Role of ARAF mutation in RAF inhibitor resistance in lung cancer cells
10.3760/cma.j.cn115355-20240118-00040
- VernacularTitle:ARAF突变在肺癌细胞RAF抑制剂耐药中的作用
- Author:
Yaoyuan WANG
1
;
Juan CHEN
;
Wei HOU
Author Information
1. 陕西省核工业二一五医院呼吸科，咸阳 712000
- Keywords:
Lung neoplasms;
Proto-oncogene proteins A-raf;
Mutation;
Drug resistance, neoplasm;
RAF inhibitor
- From:
Cancer Research and Clinic
2024;36(10):728-733
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the role of ARAF mutation in RAF inhibitor resistance in lung cancer cells.Methods:The lung cancer cell lines A549 and NCI-H1688 with ARAF gene knockout were constructed using CRISPR-Cas9 method. Overexpression of wild-type ARAF (ARAF WT) and mutant ARAF (ARAF V145L, S214F, S214C, or D429A) in lung cancer cells with ARAF gene knockout was achieved through lentiviral packaging infection method, while the control group consisted of untreated A549 or NCI-H1688 cells with ARAF gene knockout. A549 or NCI-H1688 cells in each group were treated with different concentrations of RAF inhibitor belvarafenib or AZ-628. The CCK-8 method was used to detect cell viability, and the half maximal inhibitory concentration ( IC50) of cells in each group against belvarafenib or AZ-628 was calculated. A549 or NCI-H1688 cells in each group were treated with IC50 (263 nmol/L) of belvarafenib in the ARAF WT group, and the expression levels of key proteins in the ARAF and MEK/ERK/RSK signaling pathways were detected by Western blotting. The 6-8 week old male BALB/c nude mice were selected and subcutaneously injected stable overexpression of ARAF WT or mutant ARAF A549 or NCI-H1688 cell suspension with ARAF gene knockout into the axilla to construct a xenograft tumor model, and 30 mg/kg belvarafenib or AZ-628 solution was administered continuously by gavage per day; tumor volume was compared among the groups at the 16th day. Results:Compared with the ARAF WT group, the IC50 of belvarafenib or AZ-628 in A549 or NCI-H1688 cells of the ARAF D429A group was lower, while the IC50 of the ARAF S214F and ARAF S214C groups was higher, and the differences were statistically significant (all P < 0.05). Compared with the ARAF WT group, the relative expression levels of p-MEK, p-ERK and p-RSK in A549 or NCI-H1688 cells treated with belvarafenib in the ARAF WT and ARAF D429A groups were all lower, and the differences were statistically significant (all P < 0.05), but there were no statistically significant differences in the relative expression levels of ARAF, MEK, ERK, and RSK (all P > 0.05); compared with the ARAF WT group, the ARAF S214F and ARAF S214C groups treated with belvarafenib had higher relative expression levels of p-MEK, p-ERK and p-RSK, and the differences were statistically significant (all P < 0.05), but there were no statistically significant differences in the relative expression levels of ARAF, MEK, ERK, and RSK (all P > 0.05). On the 16th day after gavage with belvarafenib or AZ-628, the tumor volume of nude mice transplanted with A549 or NCI-H1688 cells in ARAF WT and ARAF D429A groups was lower than that in the corresponding ARAF WT and ARAF D429A groups without belvarafenib or AZ-628 intervention, and the tumor volume of nude mice transplanted with A549 or NCI-H1688 cells in ARAF D429A group was lower than that in the corresponding ARAF WT group with belvarafenib or AZ-628 gavage, and the differences were statistically significant (all P < 0.05). On the 16th day after gavage with belvarafenib or AZ-628, the tumor volume of nude mice transplanted with A549 or NCI-H1688 cells in ARAF S214F and ARAF S214C groups was not significantly different from that in ARAF S214F and ARAF S214C groups without belvarafenib or AZ-628 intervention (all P > 0.05), and compared with nude mice transplanted with A549 or NCI-H1688 cells in ARAF WT group with belvarafenib or AZ-628 gavage, there was no significant difference (all P > 0.05). Conclusions:The ARAF D429A mutation increases the sensitivity of lung cancer to RAF inhibitors, while the ARAF S214F and S214C mutations increase the resistance of lung cancer to RAF inhibitors.
