Abstract 321: Autophagy is required for focal adhesion turnover, tumor cell motility and metastasis

Macro-autophagy is a catabolic process important for degradation of damaged organelles, protein aggregates and intracellular recycling of proteins and lipids. Autophagy is thought to suppress tumor initiation by promoting genome stability and limiting necrosis and inflammation, but conversely is utilized by tumor cells to survive nutrient stress, hypoxia, and cytotoxic therapies, promoting growth of established tumors. However, whether autophagy impacts the metastatic process is not well understood. To examine the role of autophagy in breast cancer metastasis, essential autophagy genes atg5 and atg7 were stably knocked down the aggressive and metastatic 4T1 mouse mammary tumor cell line using shRNA. Inhibition of autophagy did not reduce cell proliferation or survival under stress in vitro or primary tumor growth in vivo, but markedly reduced metastasis to the lungs and liver. Surprisingly, this was not due reduced survival in the circulation or at the secondary site, as atg5 and atg7-deficient cells were able to colonize the lungs to the same extent as control cells when injected into the circulation in a tail vein experimental metastasis assay, suggesting that inhibition of autophagy impairs escape from the primary tumor in this model. Indeed, inhibition of autophagy prevented cell spreading, migration and invasion through collagen in transwell assays of 4T1 cells, MDA-MB-231 human breast cancer cells, and B16.F10 melanoma cells. This was associated with failure of RhoA-mediated protrusion, impaired focal adhesion disassembly, and accumulation of the key focal adhesion component paxillin. We show that paxillin is degraded by autophagy, and siRNA-mediated knockdown of paxillin in autophagy-deficient cells rescued their spreading and motility, suggesting that impaired focal adhesion disassembly and migration in autophagy-deficient cells was the result of failure of autophagic degradation of paxillin. Intriguingly, active RhoA is localized at autophagosomes, and inhibition of RhoA activity inhibits not only cellular protrusion but also autophagic flux in metastatic cells, suggesting that RhoA may be involved in upstream regulation of autophagic degradation of paxillin to promote focal adhesion disassembly. In sum, we have identified a novel and unexpected function for autophagy in focal adhesion disassembly and tumor cell motility that is required for metastasis in vivo. This suggests that while autophagy may also promote the survival of disseminating tumor