Integrated 2D-3D Proteomic Profiling Identifies MLK4 as a Microenvironment-Responsive Regulator of Chemotherapeutic
Wannawat Khotchawan1, Pakorn Ruengket2, Pakpoom Kheolamai3,4
1Siriraj Center of Excellence for Stem Cell Research (SiSCR), Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Background:
Therapeutic resistance is a major cause of treatment failure in glioblastoma (GBM), highlighting the need for physiologically relevant models to identify actionable resistance mechanisms. While two-dimensional (2D) cultures are widely used for target discovery, they poorly represent the tumor microenvironment. In contrast, three-dimensional (3D) spheroid cultures better recapitulate spatial heterogeneity, hypoxic gradients, and stress-adaptive signaling observed in tumors.
Methods:
We applied an integrated 2D-3D quantitative proteomic approach to identify microenvironment-dependent regulators of chemoresistance in GBM. Proteomic profiling was performed in U87MG and U251MG cells grown as 2D monolayers or 3D spheroids. Differentially expressed proteins were validated by quantitative RT-PCR, and functional studies were conducted using genetic depletion followed by assessment of temozolomide (TMZ) sensitivity.
Results:
Comparative analysis identified 13 proteins consistently differentially expressed between 2D and 3D cultures: NDUFB5, RNGTT, MLK4, SYN1, DDX5, EIF2AK2, ITGA1, ZNF33B, ZNF343, WDR19, JPH3, CCT8L2, and FNDC3A. Among these, Mixed Lineage Kinase 4 (MLK4) showed strong and reproducible upregulation in 3D spheroids in both GBM cell lines. Genetic depletion of MLK4 significantly increased TMZ sensitivity without affecting basal cell viability, suggesting a specific role in therapy response. Notably, MLK4 expression was induced only under 3D conditions.
Conclusion:
MLK4 functions as a microenvironment-responsive regulator of chemoresistance in GBM. These findings demonstrate that 3D culture systems reveal clinically relevant resistance pathways not detectable in conventional 2D models and highlight 3D proteomic profiling as a powerful strategy for identifying therapeutically actionable targets.
Insights
Three-dimensional (3D) glioblastoma models reveal Mixed Lineage Kinase 4 (MLK4) as a key regulator of chemoresistance. Targeting MLK4 enhances sensitivity to temozolomide (TMZ) therapy.
Area of Science:
- Oncology
- Molecular Biology
- Proteomics
Background:
- Therapeutic resistance significantly impacts glioblastoma (GBM) treatment outcomes.
- Traditional 2D cell cultures inadequately represent the complex tumor microenvironment.
- Three-dimensional (3D) spheroid cultures offer a more physiologically relevant model for studying GBM.
Purpose of the Study:
- To identify microenvironment-dependent regulators of chemoresistance in GBM using a 2D-3D proteomic approach.
- To investigate the role of differentially expressed proteins in GBM chemoresistance.
- To validate potential therapeutic targets for overcoming treatment failure in GBM.
Main Methods:
- Integrated 2D and 3D quantitative proteomic profiling of GBM cell lines (U87MG, U251MG).
- Validation of differentially expressed proteins using quantitative RT-PCR.
- Functional studies involving genetic depletion and assessment of temozolomide (TMZ) sensitivity.
Main Results:
- Thirteen proteins were consistently differentially expressed between 2D and 3D GBM cultures.
- Mixed Lineage Kinase 4 (MLK4) was significantly upregulated in 3D spheroids.
- Genetic depletion of MLK4 enhanced TMZ sensitivity without affecting cell viability, indicating a specific role in resistance.
Conclusions:
- MLK4 acts as a microenvironment-responsive regulator of chemoresistance in GBM.
- 3D culture systems uncover clinically relevant resistance pathways missed by 2D models.
- 3D proteomic profiling is a valuable strategy for identifying novel therapeutic targets in GBM.
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