Tollip promotes hepatocellular carcinoma progression via PI3K/AKT pathway

Abstract

The activation of signaling pathways induced by Toll-like receptor (TLR) has been demonstrated to play essential roles in multiple liver diseases. Toll-interacting protein (Tollip) acts as an endogenous negative modulator of TLR signaling and is implicated in various cardio-metabolic diseases. However, the effect of Tollip in hepatocellular carcinoma (HCC) remains elusive. In the current study, enhanced Tollip expression was observed in HCC cells and tissues examined by RT-PCR, western blot, and immunohistochemistry staining. Moreover, the co-immunofluorescence staining demonstrated that increased Tollip expression was primarily located in hepatocytes. Functionally, Tollip overexpression significantly increased proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of HCC cells, which ultimately accelerated tumorigenesis. Mechanistically, Tollip overexpression dramatically promoted the activation of PI3K/AKT signaling pathway in HCC cells which was attenuated by Tollip silencing. Importantly, the inhibition of PI3K/AKT axis can abolish the promoted effects of Tollip on proliferation and EMT of HCC cells. Our current study demonstrated that Tollip played an important role in the regulation of HCC development by engaging PI3K/AKT signaling pathway. These evidences suggested that the blockade of Tollip-PI3K/AKT axis was an ideal therapeutic treatment for management of HCC.

Keywords: PI3K/AKT; Tollip; epithelial-mesenchymal transition; hepatocellular carcinoma; prognosis.

Figure 1

Upregulation of Tollip expression in HCC. (a) The expression of Tollip in HCC and lung tissues of BABL/c nude mice following subcutaneous injection of HL-7702 or Huh7 tested by immunochemistry staining. Scale bar = 50 µm. n = 5–10 fields per experimental group. (b) The immunofluorescence staining shows Tollip (red) expression and localized in hepatocyte identified by HNF4 (green) of liver tissues from BABL/c nude mice following subcutaneous injection of HL-7702 or Huh7. Scale bar = 50 µm. n = 6–8 fields per experimental group. (c and d) The expression of Tollip mRNA or protein level tested by RT-PCR or protein in HCC cell lines. n = 3 independent experiments. *p < 0.05 compared with control group.

Figure 2

The oncogenic functions of Tollip in HCC. (a and b) The efficiencies of Tollip overexpression in HCC cell lines verified by RT-PCR (a) and western blot analysis (b). n = 3 independent experiments. (c) The morphology images of tumors following subcutaneous injection of Tollip-OE and vector. n = 5 mouse number. (d) Assessment of the influence of Tollip overexpression on HCC proliferation tested by CCK-8 at the indicated time. n = 3 independent experiments. *p < 0.05 compared with control group.

Figure 3

Tollip enhanced migratory, invasive, and metastatic capacities of HCC cells. (a) Evaluations of the influence of Tollip-OE on migration activities by transwell migration assays. n = 5 fields per experimental group. (b) Matrigel invasion assays for testing the effect of Tollip-OE on invasion ability. n = 5 fields per experimental group. (c) The lung slides from nude mice subjected with Tollip-OE detected by hematoxylin-eosin staining. Scale bar = 50 µm. n = 10 fields per experimental group. (d) Expressions of EMT-related markers in HCC cell lines infected with overexpression of Tollip by western blot assays. n = 3 independent experiments. *p < 0.05 compared with control group.

Figure 4

The regulation of PI3K/AKT signaling pathway in HCC cells by Tollip. (a) Western blots were performed with Tollip or AKT antibody after co-IP of Tollip. (b) Immunoblot showed AKT and mTOR phosphorylation and total levels in Tollip-OE and vector group. n = 3 independent experiments. *p < 0.05 compared with control group.

Figure 5

PI3K/AKT activation is required for the pro-tumorigenic properties of Tollip. (a) Immunoblot showed AKT and mTOR phosphorylation and total levels in HCC cells infected with Tollip-OE pretreated with LY294002. n = 3 independent experiments. (b and c) The impact of inactivation of PI3K/AKT pathway by LY294002 in Tollip-OE mediated cell proliferation (b) and EMT (c). n = 3 independent experiments. *p < 0.05 compared with Vector group. #p < 0.05 compared with Tollip-OE group. †p < 0.05 compared with Vector with LY294002 group.

Figure A1

The regulation of PI3K/AKT signaling pathway in HL-702 cells by Tollip. Immunoblot showed AKT and mTOR phosphorylation and total levels in HL-7702 infected with Tollip-OE and vector group. n = 3 independent experiments. *p < 0.05 compared with control group.

Figure A2

Tollip enhanced proliferate, migratory and metastatic capacities of SMMC-7221 cells. (a) Assessment of the influence of Tollip overexpression on SMMC-7221 proliferation tested by CCK-8 at the indicated time. (b) Evaluations of the influence of Tollip-OE on migration activities of SMMC-7221 by transwell migration assays. n = 5 fields per experimental group. (c) Expressions of EMT-related markers in SMMC-7221 cell infected with overexpression of Tollip by western blot assays. n = 3 independent experiments. *p < 0.05 compared with control group.

Figure A3

The activation of PI3K/AKT signaling pathway in HL-7702 and Huh7 cells. Immunoblot showed AKT and mTOR phosphorylation and total levels in HL-7702 and Huh7 group. n = 3 independent experiments. *p < 0.05 compared with control group.