Chloroquine and Osimertinib Induce Synergic Reduction in Cell Viability through Autophagy/Apoptosis Crosstalk

Abstract

ObjectivesTriple‐negative breast cancer (TNBC) is an aggressive subtype of breast cancer defined by the lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Patients with relapsed/refractory TNBC tend to over‐express the epidermal growth factor receptor (EGFR) on their cancerous cells. One proposed mechanism for EGFR over‐expression is through an increased autophagic flux and decreased apoptosis. Our study aims to evaluate the effectiveness of a novel combination therapy of an anti‐EGFR drug, osimertinib (Osim), with an autophagy inhibitor, chloroquine (CQ) in TNBC.MethodsThe TNBC cell line over‐expressing the EGFR, MDA‐MB‐231, was exposed for 48 hours to Osim, CQ, and combination of both drugs. The range of concentrations were 1 to 12 μM (Osim) and 1 to 100 μM (CQ). The temporal changes from control (no‐treatment) in the expression of LC3B‐II protein, a marker for autophagic flux, was measured using Western Blot. The dynamic changes from control in the signaling proteins of intrinsic and extrinsic pathways of cell death was measured using a luminex assay with MAGPIX®. The drug‐drug interaction of Osim with CQ at various concentrations was determined by calculating the combination index (CI) using CompuSyn software. Statistical significance between various treatments arms including controls were assessed with one‐way analysis of variance followed by Tukey test using GraphPad Prism Version 5.ResultsA synergistic drug‐drug interaction between Osim and CQ was identified at concentrations of 4–6 μM for Osim and 30–75 μM for CQ with CI values <1 at all tested combinations. As single agents, both drugs increased LC3B‐II protein expression by approx. 2.5‐fold compared to control at 24 hours post drug exposure. Whereas, combination of both drugs increased further LC3B‐II protein expression from control to 6‐fold. While the former result supports a mechanism of stimulation of autophagic flux by Osim, the latter result suggests its inhibition by CQ. The changes in the activity of caspase‐3 protein were approx. 1.5‐fold higher after exposure to Osim alone, 0.5‐fold lower after exposure to CQ alone, and 2‐fold higher following exposure to the combination although these results were not significant compared to control (P>0.05). pAKT, a caspase‐3 inhibitor protein, did not change after Osim exposure, but decreased approx. 0.25‐fold after CQ exposure (P < 0.01) or combination (P < 0.05) compared to control.ConclusionsThe combination of Osim and CQ leads to a synergistic reduction in cell viability. The pAKT protein, an upstream autophagy stimulator and inhibitor of caspase‐3 protein, decreased after the addition of CQ with Osim. Considering that Osim stimulates the autophagic flux and the addition of CQ inhibits it, our suggested combination may benefit TNBC patients by killing EGFR‐positive cells via inhibition of autophagic survival mechanisms induced by Osim.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.