Purpose: Aromatase Inhibitors (AI) are standard therapy for hormone receptor positive breast cancers in post-menopausal patients. Disease recurrence is common and previous studies suggest that the altered steroid environment may be a driver of resistance. Using label-free mass-spectrometry we explored the unique androgen receptor (AR) interactome that supervenes in AI resistant breast cancer and the associated hyperandrogenic environment. Experimental Design: AR expression was evaluated in a primary breast cancer tissue-microarray (n=875) with nuclear and cytoplasmic localization quantified. Liquid-chromatography tandem mass-spectrometry (LC-MS/MS) analysis was utilized to identify proteins interacting with the AR in models of AI-resistance. Validation was carried out by co-immunoprecipitation and co-localisation studies. Live-cell imaging, Seahorse MitoStress Assays and flow cytometry were used to quantify changes in mitochondria and cell metabolism arising in models of AI-resistance. Results: Utilising digital pathology we detected that abundant cytoplasmic AR protein was associated with poor survival only in the post-menopausal cohort, and most significantly, in the therapy-refractory Luminal B subtype (p=0.0085). Models of AI-resistance and androgen excess highlight diffuse AR localisation throughout the cytoplasm and nucleus accompanied by increased mitochondrial mass and membrane potential, and increased oxidative phosphorylation and glycolysis. Exploration of the AR protein interactome identified G3BP1, SLIRP, and IGFBP5 as AR protein partners which are associated with stress, adaptive metabolic response and estrogen receptor repression. Conclusions: The findings of this study highlight the prognostic potential of cytoplasmic AR immunoreactivity in specific breast cancer subtypes and uncover novel extra-nuclear AR protein interactions that may mediate metabolic adaptations during the development of endocrine-resistance.
