MAPKAPK2 (MK2) facilitates the epithelial-mesenchymal transition in lung adenocarcinoma through activation of the AKT/MYC signaling pathway

doi:10.21203/rs.3.rs-4977046/v1

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MAPKAPK2 (MK2) facilitates the epithelial-mesenchymal transition in lung adenocarcinoma through activation of the AKT/MYC signaling pathway

https://doi.org/10.21203/rs.3.rs-4977046/v1

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Purpose

The protein kinase Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) is linked to higher risks of metastasis and mortality in some cancers. Nonetheless, its precise function in lung adenocarcinoma (LUAD) is still not well understood. Thus, our research focuses on examining MK2’s role within LUAD cells and identifying the underlying mechanisms involved.

Methods

Differences in MK2 expression among patients with LUAD were confirmed through Timer2.0 database and tissue microarrays. The activity of MK2 in LUAD cell lines A549 and H358 was inhibited using a specific MK2 inhibitor. Thereafter, the viability, migration and mobility were analyzed. Gene expression changes were confirmed through Western blotting. Additionally, an AKT activator was used to validate the role of the MK2-regulated AKT/MYC signaling pathway.

Results

MK2 shows higher expression in LUAD tissues than in surrounding normal tissues. Reducing MK2 activity not only curtails cell proliferation, migration, and EMT-related invasion in vitro but also disrupts the AKT/MYC signaling axis. Nevertheless, activating the AKT/MYC pathway can counteract the effects of MK2 inhibition.

Conclusions

Our research shows that MK2 promotes migration and invasion in LUAD through the AKT/MYC signaling pathways, highlighting MK2 as a potential therapeutic target for LUAD.

MK2

EMT

AKT/MYC Pathway

Cancer Progression

Lung Adenocarcinoma

Lung cancer holds the unfortunate distinction of being the cancer type with the highest incidence and mortality rates globally¹. Within this category, non-small cell lung cancer (NSCLC) comprises 85% of all lung cancer cases, standing as the predominant form². Among NSCLC cases, lung adenocarcinoma (LUAD) emerges as the primary histological variant, representing approximately 40% of all malignant lung tumors³. The epithelial-mesenchymal transition (EMT) is tightly linked to tumor metastasis^4,5 and is a major contributor to cancer mortality^6,7. Despite progress in targeted therapies for lung adenocarcinoma (LUAD), many patients fail to benefit from these treatments because they either lack mutations that can be targeted or they develop resistance to the drugs. Consequently, identifying new therapeutic targets remains critical.

MK2 is a serine/threonine protein kinase and serves as a downstream component of the p38 MAPK pathway. Environmental stress activates the p38/MK2 signaling axis, promoting cell migration and motility by stimulating downstream molecule expression. MK2 is believed to play a significant role in tumor metastasis, particularly in tumor invasion. Recent studies have shown that interactions between MK2 and p38α SUMOylation, which MK2 activates, contribute to the metastasis of gastric cancer⁸. Meanwhile, Shi and Henriques discovered that GAGE7B can induce the pMAPKAPK2/pHSP27 pathway, which were associated with lower clinical staging and poorer survival rates in gastric, colon, and rectal cancers^9,10. Furthermore, the ataxia-Trangit's disease group D complement gene (ATDC) is directly phosphorylated by MK2 at Ser550 in an ATM-dependent manner, thereby enhancing response to pancreatic ductal adenocarcinoma mouse xenograft experiments in vitro and in vivo. Resistance to ionizing radiation¹¹. However, it remains unclear whether MK2 affects migration and motility in LUAD cells.

The AKT/MYC signaling pathway is intimately linked to the epithelial-mesenchymal transition (EMT)¹² and plays a pivotal role in the progression of various cancers. In cancers such as bladder and nasopharyngeal, this pathway facilitates cancer advancement via the EMT process, highlighting potential avenues for developing more effective treatments¹³. Moreover, some research indicates that the MK2-regulated AKT/MYC signaling pathway enhances tumor metastasis^14,15. However, the role of MK2 in modulating this pathway in lung adenocarcinoma (LUAD) cells remains underexplored. Our study therefore focuses on examining the impact of MK2 on LUAD cell migration and invasion, and aims to define the contribution of the AKT/MYC pathway in MK2-driven tumor metastasis.

The Expression of MK2 was Increased in Lung Adenocarcinoma

Increasing researches suggest that MK2 expression is elevated in LUAD as well as in various other malignant tumors^16–18. To investigate MK2's specific function in LUAD, we utilized the TIMER 2.0 database to examine its expression profile. Bioinformatics analysis revealed that MK2's transcription levels are significantly higher in LUAD tissues compared to adjacent non-tumor tissues, underscoring its potential role in tumor development (Fig. 1A). Similarly, 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 (Fig. 1B).

Confirmatory tissue microarray analysis was conducted on 48 paired clinical LUAD samples, reinforcing the findings from previous database studies. The analysis revealed that MK2 expression levels were significantly elevated in tumor tissues compared to control tissues (Fig. 1C, D, and E). These results indicate that MK2 is involved in the pathophysiology of LUAD and may serve as a potential therapeutic target.

MK2 Inhibition Decreases the Proliferation of LUAD Cells

Previous research suggests that MK2-mediated phosphorylation of RIPK1 decreases its affinity for FADD, thereby attenuating TNF-α-induced apoptosis^19,20. Additionally, inhibiting MK2 also reduces the activation of Hsp27, thereby enhancing the sensitivity of PDAC cells to apoptosis²¹. Furthermore, inhibiting the expression of MK2 through shRNA transfection can also suppress the growth of multiple myeloma cells²². Therefore, this study was designed to explore the impact of MK2 inhibitors on the proliferation of LUAD cells using the A549 and H358 cell lines. 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 38.30 µM, respectively, at 24 hours, decreasing to 29.51 µM and 31.69 µM at 48 hours(Figure 2A). Subsequent treatment of both cell lines with 20 µM MK2-IN-1 followed by apoptosis assays at 24 hours showed a significant increase in apoptosis in MK2-IN-1 treated LUAD cells (Fig. 2B). These findings suggest that MK2 inhibitors are crucial in controlling the proliferation of LUAD cells.

Inhibiting the Activity of MK2 Reduces the EMT of LUAD Cells

Given the established correlation between MK2 expression and the metastatic potential in various cancers, including melanoma²³, gastric cancer²⁴, colorectal cancer²⁵, breast cancer²⁶ and other tumors, we explored its role in LUAD cell motility via MK2 inhibition. Transwell Matrigel invasion assay showed that the invasion ability of A549 and H358 cells treated with MK2 inhibitors was significantly lower than that of the untreated control group (Fig. 3A). At the same time, the effect of MK2 on cell migration was further evaluated through wound healing experiments. Wound healing was assessed every 24 hours using an inverted microscope, with images of cells captured before treatment (0 hours) and 24 hours after treatment. The statistical analysis results were consistent with the observations from the invasion experiments (Fig. 3B), suggesting the involvement of MK2 in regulating LUAD cell migration.

EMT stands as a fundamental process in normal embryonic development and serves as a prevalent factor initiating tumor invasion and metastasis^27–29. EMT stands as a fundamental process in normal embryonic development and serves as a prevalent factor initiating tumor invasion and metastasis. As previously documented, EMT is intricately linked with the metastatic progression of various cancers, encompassing liver, ovarian, pancreatic, and breast cancer alike^30–33. So we investigated whether MK2 modulation affects EMT in LUAD cells. Western blot analysis of EMT marker proteins revealed that inhibiting MK2 activity led to an upsurge in the expression of the epithelial marker E-cadherin, coupled with a reduction in the expression of mesenchymal markers such as N-cadherin, vimentin, and MMP2 (Fig. 3C). These findings suggest that hindering MK2 activity could potentially curtail metastasis by modulating EMT, thereby reinforcing its involvement in the progression of LUAD.

MK2 Can Modulate the AKT/MYC Signaling Pathway

Previous studies have uncovered that MK2 plays a promoting role in the progression of nasopharyngeal carcinoma by activating the AKT/MYC signaling pathway³⁴. Additionally, reports indicate the crucial involvement of the AKT/MYC signaling pathway in tumor invasion and metastasis^14,15,35, Given the pivotal role of the AKT/MYC pathway in cancer metastasis, we investigated the regulatory impact of MK2 on this pathway. Our findings revealed that diminishing MK2 activity significantly reduced the levels of phosphorylated AKT and MYC proteins in A549 and H358 cells, while the total level of AKT remained unaffected (Fig. 4A).

MK2 Regulated the EMT of LUAD Cells by the AKT/MYC Signaling Pathway

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. The results revealed that compared with the MK2 inhibitor group, cells treated with MK2 inhibitor plus SC79 restored the levels of MYC, vimentin, MMP2, and N-cadherin (Fig. 5A). Furthermore, SC79 reversed the decrease in cell migration and invasion induced by MK2 inhibitors (Fig. 5B and C). These findings underscore the involvement of MK2 in regulating the migration, invasion, and epithelial-mesenchymal transition (EMT) of LUAD cells through the AKT/MYC signaling pathway.

The p38/MAPK signaling cascade, a ubiquitous signaling enzyme in eukaryotes, is critically involved in tumorigenesis and metastasis³⁶. Previous studies have highlighted that the p38/MAPK-specific inhibitor SB203580 can impede TGF-β-induced MMP-2 expression and curb the invasion of prostate cancer cells³⁷. Furthermore, research has elucidated that IFITM3 can activate p38/MAPK signaling, thereby elevating MMP-9 expression and fostering the invasion and metastasis of hepatocellular carcinoma³⁸. Despite the pivotal role of p38/MAPK as a research target, its diverse array of upstream kinases, downstream substrates, and intricate network of regulatory factors contribute to notable side effects³⁹. Hence, identifying novel therapeutic targets within the downstream components of this pathway, particularly mitogen-activated protein kinase-activated protein kinase-2 (MK2), has emerged as a focal point of investigation. In our current study, we observed MK2 overexpression in LUAD tissues compared with adjacent normal tissues. Inhibition of MK2 activity impedes cell proliferation, migration, and invasion associated with epithelial-mesenchymal transition (EMT) in vitro. However, activation of the AKT/MYC pathway can counteract the effects of MK2 inhibition.

MK2 is a serine/threonine kinase positioned downstream of p38 MAPK, pivotal in a myriad of cellular processes including stress response, inflammation, cell proliferation, differentiation, apoptosis, and gene expression regulation^40–45. In this investigation, we identified MK2 overexpression in LUAD patients through database analysis and tissue microarray validation, implicating MK2 in LUAD pathology. Our results show that MK2 inhibition not only reduces LUAD cell proliferation (in A549 and H358 cell lines), but also diminishes EMT-associated molecular expression and invasion and migration capabilities. These findings align with Henriques et al.'s research, indicating that MK2 indeed holds significance in colon cancer development by influencing cell proliferation and migration through Hsp27 activation⁹. Moreover, in vitro experiments demonstrate that MK2 inhibition alleviates tumor inflammation and EMT, thereby impeding tumor growth and progression across various cancer types^{25,26,46–48}. However, the role of MK2 appears to diverge in glioblastoma, where it exclusively promotes temozolomide-induced cell migration through RSK-EphA2 activation⁴⁹, highlighting the cancer-specific functions of MK2.

Currently, Numerous recent studies have indicated the involvement of the PI3K/AKT pathway in the metastasis of NSCLC⁵⁰, colorectal cancer⁵¹ and hepatocellular carcinoma⁵². Concurrently, the pivotal transcription factor MYC orchestrates the expression of genes crucial for cell growth, survival, and metastasis^53–55, underscoring the significance of the AKT/MYC pathway in tumorigenesis. While research has established the potential importance of this pathway in gastric cancer and LUAD^14,15, the regulatory role of MK2 on the AKT/MYC pathway remains unclear. Hence, we conducted this study to investigate how MK2 regulates the expression of AKT and MYC proteins in LUAD cells, aiming to elucidate its impact on this signaling cascade. Our Western blot analysis revealed that reduction of MK2 activity inhibited MYC phosphorylation and altered the invasion, migration, and EMT profiles of LUAD cells, whereas AKT activation reversed these effects. Obviously, these results revealed that although MK2 can regulate many of signal pathway, AKT/MYC signal pathway is one of major downstrean signal pathway in LUAD cells. In hepatocellular carcinoma, treatment with MK2 inhibitor also can block the proliferation and induce the apoptosis via downregulating c-Myc and AKT-1⁵⁶. Additionally, Deng et al.'s research revealed that under the negative regulation of nicotine, miR-296-3p can directly target the MK2-induced Ras/Braf/Erk/Mek/c-Myc or PI3K/AKT/c-Myc signaling pathway, thereby inhibiting the proliferation and metastasis of nasopharyngeal cancer cells³⁴. In summary, MK2 can regulate EMT through the AKT/MYC signaling pathway in LUAD cells.

In summary, our findings demonstrate that MK2 plays a pivotal role in both the induction and metastasis of LUAD through modulation of the AKT/MYC signaling pathway. This revelation introduces a novel therapeutic target for LUAD treatment, enhances our comprehension of LUAD's molecular mechanisms, and establishes groundwork for future therapeutic strategies.

Public Database Analysis

The expression profile of the target molecule, MAPKAP Kinase 2 (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.

Tissue Microarray and Immunohistochemistry

Shanghai Zhuoli Biotechnology Co., Ltd. provided LUAD tissue chip (ZL-LugA961), comprising 48 pairs of tumor tissue and adjacent non-tumor tissue samples. Microarrays underwent pretreatment with bovine serum albumin (BSA) before being incubated overnight at 4°C with MK2 antibody from Proteintech (diluted 1:200). The following day, further incubation was conducted with HRP-labeled secondary antibodies. Visualization was achieved using DAB, followed by hematoxylin counterstaining, 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).

Cell Cultivation and Handling

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 (20µmol/L, MCE). The control group received no treatment. Subsequently, cells pre-treated with the MK2 inhibitor for approximately 6 hours were exposed to the AKT/MYC pathway activator SC79 (20µg/ml) in follow-up experiments to research the function of the AKT/MYC signaling pathway.

Western Blotting

Cell lysis solution was made using RIPA buffer from Beijing Beingmate Biotechnology Co., Ltd. Proteins were isolated using SDS-PAGE (8%-15%) and transferred onto PVDF membranes. These membranes were blocked with 5% skim milk for a minimum of one hour. Primary antibodies for MYC (Proteintech, 1:1000), GAPDH (Proteintech, 1:50000), AKT (Abcam, 1:1000), E-Cadherin (Proteintech, 1:1000), Vimentin (Proteintech, 1:1000), N-Cadherin (Abcolonal, 1:1000), and MMP2 (Abcam, 1:1000) were incubated at 4°C overnight. After three washes with PBST, the HRP-labelled secondary antibody was incubated for one hour. After that, protein detection was performed using an ultra-high sensitivity ECL kit manufactured by GLPBIO, USA.

Annexin V-APC/7-AAD Double Staining

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 hours. Following a PBS wash, the cells received MK2 inhibitor treatment for an additional 24 hours. Subsequently, the cells were promptly fixed and stained in accordance with the Annexin V-APC/7-AAD apoptosis kit protocol provided by Elabscience, with immediate flow cytometry analysis thereafter.

Cell Migration Assay

Wound healing assays were employed to evaluate cell migration ability. Cells were seeded in six-well plates at a density of 4 × 10^5 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 hours after wounding. All experiments were performed in triplicate.

Invasion Assay

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 hour at 37°C. Subsequently, cells were suspended in serum-free medium at a concentration of 5×10^5 cells/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 hours, cells in the lower chamber were fixed with 4% paraformaldehyde for 30 minutes and then stained with 0.1% crystal violet for another 30 minutes. Following this, the stained cells were counted using a Leica fluorescence inverted microscope (DMI8). Each experiment was conducted in triplicate.

Statistical Analysis

We employed GraphPad Prism 9.5 for statistical analysis, presenting data as mean ± standard deviation. Differences between multiple groups were assessed using ANOVA and Tukey's post hoc test, with p values less than 0.05 considered statistically significant.

MK2:Mitogen-Activated Protein Kinase Activated Protein Kinase 2

LUAD: lung adenocarcinoma

EMT: epithelial–mesenchymal transition

NSCLC: Non small cell lung cancer

MAPK: mitogen activated protein kinase

Acknowledgments

This work was supported by the grants from Surface project of the Natural Science Foundation of Jiangxi Province (No.20212ACB206029)

Author Contributions

Conception and design: Feng Qiu, Rong Qi, Chen Fang; (II) Administrative support: Feng Qiu, Chao Shi, Guohua Zhang; (III) Provision of study materials or patients: Rong Qi, Penghui Liu; (IV) Collection and assembly of data: Feng Qiu, Weiguo Gu, Rong Qi; (V) Data analysis and interpretation: Feng Qiu, Rong Qi, Guohua Zhang; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.

Data availability

The datasets generated during and analysed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest

All authors have completed the ICMJE uniform disclosure form.

Ethical Statement

The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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No competing interests reported.

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You are reading this latest preprint version

MAPKAPK2 (MK2) facilitates the epithelial-mesenchymal transition in lung adenocarcinoma through activation of the AKT/MYC signaling pathway

Status:

Version 1

Abstract

Purpose

Methods

Results

Conclusions

Figures

Introduction

Results

The Expression of MK2 was Increased in Lung Adenocarcinoma

MK2 Inhibition Decreases the Proliferation of LUAD Cells

Inhibiting the Activity of MK2 Reduces the EMT of LUAD Cells

MK2 Can Modulate the AKT/MYC Signaling Pathway

MK2 Regulated the EMT of LUAD Cells by the AKT/MYC Signaling Pathway

Discussion

Materials and Methods

Public Database Analysis

Tissue Microarray and Immunohistochemistry

Cell Cultivation and Handling

Western Blotting

Annexin V-APC/7-AAD Double Staining

Cell Migration Assay

Invasion Assay

Statistical Analysis

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1