Crotonate suppresses breast cancer metastasis and promotes immunotherapy response by inducing ACSS2-mediated EZH2-K348 crotonylation

Crotonate, a short-chain fatty acid, generates protein crotonylation. However, the role of crotonate in cancer progression is unknown. Here, we present a crotonate-crotonyl-coenzyme A (CoA)-enhancer of zeste homolog 2 (EZH2) crotonylation cascade blocking breast cancer growth and metastasis. We demonstrated that crotonate promotes EZH2 degradation via crotonyl-CoA-mediated crotonylation at Lys³⁴⁸ in EZH2 (EZH2-K348cr). EZH2-K348cr leads to reduced genome-wide H3K27me3 (trimethylation of lysine-27 on histone-3) occupancy. Crotonate metabolizes to crotonyl-CoA by ACSS2 (acyl-CoA synthetase 2), and then, acyltransferase p300 catalyzes crotonyl-CoA and generates EZH2-K348cr. Crotonylated EZH2 triggers EZH2 ubiquitination and degradation. Administration of crotonate markedly inhibits breast cancer cell growth and metastasis via a crotonate-crotonyl-CoA-EZH2-K348cr cascade. In comparison, crotonate showed better blocking effect than EZH2 inhibitor tazemetostat in suppressing breast cancer metastasis. The combination of crotonate and anti-PD-L1 (programmed cell death ligand 1) antibody enhances responses of breast cancer cells to immunotherapy. Together, our findings indicate that crotonate is a promising anticancer drug candidate that suppresses breast cancer growth and metastasis by specifically inducing EZH2 degradation.