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. 2024 Aug 14;22(1):397.
doi: 10.1186/s12964-024-01783-8.

Activity-based protein profiling and global proteome analysis reveal MASTL as a potential therapeutic target in gastric cancer

Kyoung-Min Choi #  1 , Sung-Jin Kim #  1 , Mi-Jung Ji  2 , Eunjung Kim  3 , Jae-Sung Kim  4 , Hyun-Mee Park  2 , Jae-Young Kim  5
Affiliations

Activity-based protein profiling and global proteome analysis reveal MASTL as a potential therapeutic target in gastric cancer

Kyoung-Min Choi et al. Cell Commun Signal. .

Abstract

Background: Gastric cancer (GC) is a prevalent malignancy with limited therapeutic options for advanced stages. This study aimed to identify novel therapeutic targets for GC by profiling HSP90 client kinases.

Methods: We used mass spectrometry-based activity-based protein profiling (ABPP) with a desthiobiotin-ATP probe, combined with sensitivity analysis of HSP90 inhibitors, to profile kinases in a panel of GC cell lines. We identified kinases regulated by HSP90 in inhibitor-sensitive cells and investigated the impact of MASTL knockdown on GC cell behavior. Global proteomic analysis following MASTL knockdown was performed, and bioinformatics tools were used to analyze the resulting data.

Results: Four kinases-MASTL, STK11, CHEK1, and MET-were identified as HSP90-regulated in HSP90 inhibitor-sensitive cells. Among these, microtubule-associated serine/threonine kinase-like (MASTL) was upregulated in GC and associated with poor prognosis. MASTL knockdown decreased migration, invasion, and proliferation of GC cells. Global proteomic profiling following MASTL knockdown revealed NEDD4-1 as a potential downstream mediator of MASTL in GC progression. NEDD4-1 was also upregulated in GC and associated with poor prognosis. Similar to MASTL inhibition, NEDD4-1 knockdown suppressed migration, invasion, and proliferation of GC cells.

Conclusions: Our multi-proteomic analyses suggest that targeting MASTL could be a promising therapy for advanced gastric cancer, potentially through the reduction of tumor-promoting proteins including NEDD4-1. This study enhances our understanding of kinase signaling pathways in GC and provides new insights for potential treatment strategies.

Keywords: Activity-based Protein Profiling; Gastric cancer; MASTL; NEDD4-1; Proteomics.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Identification of HSP90 client kinases through LC–MS-based activity-based protein profiling (ABPP). (A) Workflow for HSP90 client kinome profiling via desthiobiotin-ATP probe-based ABPP in GC. GC cells were incubated with vehicle control (DMSO) or 100 nM of AUY922 for 48 h and harvested (a), followed by desthiobiotin-ATP probe labeling (b). Samples were digested with trypsin (c), labeled peptides were captured by streptavidin agarose beads (d) and analyzed by LC–MS/MS (e). MaxQuant software was used to analyze LC–MS data (f). (B) HSP90 client kinases in AUY922-sensitive GC cells were illustrated in the kinome tree. (C) Common HSP90 client kinases across GC cell lines were illustrated in a Venn diagram
Fig. 2
Fig. 2
MASTL is highly expressed in GC and knockdown of MASTL inhibited proliferation, migration and invasion of GC cells. A Differential MASTL mRNA expression was analyzed in gastric tumors (T) and normal tissues (N) using the GEPIA. B Expression analysis of MASTL in different stages of GC using UALCAN database. Statistical analyses are shown in Table S3. C GC cells were transfected with siControl or siMASTL, and the effect of MASTL knockdown on proliferation of GC cells was analyzed by clonogenic assay. D GC cells were transfected with siControl or siMASTL #2, and the effect of MASTL knockdown on GC migration was analyzed by wound healing assay. E AGS cells were transfected with siControl and siMASTL #2, and the effect of MASTL knockdown on AGS migration was analyzed by Transwell assay. F SNU484 cells were transfected with siControl and siMASTL #1, and the effect of MASTL knockdown on SNU484 invasion was analyzed by invasion assay. Representative images of migrated or invaded cells on the membrane (magnification: 200x) are shown. G Effect of MASTL knockdown on MMP-1 expression was analyzed by qPCR. Error bars indicate the standard deviation of representative triplicates from at least three experiments, which showed similar results. NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001
Fig. 3
Fig. 3
Analysis of global proteome in MASTL-deficient GC cells revealed MAST-regulated protein candidates. (A) Workflow for MASTL-regulated proteome profiling via global proteome analysis in GC. SNU484 cells were transfected with two different MASTL targeting siRNAs or control siRNA for 48 h (a), followed by in-gel digestion (b). The resulting tryptic peptides were analyzed by LC–MS/MS (c). MaxQuant software was used to analyze LC–MS data (d). (B) Differentially expressed proteins (DEPs) in MASTL-deficient GC cells were visualized by volcano plot. Non-axial vertical and horizontal lines denote twofold change and p = 0.05, respectively. (C) Commonly decreased and increased DEPs were illustrated using Venn diagrams. (D) Metascape analysis of down-regulated proteins. (E) Metascape analysis of up-regulated proteins
Fig. 4
Fig. 4
NEDD4-1 is upregulated in GC and associated with poor prognosis of patients with GC; inhibition of NEDD4-1 reduced proliferation, migration and invasion of GC cells. A Kaplan–Meier survival curves were generated to compare overall survival (OS) of stomach adenocarcinoma (STAD) patients with high and low NEDD4-1 expression using Kaplan–Meier Plotter (https://kmplot.com/analysis/). NEDD4-1 expression was categorized based on microarray data with an expression range of 32–1084, using a cutoff value of 327. Patients with NEDD4-1 expression ranging from 32 to 327 were classified as the low expression group, while those with expression from 327 to 1084 were classified as the high expression group. B GC cells were transfected with control or siRNA targeting MASTL for 48 h and expressions of MASTL, NEDD4-1, p-AKT (Ser 473) were analyzed by western blotting. C GC cells were transfected with siControl and siNEDD4-1 and the effect of NEDD4-1 knockdown on proliferation in GC cells was analyzed by clonogenic assay. D GC cells were transfected with siControl and siNEDD4-1 #1 and the effect of NEDD4-1 knockdown on GC migration was analyzed by wound healing assay. E AGS cells were transfected with siControl and siNEDD4-1 #1 and the effect of NEDD4-1 knockdown on AGS migration was analyzed by transwell migration assay. F SNU484 cells were transfected with siControl and siNEDD4-1 #1, and transwell invasion assay was performed. Representative images of migrated or invaded cells on the membrane (magnification: 200x) are shown. Error bars indicate the standard deviation of representative triplicates from at least three experiments, which showed similar results. NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001
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