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Simultaneous inhibition of FAK and ROS1 synergistically repressed triple-negative breast cancer by upregulating p53
Ximin Tan1, Deguang Kong2, Zhuoli Tao3
1Department of Oncology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Background:
Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype lacking effective targeted therapies, necessitating innovative treatment approaches. While targeting ROS proto-oncogene 1 (ROS1) with crizotinib has shown promise, resistance remains a limitation. Recent evidence links focal adhesion kinase (FAK) to drug resistance, prompting our study to assess the combined impact of FAK inhibitor IN10018 and crizotinib in TNBC and elucidate the underlying mechanisms.
Methods:
We employed the Timer database to analyze FAK and ROS1 mRNA levels in TNBC and adjacent normal tissues. Furthermore, we investigated the correlation between FAK, ROS1, and TNBC clinical prognosis using the GSE database. We conducted various in vitro assays, including cell viability, colony formation, flow cytometry, EdU assays, and western blotting. Additionally, TNBC xenograft and human TNBC organoid models were established to assess the combined therapy's efficacy. To comprehensively understand the synergistic anti-tumor mechanisms, we utilized multiple techniques, such as RNA sequencing, immunofluorescence, cell flow cytometry, C11-BODIPY staining, MDA assay, and GSH assay.
Results:
The Timer database revealed higher levels of FAK and ROS1 in TNBC tissues compared to normal tissues. Analysis of GEO databases indicated that patients with high FAK and ROS1 expression had the poorest prognosis. Western blotting confirmed increased p-FAK expression in crizotinib-resistant TNBC cells. In vitro experiments showed that the combination therapy down-regulated cyclin B1, p-Cdc2, and Bcl2 while up-regulating BAX, cleaved-Caspase-3, cleaved-Caspase-9, and cleaved PARP. In TNBC xenograft models, the tumor volume in the combination therapy group was 73% smaller compared to the control group (p < 0.0001). Additionally, the combination therapy resulted in a 70% reduction in cell viability in human TNBC organoid models (p < 0.0001). RNA sequencing analysis of TNBC cells and xenograft tumor tissues highlighted enrichment in oxidative stress, glutathione metabolism, and p53 pathways. The combined group displayed a fivefold rise in the reactive oxygen species level, a 69% decrease in the GSH/GSSG ratio, and a sixfold increase in the lipid peroxidation in comparison to the control group. Western blotting demonstrated p53 upregulation and SCL7A11 and GPX4 downregulation in the combination group. The addition of a p53 inhibitor reversed these effects.
Conclusion:
Our study demonstrates that the combination of IN10018 and crizotinib shows synergistic antitumor effects in TNBC. Mechanistically, this combination inhibits cell proliferation, enhances apoptosis, and induces ferroptosis, which is associated with increased p53 levels.
Insights
Combining FAK inhibitor IN10018 with crizotinib shows synergistic antitumor effects in triple-negative breast cancer (TNBC). This novel combination therapy inhibits proliferation, enhances apoptosis, and induces ferroptosis by increasing p53 levels.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Therapeutics
Background:
- Triple-negative breast cancer (TNBC) lacks targeted therapies, and resistance to ROS1 inhibitors like crizotinib is a challenge.
- Focal adhesion kinase (FAK) is implicated in drug resistance, suggesting it as a potential therapeutic target in TNBC.
Purpose of the Study:
- To evaluate the combined efficacy of FAK inhibitor IN10018 and crizotinib in TNBC.
- To elucidate the underlying anti-tumor mechanisms of this combination therapy.
Main Methods:
- Analysis of FAK and ROS1 expression in TNBC tissues and correlation with prognosis using TIMER and GEO databases.
- In vitro assays (viability, colony formation, flow cytometry, EdU, western blotting) and in vivo models (TNBC xenografts, organoids).
- Mechanistic studies including RNA sequencing, immunofluorescence, oxidative stress assays, and western blotting for key proteins and pathways.
Main Results:
- Higher FAK and ROS1 expression correlated with poorer TNBC prognosis.
- Combination therapy significantly reduced TNBC cell viability and tumor growth in vivo.
- The combination induced apoptosis and ferroptosis, evidenced by altered protein expression (e.g., BAX, cleaved-Caspase-3, GPX4) and increased reactive oxygen species.
Conclusions:
- The combination of IN10018 and crizotinib exhibits synergistic anti-tumor activity in TNBC.
- This combination therapy effectively inhibits proliferation, promotes apoptosis, and induces ferroptosis, partly through p53 pathway activation.
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