Investigation of a Mitochondrial to Nuclear Signaling Pathway Induced by Onc201

Abstract

Onc201 is a novel therapeutic in clinical trials that displays widespread anti-cancer activity and also has been shown to act as a radiosensitizer. Previously, we showed that Onc201 and its derivatives activate a mitochondrial protease, ClpP, resulting in mitochondrial protein degradation, induction of ATF4/CHOP and cytostatic activity in a breast cancer cell line. However, how activation of ClpP, a mitochondrial protease, signals to the nucleus has not been established. We show here that Onc201 induces GDF15, a mitochondrial stress cytokine, and further delineate the upstream signaling pathways. The aim of these studies is to better understand mechanisms of mitochondrial to nuclear signaling to allow for more effective anti-cancer strategies to be developed.To monitor mitochondrial to nuclear signaling, Sum159, a breast cancer cell line, was treated with Onc201 or its derivatives, and monitored for ATF4 or GDF15 protein expression by Western blotting. Delineation of the signaling pathways involved was achieved using siRNA directed against various targets or by use of specific kinase inhibitors.Treatment of Sum159 cells with Onc201 resulted in the induction of GDF15 and ATF4 protein as early as ∼17 hours after treatment. Using siRNA knockdowns, we found that GDF15 induction is dependent upon ClpP, ATF4, and CHOP. However, knockdown of GCN2 or HRI, which are eiF2a kinases known to regulate ATF4, had no effect on GDF15, nor did siRNA against IRE1a. Because we found that Onc201 induced Erk, Akt and AMPK activity, we targeted these pathways with specific kinase inhibitors; however, none of these pathways were required for GDF15 induction. These results suggest that other pathways may be involved in the induction of ATF4 and GDF15 and these are currently under investigation.We and others have shown that Onc201 and related molecules, activate ClpP, a mitochondrial protease resulting in rapid mitochondrial protein degradation, loss of mitochondrial function, and cytostasis or apoptosis in a variety of cancer cell lines. Importantly, Onc201 shows very low toxicity to normal cell lines and is well tolerated in patients. However, additional stratagems need to be explored to improve the efficacy of Onc201. To this end, we sought to understand how mitochondrial to nuclear signaling occurs as a way to target this pathway. These results establish that ATF4/CHOP signaling is induced and this subsequently signals to induce GDF15. However, it still remains unclear how ATF4/CHOP expression is induced and this area is currently being investigated.