Abstract
The mechanism(s) in which transforming growth factor beta 1 (TGFβ) modulates autophagy in cancer remain unclear. Here, we characterized the TGFβ signaling pathways that induce autophagy in non-small cell lung cancer cells, using cells lines stably expressing GFP-LC3-RFP-LC3ΔG constructs that measure autophagic flux. We demonstrated that TGFβ1 increases Unc 51-like kinase 1 (ULK1) protein levels, 5′ adenosine monophosphate-activated protein kinase (AMPK)-dependent ULK1 phosphorylation at serine (S) 555 and ULK1 complex formation but decreases mechanistic target of rapamycin (mTOR) activity on ULK1. Further analysis revealed that the canonical Smad4 pathway and the non-canonical TGFβ activated kinase 1/tumor necrosis factor receptor-associated factor 6/P38 mitogen activated protein kinase (TAK1-TRAF6-P38 MAPK) pathway are important for TGFβ1-induced autophagy. The TAK1-TRAF6-P38 MAPK pathway was essential for downregulating mTOR S2448 phosphorylation, ULK1 S555 phosphorylation and autophagosome formation. Furthermore, although siRNA-mediated Smad4 silencing did not alter mTOR-dependent ULK1 S757 phosphorylation, it did reduce AMPK-dependent ULK1 S555 phosphorylation and autophagosome formation. Additionally, Smad4 silencing and inhibiting the TAK1-TRAF6-P38 MAPK pathway decreased autophagosome-lysosome co-localization in the presence of TGFβ. Our results suggest that the Smad4 and TAK1-TRAF6-P38 MAPK signaling pathways are essential for TGFβ-induced autophagy and provide specific targets for the inhibition of TGFβ in tumor cells that utilize autophagy in their epithelial-mesenchymal transition program.
Highlights
Macroautophagy, hereafter referred to as autophagy, is a catabolic process facilitated by lysosomes and acidic late endosomes that degrade macromolecules and organelles to replenish the building blocks for nucleic acids, proteins, carbohydrates, and lipids (Feng et al, 2014)
Since a low P-mechanistic target of rapamycin (mTOR)/mTOR ratio increases the amount of ULK1 available for 5 adenosine monophosphate-activated protein kinase (AMPK)-dependent S555 phosphorylation and a high phospho-S555-ULK1/ULK1 ratio indicates an increase of active ULK1, we postulated that transforming growth factor beta 1 (TGFβ1) increases the amount of post-translationally modified ULK1 to initiate autophagy
We observed that LC3B-II protein levels are limited in measuring autophagy and methods that investigate autophagic flux should be used to measure TGFβ1dependent autophagy
Summary
Macroautophagy, hereafter referred to as autophagy, is a catabolic process facilitated by lysosomes and acidic late endosomes that degrade macromolecules and organelles to replenish the building blocks for nucleic acids, proteins, carbohydrates, and lipids (Feng et al, 2014). All cells increase the rate of autophagy (autophagic flux) to eliminate the influx of damaged cellular materials mediated by cell stress to survive (Ding et al, 2007). Cells have mechanisms to dampen autophagic flux because excessive degradation may initiate cell death (Shi et al, 2012). Cells modulate autophagic flux through post-translational modifications of autophagy related protein 1 (ATG1) (Yang and Klionsky, 2020). When the rate of autophagy is detrimental to cells, mTOR phosphorylates ULK1 at serine (S)757 to disrupt ULK1-AMPK interactions (Kim et al, 2011). Cell stressors impede mTOR and activate AMPK to directly phosphorylate ULK1 at S317, S555 and S778 (Dorsey et al, 2009). AMPK-dependent phosphorylation of ULK1 results in the formation of the ULK1 complex (Zachari and Ganley, 2017)
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