Dried Blood Spot analysis as a sustainable and innovative approach for the detection of psychoactive substances and markers of alcohol abuse

Signal Transducer and Activator of Transcription 3 (STAT3) plays a plethora of different roles during tumor transformation and progression via both canonical and non-canonical, non-transcriptional functions. Among these, we have recently described STAT3 ability to control Ca2+ release from the Endoplasmic Reticulum (ER) and Ca2+-mediated apoptosis in STAT3-dependent Triple Negative Breast Cancer (TNBC) cells. This function is exerted via STAT3 localization to the ER and Mitochondrial Associated Membranes (MAMs), where it interacts with the Ca2+ channel IP3R3, mediating its degradation when phosphorylated on Serine 727 (S-P). Accordingly, STAT3 and IP3R3 protein levels are inversely correlated in the highly aggressive human basal-like breast tumors, where STAT3 is often constitutively activated. To elucidate the mechanisms regulating STAT3 S-P at the ER, we investigated the role of its interactor mTOR, which has also been reported to localize to the ER. We confirmed STAT3-mTOR ER co-localization by immunofluorescence, and their interaction by co-immunoprecipitation from purified ER fractions and Proximity Ligation Assay (PLA) in MDA-MB-468 and MDA-MB-231 TNBC cells. Pan-mTOR inhibition by Torin-1 – but not mTOR Complex 1 inhibition via Rapamycin – could prevent both STAT3-mediated IP3R3 degradation and Ca2+-mediated apoptosis, mimicking the effects of STAT3 silencing. Accordingly, mTOR inhibition abolished STAT3 S-P within the ER, increasing intracellular Ca2+ concentration. These data demonstrate for the first time localized STAT3 Serine phosphorylation occurring at the ER, establishing an important correlation with mTOR leading to enhanced apoptosis resistance via IP3R3 degradation. Dissecting these molecular details may reveal novel therapeutic targets to disrupt apoptotic resistance in STAT3-dependent TNBC cells. This thesis focuses on the regulation of calcium-mediated apoptosis in cancer and particularly triple negative breast cancer, via a newly discovered mTOR-STAT3 pathway that takes place at the ER and in turn regulates the levels of the Ca2+ channel IP3R3 thus modulating the release of Ca2+ from the ER towards the mitochondria.