Abstract 1021: Superoxide anion (O2.-) mediated activation of mTORC2 by estrogen receptor in breast cancer cells: Role of acetylation dependent inhibition of MnSOD

Introduction: We recently reported a hitherto unknown nongenomic pathway unique to estrogen responsive breast-cancer cells wherein, following stimulation by estrogen (E2), Estrogen-receptor-alpha (ER-α) may redox regulate mTORC2 activity by affecting transient up-regulation of O2.- levels particularly within mitochondria. Our study established O2.- not only as an upstream regulator of mTORC2 but a critical mediator of ER signaling. Precise knowledge of the mechanisms underlying aforementioned O2.- potentiation may lead to development of novel and more efficacious measures to intervene the breast-cancer progression. The principle enzyme that regulates mitochondrial superoxide anion levels is MnSOD (SOD2), thus current study was planned to determine whether ER may affect transient superoxide anion O2.- upregulation in E2 stimulated breast-cancer cells by modulating MnSOD activity, if so, how? Methodology/Principle findings: Evaluation of MnSOD specific activity in accordance with Mishra and Fridovich (1971) revealed time dependent inhibition of MnSOD enzyme in MCF-7 breast-cancer cells following their stimulation with E2. Detailed analysis revealed that the transient inhibition of MnSOD was a receptor dependent phenomenon. Western-blotting analysis revealed potentiation of inhibitory Lysine-K68 acetylation of MnSOD in a characteristic ER dependent manner. Co-immunoprecipitation and reverse co-immunoprecipitation studies revealed physical-interaction between ER and MnSOD which in-turn was associated with impaired association of MnSOD with key deacetylase SIRT3. Furthermore, knocking down SIRT3 levels through SIRT3 directed siRNA resulted in elevated superoxide anion O2.- levels and potentiated mTORC2 signaling. In-vivo experiments employing 4T1-BALB/c mouse model of breast-cancer corroborated these findings Summary of the results: Collectively our results demonstrate for the first time direct physical-interaction between ER and MnSOD following E2 stimulation; physical-interaction of ER with MnSOD in-turn resulted in disengagement of SOD2 with SIRT3. Resultant compromised deacetylation of SOD2 at critical K68 may lead to its inhibition and thus cause a transient buildup of superoxide anion O2.- within mitochondria leading to activation of mTORC2 signaling. Conclusion Our findings unravel a new role of MnSOD as an important control-switch through which ER might affect its downstream non-genomic signaling cascades in a redox dependent manner particularly potenti