TGF-β2-induced EMT is dampened by inhibition of autophagy and TNF-α treatment
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Subhra Dash1, Prasad M. Sarashetti2, Balaji Rajashekar2,3, Rajdeep Chowdhury1 and Sudeshna Mukherjee1
1Department of Biological Sciences, Birla Institute of Technology and Science (BITS), Pilani, Pilani Campus, Rajasthan, India
2Genotypic Technology Pvt. Ltd., Bangalore, India
3Institute of Computer Science, University of Tartu, Estonia
Sudeshna Mukherjee, email: [email protected]
Rajdeep Chowdhury, email: [email protected]
Keywords: autophagy; ROS; EMT; TGF-β2; TNF-α
Received: June 16, 2017 Accepted: December 23, 2017 Published: January 04, 2018
Hepatocellular carcinoma (HCC) typically develops in a chronic inflammatory setting causal to release of a plethora of growth factors and cytokines. However, the molecular effect of these cytokines on HCC progression is poorly understood. In this study, we exposed HCC cells to TGF-β2 (Transforming Growth Factor-β2), which resulted in a significant elevation of EMT (Epithelial to Mesenchymal Transition) like features. Molecular analysis of EMT markers showed an increase at both RNA and protein levels upon TGF-β2 administration along with up-regulation of TGF-β-induced Smad signaling. Induction of EMT was associated with a simultaneous increase in reactive oxygen species (ROS) and cytostasis of TGF-β2-treated cells. Importantly, quenching of ROS resulted in a significant promotion of TGF-β2-induced EMT. Furthermore, cells treated with TGF-β2 also showed an enhanced autophagic flux. Interestingly, inhibition of autophagy by chloroquine-di-phosphate (CQDP) or siRNA-mediated ablation of ATG5 drastically inhibited TGF-β2-induced EMT. Autophagy inhibition significantly increased ROS levels promoting apoptosis. It was further observed that pro-inflammatory cytokine like, TNF-α (Tumor Necrosis Factor-α) can antagonize TGF-β2-induced response by down-regulating autophagy, increasing ROS levels and thus inhibiting EMT in HCC cells. This inhibitory effect of TNF-α is serum-independent. Transcriptomic analysis through RNA sequencing was further performed which validated that TGF-β2-induced autophagic genes are inhibited by TNF-α treatment suppressing EMT. Our study suggests that autophagy plays a pro-metastatic role facilitating EMT by regulating ROS levels in HCC cells and TNF-α can suppress EMT by inhibiting autophagy. We provide unique mechanistic insights into the role of TGF-β2 in HCC cells, along with appropriate cues to effectively control the disease.
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