G protein subtype preference dictates paroxetine-enhanced serotonin receptor response in normal breast epithelial cells

In the healthy mammary gland serotonin (5-HT) has a key role in shaping tissue morphology and regulating lactation and involution. Serotonin re-uptake inhibitors (SSRIs) alter the distribution of 5-HT across the cell membrane, but how increased extracellular 5-HT may impact breast cell homeostasis is unclear. We demonstrate that paroxetine reduces 5-HT levels in immortalized primary breast epithelial (HME-hTert) cells, mitigates free oxygen radical formation and decreases cell migration and proliferation. Accordingly, pathways related to cell cycle and DNA damage repair were underrepresented in the transciptomic profile of paroxetine-treated cells. On the other hand, enriched transcipts overrepresented genes affecting neural transmission and GPCR signaling, suggesting an increase in 5-HT receptor (5HTR) activation. As 5-HT induced the levels of both cAMP and inositol triphosphate (IP3), the contribution of individual 5HTRs were decyphered using receptor-selective agonists and antagonists. 5HTRs coupled to each Gα protein subtype were expressed and functional in HME-hTert cells. The activation of G S -coupled receptor 5HTR7 and antagonists of G i - and G q -coupled receptors 5HTR1D and 5HTR2B generally suppressed proliferation. The induction of cAMP levels by 5-HT was reduced by the 5HTR7 antagonist and IP3 induction was blocked by an 5HTR2B inhibitor, while the 5HTR1D antagonist further increased cAMP levels induced by 5-HT. Paroxetine-dependent growth suppression was reversed by inhibitors of G S -coupled 5HTR7, protein kinase A or adenylyl cyclase, and agonists of G i or G q -coupled receptors. These results suggest that the anti-proliferative responses to 5-HT in non-malignant breast cells align with the G protein preference of the receptors, and reveal potential benefits of repurposing receptor subtype-selective agents and SSRIs for cancer risk reduction. Graphical abstract