Abstract A05: Targeting autophagy through novel anionophores that induce lysosomal dysfunction

Abstract

Abstract Macroautophagy, herein referred to as autophagy, is a biological process activated under diverse cellular stress conditions, such as starvation or oxidative stress. It involves the internalization of cytoplasmic portions, with or without organelles, within double-membrane vesicles called autophagosomes. These vesicles finally fuse with lysosomes, forming the autolysosomes, where the cargo is degraded due to the activity of the lysosomal enzymes. This catabolic process has primarily a cellular protective role but, depending on the context, it can also mediate an autophagic cell death. In fact, research related to the activation and impairment of autophagy is gaining importance as a novel anticancer therapeutic strategy in recent times. Therefore, identification of new potent and selective anticancer compounds is eagerly needed. In this view, we have synthesized novel and effective anion transporter molecules (anionophores), designed to disrupt the cellular ion homeostasis, therefore triggering cell death. The molecular mechanism implicated in their cytotoxic properties in lung cancer has been deeply analyzed in the present work. First, cytotoxic effects of our candidate compounds were evaluated by the MTT assay in four lung cancer cell lines representative of the main clinical histological subtypes as well as in cancer stem cells, a tumor cell subpopulation related to tumor initiation, progression and chemoresistance. A significant decrease in cell viability was observed in all of them, being compound 2 the most cytotoxic in all the cell lines and cancer stem cells tested. In order to characterize the cell death mechanisms involved in its cytotoxic activity, different molecular markers related to apoptosis and autophagy were analyzed. At low concentrations, we observed a very significant autophagosomal accumulation, detected through a high LC3II increase by Western blot and immunofluorescence, while some evidence of caspases activation is observed at higher concentrations. Moreover, p62 protein was also accumulated after treatment, suggesting impairment of the autophagic process and blockade of autophagosome processing. Interestingly, acidic lysosomal pH was totally disrupted by the compound, measured by acridine orange staining, which might be the cause of this autophagosomal accumulation. On the other hand, it was observed, by phase contrast and electron microscopy, that the compound also induced massive cytoplasmic vacuolization. We tested several markers to distinguish among different potential organelles that could have undergone this phenomenon. We used LAMP1, LC3II and EEA1 by immunofluorescence and we could exclude lysosomes, autophagosomes and early endosomes, respectively. Finally TOMM20, a mitochondrial marker, proved to be positive. That was subsequently corroborated by transfecting A549 cells with the subunit VIII of human cytochrome C oxidase mCherry-MITO7 plasmid. Therefore, mitochondrial swelling induced by the compound might be involved in the triggering of the autophagic process and provoking an energy failure which might participate in the cytotoxic effect observed. Altogether, these results show that these anion transporters have potent cytotoxic effects in lung cancer and cancer stem cells. They induce an imbalance in cellular ion homeostasis, which triggers mitochondrial swelling and lysosomal dysfunction leading to autophagosomal accumulation and finally cell death. Acknowledgements: This work was partially supported by grants from the Spanish government and the EU (FIS PI13/00089), La Marató TV3 Foundation (20132730) and SEPAR foundation (17/2014). LKG holds a FCT (SFRH/BPD/91766/2012) fellowship. Citation Format: Ananda Marina Rodilla, Luis Korrodi-Gregório, Pilar Manuel-Manresa, Roberto Quesada, Ricardo Pérez-Tomás, Vanessa Soto-Cerrato. Targeting autophagy through novel anionophores that induce lysosomal dysfunction. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A05.