The Formation Of Redox Active Complexes Is Important For The Induction Of The Upr By Dp44mt

Activation of the unfolded protein response (UPR) has been demonstrated in several cancer types. This has been attributed to two main factors, namely: (1) intrinsic factors (e.g., hyperactivation of oncogenes) and (2) extrinsic factors (i.e., the hostile microenvironment of tumors). This activation of the UPR has been linked to oncogenic transformation and cancer development. Considering the UPR has been identified as an adverse marker for prognosis, a better understanding of the role of the UPR in the outcome of cancer therapeutics will offer new opportunities to improve existing treatment strategies. Notably, the anticancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazones (Dp44mT), has been shown to activate the UPR, specifically PERK, IRE1α, and ATF6. This agent has been demonstrated to possess potent and selective antitumor and antimetastatic activity. In fact, this class of agents is currently in clinical trials for the treatment of advanced solid tumors (NCT02688101). Hence, the role of the UPR in the anticancer activity of Dp44mT and the importance of the formation of redox active complexes by Dp44mT was assessed. Studies have demonstrated that Dp44mT activates the main arms of the UPR, namely PERK, IRE1α, and ATF6, inducing proapoptotic pathways (e.g., CHOP, caspase 3), while inhibiting the prosurvival signals, i.e., XBP1s, p58 IPK . Considering this and the induction of ROS by Dp44mT, experiments assessed the role of the formation of redox active complexes by Dp44mT in inducing UPR activation using the glutathione synthesis inhibitor, buthionine sulfoximine (BSO), and the antioxidant, N-acetyl-L-cysteine (NAC), that potentiates and inhibits oxidative stress, respectively, in human SK-N-MC neuroepithelioma cells. Importantly, Western blot analysis demonstrated that BSO or NAC alone did not significantly induce or alter the expression of the UPR mediators, p-eIF2α, p-IRE1α, cleaved ATF6, and p-CaMKII. Incubation of cells with Dp44mT and BSO potentiated Dp44mT-mediated activation of the UPR, increasing levels of p-eIF2α, p-IRE1α, cleaved ATF6, and p-CaMKII relative to the control. In contrast, NAC and Dp44mT treatment reduced the expression of these UPR mediators in comparison to cells treated with Dp44mT alone. Hence, BSO enhanced the activation of the UPR pathways by Dp44mT, while the antioxidant NAC inhibited this process. This suggested that the formation of ROS is important for Dp44mT-mediated UPR activation. MTT studies were also performed to determine the role of the main UPR arms, PERK or IRE1α, in the antiproliferative activity of Dp44mT. PERK or IRE1α silencing increased the effectiveness of Dp44mT by significantly (p 50 , suggesting these proteins are important for cell survival and are indicators of therapeutic outcomes. In conclusion, the formation of redox active complexes is important for the induction of the UPR by Dp44mT. Additionally, PERK and IRE1α are important mediators that enhance cell survival and silencing these proteins increases the antiproliferative activity of Dp44mT. This may be related to their role in autophagy. Considering the activation of the UPR in cancers, it is crucial to continue to characterize the effect of the UPR on cancer therapeutics to better predict clinical outcomes and the subpopulations of patients that will greatly benefit from this type of therapy. Citation Format: Angelica Merlot, Nurul Shafie, Georgia Porter, Sumit Sahni, Des Richardson. The formation of redox active complexes is important for the induction of the UPR by Dp44mT [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B143.