Introduction: We recently reported a hitherto unknown nongenomic pathway unique to estrogen responsive breast cancer cells wherein, following stimulation by E2, ER may redox regulate mTORC2 activity by affecting transient upregulation of O2.- levels particularly with in mitochondria. Our study established O2.- not only as an upstream regulator of mTOR signaling complex 2 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 Manganese superoxide dismutase 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 mTOR signaling complex 2. In-vivo experiments employing 4T1 BALB/c mouse model of breast cancer corroborated these findings

Summary of the results: Collectively our results demonstrated for the first time direct physical interaction between ER and MnSOD following E2 stimulation. Our results indicate the 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.- with in mitochondria leading to activation of mTORC2 signaling.

Conclusion: Our finding unravel a new role of MnSOD as important control switch through which ER might affect its downstream non genomic signaling cascades in a redox dependent manner particularly potentiation of mTOR signaling complex 2. We present data in support of MnSOD being responsible for previously reported ER dependent superoxide anion O2.- potentiation in breast cancer cells following E2 exposure. We showed that MnSOD interacts with ER alpha which in turn is associated with its diminished SIRT 3 dependent deacetylation, leading to its inhibition and superoxide anion O2.- build up and consequent activation of mTORC2

Note: This abstract was not presented at the conference.

Citation Format: Mehraj U din Lone, Ranjana Km Kanchan, Chakrapani Tripathi, Khemraj Singh Baghel, Brijnath Tiwari, Smrati Bhadauria. Superoxide anion O2.-mediated activation of mTORC2 by estrogen receptor in breast cancer cells: Role of acetylation dependent inhibition of MnSOD. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B27.