Abstract C19: The effect of autophagy inhibition on anchorage-independent cell growth
Authors: Barnard Rebecca A. , Maycotte Paola , Hansen Ryan J. , Gustafson Daniel L. , Thorburn Andrew 
Published: August 2014

Abstract
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. Autophagy has also been shown to play a role in tumorigenesis, yet the role of autophagy in metastasis progression is poorly understood. Some of the key steps in metastasis progression are the ability of tumor cells to detach from the extracellular matrix, enter circulation, and seed a distal site. Non-tumorigenic cells undergo apoptosis upon detachment, referred to as anoikis. Some evidence suggests that autophagy is induced after detachment and facilitates anchorage independent growth and anoikis evasion. Therefore, we studied the effect of autophagy inhibition on anchorage independent growth using multiple cancer models both in vitro and in vivo.
Material and Methods: Murine cell lines used were 4T1, mammary carcinoma, B16-F10, melanoma, and DLM8, osteosarcoma. Autophagy was inhibited by multiple methods. The first method was pharmacologic inhibition using 10uM chloroquine (CQ) or 1nM bafilomycin A1 (BafA1). 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 Bcn-1 or Atg7 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. To determine anchorage independent proliferation, PolyHEMA coated plates were used to prevent attachment. For cell cycle analysis, cells were grown for 48h, stained with propidium iodine and analyzed by flow cytometry. For the in vivo model, mice were pretreated with CQ for 72h at 60mg/kg daily IP, allowing for systemic autophagy inhibition. 4T1 cells transfected with a luciferase reporter, or B16-F10 cells were injected via the tail vein of syngeneic Balb/c or C57Bl/6J mice. Mice continued to receive CQ or vehicle daily and were monitored thrice weekly until the development of luciferase positive metastases or 10% weight loss. Blood and liver samples were also collected to assess autophagy inhibition by measuring LC3 expression as determined by flow cytometry or Western blot.
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. Pharmacologic inhibition had no significant effect on 4T1 proliferation under any culture condition and failed to delay metastasis development in vivo. However, CQ did inhibit proliferation for B16-F10 and DLM8 cells, and this effect was more pronounced in B16-F10 cells grown in suspension. To demonstrate if a similar affect can be seen in vivo, mice will also be challenged with B16-F10 cells and treated with CQ. Conversely, Bcn-1 knockdown was able to significantly inhibit proliferation for all cell types and under all culture conditions, but there was no difference whether cells were attached or suspended. Further study with the Atg7 cells and BafA1 will reveal if this effect is merely an off target effect of Bcn-1 or truly autophagy inhibition. These studies suggest that the role autophagy plays in anchorage independent growth and survival of tumor cells is context dependent and thus broad use of autophagy inhibitors in cancer treatment is a questionable strategy.
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. The effect of autophagy inhibition on anchorage-independent cell growth. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C19.