SSTP1, A Host Defense Peptide, Exploits the Immunomodulatory Il6 Pathway to Induce Apoptosis in Cancer Cells

Abstract Background: The anticancer activities of host defense peptides (HDPs) are mainly attributed to their cell penetrating activity. Accordingly, several approaches based on machine learning, calculating the membrane pore formation ability, have been proposed for the anti-cancer peptide identification. Since the membranolytic activity can lead to nonspecific effects, like hemolysis, the therapeutic application of such molecules is limited. In this context, we considered the immunomodulatory activity of HDPs, which is regulated by specific membrane targets and immunomodulatory pathways in immune cells. As many immunomodulators are aberrantly expressed in cancer cells, the possibility of the activation of an immunomodulatory pathway was investigated for a novel anticancer HDP, SSTP1.Methods: The fourteen mature peptides identified by shotgun cloning from the frog-skin secretions were screened for cytotoxicity in oral cancer cells. The mechanism of action of the selected peptide, SSTP1 was investigated in comparison to its inactive mutant, SSTP2. An RNA-Seq coupled with pathway enrichment analysis was performed to identify the upstream signaling leading to the mitochondrial pathway of apoptosis. Since the activation of the IL6/IL6R pathway was suggested, we performed in silico docking studies to find the binding of SSTP1 to the IL6/IL6Rα/gp130 complex. The dynamic simulation predicted the conformational changes in the active site residues. The confocal co-localization studies, pull-down assay, FRET analysis, western blot and reporter assays were performed to elucidate the role of the IL6/IL6R pathway in SSTP1-induced apoptosis. Specific small molecule inhibitors and neutralizing antibodies were used to ascertain the role of the IL6/IL6Rα/gp130 complex in the activation of JNK/AP1 pathway-dependent cell death.Results: SSTP1, a novel temporin, modulates the IL6 pathway and induces apoptosis when it binds to IL6Rα on the active IL6/IL6Rα/gp130 complex, rearranging the active site residues. In contrast to the IL6 blockers inhibiting JAK/STAT activity, SSTP1 shifts the proliferative IL6/JAK/STAT signaling to the apoptotic IL6/JNK/AP1 pathway. In IL6Rα-overexpressing cancer cells, apoptosis is preferred over the membranolytic activity, upon SSTP1 treatment.Conclusions: Here, we provide the evidence of an HDP-induced signaling through immunomodulators, leading to apoptosis in cancer cells. Our study also implies the importance of identifying the targets of HDPs for their clinical application