Protein Kinase A-induced tamoxifen resistance is mediated by anchoring protein AKAP13

Background: Estrogen Receptor alpha (ERaα)-positive breast cancer patients receive endocrine therapy, often in the form of tamoxifen. However, resistance to tamoxifen is frequently observed. A signalling cascade that leads to tamoxifen resistance is dictated by activation of the Protein Kinase A (PKA) pathway, which leads to phosphorylation of ERaα on Serine 305 and receptor activation, following tamoxifen binding. Thus far, it remains elusive what protein complexes enable the PKA-ERaα interaction resulting in ERaα Serine 305 phosphorylation. Methods: We performed immunohistochemistry to detect ERaαSerine 305 phosphorylation in a cohort of breast cancer patients who received tamoxifen treatment in the metastatic setting. From the same tumor specimens, Agilent 44 nK gene expression analyses were performed and integrated with clinicopathological data and survival information. In vitro analyses were performed using MCF7 breast cancer cells, which included immunoprecipitations and Fluorescence Resonance Energy Transfer (FRET) analyses to illustrate ERaα complex formation. siRNA mediated knockdown experiments were performed to assess effects on ERaαSerine 305 phosphorylation status, ERaα/PKA interactions and downstream responsive gene activity. Results: Stratifying breast tumors on ERaα Serine 305 phosphorylation status resulted in the identification of a gene network centered upon AKAP13. AKAP13 mRNA expression levels correlate with poor outcome in patients who received tamoxifen treatment in the metastatic setting. In addition, AKAP13 mRNA levels correlate with ERaαSerine 305 phosphorylation in breast tumor samples, suggesting a functional connection between these two events. In a luminal breast cancer cell line, AKAP13 was found to interact with ERaα as well as with a regulatory subunit of PKA. Knocking down of AKAP13 prevented PKA-mediated Serine 305 phosphorylation of ERaα and abrogated PKA-driven tamoxifen resistance, illustrating that AKAP13 is an essential protein in this process. Conclusions: We show that the PKA-anchoring protein AKAP13 is essential for the phosphorylation of ERaαS305, which leads to tamoxifen resistance both in cell lines and tamoxifen-treated breast cancer patients.

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