Assessing methods to quantitatively validate TGFβ-dependent autophagy.

Abstract

ABSTRACTTransforming growth factor beta (TGFβ) promotes tumorigenesis by suppressing immune surveillance and inducing epithelial to mesenchymal transition (EMT). TGFβ may augment tumorigenesis by activating autophagy, which protects cancer cells from chemotherapy and promotes invasive and anti-apoptotic properties. Here, we assess how TGFβ1 modulates autophagy related (ATG) gene expression and ATG protein levels. We also assessed microtubule-associated protein light chain 3 (LC3) lipidation, LC3 puncta formation and autophagosome-lysosome co-localization in non-small cell lung cancer (NSCLC) cell lines. These experimental approaches were validated using pharmacological autophagy inhibitors (chloroquine and spautin-1) and an autophagy activator (MG132). We found that TGFβ1, chloroquine and MG132 had little effect on ATG protein levels but increased LC3 lipidation, LC3 puncta formation and autophagosome-lysosome co-localization. Since similar outcomes were observed using chloroquine and MG132, we concluded that several techniques employed to assess TGFβ-dependent autophagy may not differentiate between the activation of autophagy versus lysosomal inhibition. Thus, NSCLC cell lines stably expressing a GFP-LC3-RFP-LC3ΔG autophagic flux probe were used to assess TGFβ-mediated autophagy. Using this approach, we observed that TGFβ, MG132 and serum starvation increased autophagic flux, whereas chloroquine and spautin-1 decreased autophagic flux. Finally, we demonstrated that ATG5 and ATG7 are critical for TGFβ-dependent autophagy in NSCLC cells. The application of this model will fuel future experiments to characterize TGFβ-dependent autophagy, which is necessary to understand the molecular processes that link, TGFβ, autophagy and tumorigenesis.