The expression profile of the target molecule, MK2, in LUAD was derived from online analysis using immune infiltration data for various cancer types (pan-cancer) sourced from the TIMER 2.0 database [http://timer.compgenomics.org/].

Additionally, survival analysis was conducted using the Kaplan-Meier method [https://kmplot.com/analysis/] on patient data extracted from the Kaplan-Meier plotter database for LUAD. Patients were classified according to the expression level of MK2 in their tumors, with the median value using the median value as a threshold to separate those with high expression from those with low expression.

Shanghai Zhuoli Biotechnology Co., Ltd. provided LUAD tissue chip (ZL-LugA961), comprising 48 pairs of tumor tissue and adjacent non-tumor tissue samples. However, after analysis, only 47 pairs had sufficient data for immunohistochemistry (IHC) analysis, as one pair failed to exhibit adequate staining or could not be scored. Therefore, the final number of analyzable samples was 47. Microarrays underwent pretreatment with bovine serum albumin (BSA, Elabscience Biotechnology Co.,Ltd., China, Cat. No. E-IR-R107, validated for IHC use) before being incubated overnight at 4 °C with MK2 antibody from Proteintech (Cat. No. 13949-1-AP, validated for IHC, diluted 1:200). The following day, further incubation was conducted with HRP-labeled secondary antibodies. Visualization was achieved using DAB (ZSGB-BIO, Cat. No. ZLI-9017), followed by hematoxylin counterstaining (Beyotime, Cat. No. C0107), and images were captured utilizing a microscope. Finally, Visiopharm software facilitated quantitative analysis of staining intensity. The HDAB-DAB filter was used to segment regions of interest (ROIs) based on staining intensity. Intensity categories are as follows: 0–75 (strong), 76–120 (moderate), 121–160 (weak), and 161–212 (negative). The staining area is measured in square micrometers (μm²). Staining intensity was measured using the H-Score (H-SCORE = ∑(pi × i)), calculated by summing the products of the percentages of positively stained cells at each intensity level and their respective intensity levels (H-SCORE = ∑ (pi × i)). Staining intensities are categorized as weak (1), medium (2), or strong (3).

Tissue samples were fixed in 10% formalin, dehydrated through graded ethanol, embedded in paraffin, and sectioned at 4 µm thickness. Sections were stained with hematoxylin and eosin (H&E) to evaluate histological morphology under a light microscope, facilitating the assessment of structural and pathological features.

Clinical specimens were collected from LUAD patients, cryopreserved, and promptly processed for digestion. The primary tumor cells obtained were embedded in Matrigel (bioGenous Biotechnology Co. Ltd, China, Cat. No. M315066, validated for organoid culture) for three-dimensional culture, with media changes every 2–3 days. Once the organoids reached an appropriate size, they were passaged and collected. The organoids were then fixed in 4% paraformaldehyde, embedded in paraffin blocks, and prepared for subsequent sectioning and experimentation. Detailed culture steps were performed as previously described (Kim et al., 2019). All culture reagents were sourced from bioGenous Biotechnology Co., Ltd. (Suzhou, China), and are certified for clinical and research use with quality validation provided by the manufacturer.

The H358 and A549 LUAD cell lines were procured from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). These cells were maintained in RPMI 1640 medium (Solarbio, China) supplemented with 10% FBS. Culturing was performed at 37 °C under a humidified atmosphere containing 5% CO2. To assess MK2’s function, cells in the inhibitor group were treated with a specific MK2 inhibitor (Huang et al., 2011; Zhang et al., 2021). The MK2 inhibitor used in this study, MK2-IN-1 (MCE, Cat.No. HY-12834; CAS No. 1314118–92–7; molecular formula: C₂₇H₂₅ClN₄O₂), is a potent, non-ATP-competitive inhibitor designed to achieve high selectivity for MK2. For all experiments, MK2-IN-1 was dissolved in PBS, and cells in the MK2 inhibitor treatment groups were exposed to 20 μM MK2-IN-1 for 24 h prior to subsequent assays. The control group received no treatment. Subsequently, cells pre-treated with the MK2 inhibitor for approximately 6 h were exposed to the AKT/MYC pathway activator SC79 (20 μg/mL) in follow-up experiments to research the function of the AKT/MYC signaling pathway.

Cell lysis solution was made using RIPA buffer (Beyotime, China, Cat. No. P0013B) supplemented with protease and phosphatase inhibitors (Beyotime, Cat. No. P1045 and P1081). Proteins were isolated using SDS-PAGE (8%–15%) and transferred onto PVDF membranes (Millipore, Cat. No. IPVH00010). These membranes were blocked with 5% skim milk for a minimum of 1 hour. Primary antibodies for MYC (Proteintech, Cat. No. 10828-1-AP, 1:1000), GAPDH (Proteintech, Cat. No. 60004-1-Ig, 1:50,000), AKT (Proteintech,Cat. No. 10176-2-AP, 1:2000), P-AKT (Ser-473) (HUABIO, Cat. No. ET1607-73, 1:1000), E-Cadherin (Proteintech, Cat. No. 60335-1-Ig, 1:2000), Vimentin (Proteintech, Cat. No. 60330-1-Ig, 1:20,000), N-Cadherin (Abcolonal, Cat. No. A0433, 1:500), and MMP2(Proteintech, Cat. No. 66366-1-Ig, 1:1000) were incubated at 4 °C overnight. After three washes with PBST, the HRP-labelled secondary antibody was incubated for 1 hour. After that, protein detection was performed using an ultra-high sensitivity ECL (GLPBIO, USA, Cat. No. GK10008).

Total RNA was extracted from cells using Eastep total RNA extraction kit (Promega, Cat. No. LS1040) following the manufacturer’s instructions, and RNA quality was assessed by spectrophotometry. cDNA synthesis was performed with Superscript III reverse transcriptase (Applied Biosystems) using 1 μg of RNA as the template, under conditions specified by the manufacturer. Real-time quantitative PCR (qPCR) was carried out using a PerfectStart^® Green qPCR Super Mix on a Roche with specific primers for target genes and we quantified GAPDH mRNA levels as an internal quantity control. Each reaction included 2 μL of cDNA template in a final reaction volume of 20 μL. Cycling conditions included an initial denaturation step, followed by 40 cycles of denaturation, annealing, and extension. Specificity was confirmed with melt curve analysis. Relative gene expression was calculated using the 2^(-^ΔΔCt) method, normalized to GAPDH. Reactions were performed in triplicates, and results were analyzed using GraphPad Prism to determine statistical significance. All primers used for RT-qPCR were obtained from Sangon Biotech (Shanghai, China). RT-qPCR products were then subjected to electrophoresis. The primer sequences of target genes are listed in Table 1.

Cells were lysed in ice-cold IP lysis (Beyotime, China, Cat. No. P0013) buffer containing protease inhibitors. The lysates were incubated with specific antibodies against AKT (Proteintech, Cat. No. 10176-2-AP), followed by the addition of magnetic beads (Protein A or G Magnetic Beads, BeaverBio™). After incubation and washing, protein complexes were eluted and separated by SDS-PAGE. Western blotting with anti-MK2 and anti-AKT antibodies confirmed the interaction.

The organization referred to as “LUAD cells” typically denotes lung adenocarcinoma cells. They were cultured in six-well plates with a density of 100,000 cells per well for 24 h. Following a PBS wash, the cells received MK2 inhibitor treatment for an additional 24 h. Subsequently, the cells were promptly fixed and stained in accordance with the Annexin V-APC/7-AAD apoptosis kit protocol (Elabscience, Cat. No. E-CK-A218, validated for flow cytometry), with immediate flow cytometry analysis thereafter.

Wound healing assays were employed to evaluate cell migration ability. Cells were seeded in six-well plates at a density of 4 × 10⁵ cells per well and permitted to grow overnight to establish a monolayer. After making scratches using 200 µL pipette tips, they were washed with PBS and incubated in RPMI 1640 medium with 1% FBS for 24 h. Wound closure was measured using a Leica DMI8 fluorescence microscope at 0 and 24 h after wounding. All experiments were performed in triplicate.

We utilized Matrigel matrix provided by BD Biosciences (BD Biosciences, USA) diluted at a ratio of 1:6 in serum-free RPMI 1640 medium, and stored it at 4 °C. After evenly spreading 100 μL of the matrix solution onto the surface of the upper chamber, we allowed it to dry for 1 h at 37 °C. Subsequently, cells were suspended in serum-free medium at a concentration of 5 × 10⁵cells/mL. Then, we added 200 μL of the cell suspension to the upper chamber coated with the matrix solution, while filling the lower chamber with RPMI 1640 medium containing 10% FBS. After 24 h, cells in the lower chamber were fixed with 4% paraformaldehyde for 30 min and then stained with 0.1% crystal violet for another 30 min. Following this, the stained cells were counted using a Leica fluorescence inverted microscope (DMI8). Each experiment was conducted in triplicate.

Cells were treated with 10 μM EdU for 2 h, fixed, and permeabilized. EdU incorporation was detected using EdU Cell Proliferation Kit with Alexa Fluor 555(CX003,CellorLab), followed by DAPI counterstaining. The percentage of EdU-positive cells was quantified by fluorescence microscopy or flow cytometry.

All data were analyzed using GraphPad Prism 9. For comparisons between two groups, unpaired Student’s t-tests were used. For multiple group comparisons, one-way or two-way ANOVA followed by Tukey’s post hoc test was applied. P values <0.05 were considered statistically significant. All quantitative data are presented as mean ± standard deviation (SD). Each experiment was independently repeated at least three times (n = 3). Error bars in bar graphs represent SD.

Emerging evidence has established MK2 as a commonly dysregulated signaling node in LUAD and multiple human malignancies (Nguyen Ho-Bouldoires et al., 2015; Soni et al., 2019; Jacenik et al., 2023). To systematically characterize its pathobiological relevance, we performed multi-platform bioinformatics validation using the TIMER 2.0 database. Bioinformatics analysis revealed that MK2’s transcription levels are significantly higher in LUAD tissues compared to adjacent non-tumor tissues, suggesting its functional involvement in pulmonary carcinogenesis (Figure 1A). To validate these findings at the protein level, we conducted IHC analysis on 47 matched LUAD-normal tissue pairs using clinically annotated tissue microarrays. Quantitative histoscore analysis confirmed significant elevation of MK2 protein expression in tumor tissues, with representative IHC staining shown in (Figure 1B–C). Further stratification by tumor stage demonstrated markedly higher MK2 expression in advanced-stage (III-IV) LUAD compared to early-stage (I-II) disease (Figure 1D). Clinically, data from the Kaplan-Meier Plotter database indicated a negative correlation between elevated MK2 mRNA levels and overall survival rates in LUAD patients, underscoring MK2’s prognostic significance (Figure 1E).

Collectively, these multi-omics concordant data demonstrate MK2 overexpression at both transcriptional and translational levels in LUAD, mechanistically implicating this kinase in disease progression. The clinical correlation between MK2 overexpression and adverse outcomes warrants functional investigation of its therapeutic targeting potential.

Previous research suggests that MK2-mediated phosphorylation of RIPK1 decreases its affinity for FADD, thereby attenuating TNF-α-induced apoptosis (Jaco et al., 2017; Menon et al., 2017). The therapeutic potential of MK2 inhibition extends beyond direct apoptosis modulation, as it synergistically disrupts oncogenic signaling through dual pathways: sensitizing pancreatic ductal adenocarcinoma (PDAC) to apoptosis via Hsp27 inactivation and restricting breast cancer metastasis by suppressing stromal IL-6 production (Grierson et al., 2021; Murali et al., 2018). To further investigate the role of MK2 in LUAD proliferation, we established in vitro models using A549 and H358 cell lines. MK2 expression or activity was inhibited using both siRNA and an MK2 inhibitor (Huang et al., 2011). Comparative analysis of genetic (siRNA) versus pharmacological (MK2-IN-1) MK2 suppression revealed comparable efficacy in modulating proliferation and invasion (Supplementary Material 1A-E), prompting subsequent focus on MK2-IN-1 for target specificity validation and translational relevance. Preliminary determination of IC50 values for the MK2 inhibitor (MK2-IN-1) revealed that the IC50 for A549 and H358 were 40.18 µM and 38.30µM, respectively, at 24 h, decreasing to 29.51µM and 31.69  μM at 48 h (Figure 2A). Crucially, 20 μM MK2-IN-1 exhibited no significant cytotoxicity in non-transformed BEAS-2B lung cells (Supplementary Material 1F), underscoring its tumor-selective targeting potential. Functional characterization through EdU incorporation assays revealed marked reduction in LUAD proliferative capacity following MK2 inhibition (Figure 2B), paralleled by a 2fold increase in apoptosis rates as quantified by Annexin V/PI dual staining (Figure 2C). These findings collectively establish that MK2 inhibition disrupts malignant homeostasis by concurrently suppressing proliferation and activating apoptosis in LUAD models, with tumor cell-selective efficacy highlighting its therapeutic promise. These findings show that MK2 inhibitors significantly inhibit the proliferation of LUAD cells, suggesting that MK2 may play an important role in LUAD cell proliferation.

Given the established correlation between MK2 overexpression and the metastatic potential in various cancers, including melanoma (Wenzina et al., 2020), gastric cancer (Qeadan et al., 2020), colorectal cancer (Ray et al., 2018), breast cancer (Murali et al., 2018) and other tumors; this study investigates MK2-driven invasion-metastasis cascades in LUAD. Functional validation using a homologous LUAD cell model (A549 and H358 cells)showed that the use of MK2 inhibitors greatly reduced tumor cell invasiveness in the Transwell Matrigel assay (Figure 3A). Consistent with these findings, wound healing assays showed a significant inhibition of migratory capacity following inhibition of MK2 viability, and quantitative analysis confirmed a time-dependent inhibition of wound closure (Figure 3B). Next, we further validated the correlation between MK2 and lung adenocarcinoma metastasis at the organoid level. We collected early surgical specimens and advanced malignant pleural effusions for patient-derived carcinoid (PDLCOs) cultures, and verified the biological properties of PDLCOs by a multidimensional technique, where lung adenocarcinoma carcinoids showed cell clustering at the early stage of culture (D3), and in mature carcinoids (D12), the carcinoids were observed to show a dense three-dimensional structure. Subsequently, the lung adenocarcinoma cell origin was further verified by HE staining and the molecular marker for lung adenocarcinoma, Napsin (Figure 3C). Patient-derived organoids from early-stage surgical specimens and advanced malignant pleural effusions demonstrated progression-dependent MK2 upregulation, with advanced-stage organoids exhibiting higher MK2 expression via immunohistochemical quantification (Figure 3D).

EMT stands as a fundamental process in normal embryonic development and serves as a prevalent factor initiating tumor invasion and metastasis (Christofori, 2003; Wheelock and Johnson, 2003; Hazan et al., 2004). As previously documented, EMT is intricately linked with the metastatic progression of various cancers, encompassing liver, ovarian, pancreatic, and breast cancer alike (DiMeo et al., 2009; Cano et al., 2010; Ponnusamy et al., 2010; Xu et al., 2017). To investigate MK2’s role in LUAD-associated EMT, we analyzed transcriptional and translational dynamics of EMT markers following MK2 inhibition. qRT-PCR profiling revealed coordinated transcriptional reprogramming, with significant upregulation of epithelial marker E-cadherin and marked downregulation of mesenchymal regulators N-cadherin, vimentin, MMP2, and Snail following inhibition of MK2 activity (Figure 3E). Consistent with transcriptional changes, Western blot analysis demonstrated protein-level restoration of epithelial phenotypes, evidenced by enhanced E-cadherin expression and reduced mesenchymal marker abundance (Figure 3F; Supplementary Material 2A-D).

These multi-omics findings establish MK2 as a pivotal regulator of EMT plasticity in LUAD. The conserved capacity of MK2 to orchestrate EMT-driven malignancy across LUAD positions it as a therapeutic target for disrupting metastasis-initiating pathways.

Previous studies have uncovered that MK2 plays a promoting role in the progression of nasopharyngeal carcinoma by activating the AKT/MYC signaling pathway, which is known to be a key driver in tumorigenesis (Deng et al., 2018). Specifically, the activation of this pathway by MK2 has been associated with enhanced cellular processes that facilitate tumor growth and progression. Additionally, accumulating evidence has emphasized the critical involvement of the AKT/MYC signaling pathway in promoting tumor invasion and metastasis (Wei et al., 2019; Su et al., 2024; Liang et al., 2020), two hallmark features of aggressive cancers. These findings underscore the importance of understanding how MK2 interacts with this pathway to regulate these malignant behaviors.

Given the central role of the AKT/MYC pathway in cancer metastasis, we sought to further elucidate the regulatory effects of MK2 on this signaling cascade. Our experimental data demonstrated that the suppression of MK2 activity led to a marked decrease in the levels of phosphorylated AKT (p-AKT Ser-473) and c-MYC proteins in both A549 and H358 cell lines. This suggests that MK2 modulates the pathway primarily through influencing the phosphorylation state of AKT and the stability or expression of c-MYC. Notably, total AKT protein abundance remained unaltered, pinpointing MK2’s modulatory role in AKT activation status rather than proteostatic regulation (Figure 4A). To confirm a mechanistic interaction, we performed co-immunoprecipitation (Co-IP) assays in A549 cells. The results revealed a direct interaction between MK2 and AKT (Figure 4B), supporting MK2’s involvement in regulating AKT phosphorylation status within this signaling axis. The signaling activation observed over a short period of time (e.g., 24 h) may not adequately represent the persistence of the signaling pathway, so we again extended our analysis to 48 h and 72 h of MK2 inhibitor treatment (Figures 4C–D), and we observed that, in both cell lines, the reduction of p-AKT and MYC was most pronounced at 48 h and 72 h, which confirms that MK2 activity Inhibition of MK2 activity leads to sustained inhibition of AKT phosphorylation and MYC expression over time, implying that MK2 does not only activate the AKT/MYC signaling pathway initially, but that it may also play an important role in maintaining the persistence and strength of these signals. These findings collectively establish MK2 as a kinase-dependent gatekeeper of AKT/MYC-driven oncogenic signaling, coupling post-translational modification to transcriptional reprogramming in metastatic progression.

To further ascertain whether the anti-invasive and EMT-modulating effects of MK2 inhibitors in LUAD are mediated through the AKT/MYC pathway, we employed SC79, an activator of the AKT/MYC pathway. Notably, co-administration of SC79 with MK2 inhibitors completely abrogated the inhibitor-induced suppression of MYC and mesenchymal markers (vimentin, MMP2, N-cadherin), effectively reinstating their baseline expression profiles (Figure 5A). Functional complementation assays further demonstrated that AKT/MYC pathway activation rescued the impaired metastatic potential of LUAD cells, as evidenced by restored migratory and invasive capacities in MK2 inhibitor-treated populations (Figures 5B–C). Taken together, our results confirm that MK2 interacts with AKT, promoting AKT phosphorylation and, in turn, enhancing C-MYC expression. The dose-responsive reversibility of MK2 inhibitor effects through pathway activation definitively positions this signaling axis as the mechanistic linchpin connecting MK2 to LUAD progression (Figure 6). These findings establish the AKT/MYC pathway as the dominant downstream effector mediating MK2’s pro-metastatic functions in LUAD, bridging kinase activity to EMT-driven metastatic reprogramming.