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

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.

Transforming growth factor-β in tumour development

“…In non-small cell lung cancer cells transfected with a pMRX-IP-green fluorescent protein (GFP)-LC3-red fluorescent protein (RFP)-LC3ΔGly construct, TGFβ decreased the GFP/RFP ratio, which verified that TGFβ upregulated autophagic flux ( Trelford and Guglielmo, 2020 ). However, the TGFβ-dependent increase in autophagic flux was attenuated by Smad4 knockdown or TAK1/TRAF6/p38 MAPK pathway disruption ( Trelford and Di Guglielmo, 2021 ). In the same cell line system, TGFβ increased the proportion of phosphorylated ULK1 mediated by AMPK and further investigation showed that ULK1 inhibition blocked TGFβ-dependent autophagy ( Trelford and Di Guglielmo, 2021 ; Trelford and Guglielmo, 2021 ).…”

“…However, the TGFβ-dependent increase in autophagic flux was attenuated by Smad4 knockdown or TAK1/TRAF6/p38 MAPK pathway disruption ( Trelford and Di Guglielmo, 2021 ). In the same cell line system, TGFβ increased the proportion of phosphorylated ULK1 mediated by AMPK and further investigation showed that ULK1 inhibition blocked TGFβ-dependent autophagy ( Trelford and Di Guglielmo, 2021 ; Trelford and Guglielmo, 2021 ). In summary, Smad-dependent and -independent TGFβ signalling activate autophagy in a ULK1-dependent manner ( Trelford and Di Guglielmo, 2021 ).…”

The Bright and the Dark Side of TGF-β Signaling in Hepatocellular Carcinoma: Mechanisms, Dysregulation, and Therapeutic Implications

“…More importantly, the circuit between TGFβ signaling and autophagy provokes apoptosis in Huh7 cells by modulating the expression of proapoptotic Bcl-2 family members, Bmf and Bim [ 125 ]. More recently, a distinct mechanism of autophagy induction has been described which, in particular, entails the activation of Unc-51-like kinase 1 (ULK1) by the crosstalk between TGF-β signaling and the TAK1-TRAF6-p38-MAPK pathway [ 126 ]. Whether this mode of autophagy activation induces apoptosis remains to be elucidated.…”

Proton pump inhibitors display anti‐tumour potential in glioma

“…MAPK inhibits mTORC1 activity, which leads to suppression of autophagy activating unc‐51‐like kinase 1 (ULK1). ULK1 induces the Beclin‐1 phosphorylation, which can result in autophagy 138–140 . At the same time, autophagy is an adaptable response under the stimulation of the endoplasmic reticulum (ER).…”