Synonymous codon usage shapes ERα function through specialized translational programs during breast cancer progression.

Long considered neutral, synonymous codon usage is increasingly recognized as a regulatory layer of mRNA translation. Proteins expressed during proliferation are translated from mRNAs with codon compositions distinct from those encoding proteins in differentiated states, suggesting the existence of specialized translational programs. In hormone-receptor-positive breast cancer, estrogen receptor-alpha (ERα/ESR1) acts both as a driver of proliferation and as a maintainer of luminal identity, making its translational regulation particularly relevant. Here, we show that the transition of breast cancer cells from quiescent, differentiated states towards proliferative, less differentiated phenotypes, is accompanied by a systematic shift from G/C3- to A/U3-biased codon usage. ERα coding sequence architecture mirrors these translational programs through both global codon composition and position-dependent codon patterns. These features influence translational efficiency and likely modulate co-translational folding dynamics, thereby adjusting receptor abundance and functional outputs according to the prevailing translational program. Consistent with their functional importance, synonymous codon choices are subject to increased selective constraints within regions encoding structural and functional domains. Using engineer synonymous ERα variants, we demonstrate that codon usage fine-tunes receptor function across distinct cellular states. Together, our findings identify synonymous codon usage as a previously unrecognized mechanism linking translational programs to ERα expression and function.