Abstract
Several antitumor therapies work by increasing reactive oxygen species (ROS) within the tumor micromilieu. Here, we reveal that L-plastin (LPL), an established tumor marker, is reversibly regulated by ROS-induced thiol oxidation on Cys101, which forms a disulfide bridge with Cys42. LPL reduction is mediated by the Thioredoxin1 (TRX1) system, as shown by TRX1 trapping, TRX1 knockdown and blockade of Thioredoxin1 reductase (TRXR1) with auranofin. LPL oxidation diminishes its actin-bundling capacity. Ratiometric imaging using an LPL-roGFP-Orp1 fusion protein and a dimedone-based proximity ligation assay (PLA) reveal that LPL oxidation occurs primarily in actin-based cellular extrusions and strongly inhibits cell spreading and filopodial extension formation in tumor cells. This effect is accompanied by decreased tumor cell migration, invasion and extracellular matrix (ECM) degradation. Since LPL oxidation occurs following treatment of tumors with auranofin or γ-irradiation, it may be a molecular mechanism contributing to the effectiveness of tumor treatment with redox-altering therapies.
Highlights
Several antitumor therapies work by increasing reactive oxygen species (ROS) within the tumor micromilieu
We found that Cys[101] and Cys[42] were strongly alkylated with d5NEM and that the signal intensity increased with increasing H2O2 concentrations (Fig. 1c–e, Supplementary Fig. 1a–b)
While LPL reduction is mediated by the TRX1 system, LPL oxidation is provoked by locally high intracellular ROS levels and preferentially takes place at spatially confined cellular extrusions, such as filopodia and invadopodia
Summary
Several antitumor therapies work by increasing reactive oxygen species (ROS) within the tumor micromilieu. Ratiometric imaging using an LPL-roGFPOrp[1] fusion protein and a dimedone-based proximity ligation assay (PLA) reveal that LPL oxidation occurs primarily in actin-based cellular extrusions and strongly inhibits cell spreading and filopodial extension formation in tumor cells. This effect is accompanied by decreased tumor cell migration, invasion and extracellular matrix (ECM) degradation. ROS are physiologically produced in response to several stimuli, such as cytokines and growth factors, both in tumor cells[1] and in immune cells[2] Their major cellular sources are NADPH oxidases (NOXes) and the incomplete reduction of oxygen to water in the mitochondrial electron transport chain system. We have shown that the ectopic expression and phosphorylation of LPL in human melanoma cells or prostate cancer cells enhances their migration and invasion in vitro[21] and leads to an enhanced metastatic capacity of these tumor cells in vivo[21,28]
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