STAT3 is a pleiotropic transcription factor often constitutively activated in Triple-Negative Breast Cancer (TNBC). STAT3 canonical transcriptional activity is mediated by Y705 phosphorylation (pY); in contrast, phosphorylation of S727 (pS) orchestrates transcription-independent activities in distinct cellular compartments. The mechanisms that orchestrate the sub-cellular distribution of pS-STAT3 are however unknown. We have recently described pS-STAT3's ability to control Ca²⁺ release and Ca²⁺-mediated apoptosis at the Endoplasmic Reticulum (ER) in TNBC cells, by promoting proteasomal degradation of the Ca²⁺ channel IP3R3. Consistently, STAT3 and IP3R3 protein levels are inversely correlated in highly aggressive human basal-like breast tumors, where STAT3 is often constitutively activated.

To elucidate the mechanisms regulating pS-STAT3 at the ER, a network of ER-STAT3 interactors was generated, identifying mTOR as a potential STAT3 S-kinase. Accordingly, mTOR and STAT3 interact at the ER, as shown by both co-IP and PLA and pan mTOR inhibition by Torin-1, but not by mTORC1 inhibition via Rapamycin, which could hinder pS-STAT3 within the ER, IP3R3 degradation, and Ca²⁺-mediated apoptosis, mirroring STAT3's silencing effects. Our data suggest that STAT3–mTOR interaction does not require either mTORC1 nor mTORC2, since co-IP is still observed upon RAPTOR and RICTOR ablation, respectively. Accordingly, AlphaFold 3 modelling indicates that STAT3 interacts with the Armadillo region of mTOR via its coiled-coil domain, and with the mTOR catalytic pocket via its carboxyterminal region, which contains S727. The assessment of mTOR and pS-STAT3 protein levels in patient-derived tumor samples is in progress.

This data demonstrates for the first time localized control of pS-STAT3, in this case occurring at the ER downstream of mTOR, leading to enhanced apoptosis resistance via IP3R3 degradation. Dissecting these molecular details may provide clues as to how to disrupt apoptotic resistance in STAT3-dependent TNBC cells as well as in other tumors carrying constitutively active pS-STAT3.