The Effects Of Autophagy And Proteasomes On TGFβ Signalling And EMT
Abstract
Transforming growth factor-β (TGFβ) signalling regulates growth, proliferation, immunity, and development. Although TGFβ typically antagonizes tumour formation, tumour cells often acquire mutations within the TGFβ signalling pathway that activate epithelial-mesenchymal transition (EMT). During EMT, epithelial tumour cells lose epithelial-like properties and acquire mesenchymal-like characteristics, which allows tumour cells to detach from the primary tumour and establish metastatic colonies. In addition to EMT, TGFβ augments tumourigenesis by increasing the degradation of damaged macromolecules and organelles via autophagy. Autophagy contributes to radiotherapy and chemotherapy resistance by mitigating the damages inflicted on tumour cells. Currently, there is a growing interest in the relationship between TGFβ signalling and protein degradative pathways as inhibitors of both autophagy and proteasomes block TGFβ-dependent EMT. Therefore, understanding pro-tumourigenic TGFβ signalling and its relationship with degradative processes has become a topic of interest for identifying novel therapeutic targets.
Since the mechanism of TGFβ-dependent autophagy activation was unknown, this work utilized immunoblotting and fluorescence microscopy of non-small cell lung cancer (NSCLC) cells that stably expressed a green fluorescent protein and red fluorescent protein conjugated to microtubule-associated protein light chain 3 (LC3) and LC3 with a glycine deletion, respectively, to elucidate TGFβ signalling branches that activate autophagy. Indeed, both Smad-dependent and -independent TGFβ signalling, activate autophagy by increasing the proportion of active uncoordinated 51-like autophagy activating kinase 1. This work also demonstrated that impeding autophagy using small interfering RNA targeting autophagy-related genes as well as pharmacological inhibitors including chloroquine, spautin-1, and ULK-101 blocked TGFβ receptor endocytosis, Smad phosphorylation, Smad nuclear translocation, EMT, and cell migration. Likewise, proteasome inhibitors, such as MG132 and lactacystin, promoted lysosomal-targeting of TGFβRII and dampened TGFβ-induced R-Smad phosphorylation, R-Smad nuclear translocation, and EMT. Since protein 62/sequestosome 1 (p62/SQSTM1) delivers cargo to both degradative pathways, its expression was silenced using small interfering RNA. Silencing p62/SQSTM1 disrupted TGFβ signalling and promoted EMT; however, there were no changes to autophagy.
In conclusion, this work discovered that altering autophagy or proteasome activity attenuated TGFβ signalling and blocked the pro-tumourigenic properties of TGFβ in NSCLC.