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TGF-?2-induced EMT is dampened by inhibition of autophagy and TNF-? treatment.

ABSTRACT: 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-?2 (Transforming Growth Factor-?2), 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-?2 administration along with up-regulation of TGF-?-induced Smad signaling. Induction of EMT was associated with a simultaneous increase in reactive oxygen species (ROS) and cytostasis of TGF-?2-treated cells. Importantly, quenching of ROS resulted in a significant promotion of TGF-?2-induced EMT. Furthermore, cells treated with TGF-?2 also showed an enhanced autophagic flux. Interestingly, inhibition of autophagy by chloroquine-di-phosphate (CQDP) or siRNA-mediated ablation of ATG5 drastically inhibited TGF-?2-induced EMT. Autophagy inhibition significantly increased ROS levels promoting apoptosis. It was further observed that pro-inflammatory cytokine like, TNF-? (Tumor Necrosis Factor-?) can antagonize TGF-?2-induced response by down-regulating autophagy, increasing ROS levels and thus inhibiting EMT in HCC cells. This inhibitory effect of TNF-? is serum-independent. Transcriptomic analysis through RNA sequencing was further performed which validated that TGF-?2-induced autophagic genes are inhibited by TNF-? treatment suppressing EMT. Our study suggests that autophagy plays a pro-metastatic role facilitating EMT by regulating ROS levels in HCC cells and TNF-? can suppress EMT by inhibiting autophagy. We provide unique mechanistic insights into the role of TGF-?2 in HCC cells, along with appropriate cues to effectively control the disease.

SUBMITTER: Dash S

PROVIDER: S-EPMC5814223 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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TGF-β2-induced EMT is dampened by inhibition of autophagy and TNF-α treatment.

Dash Subhra S Sarashetti Prasad M PM Rajashekar Balaji B Chowdhury Rajdeep R Mukherjee Sudeshna S

Oncotarget 20180104 5

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 le ...[more]

PMID: 29464083

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Project description:Hepatocellular carcinoma (HCC) is the most common type of primary hepatic malignancy. HCC is one of the leading causes of cancer deaths worldwide. The oral multi-tyrosine kinase inhibitor Sorafenib is the standard first-line therapy in patients with advanced unresectable HCC. Despite the significant survival benefit in HCC patients post treatment with Sorafenib, many patients had progressive disease as a result of acquiring drug resistance. Circumventing resistance to Sorafenib by exploring and targeting possible molecular mechanisms and pathways is an area of active investigation worldwide. Epithelial-to-mesenchymal transition (EMT) is a cellular process allowing epithelial cells to assume mesenchymal traits. HCC tumour cells undergo EMT to become immune evasive and develop resistance to Sorafenib treatment. Immune checkpoint molecules control immune escape in many tumours, including HCC. The aim of this study is to investigate whether combined inhibition of EMT and immune checkpoints can re-sensitise HCC to Sorafenib treatment. Post treatment with Sorafenib, HCC cells PLC/PRF/5 and Hep3B were monitored for induction of EMT and immune checkpoint molecules using quantitative reverse transcriptase (qRT)- PCR, western blot, immunofluorescence, and motility assays. The effect of combination treatment with SB431542, a specific inhibitor of the transforming growth factor (TGF)-β receptor kinase, and siRNA mediated knockdown of programmed cell death protein ligand-1 (PD-L1) on Sorafenib resistance was examined using a cell viability assay. We found that three days of Sorafenib treatment activated EMT with overexpression of TGF-β1 in both HCC cell lines. Following Sorafenib exposure, increase in the expression of PD-L1 and other immune checkpoints was observed. SB431542 blocked the TGF-β1-mediated EMT in HCC cells and also repressed PD-L1 expression. Likewise, knockdown of PD-L1 inhibited EMT. Moreover, the sensitivity of HCC cells to Sorafenib was enhanced by combining a blockade of EMT with SB431542 and knockdown of PD-L1 expression. Sorafenib-induced motility was attenuated with the combined treatment of SB431542 and PD-L1 knockdown. Our findings indicate that treatment with Sorafenib induces EMT and expression of immune checkpoint molecules, which contributes to Sorafenib resistance in HCC cells. Thus, the combination treatment strategy of inhibiting EMT and immune checkpoint molecules can re-sensitise HCC cells to Sorafenib.

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TGF-?2-induced EMT is dampened by inhibition of autophagy and TNF-? treatment.

Publications

TGF-β2-induced EMT is dampened by inhibition of autophagy and TNF-α treatment.

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