Related Experiment Video
Updated: Aug 6, 2026
Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure
Published on: August 11, 2017
Targeting the adaptor protein Gab1 overcomes MET/Src-driven alectinib resistance in ALK-rearranged NSCLC
Hengyi Chen1, Caiyu Lin2, Chen Hu2
1Department of Pulmonary and Critical Care Medicine, The Seventh People's Hospital of Chongqing, Chongqing, China.
Background:
In non-small cell lung cancer (NSCLC) with anaplastic lymphoma kinase (ALK) rearrangement, bypass signaling activation commonly leads to resistance against alectinib. Identifying key molecular targets that integrate signals from resistance-driving kinases is crucial for overcoming this resistance. However, these targets have not yet been identified.
Methods:
We generated alectinib-resistant (AR) subclones from H3122 and H2228 cells. Subsequently, we analyzed bypass signaling pathways using western blotting, evaluated drug sensitivity with Cell Counting Kit-8 (CCK-8) assays. To assess the function of Grb2-associated binder 1 (Gab1), we employed siRNA-mediated knockdown and lentiviral shRNA both in vitro and in xenograft models. Additionally, we determined the stability of Gab1 through cycloheximide chase assays.
Results:
AR cells demonstrated concurrent activation of the mesenchymal epithelial transition receptor tyrosine kinase (MET) and Rous sarcoma oncogene cellular homolog non-receptor tyrosine kinase (Src), accompanied by upregulation of Gab1. The combined inhibition of MET and Src, as opposed to single-agent blockade, effectively suppressed Gab1/protein kinase B (AKT) signaling and restored sensitivity to alectinib. Gab1 knockdown mirrored the effects of dual kinase inhibition, by disrupting MET/Src/AKT signaling and resensitizing cells to alectinib. Mechanistically, the elevation of Gab1 resulted from post - translational stabilization, with a significantly extended half - life in resistant cells. In vivo, silencing Gab1 inhibited the growth of H3122-AR2 xenografts without causing systemic toxicity, which correlated with decreased phosphorylation of MET, Src and AKT in tumor tissues.
Conclusion:
In ALK-positive NSCLC with acquired resistance to alectinib, Gab1 emerged as a crucial downstream signaling convergence target of MET and Src co-activation. Targeting this adaptor protein presented a promising therapeutic strategy to overcome bypass-mediated resistance.
Insights
Grb2-associated binder 1 (Gab1) is a key target in overcoming alectinib resistance in non-small cell lung cancer (NSCLC). Silencing Gab1 resensitizes cancer cells to alectinib by disrupting MET/Src/AKT signaling.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Therapeutics
Background:
- Acquired resistance to alectinib in anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) is often driven by bypass signaling pathways.
- Identifying molecular targets that integrate signals from resistance-driving kinases is critical for developing effective treatment strategies.
- Current therapeutic approaches have not yet identified key targets for overcoming alectinib resistance.
Purpose of the Study:
- To identify and characterize key molecular targets involved in alectinib resistance in ALK-positive NSCLC.
- To investigate the role of Grb2-associated binder 1 (Gab1) in mediating resistance to alectinib.
- To evaluate Gab1 as a potential therapeutic target for overcoming acquired resistance.
Main Methods:
- Generation of alectinib-resistant (AR) NSCLC cell lines (H3122, H2228).
- Analysis of bypass signaling pathways via western blotting and drug sensitivity assays (CCK-8).
- Functional assessment of Gab1 using siRNA/shRNA knockdown and in vivo xenograft models; Gab1 stability determined by cycloheximide chase assays.
Main Results:
- Alectinib-resistant cells exhibited co-activation of MET and Src kinases, with increased Gab1 levels.
- Combined inhibition of MET and Src, or Gab1 knockdown, suppressed Gab1/AKT signaling and restored alectinib sensitivity.
- Gab1 elevation resulted from post-translational stabilization, increasing its half-life in resistant cells.
- In vivo, Gab1 silencing inhibited tumor growth and decreased MET, Src, and AKT phosphorylation without systemic toxicity.
Conclusions:
- Gab1 serves as a critical downstream signaling convergence target of MET and Src co-activation in acquired alectinib resistance in ALK-positive NSCLC.
- Targeting Gab1 represents a promising therapeutic strategy to overcome bypass signaling-mediated resistance.
- Gab1's role as an adaptor protein makes it a viable target for novel combination therapies.
More Related Videos
04:36Comet Assay to Quantify DNA Damage in FLT3 Mutant-expressing 32D Cells after Exposure to Type I and Type II FLT3 Inhibitors
Published on: October 17, 2025
09:38Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib
Published on: June 26, 2019
