Activation of the mTORC2/Akt axis plays a crucial role in celastrol- induced paraptosis of breast cancer cells Celastrol, a quinone methide triterpene derived from the Chinese medicinal plant Tripterygium wilfordii, has been shown in our previous study to induce paraptosis in breast cancer cells by perturbing Ca2+ homeostasis. In this study, we investigate the molecular mechanisms underlying celastrol-induced paraptosis, with a focus on the mTORC2/Akt signaling axis. My investigation revealed the transient activation of mTORC2/Akt is crucial for celastrol-induced paraptosis, as pharmacological inhibition of mTOR with PP242 and PI3K/Akt with LY294002, as well as genetic knockdown of mTOR and Akt, significantly reduced celastrol-induced cytotoxicity by delaying mitochondrial Ca2+ overload and alleviating proteotoxic stress. Notably, mTORC2, rather than mTORC1, plays a central role in this process. Akt inhibition, compared to mTOR inhibition, less effectively blocks celastrol-induced paraptosis, possibly due to the mitigation of the toxic effect by JNK activation, which is known to be a critical signal for paraptosis. Furthermore, the study shows that mTORC2- mediated mitochondrial Ca2+ overload and subsequent proteotoxic stress are pivotal in celastrol-induced paraptosis. This study also shows that celastrol selectively induces death in cancer cells while sparing non- transformed cells, highlighting its potential as a targeted anticancer agent. These insights emphasize the importance of targeting the mTORC2/Akt in cancer therapy, exploiting the vulnerabilities in cancer cells while minimizing harm to normal cells. This research opens new avenues for developing therapeutic strategies that enhance the efficacy of paraptosis inducers like celastrol by specifically targeting mTORC2/Akt-associated mechanisms. Further mechanistic studies are warranted to fully understand these processes and optimize celastrol’s therapeutic potential in cancer treatment. ____________________________________________________________ Keywords: paraptosis, celastrol, mTORC2, Akt, ER stress, mitochondrial Ca2+
