Formononetin Inhibits Tumor Growth by Suppression of EGFR Signaling and Destabilization of Mcl-1 Protein in Non-Small Cell Lung Cancer

Background: Epidermal growth factor receptor (EGFR) activating mutations play crucial roles in the tumorigenesis of human non-small cell lung cancer (NSCLC). The mechanism regarding how EGFR signaling regulates myeloid cell leukemia sequence 1 (Mcl-1) protein stability and ubiquitination remains undefined. Methods: MTS assay was used for natural product library screening. The effect of formononetin on NSCLC cells was determined by MTS assay and soft agar assay. Molecular modeling was performed to analyze the potential different binding modes between formononetin and EGFR WT or mutants. The inhibitory effect of formononetin on EGFR signaling and Mcl-1 expression were examined by immunoblot, in vitro kinase assay, in vitro pulldown and ATP competition assays, co-immunoprecipitation assay, ubiquitination analysis, in vivo xenograft model, and immunohistochemical staining. Findings: Formononetin was identified as an EGFR inhibitor by a 98 commercially available natural product screening. Formononetin suppresses WT and mutant EGFR kinases activity in vitro, ex vivo, and in vivo. Molecular modeling indicates that formononetin docks into the ATP-binding pocket of both WT and mutant EGFR. Formononetin inhibits EGFR-Akt signaling, which in turn activates GSK3β and promotes Mcl-1 phosphorylation in NSCLC cells. Treatment with formononetin enhances the interaction between Mcl-1 and SCFFbw7, which eventually promotes Mcl-1 ubiquitination and degradation. Depletion of either GSK3β or SCFFbw7 compromised formononetin-induced Mcl-1 downregulation. Finally, formononetin inhibits the in vivo tumor growth in a xenograft mouse model. Interpretation: This study highlights the importance of promoting ubiquitination-dependent Mcl-1 turnover might be an alternative strategy to enhance the anti-tumor efficacy of EGFR-TKI. Funding Statement: This work was supported by the National Natural Science Foundation of China (No.81904262, and No.81972837), the Natural Science Foundation of Hunan Province (2018JJ3787, 2018JJ2604, 2019JJ50682). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: All mice experiments were performed according to strict guidelines established by the Medical Research Animal Ethics Committee, Central South University, China.