Abstract 3537: Cisplatin refractory lung cancer cells exhibit an increased autophagic flux at baseline and following exposure to cisplatin
Authors: Magdalena Circu, James Cardelli, Martin Barr, Hazem E. El-Osta
Journal: Cancer Research (2016)

Abstract Introduction: Lung cancer is the leading cause of cancer-related death. Platinum-based therapy is a standard treatment for advanced lung cancer. However, most patients develop resistance to treatment within few months. Autophagy is a catabolic degradation process whereby cellular proteins and organelles are engulfed by autophagosomes, degraded in lysosomes, and recycled to sustain cellular metabolism. Activation of autophagy in response to cellular stress enables cancer cell survival, which can lead to tumor growth and therapeutic resistance. Materials and Methods: Cisplatin-resistant (cisR A549) lung cancer cells, obtained by treating parental A549 cells with incremental concentrations of cisplatin, were kindly provided by Dr. Martin Barr. To measure autophagy, cells were stained for CytoID and mounted on the Cellometer vision CBA platform, an automated image-based fluorescence microscope equipped with bio-analysis system. Autophagy was confirmed by immunoblotting for LC3II/I and p62 in cell lysates. Lysosomal membrane permeabilization (LMP) was detected after loading lysosomes with FITC-dextran 40KDa and detecting dextran leakage into the cytosol on confocal microscopy. Cell viability was evaluated using CellTiter blue assay. Results: We observed an increase in baseline autophagic flux in cisR A549 lung cancer cells as compared to parental cells in full media condition. This was demonstrated by an increase in LC3II/I ratio, and reduction in p62 level in untreated cells. Compared with untreated cells, cisplatin induced a time-dependent increase in autophagosome formation in cisR A549 cells as manifested by the enhanced cytoID signal. To examine whether the increase in CytoID signal was due to autophagic flux stimulation or autophagosome accumulation secondary to blockage of the last step of autophagy, cells were treated with cisplatin in combination with chloroquine, a known lysosomal inhibitor. This had led to a significant increase in cytoID signal as compared to each drug alone, suggesting that autophagy was induced following exposure to cisplatin in cisR cells. Interestingly, increased CytoID signal following exposure to cisplatin was less pronounced in cisplatin-sensitive cells, suggesting that autophagy stimulation following exposure to the drug is inherent for resistant cells. Inhibition of autophagy using chloroquine sensitized resistant cells to cisplatin. Chloroquine was found to induce LMP. Indeed, neutralization of leaked lysosomal proteases with E64, a pan-protease inhibitor, attenuated the synergistic effect of chloroquine. Further works evaluating whether specific autophagy inhibition employing siRNA ATG3 or 3-MA can restore susceptibility to cisplatin are ongoing. Conclusion: Our findings suggest that increased autophagy may be involved in acquired resistance to cisplatin in lung cancer. Targeting autophagy is a viable strategy to reverse chemoresistance. Citation Format: Magdalena Circu, James Cardelli, Martin Barr, Hazem E. El-Osta. Cisplatin refractory lung cancer cells exhibit an increased autophagic flux at baseline and following exposure to cisplatin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3537.