B07040473].

Artificial Intelligence (AI) Disclosure
During the preparation of this manuscript, a large language model (ChatGPT 4.5, OpenAI) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning–based image enhancement tools.