Assessing methods to quantitatively validate TGFβ-dependent autophagy

Abstract

Transforming 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.

Canonical and Non-canonical TGFβ Signaling Activate Autophagy in an ULK1-Dependent Manner

“…We previously reported that TGFβ1 induced ULK1 protein levels and stimulated autophagy in NSCLC cells ( Trelford and Di Guglielmo, 2020 ), however the mechanism of how this was achieved remained unknown. To this end, we first investigated if TGFβ1 alters AMPK and mTOR activity by following site-specific ULK1 phosphorylation.…”

“…A549 cells stably expressing GFP-LC3 were treated with si-RNA against Smad4 or si-RNA targeting TRAF6 and TAK1 in combination with a p38 MAPK inhibitor for 24 h. Each experiment was conducted in the presence and absence of TGFβ1 for 24 h. LysoTracker Deep Red labeled lysosomes and Hoechst stain labeled the nucleus 2 h and 10 min prior to imaging, respectively. Imaging and quantitation was performed as previously described ( Trelford and Di Guglielmo, 2020 ).…”

“…As such, there is a need to understand the signaling pathways that may activate autophagy to promote tumorigenesis. In the past decade, several reports have suggested that transforming growth factor beta (TGFβ) activates autophagy ( Kiyono et al, 2009 ; Suzuki et al, 2010 ; Xu et al, 2012 ; Fu et al, 2014 ; Alizadeh et al, 2018 ; Trelford and Di Guglielmo, 2020 ). Interestingly, like autophagy, TGFβ signaling impedes tumor formation in normal cells, yet promotes metastatic potential in tumor cells ( Katsuno et al, 2013 ).…”

“…However, the specific TGFβ signaling pathway responsible for autophagy remain(s) unclear. Furthermore, many studies investigating TGFβ-dependent autophagy relied on LC3 protein levels as a readout for autophagy, which provides an incomplete picture ( Klionsky et al, 2016 ; Trelford and Di Guglielmo, 2020 ). For this reason, our previous work verified that TGFβ increased autophagic flux using cells stably expressing green fluorescent protein (GFP)-LC3-red fluorescent protein (RFP)-LC3ΔG ( Trelford and Di Guglielmo, 2020 ).…”

“…Furthermore, many studies investigating TGFβ-dependent autophagy relied on LC3 protein levels as a readout for autophagy, which provides an incomplete picture ( Klionsky et al, 2016 ; Trelford and Di Guglielmo, 2020 ). For this reason, our previous work verified that TGFβ increased autophagic flux using cells stably expressing green fluorescent protein (GFP)-LC3-red fluorescent protein (RFP)-LC3ΔG ( Trelford and Di Guglielmo, 2020 ). After ATG4 cleaves GFP-LC3-RFP-LC3ΔG to generate GFP-LC3 and RFP-LC3ΔG, RFP-LC3ΔG cannot be conjugated to a phosphatidylethanolamine nor be incorporated into the autophagosome membrane.…”