Proteomic profiling reveals that ESR1 mutations enhance cyclin-dependent kinase signaling

Abstract Three quarters of all breast cancers express the estrogen receptor (ER, ESR1 gene), which promotes tumor growth and constitutes a direct target for endocrine therapies. ESR1 mutations have been implicated in therapy resistance in metastatic breast cancer, in particular to aromatase inhibitors. ESR1 mutations promote constitutive ER activity and affect other signaling pathways, allowing cancer cells to proliferate by employing mechanisms within and without direct regulation by the ER. Although subjected to extensive genetic and transcriptomic analyses, understanding of protein alterations remains poorly investigated. Towards this, we employed an integrated mass spectrometry based proteomic approach to profile the protein and phosphoprotein differences in breast cancer cell lines expressing the frequent Y537N and Y537S ER mutations. Global proteome analysis revealed enrichment of mitotic and immune signaling pathways in ER mutant cells, while phosphoprotein analysis evidenced enriched activity of proliferation associated kinases, in particular CDKs and mTOR. Integration of protein expression and phosphorylation data revealed pathway-dependent discrepancies (motility vs proliferation) that were observed at varying degrees across mutant and wt ER cells. Additionally, protein expression and phosphorylation patterns, while under different regulation, still recapitulated the estrogen-independent phenotype of ER mutant cells. Our study is the first proteome-centric characterization of ESR1 mutant models, out of which we confirm estrogen independence of ER mutants and reveal the enrichment of immune signaling pathways at the proteomic level.