ASIC3-PLCG1 axis-driven macropinocytosis promotes osimertinib drug-tolerant persistence in malignant pleural
Wen-Chien Huang1, Chieh-Yung Wang2, Ying Chen3
1Department of Medicine, MacKay Medical College, New Taipei City 252, Taiwan, Republic of China; Division of Thoracic Surgery, Department of Surgery, MacKay Memorial Hospital, Taipei 104, Taiwan, Republic of China.
Background:
Non-small cell lung cancer (NSCLC) frequently develops resistance to EGFR-tyrosine kinase inhibitors (TKIs), particularly in malignant pleural effusion (MPE), where a highly acidic tumor microenvironment promotes the emergence of drug-tolerant persister (DTP) cells. Building upon previous evidence that MPE acidity drives metabolic plasticity, this study investigates how acid-sensing ion channel 3 (ASIC3) coordinates PLCG1-dependent macropinocytosis to maintain osimertinib tolerance.
Methods:
Primary MPE-derived NSCLC cultures underwent next-generation sequencing to define mutational heterogeneity. Acidic (pH 6.5-6.8) conditions were used to generate osimertinib-induced DTP (Osi-DTP) models. Functional assays assessed viability, invasion, colony formation, autophagy, and macropinocytosis. ASIC3 was silenced using shRNA, followed by Seahorse metabolic analysis. Transcriptomic profiling identified differentially expressed genes. ASIC3-targeted therapeutic interventions were evaluated in vitro and in xenograft models.
Results:
MPE-derived NSCLC cultures showed substantial genomic diversity, including EGFR exon 19 deletion and T790M mutations. Acid-adapted Osi-DTP cells exhibited EMT-like phenotypes, reduced proliferation, elevated stemness markers, and strong activation of stress-response pathways. ASIC3 was consistently upregulated under acidic conditions and drove PLCG1-mediated macropinocytosis to support nutrient scavenging and survival. ASIC3 knockdown markedly reduced DTP cell viability, invasiveness, colony formation, autophagy, and glycolysis, while inducing a metabolic shift toward oxidative phosphorylation. In vivo, combining an ASIC3 inhibitor with osimertinib significantly delayed tumor progression and improved survival without added toxicity. Clinically, high ASIC3 expression correlated with poor overall survival and increased autophagy-associated markers.
Conclusion:
ASIC3 is a central regulator of acidosis-driven drug persistence in MPE-associated NSCLC, sustaining osimertinib tolerance through PLCG1-dependent macropinocytosis and metabolic reprogramming. Targeting ASIC3 restores TKI sensitivity by disrupting nutrient acquisition, autophagy, and metabolic adaptation, representing a promising therapeutic strategy to prevent or delay EGFR-TKI resistance.
Insights
Acid-sensing ion channel 3 (ASIC3) drives drug resistance in non-small cell lung cancer (NSCLC) with malignant pleural effusion by promoting nutrient uptake. Targeting ASIC3 may restore sensitivity to EGFR-tyrosine kinase inhibitors (TKIs).
Area of Science:
- Oncology
- Molecular Biology
- Cancer Research
Background:
- Non-small cell lung cancer (NSCLC) often develops resistance to EGFR-tyrosine kinase inhibitors (TKIs).
- Malignant pleural effusion (MPE) presents a highly acidic tumor microenvironment that promotes drug-tolerant persister (DTP) cells.
- MPE acidity influences metabolic plasticity, necessitating investigation into mechanisms of TKI resistance.
Purpose of the Study:
- To investigate the role of acid-sensing ion channel 3 (ASIC3) in coordinating PLCG1-dependent macropinocytosis.
- To understand how ASIC3 maintains osimertinib tolerance in NSCLC within an acidic MPE environment.
- To explore ASIC3 as a potential therapeutic target for overcoming TKI resistance.
Main Methods:
- Generated osimertinib-induced DTP (Osi-DTP) models under acidic conditions (pH 6.5-6.8).
- Assessed functional assays including viability, invasion, colony formation, autophagy, and macropinocytosis.
- Utilized ASIC3 silencing (shRNA), Seahorse metabolic analysis, transcriptomic profiling, and in vivo xenograft models.
Main Results:
- Acid-adapted Osi-DTP cells exhibited EMT-like phenotypes, reduced proliferation, and activated stress-response pathways.
- ASIC3 upregulation under acidic conditions drove PLCG1-mediated macropinocytosis for nutrient scavenging and survival.
- ASIC3 inhibition reduced DTP cell viability, invasiveness, and glycolysis, shifting metabolism to oxidative phosphorylation; combination therapy improved outcomes in vivo.
- High ASIC3 expression correlated with poor survival and increased autophagy markers in patients.
Conclusions:
- ASIC3 is crucial for acidosis-driven drug persistence in MPE-associated NSCLC, mediating osimertinib tolerance via macropinocytosis and metabolic reprogramming.
- Targeting ASIC3 disrupts nutrient acquisition and metabolic adaptation, restoring TKI sensitivity.
- ASIC3 inhibition represents a promising strategy to combat EGFR-TKI resistance in NSCLC.
