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) 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-1alpha) 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 GATE16 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-1alpha) or separate (MMPs, cytoskeleton) targets differently regulate cell invasion, MMPs activity, and survival.
Indelicato
Manuela
M
Department of Cellular and Molecular Pathology, IRCCS San Raffaele Pisana, 00163 Rome, Italy. manuela.indelicato@sanraffaele.it
Pucci
Bruna
B
Schito
Luana
L
Reali
Valentina
V
Aventaggiato
Michele
M
Mazzarino
Maria C
MC
Stivala
Franca
F
Fini
Massimo
M
Russo
Matteo A
MA
Tafani
Marco
M
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D054443:Cell Migration Assays; D002465:Cell Movement / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D003599:Cytoskeleton / Q000378:metabolism; D004220:Disulfides / Q000494:pharmacology; D018492:Dopamine Antagonists / Q000494:pharmacology; D005109:Extracellular Matrix / Q000201:enzymology; D005260:Female; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000378:metabolism; D026121:Indole Alkaloids / Q000494:pharmacology; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics; D009361:Neoplasm Invasiveness / Q000503:physiopathology*; D009369:Neoplasms / Q000378:metabolism* / Q000503:physiopathology*; D010101:Oxygen Consumption / Q000502:physiology; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism; D034622:RNA Interference; D014268:Trifluoperazine / Q000494:pharmacology
D004220:Disulfides; D018492:Dopamine Antagonists; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D026121:Indole Alkaloids; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; C001598:chetomin; D014268:Trifluoperazine; C025946:3-methyladenine; D020782:Matrix Metalloproteinases; D020778:Matrix Metalloproteinase 2; D000225:Adenine