Melanoma is the deadliest form of skin cancer in the US. Although immunotherapeutic checkpoint inhibitors and small-molecule kinase inhibitors have dramatically increased the survival of patients with melanoma, new or optimized therapeutic approaches are still needed to improve outcomes. 15-deoxy-Δ 12,14 -prostamide J 2 (15d-PMJ 2 ) is an investigational small-molecule that induces ER stress-mediated apoptosis selectively in tumor cells. Additionally, 15d-PMJ2 reduces melanoma growth in vivo . To assess the chemotherapeutic potential of 15d-PMJ 2 , the current study sought to uncover molecular pathways by which 15d-PMJ 2 exerts its antitumor activity. B16F10 melanoma and JWF2 squamous cell carcinoma cell lines were cultured in the presence of pharmacological agents that prevent ER or oxidative stress as well as Ca 2+ channel blockers to identify mechanisms of 15d-PMJ 2 cell death. Our data demonstrated the ER stress protein, PERK, was required for 15d-PMJ 2 -induced death. PERK activation triggered the release of ER-resident Ca 2+ through an IP 3 R sensitive pathway. Increased calcium mobilization led to mitochondrial Ca 2+ overload followed by mitochondrial permeability transition pore (mPTP) opening and the deterioration of mitochondrial respiration. Finally, we show the electrophilic double bond located within the cyclopentenone ring of 15d-PMJ 2 was required for its activity. The present study identifies PERK/IP3R/mPTP signaling as a mechanism of 15d-PMJ 2 antitumor activity.

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Melanoma is the most aggressive and deadly form of cutaneous cancer in the United States, representing a major clinical challenge. Our lab previously demonstrated that the endocannabinoid metabolite, 15-deoxy, Δ12,14 prostamide J2 (15d-PMJ2), was an effective and selective inducer of apoptotic cell death in B16F10 murine melanoma cells. Furthermore, we found that 15d-PMJ2 mediated apoptosis was regulated by endoplasmic reticulum (ER) stress. Numerous studies have determined that ER stress causes Ca+2 flux, and that this action initiates cellular apoptosis. As such, we investigated Ca+2 mobilization in the context of 15d-PMJ2-mediated ER stress. In B16F10 melanoma cells, 15d-PMJ2 significantly increased cytosolic Ca+2 levels. Blockade of Ca+2 channels using ruthenium red (a pan Ca+2 channel inhibitor) or 2-APB (an IP3 selective inhibitor) decreased the cytotoxicity of 15d-PMJ2. To investigate the role of ER stress in Ca+2 mobilization, cells were pretreated with the ER stress inhibitors, phenylbutyric acid (PBA) and GSK2602 (a PERK-selective inhibitor). Both inhibitors significantly lowered cytosolic Ca+2 levels suggesting Ca+2 flux is mediated by ER stress. Moreover, BAP31, an important regulator of endoplasmic Ca+2 release and ER stress was activated following exposure to 15d-PMJ2. To understand the impact of elevated Ca+2 levels, we investigated cysteine proteases (calpains) and mitochondrial Ca+2, both of which induce apoptosis in a Ca+2-dependent manner. Our results show that mitochondrial Ca+2 levels were significantly increased by 15d-PMJ2 and that this effect was prevented by blocking ER stress. In contrast, 15d-PMJ2 increased calpain-2 activity however, the inhibition of calpain had no effect on the cytotoxicity of 15d-PMJ2. These findings indicate that mitochondrial Ca2+ flux, but not calpains regulate ER stress apoptosis induced by 15d-PMJ2. It has been reported that the activity of J-series prostaglandins is conferred by an electrophilic double bond contained within its cyclopentenone ring. To evaluate the significance of this moiety in 15d-PMJ2, a neutral analog (neutral-15d-PMJ2) lacking the double bond was synthesized. We determined that that the accumulation of intracellular and mitochondrial Ca2+ was dependent on the electrophilic double bond, suggesting it is a critical structural component required for activity. Taken together, these data indicate that the mobilization of calcium to the cytoplasm and mitochondria may critically regulate ER stress apoptosis that is mediated by 15d-PMJ2. Citation Format: Daniel Alexander Ladin, Estefani Cota, Rukiyah Van Dross. Regulation of ER stress-mediated cell death by calcium mobilization: A potential mitochondrial pathway for 15-deoxy-Δ12,14prostamide J2cytotoxicity in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2663.