Dash Subhra, Sarashetti Prasad M, Rajashekar Balaji, Chowdhury Rajdeep, Mukherjee Sudeshna
Department of Biological Sciences, Birla Institute of Technology and Science (BITS), Pilani, Pilani Campus, Rajasthan, India.
Genotypic Technology Pvt. Ltd., Bangalore, India.
Oncotarget. 2018 Jan 4;9(5):6433-6449. doi: 10.18632/oncotarget.23942. eCollection 2018 Jan 19.
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.
肝细胞癌（HCC）通常在慢性炎症环境中发生，这种环境会导致大量生长因子和细胞因子的释放。然而，这些细胞因子对HCC进展的分子效应尚不清楚。在本研究中，我们将HCC细胞暴露于转化生长因子-β2（TGF-β2），这导致了类似上皮-间质转化（EMT）特征的显著升高。对EMT标志物的分子分析显示，给予TGF-β2后，RNA和蛋白质水平均增加，同时TGF-β诱导的Smad信号上调。EMT的诱导与活性氧（ROS）的同时增加以及TGF-β2处理细胞的细胞停滞有关。重要的是，ROS的淬灭导致TGF-β2诱导的EMT显著促进。此外，用TGF-β2处理的细胞也显示出自噬通量增强。有趣的是，用二磷酸氯喹（CQDP）抑制自噬或通过siRNA介导的ATG5缺失可显著抑制TGF-β2诱导的EMT。自噬抑制显著增加ROS水平，促进细胞凋亡。进一步观察到，促炎细胞因子如肿瘤坏死因子-α（TNF-α）可通过下调自噬、增加ROS水平从而抑制HCC细胞中的EMT来拮抗TGF-β2诱导的反应。TNF-α的这种抑制作用不依赖于血清。通过RNA测序进行的转录组分析进一步证实，TNF-α处理可抑制TGF-β2诱导的自噬基因，从而抑制EMT。我们的研究表明，自噬通过调节HCC细胞中的ROS水平发挥促进转移的作用，促进EMT，而TNF-α可通过抑制自噬来抑制EMT。我们为TGF-β2在HCC细胞中的作用提供了独特的机制见解，以及有效控制该疾病的适当线索。
