Autophagy regulates transforming growth factor β signaling and receptor trafficking

Abstract

Transforming growth factor beta (TGFβ) stimulates tumorigenesis by inducing epithelial to mesenchymal transition (EMT) and cell migration. TGFβ signaling is regulated by the endocytosis of cell surface receptors and their subcellular trafficking into the endo-lysosomal system. Here we investigated how autophagy, a cellular quality control network that delivers material to lysosomes, regulates TGFβ signaling pathways that induce EMT and cell migration. We impaired autophagy in non-small cell lung cancer cells using chloroquine, spautin-1, ULK-101, or small interfering RNA (siRNA) targeting autophagy-related gene (ATG)5 and ATG7 and observed that inhibiting autophagy results in a decrease in TGFβ1-dependent EMT transcription factor and cell marker expression, as well as attenuated stress fiber formation and cell migration. This correlated with decreased internalization of cell surface TGFβ receptors and their trafficking to early/late endosomal and lysosomal compartments. The effects of autophagy inhibition on TGFβ signaling were investigated by Smad2/Smad3 phosphorylation and cellular localization using western blotting, subcellular fractionation, and immunofluorescence microscopy. We observed that inhibiting autophagy decreased the amount and timeframe of Smad2/Smad3 signaling. Taken together, our results suggest that inhibiting autophagy attenuates pro-tumorigenic TGFβ signaling by regulating receptor trafficking, resulting in impaired Smad2/Smad3 phosphorylation and nuclear accumulation.