Activating NEDD4L suppresses EGFR-driven lung adenocarcinoma growth via facilitating EGFR proteasomal degradation.

Abstract

Background: Resistant mutations and amplification of the epidermal growth factor receptor (EGFR), followed by the upregulation of its translated protein undermines the efficacy of EGFR-tyrosine kinase inhibitors (TKIs) in EGFR-mutant lung adenocarcinoma (LUAD). This underscores that promoting EGFR protein degradation may be a promising strategy for treatment.

Methods: Ubiquitin ligases database analysis identified NEDD4L as a mediator of EGFR proteasomal degradation, which was further confirmed by qPCR, western blot, immunofluorescence staining and CO-IP. The upstream regulatory role of FOXM1 on NEDD4L was elucidated through bioinformatics analyses and validated using dual luciferase reporter assay, ChIP, qPCR, western blot and immunohistochemistry. Virtual screening and molecular docking were used to identify inhibitors of FOXM1. Functional studies and therapeutic strategies were conducted using gain- and loss-of-function assays, and evaluated through in vitro and in vivo experiments.

Results: We identified the E3 ubiquitin ligase NEDD4L that targets both wild-type EGFR and osimertinib-sensitive/resistant EGFR mutants for proteasomal degradation, thereby effectively inhibiting EGFR-driven LUAD growth. We found FOXM1 as a critical upstream transcription factor that binds to the promoter of NEDD4L and represses its expression, further promoting tumor growth and osimertinib resistance in LUAD by increasing EGFR protein level. High FOXM1 expression correlates with low NEDD4L expression in LUAD patients, which is associated with poor clinical outcomes. Notably, we further identified that verteporfin, an FDA-approved small molecule drug, as a FOXM1 inhibitor. Verteporfin suppresses FOXM1 to upregulate NEDD4L expression and facilitate EGFR proteasomal degradation, thereby inhibiting EGFR-driven LUAD growth and overcoming osimertinib resistance. Remarkably, the combination of verteporfin and osimertinib shows an additively inhibitory effect on EGFR-mutated LUAD growth compared to monotherapy, both in post-TKI resistance and upfront treatment settings.

Conclusions: This study demonstrates that FOXM1/NEDD4L axis impairs EGFR proteasomal degradation, thus contributing to EGFR-driven LUAD growth and osimertinib resistance. Combination therapy incorporating NEDD4L activation may represent a new valued therapeutic strategy for EGFR-driven LUAD and osimertinib resistance.