Loss of ETV4 triggers PPM1E downregulation and AMPK-ULK1-dependent autophagy to promote intrinsic trametinib resistance in breast cancer.

Trametinib, a selective MEK1/2 inhibitor, is approved for melanoma, BRAF-mutant non-small cell lung cancer, and thyroid cancer. Its favorable pharmacologic profile has prompted broader evaluation across cancers driven by MAPK/ERK signaling. However, its efficacy as monotherapy in breast cancer remains limited due to intrinsic resistance. Here, we investigated the molecular basis of intrinsic trametinib resistance and sought strategies to enhance therapeutic response. Trametinib responsiveness was associated with ETV4 expression. Notably, trametinib reduced ETV4 expression in three cell lines with high basal ETV4 expression (MDA-MB-453, SKBR3, and T47D). Consistent with ERK/MAPK-dependent regulation of ETV4, trametinib-mediated MEK inhibition was associated with stabilization of Capicua (CIC), a transcriptional repressor of ETV4, thereby suppressing ETV4 expression. In ETV4-high cells, trametinib-induced ETV4 downregulation promoted autophagic flux. Mechanistically, trametinib treatment and ETV4 silencing induced AMPK Thr172 phosphorylation, leading to ULK1 Ser555 phosphorylation and mTOR inhibition, thereby activating protective autophagy. RNA-seq analysis revealed that trametinib treatment and ETV4 knockdown produced highly overlapping transcriptomic profiles. Notably, trametinib reduced the expression of PPM1E, a phosphatase that negatively regulates AMPK, along with canonical MAPK effector genes. ChIP-PCR analysis and public ChIP-seq data demonstrated that ETV4 directly occupies the PPM1E promoter region and enhances PPM1E transcription, whereas trametinib-induced ETV4 suppression reduced PPM1E expression, limiting AMPK dephosphorylation and thereby promoting AMPK activation. Pharmacological inhibition of autophagy using chloroquine (CQ) or 3-methyladenine (3-MA) enhanced trametinib-induced apoptosis in vitro and suppressed T47D xenograft tumor growth in vivo. Collectively, our findings define a CIC-ETV4-PPM1E-AMPK signaling cascade through which trametinib-induced ETV4 downregulation drives AMPK-ULK1-dependent protective autophagy, thereby conferring a survival advantage in ETV4-high breast cancer. Autophagy blockade restores trametinib sensitivity and induces apoptosis, supporting a combinatorial strategy to improve MEK1/2-targeted therapy.