Background: Autophagy is a cellular catabolic which is known to be activated by cellular stress and acts as a critical cell survival pathway. Solid tumours experience different microenvironmental metabolic stresses such as hypoxia and nutrient/energy deprivation. These highly stressful microenvironmental conditions could lead to activation of the pro-survival autophagic pathway, leading to cancer progression. Currently, clinically available autophagy inhibitors (e.g., chloroquine) only provides modest inhibitory activity. Hence, development of novel strategies to potently inhibit the autophagic pathway are warranted.
Methods: Triple negative breast cancer (HCC 1806 and MDA-MB-231) and oral cancer (SCC 25) were used for this study. The levels of a well-established autophagic marker, LC3-II, were determined by immunoblotting under different microenvironmental stressors (i.e., serum and glucose starvation). We also utilized different autophagy inhibitors inhibiting two major regulatory complexes (i.e., ULK-1 and Beclin-1 complexes) in the core autophagic machinery. IC50 of the inhibitors was determined using cellular proliferation assay. The synergy between different autophagy inhibitors and also, between autophagy inhibitors and standard chemotherapeutics (e.g., Paclitaxel and Cisplatin) was determined using Chou-Talalay method.
Results: Autophagy marker LC3-II was upregulated after incubation of cells under microenvironmental stressors in the three cell models. Increase in the IC50 for standard chemotherapeutics (e.g., cisplatin) was observed under incubation with microenvironmental stressors. In contrast, there was marked decrease in IC50 of the autophagy inhibitors (Spautin-1, MRT68921 and SAR405) after incubation under microenvironmental stressors. Notably, Beclin-1 complex inhibitor (i.e., SAR405) and ULK-1 complex inhibitor (i.e., MRT68921) demonstrated potent synergism (Combination Index (CI) < 1.0) for all three cell models used (CIHCC1806: 0.52±0.23; CIMDA-MB-231: 0.6±0.14; and CISCC25: 0.51±0.08). Synergism was also observed between autophagy inhibitors and chemotherapeutics (i.e., SAR405 with Paclitaxel or cisplatin) for all three cell models used (CIHCC1806: 0.82±0.05; CIMDA-MB-231: 0.717±0.14; and CISCC25: 0.85±0.1).
Conclusion: These studies demonstrate potential of utilizing autophagy inhibitors as a novel anti-cancer combination therapy.