Abstract 1863: Differing sensitivity to stage-specific autophagy inhibition.
Authors: Barnard Rebecca A. , Maycotte Paola , Hansen Ryan J. , Gustafson Daniel L. , Thorburn Andrew 
Published: August 2014

Background: Autophagy is an intracellular process that involves the sequestration of cytosolic proteins and organelles into double-membraned vesicles called autophagosomes and their subsequent degradation in the lysosome. This process is induced during cellular stress and is crucial for maintaining proper homeostasis. Recently, autophagy has been identified as a potential mechanism of chemoresistance. Thus, autophagy inhibition has become an attractive anti-cancer therapy. However, autophagy is a multi-step process and it is still unclear if blocking autophagy at one particular stage is more advantageous than another. Therefore, we inhibited autophagy at various points in the pathway using genetic and pharmacologic means in multiple cancer models.

Material and Methods: Murine cell lines used were 4T1, mammary carcinoma, B16-F10, melanoma, DLM8, osteosarcoma, and A20, lymphoma. Autophagy was inhibited by multiple methods. The first method was pharmacologic inhibition using 10uM chloroquine (CQ) or 1nM bafilomycin A1 (Baf A1), doses sufficient to inhibit autophagy. The second method was genetic knockdown by decreasing expression of critical autophagy proteins Beclin-1 (Bcn-1) or Atg7 by shRNA lentiviral delivery. Cells were assessed for protein knockdown by western blot and qPCR analysis. Proliferation of cells in the presence or absence of active autophagy was assessed using an Alamar Blue assay. For cell cycle analysis, cells were grown for 48h, stained with propidium iodine and analyzed by flow cytometry.

Results and Conclusions: A significant decrease in expression of Bcn-1 and Atg7 was observed after lentiviral transduction. Autophagy was also successfully inhibited as indicated by a failure to induce elevations in autophagy marker LC3 after serum starvation, a positive autophagy control. CQ and Baf A1, which inhibit late stage autophagy, had little effect on the 4T1 cells, but CQ did inhibit the other three lines. Bcn-1 knockdown, preventing nucleation of the autophagosome, was able to inhibit proliferation in all lines at a level that was even more pronounced than CQ inhibition. In addition, the percentage of cells in G1 significantly increased in the Bcn-1 knockdown cells. Continued study with the Atg7 cells and Baf A1 will reveal if the effects of CQ and Bcn-1 knockdown are merely off target effects or truly autophagy inhibition. The inclusion of Vps 34 inhibitors and late stage mediator knockdown lines will also be useful in determining if the early, nucleation stage of autophagy is the critical step(s) to target. Lastly, it will be important to determine if these effects differ when cells experience stress, such as the presence of chemotherapy. These studies suggest that the sensitivity toward autophagy inhibition may depend on the stage of the pathway that is targeted and cancer type.

Acknowledgements:

Role of Autophagy in Tumor Cell Death

National Cancer Institute 1R01CA150925

Citation Format: Rebecca A. Barnard, Paola Maycotte, Ryan J. Hansen, Daniel L. Gustafson, Andrew Thorburn. Differing sensitivity to stage-specific autophagy inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1863. doi:10.1158/1538-7445.AM2013-1863