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. 2026 May 27;45(26):2567–2578. doi: 10.1038/s41388-026-03824-7

MST4 plays dual roles in lung adenocarcinoma by regulating homeostasis of wild-type and mutant p53 protein

Minghui Zhang 1,✉,#, Changwei Hu 2,#, Huilong Liu 2,#, Chenxi Zhang 2, Qipeng Shu 2, Yu-Jian Kang 2, Yuetian Ding 3, Jin Zhang 2, Zhenghui Hu 4, Haokai Wang 4, Youfan Zhang 2, Ze-Yan Zhang 4,✉, Shangze Li 2,3,✉
PMCID: PMC13286987  PMID: 42204266

Abstract

Mutations are highly prevalent in the TP53 tumor suppressor gene, which encodes p53, in lung adenocarcinoma (LUAD). However, targeted therapies centered on p53 mutations remain challenging. Here, we found MST4 highly expressed in LUAD and linked to patient survival. Functional assays using CRISPR-generated MST4-knockout LUAD cell lines revealed context-dependent dual roles of MST4: tumor-suppressive in wild-type p53 (wtp53) cells (inhibiting proliferation, colony formation, migration) and oncogenic in gain-of-function (GOF) p53 cells. Mechanistically, MST4 directly interacts with p53, competing with MDM2 to prevent K48-linked ubiquitination and degradation of both wtp53 and GOF-mutant p53, which explains cell-context-dependent phenotypes of MST4 suppression. Preclinically, targeting MST4 significantly repressed tumor formation of xenograft model with GOF-mutant p53. Collectively, our findings identify MST4 as a context-dependent regulator of LUAD, whose function is dictated by p53 mutation status. MST4 stabilizes p53 via a novel MDM2-competitive, kinase-independent mechanism, and targeting MST4 represents a promising therapeutic strategy for GOF-mutant p53-driven LUAD.

Subject terms: Cancer, Cell signalling

Introduction

Lung adenocarcinoma (LUAD), the most prevalent subtype of non-small cell lung cancer, remains a leading cause of cancer-related mortality globally, with limited therapeutic options for advanced or recurrent disease [1]. The identification of molecular regulators with context-dependent functions has become critical for developing precision therapies, as tumor progression often relies on dysregulated signaling pathways that exhibit variable roles based on genetic backgrounds (e.g., p53 mutation status).

p53, often referred to as the “guardian of the genome”, is the most frequently mutated gene in human cancers [2], including LUAD [2]. Wild-type p53 (wtp53) exerts tumor-suppressive effects by regulating cell cycle arrest [3], apoptosis [4], and DNA repair [5], while gain-of-function (GOF) p53 mutations (e.g., R273H) [6, 7] drive oncogenesis via promoting proliferation [8], metastasis, and therapeutic resistance [9–13]. Despite its clinical significance, p53 has long been considered “undruggable” due to the lack of typical drug target features (e.g., enzymatic active sites) [14–16]. Given that, people turned their attention to the p53 regulatory genes [17–20], such as an E3 ubiquitin ligase–MDM2 [21]. MDM2, which was reported to promote K48-link ubiquitination and proteasomal degradation of p53, plays a key role of negatively regulating the activity of p53 [22–24]. Several MDM2 inhibitors have been developed to inhibit the interaction between MDM2 and p53 and then rescue the activity of wtp53 (e.g., RG7112, APG-115, and AMG232) [25–28], however, leaving GOF-mutant p53-driven tumors with unmet therapeutic needs. Given the urgent need for targeted therapies against GOF-mutant p53 LUAD, identifying novel regulators that interact with p53 (especially mutant p53) and modulate its stability or function could open new therapeutic avenues.

The MST kinase family (Mammalian Sterile 20-like kinases) plays essential roles in cell proliferation, apoptosis, and metabolism, with dysregulation linked to multiple cancers [29]. MST4, a member of this family, is involved in several cancers [30]. On one hand, MST4 has been reported to promote tumorigenesis in glioblastoma and colorectal cancer by activating oncogenic signaling cascades [31, 32]. On the other hand, it was also reported to suppress gastric tumorigenesis via phosphorylating YAP [33]. Notably, emerging evidence suggests cross-talk between the MST kinase family and p53 signaling—for instance, MST1/2 have been shown to regulate p53 stability via phosphorylation [34, 35]—yet whether MST4 interacts with p53 or modulates its function remains unknown. Moreover, the context-dependent role of MST4 in cancers (tumor-suppressive vs. oncogenic) raises the hypothesis that its function in LUAD may be dictated by p53 mutation status, particularly in GOF-mutant p53 cells where therapeutic options are scarce. However, its expression pattern, functional roles, and regulatory mechanisms in LUAD remain largely unexplored—especially whether its function is influenced by key genetic alterations (e.g., p53 mutations) that define LUAD subtypes.

Here, we systematically investigated the role of MST4 in LUAD. We first confirmed MST4 upregulation in LUAD tissues and its association with patient survival. Unexpectedly, MST4 exhibited dual roles in LUAD tumorigenesis—tumor-suppressive in wtp53 cells (A549) and oncogenic in GOF-mutant p53 cells (NCI-H1975). Mechanistically, MST4 stabilizes both wtp53 and GOF-mutant p53 in a kinase-independent manner by competing with MDM2 for binding to the p53, thereby inhibiting MDM2-mediated K48-linked ubiquitination and proteasomal degradation. Importantly, targeting MST4 suppressed tumor growth in GOF-mutant p53 LUAD xenografts and enhanced chemosensitivity. Our findings reveal MST4 as a context-dependent regulator of LUAD and a potential therapeutic target for GOF-mutant p53-driven LUAD.

Materials and methods

Cell lines and cell culture

Normal lung epithelial cell (BEAS-2B), human lung cancer cell lines (A549, NCI-H1975, NCI-H460) and HEK293T cells were maintained in DMEM (PM150210, Procell, China) supplemented with 10% fetal bovine serum (FBS, 164210–50, Procell, China) and 1% penicillin/streptomycin (C0222, Beyotime, China) at 37°C in a 5% CO2 incubator. All cell lines were obtained from ATCC.

Production and infection of lentivirus

To generate lentiviruses expressing the indicated single guide RNAs (sgRNAs), HEK293T cells were transfected with sgRNA (cloned in lentiCRISPRv2), pMD2G and SPAX. For generation of the control virus, pSin empty vector, scramble shRNA cloned in PLKO.1 and scramble sgRNA cloned in lentiCRISPRv2 were used. Then, 12 h after transfection, cells were cultured in fresh medium for another 36 h. The culture medium containing lentivirus particles was filtered through a 0.45-μm PVDF filter (Millipore) and used for infection. Target cells were infected with the indicated lentiviruses in the presence of 10 μg/ml polybrene. The sgRNA target sequences used in this study are listed in Supplementary Table 2.

RNA sequencing analysis

Total RNA was extracted from MST4-deficient A549 and empty vector control A549 cells using a Ribospin RNA extraction kit (GeneAll Biotechnology, Seoul, Korea). After quality evaluation, libraries were generated using a TruSeq RNA Library Prep Kit V2 and sequenced on an Illumina platform (San Diego, CA, USA). LAS, Inc. (Seoul, Korea) carried out all sequencing analyses. Hisat2 was used to map the mRNA-sequencing (mRNA-seq) data to the human reference genome (GRChg38), and String Tie was used to calculated gene expression. DESeq2 was employed to perform differential gene expression analysis. To identify differentially expressed genes, we selected genes with an average of at least one for each normalized read. Genes with a p-value of 0.001 or less and an absolute log2 fold-change value of 2 were considered differentially expressed compared to the control samples.

Plasmids

The plasmids pGEX6p-1-GST-p53, pGEX6p-1-GST-MST4, pHAGE-3×Flag-MST4, pHAGE-3×Flag-MST4K53R were constructed according to the “Molecular Cloning Experiment Guide”. PCR primers are listed in the Supplementary Table 2.

Reagent and antibodies

The primary antibodies were used as follows: anti-MST4 antibody (10847-1-AP, proteintech), anti-p53 antibody (10442-1-AP, proteintech & 3822S, CST), anti-MDM2 antibody (66511-1-Ig, proteintech), anti-p21 antibody (2947T, CST), anti-Cyclin D antibody (2922S, CST), anti-puma antibody (4976T, CST), anti-HA antibody (HRP-81290, proteintech), anti-Flag antibody (66008-4-Ig, proteintech), anti-Myc antibody (60003-2-Ig, proteintech), anti-β-actin antibody (66009-1-Ig, proteintech).

Cell proliferation assay

Cell proliferation was measured by cell counting kit (CCK8) (HY-K0301, MCE, China). Each group of cells (2 × 103 cells/well) were plated into 96-well plates, after indicated time, the CCK8 reagent (10 μl/well) was added into wells according to the instruction and then incubated at 37°C, 5% CO2 for 1 h. The absorbance was measured at 450 nm with Thermo Scientific™ Multiskan™ FC instrument (Thermo Fisher, Shanghai, China).

Colony formation assay

After 800 cells were plated into 6-well plates for 14 days, the cells were stained with 0.2% crystal violet (C0121, Beyotime, China) for 20 min, then the 0.2% crystal violet was discarded and cells were washed with water, later stained colonies images were captured and number was counted.

Cell migration analysis

In order to measure the ability of cell migration, 1 × 104 cells/well containing medium with 1% FBS were seeded in the transwell and placed into 24-well plates with medium containing 40% FBS. After 36 h, cells that had migrated outside the chamber were fixed and stained with 0.2% crystal violet.

Immunohistochemistry

Lung adenocarcinoma tissue microarrays were purchased from Shanghai WEIAO Biotech Company (ZL-LugA961) and Wuhan Service Biotech Company (IWLT-N-70L43). Protein expression was measured with immune peroxidase and scored by the staining intensity and area. Antibody against MST4 were used to determine their expression levels.

Immunofluorescence assay

A549 cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Then, the cells were washed with PBS and blocked with 5% BSA in PBS for 45 min. To visualize the expression of MST4 and p53, rabbit anti-MST4 antibody and mouse anti-p53 antibody were used as primary antibodies, and mouse Alexa594- and rabbit Alexa488-conjugated antibodies (Service) were used as secondary antibodies. DAPI (C0065, Beijing, China), was used to stain the nuclei. Finally, the coverslips were observed and digitally photographed using a confocal microscope with a 60× oil objective.

RNA isolation for real-time quantitative PCR

The cells were harvested with TRIzol reagent (Takara), and the extraction of total RNA is followed by a standard protocol. cDNA was obtained with a reverse transcription kit according to the instructions (Takara). The sequences of the primers used in the study are listed in Supplementary Table 1.

Coimmunoprecipitation (Co-IP) and western blot analysis

The transfected cells were lysed in 800 μL lysis buffer (20 mM Tris-HCl, pH 7.4; 150 mM NaCl) with proteinase and phosphatase inhibitor cocktail (Roche) for 15–30 min at 4°C in reversing shaker before ultrasonication. Then 80 μL lysates were used as input after added with 20 μL 5× loading buffer at 95°C for 10 min. The rest supernatant of lysates was incubated with 1–3 μg of the indicated antibody with the addition of protein A/G agarose beads (Yeasen, Shanghai) at 4°C for 12 h in reversing shaker. After that, the complexes were collected and added 100 μL of 1× SDS loading buffer at 95°C for 10 min, which making elution of target proteins from beads. The samples were subjected to western blot analysis. Total protein (20–40 μg) was separated by 8–12% SDS-PAGE and transferred to the polyvinylidene fluoride (PVDF) membrane. The membranes were blocked in 5% nonfat milk in Tris-buffered saline with Tween 20 (TBST) for 1 h before incubation with the desired primary antibodies overnight at 4°C. Then, the membranes were incubated with secondary antibodies at room temperature for 1 h. Finally, the signals were visualized on a Tanon 5200 Image System.

GST pull down assay

The GST fusion protein of MST4 and p53 purified by Glutathione-Sepharose beads (Smart-Lifesciences Biotech, China) were incubated with cell lysates at 4°C overnight, and then the beads were washed by GST binding buffer for three times, then the bound protein was submitted for western blot analysis.

Dual luciferase reporter assay

HEK293T cells (30% confluence) were seeded into 24-well plates and then transfected with some tumor-relevant luciferase reporter plasmids (contains signaling pathways: ATF2/3/4, E2F, p53, STAT1/2, C/EBP, CREB, ER, GATA, GLI, HIF1A, c-Myc, Pax6 and PPAR), 5 ng pPL-CMV together with the indicated gene-expressing plasmids (pHAGE-3×Flag-USP14) or empty vector (pHAGE-3×Flag). After 48 h, the luciferase reporter experiment was carried out using a dual-specific luciferase assay kit from Promega (Fitchburg, WI, USA).

Xenograft assay

Given the effect size and standard deviation, the method for determining the sample size in animal studies was chosen in accordance with the animal research committee’s suggestion. Control (Vector) and MST4-deficient (MST4-sg1 or MST4-sg2) NCI-H1975 cells were collected and washed twice with PBS. A total of 1–10 × 106 cells were resuspended in 0.2 ml of PBS and inoculated into the flanks of ten 6-week-old male nude mice randomly divided into three groups. Tumor volume was measured three times every 5 days and calculated according to the following formula: [length × width2] × 0.5. Mice were killed 30 days post inoculation. Tumors were weighed and fixed in 4% paraformaldehyde. All animal studies were conducted in accordance with the Guidelines of the China Animal Welfare Legislation and approved by the Committee on Ethics in the Care and Use of Laboratory Animals of Chongqing University (permit number: CQU-IACUC-RE-202501-001). All efforts were made to minimize animal suffering.

UALCAN

By obtaining data from TCGA database, UALCAN can be used not only to assess the expression of protein-coding genes, but also to conduct in-depth analysis of clinical data in 33 cancers. In this study, differences in MST4 mRNA expressions between normal and tumor tissues were obtained through “Expression Analysis” module and Lung Adenocarcinoma (LUAD) dataset, and t test was used for analysis, with p < 0.05 as the significance threshold.

GEPIA

GEPIA is an analysis tool based on TCGA and GTEx data, which contains RNA sequence expression data of 9736 tumors and 8587 normal tissue samples. In this study, GEPIA single gene analysis was used to analyze both the difference of MST1, MST2, MST3, MST4, STK25 mRNA expression LUAD dataset and TP53 mutation data were obtained from the GEPIA 3 database [36] T test analysis was used, with p < 0.05 as the significant threshold, and Kaplan–Meier curve was used for prognosis analysis.

Kaplan–Meier plotter

The KM plotter (http://kmplot.com) database evaluates survival of different genes in 21 cancer types including breast (n = 6234), ovarian (n = 2190), lung (n = 3452) and gastric (n = 1440) cancer. Prognostic values including OS (overall survival) of MST4 were evaluated in Lung Adenocarcinoma (LUAD). Hazard ratios (HRs) with 95% confidence intervals and logrank P-value were determined.

Statistical analysis

The experiments were repeated at least three times beside mice experiments. Statistical analyses were performed using GraphPad Prism 9.5 software (GraphPad Prism Software Inc.). The data are expressed as the means ± SEM. Student’s t test and analysis of variance (ANOVA) were performed. p < 0.05 was regarded to be indicative of statistical significance.

Results

MST4 is upregulated in lung adenocarcinoma and associated with patient survival

Studies have shown an association of MST4 with many cancers, such as glioblastoma and colorectal cancer [30, 37]. To explore its relationship with lung cancer, we first analyzed public data in GEPIA online tool and found that MST4, but not other MST family kinases, was upregulated in LUAD tissue compared with non-tumoral controls (Fig. 1a). Then, immunohistochemistry (IHC) was employed to examine MST4 protein levels. After analyzing a set of LUAD tissue microarrays containing 83 patients’ tumor tissues and adjacent non-tumoral tissues (Fig. 1b, c), we confirmed the upregulation of MST4 in LUAD tissues. In addition, MST4 protein levels were also detected and found that MST4 expression was increased in lung cancer cell lines (A549, NCI-H1975, NCI-H460 and NCI-H1299) compared to normal lung epithelial cells BEAS-2B (Fig. 1d). Higher expression of MST4 was associated with higher grades, metastasis and worse patient survival (Fig. 1e–g). Of note, MST4 expression is also upregulated in various histological subtypes of lung adenocarcinoma (Supplementary Fig. 1a). Overall, these results demonstrated the upregulated expression pattern of MST4 in LUAD and it was associated with patient survival.

Fig. 1. MST4 is upregulated in lung adenocarcinoma and associated with patient survival.

Fig. 1

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a The expression of MST family (including MST1/2/3/4 and STK25) in various cancers (red) compared with the adjacent tissues (gray) obtained from GEPIA online tool (T = Tumor tissue and N = Normal tissue). b The representative images of IHC staining for MST4 in lung adenocarcinoma including tumor and adjacent tissues. The scale bar is 100 μm. c The analysis of MST4 expression in lung adenocarcinoma tissue microarrays from 83 tumor and adjacent tissues. d The expression of MST4 is upregulated in lung cancer cells. e Box plot of MST4 mRNA expression among groups of different tumor grades in lung adenocarcinoma patients. f Box plot of MST4 mRNA expression among groups of different nodal metastasis status in lung adenocarcinoma patients. g The expression of MST4 was assessed using Kaplan–Meier survival analysis in patients with lung adenocarcinoma. The data are presented as the means ± SEM. Statistical significance was analyzed by Student’s t test and ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Context-dependent dual roles of MST4 in LUAD tumorigenesis

Since lung cancer cell lines recapitulated high MST4 expression as in LUAD (Fig. 1d), we hypothesized that MST4 might play oncogenic roles in LUAD cells. To test this hypothesis, we generated MST4 knockout lines from different LUAD cell lines by utilizing CRISPR/Cas9 with two sgRNAs for each line, and the knockout efficiency was confirmed by immunoblotting (Supplementary Fig. 2a–d). Then, the colony formation, CCK8, and transwell assays were employed to examine tumorigenesis-related phenotypes with or without MST4. As expected, MST4 exerted oncogenic roles in NCI-H1975, as evidenced by repressed cell colony formation, proliferation, and migration upon MST4 knockout (Fig. 2a–d). Surprisingly, MST4 knockout in A549 cells significantly promoted cell colony formation, proliferation, and migration (Fig. 2e–h), which exhibits opposite phenotypes with NCI-H1975, indicating that MST4 also plays tumor-suppressive roles in LUAD. Consistent with this observation, MST4 played tumor suppressive role in NCI-H460 but oncogenic role in PC-9 cells (Supplementary Fig. 3a–f). Taken together, these data suggest that MST4 plays complex roles in LUAD, and may depend on different cell contexts.

Fig. 2. Context-dependent dual roles of MST4 in LUAD tumorigenesis.

Fig. 2

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a Control (Vector) or MST4-deficient (MST4-sg1 or MST4-sg2) NCI-H1975 cells were seeded to perform colony formation assay. The stained colonies were showed by the representative images. b A bar chart showing quantification results of (a). c Curves showing cell proliferation of NCI-H1975 cells the same as used in (a). d Cell migration results of Control (Vector) or MST4-deficient (MST4-sg1 or MST4-sg2) NCI-H1975. Representative images of staining were shown on the left, and quantification results were shown on the right. e Control (Vector) or MST4-deficient (MST4-sg1 or MST4-sg2) A549 cells were seeded to perform colony formation assay. The stained colonies were showed by the representative images. f A bar chart showing quantification results of (e). g Curves showing cell proliferation of A549 cells the same as used in (e). h Cell migration results of Control (Vector) or MST4-deficient (MST4-sg1 or MST4-sg2) A549. Representative images of staining were shown on the left, and quantification results were shown on the right. The data are presented as the means ± SEM. Statistical significance was analyzed by ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

MST4 regulates p53 at post-transcriptional level

To investigate the complex functions of MST4 and figure out the underlying mechanism of its dual-role in LUAD, we next explored MST4-mediated signal transduction via screening signal pathway reporters. Among the screened pathways, MST4 expression strongly activated the p53 signaling pathway luciferase reporter in HEK293T cells (Fig. 3a). In addition, validation experiments showed that MST4 activated the p53 luciferase reporter in a dose-dependent manner (Fig. 3b) and synergistically activated the p53 signaling pathway when co-expressed with p53 (Fig.3c). Furthermore, to confirm these findings in lung cancer model, we performed transcriptome analysis of control (vector) and MST4-deficient (MST4-sg1 or MST4-sg2) A549 cells via RNA sequencing (RNA-Seq) (Supplementary Table 1). As shown in the volcano plot and heatmap (Fig. 3d, e), p53 target genes (e.g., p21, PUMA, and 14-3-3σ) were among the most significantly downregulated genes in MST4 KO compared to WT A549 cells. Gene set enrichment analysis (GSEA) of the RNA-Seq results also demonstrated positive enrichment of the p53 signaling pathway in WT cells (Fig. 3f). Meanwhile, the mRNA levels of p53 target genes—including p21, MDM2, CCND1, 14-3-3σ, PUMA, and Fas—were dramatically downregulated in MST4-deficient A549 cells (Fig. 3g). Consistently, the protein levels of these genes were also downregulated in MST4-deficient A549 cells. Interestingly, p53 protein (but not mRNA) levels were decreased in MST4-deficient A549 cells (Fig. 3h, Supplementary Fig. 4a), indicating post-transcriptional regulation of p53 by MST4. Notably, however, MST4 knockdown led to distinct alterations in the p53 signaling pathway in NCI-H1975 cells (Supplementary Fig. 5a). Given the mutant status of TP53, MST4 is highly expressed in both subtypes of lung cancer yet displays divergent prognostic curves, which may underlie its dual functions in LUAD (Supplementary Fig. 6a–c). In summary, these results demonstrated that MST4 modulates the p53 signaling pathway by regulating p53 at post-transcriptional level.

Fig. 3. MST4 promotes the transcriptional activity of p53.

Fig. 3

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a Bar charts showing results of effects of MST4 overexpression on indicated signal pathway reporters HEK293T cells (n = 3). b Bar charts showing the activation of 53 signaling pathway by increasing MST4 transfection in HEK293T (n = 3) c The synergistic transactivation of p53 signaling pathway was shown when p53 and MST4 plasmids co-transfected into HEK293T cells (n = 3). d A volcano plot showing the differential expressed genes between control and MST4-deficient A549 cells from RNA-seq analysis. e A heatmap showing top differential expressed genes between wild type (WT) and MST4-knockout (KO) A549 cells. f GSEA analysis of p53 signaling pathway target genes using the RNA-seq data. g The relative mRNA expression of p53 target genes of MST4-deficient (MST4-sg1 or MST4-sg2) A549 cells when compared to control (Vector) A549 determined by qPCR. h Data validating the results at protein levels. The β-actin was used as the loading control. The data are presented as the means ± SEM. Statistical significance was analyzed by ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

MST4 stabilizes both wild-type and GOF mutant p53 independent on its kinase activity

Considering that the p53 signaling pathway plays critical roles in tumorigenesis [17] and our findings that MST4 exerts dual roles in LUAD tumorigenesis, we speculated that the complex phenotypes induced by MST4 knockout in lung cancer cells were related to p53. By examining p53 status, we found that both A549 and NCI-H460 cells harbor wild-type p53 (wtp53), while NCI-H1975 cells harbor p53 with a gain-of-function (GOF) mutation (R273H). Given that MST4 activates the p53 signaling pathway and modulates p53 protein levels, we hypothesized that MST4 stabilizes both wild-type p53 and GOF-mutant p53, thereby exerting tumor-suppressive functions in LUAD cells (A549) with wtp53 and oncogenic roles in LUAD cells with GOF-mutant p53 (NCI-H1975).

To test this hypothesis, we first explored how MST4 (as a kinase) regulates p53 protein levels. We co-transfected HA-p53, Flag-GFP, and increasing amounts of Flag-MST4 into cells; Western blot results showed that MST4 dose-dependently increased p53 protein levels driven by the exogenous promoter in the overexpression vector (Fig. 4a), confirming the post-transcriptional regulation of p53 by MST4. Interestingly, this regulation was kinase-independent, as the MST4 kinase-dead mutant (MST4K53R) still induced p53 protein accumulation (Fig. 4b). in both A549 and NCI-H1975 cell lines, overexpression of MST4 also upregulated p53 expression (Supplementary Fig. 7a). To further confirm this regulation reflects protein stabilization rather than mRNA stabilization, a cycloheximide chase (CHX) assay was performed, which showed that both MST4 and MST4K53R extended the protein half-life of p53 (Fig. 4c). Moreover, CHX chase assays were also performed in A549 and NCI-H1975 MST4 deficient cells, confirming that MST4 was able to stabilize both endogenously expressed wild-type and GOF-mutant p53 (Fig. 4d).

Fig. 4. MST4 stabilizes both wild-type and GOF mutant p53 independent on its kinase activity.

Fig. 4

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a The HEK293T cells were transfected with Myc-p53, Flag-GFP and increasing amounts of Flag-MST4 as indicated, 48 h after transfection, cell lysates were subjected to western blot analysis. GFP was used as transfection control and b Myc-p53, Flag-MST4 and Flag-MST4K53R were transfected into HEK293T, cell lysates were harvested 48 h after transfection for western blot analysis. c After transfecting with indicated expression constructs, CHX (50 μg/ml) was used to treated the cells for the indicated time, and then cells lysates were analyzed by western blot. d The relative protein level of p53 of MST4-deficient (MST4-sg1 or MST4-sg2) 239T cells when compared to control (Vector) 239T following CHX treatment. e Wild type and MST4-dificient A549/NCI-H1975 cells were treated with CHX (50 μg/ml) for the indicated time, followed by western blot analysis. f The relative protein level of p53 of MST4-sg A549/NCI-H1975 cells when compared to Vector A549/NCI-H1975 cells following CHX treatment. g Treated with MG132 (20 μM) for 12 h in wild type and MST4 deficient A549/NCI-H1975 cells, western blot analysis was employed to analyze cell lysates, and then the relative expression of p53 was determined. h In HEK293T cells, HA-p53, Flag-MST4 and Myc-Ub were transfected for 48 h accompanied with the treatment of MG132 (20 μM) for 12 h, and then cell lysates were submitted to western blot analysis. i Wild type and MST4-deficient A549/NCI-H1975 cells were treated with MG132 (20 μM) for 12 h, and then cells were lysed by RIPA followed with western blot analysis. j Myc-p53, Flag-MST4 and HA-Ub K48O were transfected into HEK293T cell, after treated by MG132 (20 μM) for 12 h, cells were lysed by RIPA followed by western blot analysis.

The regulation of p53 protein stability is largely mediated by the ubiquitin-proteasome system [38]. Immunoblotting results showed that proteasome inhibitor MG132 blocked the decrease of p53 protein in MST4-deficient A549 and NCI-H1975 cells (Fig. 4e), indicating that MST4 may prevent ubiquitin-proteasome-mediated degradation of both wild-type and GOF-mutant p53. Therefore, we also examined the effects of MST4 on p53 ubiquitination. Consistent with MST4-mediated p53 stabilization, p53 ubiquitination was reduced when MST4 was overexpressed (Fig. 4f). Meanwhile, the endogenous ubiquitin assays exhibited the increased ubiquitination of p53 in MST4-deficient A549 and NCI-H1975 cells (Fig. 4g), confirming that MST4 prevents ubiquitin-proteasome-mediated degradation of both wild-type and GOF-mutant p53. In addition, K48-linked ubiquitination is the major type of ubiquitination mediating p53 degradation [39], and our result showed that overexpression of MST4 prevented the K48-linked ubiquitination level of p53 (Fig. 4h). Collectively, these data demonstrated that MST4 prevents K48-linked ubiquitination and subsequent proteasome degradation of both wild-type and GOF-mutant p53 in a kinase-independent manner—this explains the cell-context (p53 status)-dependent dual roles of MST4 in LUAD tumorigenesis.

MST4 prevents MDM2-mediated p53 degradation by antagonizing their interaction

So far, we have found that MST4 stabilizes wild-type and GOF-mutant p53 via kinase-independent inhibition of K48-linked ubiquitination and degradation. However, the specific regulatory mechanism by which MST4 modulates p53 ubiquitination remains unclear. Given that MDM2 is the E3 ubiquitin ligase that mediates K48-linked ubiquitination and degradation of p53 [40], we first explored whether an interaction exists between MST4 and MDM2. While overexpressed MST4 stabilized p53 in a dose-dependent manner (Fig. 4a), it did not alter the protein level of overexpressed MDM2 (Supplementary Fig. 8a). Furthermore, a co-immunoprecipitation (Co-IP) assay demonstrated no interaction between MST4 and MDM2 (Supplementary Fig. 8b), indicating that MST4 does not directly affect MDM2. We therefore next examined whether MST4 interacts with p53. The exogenous Co-IP was conducted in HEK-293T cells, which demonstrated that MST4 interacts with p53 (Fig. 5a). Moreover, endogenous Co-IP assays in A549 and NCI-H1975 cells confirmed the physiological interaction between MST4 and wild-type p53 (in A549) or GOF-mutant p53 (in NCI-H1975), respectively (Fig. 5b, c). GST pull-down assays further supported a direct interaction between MST4 and p53 (Fig. 5d, e). Additionally, immunofluorescence experiments showed subcellular colocalization of MST4 and p53 in A549 cells (Fig. 5f), providing further evidence of their interaction. Interestingly, domain-mapping Co-IP experiments revealed that MST4 interacts with the DNA-binding domain (DBD; Δ100-200) of p53 (Fig. 5g), a region known to mediate MDM2 binding to p53 [41]. Combined with the above results, we thus hypothesized that the interaction between MST4 and p53 would antagonize the interaction between MDM2 for p53. As expected, exogenous Co-IP assays showed that the interaction between MDM2 and p53 was weakened in the presence of MST4 (Fig. 5h). To further validate this finding, we performed an endogenous Co-IP in NCI-H1975 cells, which confirmed that MST4 strongly impaired the endogenous interaction between MDM2 and p53 in physiological condition (Fig. 5i). In conclusion, these results suggest that MST4 prevents MDM2-mediated p53 degradation by competing with MDM2 for binding to the p53 (Fig. 5j).

Fig. 5. MST4 prevents MDM2-mediated p53 degradation by antagonizing their interaction.

Fig. 5

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a The HEK293T cells were transfected with either Myc-p53 or together with Flag-MST4, 48 h later, cell lysates were subjected to immunoprecipitated with anti-Flag antibody. Lysates from NCI-H1975 or A549 were immunoprecipitated with anti-MST4 antibody (b) or anti-p53 antibody (c), and IgG was used as a control (* Represents the mouse anti-heavy chain band). The HEK293T cells transfected with HA-p53 (d) or Flag-MST4 (e) for 48 h were lysed by RIPA, and then lysates were incubated with recombinant GST and GST-MST4 or GST-p53 proteins purified by glutathione beads, followed by western blot analysis. f Immunofluorescence staining of A549 cells with the indicated antibodies. The images were obtained using fluorescence microscope (Nikon). Scale bar (25 μm). g Co-IP experiments were performed to determine the interaction between full-length p53 or those with indicated deletions (showed in the topper part of this panel). Co-IP experiments were performed to determine the impact of MST4 expression on the interaction between overexpressed (h) or endogenous (i) p53 and MDM2. j A diagram illustrates the model of how MST4 stabilizes p53 by competing with MDM2 for p53 binding.

Targeting MST4 inhibits tumor growth in LUAD with GOF-mutant p53 by modulating p53 stability

As the most frequently mutated gene in cancer, p53 was identified as a key therapeutic target for tumors decades ago [17]. However, as a nuclear transcription factor, p53 lacks typical drug target characteristics (e.g., enzymatic active sites or extracellular binding domains) and has thus long been regarded as “undruggable.” Therefore, p53 regulatory genes have gained increased attention and several targeted therapeutic strategies have emerged in recent years.

Building on our earlier finding that MST4 promotes tumorigenesis by stabilizing gain-of-function (GOF)-mutant p53, we hypothesized that targeting MST4 could serve as a complementary therapeutic strategy for cancers harboring GOF-mutant p53. To test this hypothesis, we first validated the in vivo efficacy of MST4 targeting in cancer cells with GOF-mutant p53: we established xenograft models in nude mice by injecting either MST4-wild-type (WT) or MST4-knockout (KO) NCI-H1975 cells (which express GOF-mutant p53). As shown in Fig. 6a–c, tumors derived from MST4-KO NCI-H1975 cells exhibited significantly reduced size, weight, and volume compared to those from MST4-WT NCI-H1975 cells—supporting that MST4 is a potential therapeutic target for lung adenocarcinoma (LUAD) with GOF-mutant p53 in vivo.

Fig. 6. Targeting MST4 inhibits tumor growth in LUAD with GOF-mutant p53 by modulating p53 stability.

Fig. 6

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a The images of tumors from xenograft assay with control (Vector) and MST4-deficient (MST4-sg1 or MST4-sg2) NCI-H1975 cells. b Curves showing quantification results of the tumor volumes of tumors measured at indicated days after implantation from the xenograft assay. c Quantification of the harvested tumors from the xenograft assay. d Immunoblotting results showing the p53 and MST4 protein in NCI-H1975 cells transfected with indicated plasmids. Vec Vector, sg single guide RNA, LE long exposure, SE short exposure. e Curves showing cell proliferation of cells the same as used in (d). f Representative colony formation images of the cells the same as used in (d). The data are presented as the means ± SEM. Statistical significance was analyzed by ANOVA. ****p < 0.0001.

To confirm that the tumor-suppressive effect of MST4 targeting is mediated by inhibiting GOF-mutant p53, we generated p53-knockdown (KD) NCI-H1975 cells based on both MST4-WT and MST4-KO backgrounds. Immunoblotting results showed that MST4 targeting (i.e., MST4 KO) downregulated GOF-mutant p53 protein levels; notably, this effect was largely abrogated in p53-KD cells (Fig. 6d). Consistent with the changes in GOF-mutant p53 levels, the antiproliferative effect of MST4 targeting in NCI-H1975 cells was also significantly diminished when p53 was knocked down (Fig. 6e). Similar results were observed in colony formation assays (Fig. 6f). Collectively, these data demonstrate that MST4 is a potential therapeutic target for treating LUAD with GOF-mutant p53, as its targeting modulates the stability of GOF-mutant p53.

Discussion

In this study, we identify MST4 as a key cell-context-dependent regulator of LUAD, with its function determined by p53 mutation status. We demonstrate that MST4 stabilizes both wtp53 and GOF-mutant p53 via a kinase-independent, MDM2-competitive mechanism, and targeting MST4 provides a novel therapeutic strategy for GOF-mutant p53 LUAD. These findings address critical gaps in our understanding of MST4 biology and p53-targeted therapy.

A major unexpected finding is MST4’s dual roles in LUAD: tumor-suppressive in A549 and NCI-H460 (wtp53) cells and oncogenic in NCI-H1975 (GOF-mutant p53) cells. This contrasts with previous reports of MST4’s exclusive oncogenic function in other cancers [30, 33], highlighting the importance of tumor type and genetic background in defining kinase function. The key driver of this duality is MST4’s ability to stabilize both wtp53 and GOF-mutant p53: in wtp53 cells, MST4-enhanced wtp53 activity restrains proliferation (tumor suppression), while in GOF-mutant p53 cells, MST4-stabilized GOF p53 promotes oncogenesis. This aligns with growing evidence that “contextual oncogenes” or “conditional tumor suppressors” are common in cancer, with their function dictated by co-occurring genetic alterations [42–44].

A critical mechanistic novelty is MST4’s kinase-independent regulation of p53. MST kinases typically exert their functions via phosphorylation [29], but our data show that the MST4 kinase-dead mutant (MST4K53R) retains full ability to stabilize p53. This is consistent with recent reports of kinase-independent roles for MST family members [45], expanding our understanding of MST4’s functional repertoire.

We further demonstrate that MST4 stabilizes p53 by competing with MDM2 for binding to the p53 DBD. This mechanism is distinct from existing p53 stabilizers (e.g., MDM2 inhibitors, which block MDM2 activity directly) [17], as MST4 acts as a “physical competitor” rather than an enzymatic inhibitor. Importantly, this competition inhibits K48-linked ubiquitination of both wtp53 and GOF-mutant p53, explaining MST4’s ability to stabilize both p53 isoforms. Domain-mapping studies confirm MST4’s interaction with the p53 DBD, and GST pull-down assays validate a direct MST4-p53 interaction—strengthening the mechanistic rigor.

Our preclinical data highlight MST4 as a promising therapeutic target for GOF-mutant p53 LUAD. Targeting MST4 (via CRISPR knockout) suppressed tumor growth in NCI-H1975 (R273H p53) xenografts, and this effect was abrogated by p53 knockdown—confirming that MST4’s oncogenic function is GOF-mutant p53-dependent. This is clinically relevant because GOF-mutant p53 was frequently observed in LUAD patients [46], and no approved therapies specifically target GOF-mutant p53. MST4 targeting offers a complementary strategy to existing MDM2 inhibitors (which only work in wtp53 tumors), addressing an unmet need.

This study has several limitations. First, our functional assays rely on cell lines and xenografts; future studies using genetically engineered mouse models (GEMMs) of LUAD with defined p53 status will better recapitulate the in vivo tumor microenvironment and validate MST4’s role. Second, we focus on three LUAD cell lines (A549, NCI-H1975 and NCI-H460); expanding to additional cell lines with diverse p53 mutations (e.g., R175H, G245S) will confirm the generality of MST4’s mechanism. Third, while we demonstrate MST4’s potential as a target, small-molecule MST4 inhibitors are not yet available; developing such inhibitors (especially those targeting MST4-p53 interaction) will be critical for clinical translation.

Future directions also include exploring MST4’s role in other p53-mutant cancers (e.g., breast, ovarian cancer) to determine if its mechanism is conserved. Additionally, investigating the upstream regulators of MST4 in LUAD (e.g., growth factors, oncogenic signaling) could reveal strategies to indirectly target MST4. Finally, integrating MST4 expression and p53 mutation status into clinical decision-making may help stratify LUAD patients for personalized therapies.

In summary, our study identifies MST4 as a context-dependent regulator of LUAD, with its function determined by p53 mutation status. MST4 stabilizes both wtp53 and GOF-mutant p53 via a kinase-independent, MDM2-competitive mechanism, and targeting MST4 suppresses GOF-mutant p53 LUAD growth (Fig.7). These findings not only advance our understanding of MST4 and p53 biology but also provide a novel therapeutic target for GOF-mutant p53-driven LUAD—addressing a critical unmet clinical need.

Fig. 7.

Fig. 7

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Schematic model on how does MST4 play dual-roles in lung adenocarcinoma by regulating homeostasis of wild-type and mutant p53.

Supplementary information

Supplementary Figs. 1-8 (737KB, pdf)
Supplementary Tables 1-3 (1.3MB, xlsx)

Acknowledgements

The authors would like to thank Prof. Yide Mei (School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China) for assistance with experimental skills and instrumental help.

Author contributions

MZ, ZZ, and SL conceived the study design and performed the literature search. MZ, CH, ZZ and HL drafted the manuscript. MZ, CH, HL, and CZ designed and performed the experiments. MZ, CH, HL, CZ, QS, YD, KY, JZ, and YZ revised the manuscript. ZH and HW conducted data collection and analysis. SL and ZZ supervised the study. All authors read and approved the final version of the manuscript.

Funding

This work was supported by: the National Natural Science Foundation of China (grant No. 32270760); the Fundamental Research Funds for the National Key R&D Project (grant No. 2022YFF0710700).

Data availability

All datasets and raw data generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Minghui Zhang, Changwei Hu, Huilong Liu.

Contributor Information

Minghui Zhang, Email: zhangminghui160@163.com.

Ze-Yan Zhang, Email: zhangzeyan@ncu.edu.cn.

Shangze Li, Email: shangze.li@cqu.edu.cn.

Supplementary information

The online version contains supplementary material available at 10.1038/s41388-026-03824-7.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figs. 1-8 (737KB, pdf)
Supplementary Tables 1-3 (1.3MB, xlsx)

Data Availability Statement

All datasets and raw data generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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