Abstract A175: Role of MnSOD and hydrogen peroxide in rapamycin-induced cytostasis.

Existing treatments for mantle cell lymphoma (MCL) are non-curative, demonstrating a need for a refined treatment approach. Recent clinical trials have shown promising results with the use of mammalian target of rapamycin (mTOR) inhibitors. While cyclin D1 can be regulated through the mTOR pathway, treatment with mTOR inhibitors often inhibits MCL growth without altering cyclin D1 levels of expression. Thus, the anti-tumor activity of mTOR inhibitors is likely mediated through other key targets. In other malignancies, recent data suggests that increased oxidative stress plays a role in the growth suppressive mechanisms of mTOR inhibitors. Initial studies in our lab indicated that a 48 hour rapamycin treatment of MCL cell lines increased overall levels of reactive oxygen species (ROS). Specifically, we found evidence of increased hydrogen peroxide (H2O2) levels in the cells. One potential source of H2O2 is manganese superoxide dismutase (MnSOD), which is a known tumor suppressor in several cell types. MnSOD catalyzes the dismutation of superoxide to H2O2 in the mitochondria. Thus, it removes one form of ROS while generating another. We hypothesized that the anti-tumor effect of mTOR inhibitors in MCL is mediated by an accumulation of ROS, specifically H2O2, due to an increase in MnSOD protein expression. We investigated the effect of a 48 hour rapamycin treatment on MnSOD in two MCL cell lines and genetic manipulation of MnSOD in Jurkat cells, a T lymphocyte cell line. Rapamycin treatment increased MnSOD in the MCL cell lines as well as Jurkat cells. Overall, sensitivity of cells to rapamycin correlated with high MnSOD levels. Analysis of the results showed that rapamycin treatment increased MnSOD expression in parallel with H2O2. Over-expression of MnSOD in Jurkat cells elevated the level of H2O2 and resulted in a cytotoxic, rather than cytostatic, effect of rapamycin. Treatment with the general ROS scavenger, N-acetyl cysteine, reduced overall ROS, cellular levels of H2O2, and the growth inhibitory effect of rapamycin. These findings indicate that the rapamycin-induced cytostatic effect is characterized by increased levels of MnSOD and H2O2, which is necessary for the full growth inhibitory effect of rapamycin. Rapamycin and its derivatives are generally well accepted as inhibitors of the mTORC1 complex upon initial treatment, but recent studies in other cell lines have shown that long term treatment can have an inhibitory effect on the mTORC2 complex and its downstream targets, such as Akt. Our results found that treatment with rapamycin resulted in a loss of S473 phosphorylation of Akt in the MCL cell lines. Increased levels of MnSOD following treatment of Jurkat cells were found to be due to inhibition of the mTORC2 complex. These results suggest that in MCL cells treated with rapamycin for 48 hours the mTORC2 complex is inhibited. Multiple transcription factors that have been linked to MnSOD regulation are affected by the Akt pathway. By understanding the key signaling molecules and affected pathways in the anti-tumor effects of rapamycin, we may be able to identify additional predictive markers to improve the therapeutic value of mTOR inhibitors, or study drug combinations that will enhance the effect of ROS-induced cytotoxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A175.