Role of Hypoxia and Autophagy in MDA-MB-231 Invasiveness

Survival strategies adopted by tumor cells in response to a hypoxic stress include activation of hypoxia-inducible factor 1 (HIF-1) andautophagy. However, the importance and the function of each molecular response is not well defined. In the present study, we investigatedinvasiveness, migration, matrix metalloproteinases (MMPs) activity, and cell survival of MDA-MB-231 cells under normoxia, hypoxia, andhypoxia/reoxygenation (H/R). Moreover, to assess the importance of hypoxia and autophagy on the parameters studied, cells were eitherleft untreated or treated with Chetomin (a selective inhibitor of HIF-1a) or trifluoperazine (TFP, an activator of autophagy).Wefound thathypoxia and H/R stimulated invasiveness and migration of MDA-MB-231 cells with an increased MMP-2 activity. Chetomin and TFPdifferently regulated the cellular behavior under the oxygenation conditions studied. In fact, Chetomin was most effective in inhibiting cellinvasion, MMPs activity, and cell survival under hypoxia but not normoxia or H/R. By contrast, TFP inhibition of cell invasion, migration, andcell survival was independent from oxygenation conditions. TFP-induced autophagy was inhibited by light chain protein 3 (LC3) silencing or3-methyladenine (3MA) treatment. In fact, LC3-silenced cells were able to invade in the presence of TFP without any GATE16 processingand p62 degradation. Immunofluorescence assay showed that LC3 silencing inhibited TFP-induced autophagosome formation. However,we also showed that both TPF treatment and LC3 silencing caused cytoskeleton impairments suggesting a possible interaction betweenLC3 and cytoskeleton components. In conclusion, our study shows that hypoxia and autophagy by acting on common (HIF-1a) or separate(MMPs, cytoskeleton) targets differently regulate cell invasion, MMPs activity, and survival.

Survival strategies adopted by tumor cells in response to a hypoxic stress include activation of hypoxia-inducible factor 1 (HIF-1) and autophagy. However, the importance and the function of each molecular response is not well defined. In the present study, we investigated invasiveness, migration, matrix metalloproteinases (MMPs) activity, and cell survival of MDA-MB-231 cells under normoxia, hypoxia, and hypoxia/reoxygenation (H/R). Moreover, to assess the importance of hypoxia and autophagy on the parameters studied, cells were either left untreated or treated with Chetomin (a selective inhibitor of HIF-1 alpha) or trifluoperazine (TFP, an activator of autophagy). We found that hypoxia and H/R stimulated invasiveness and migration of MDA-MB-231 cells with an increased MMP-2 activity. Chetomin and TFP differently regulated the cellular behavior under the oxygenation conditions studied. In fact, Chetomin was most effective in inhibiting cell invasion, MMPs activity, and cell survival under hypoxia but not normoxia or H/R. By contrast, TFP inhibition of cell invasion, migration, and cell survival was independent from oxygenation conditions. TFP-induced autophagy was inhibited by light chain protein 3 (LC3) silencing or 3-methyladenine (3MA) treatment. In fact, LC3-silenced cells were able to invade in the presence of TFP without any GATE 16 processing and p62 degradation. Immunofluorescence assay showed that LC3 silencing inhibited TFP-induced autophagosome formation. However, we also showed that both TPF treatment and LC3 silencing caused cytoskeleton impairments suggesting a possible interaction between LC3 and cytoskeleton components. In conclusion, our study shows that hypoxia and autophagy by acting on common (HIF-1 alpha) or separate (MMPs, cytoskeleton) targets differently regulate cell invasion, MMPs activity, and survival. J. Cell. Physiol. 223: 359-368, 2010. (c) 2010 Wiley-Liss, Inc.