TRAF4 Regulates Migration, Invasion, and Epithelial-Mesenchymal Transition via PI3K/AKT Signaling in Hepatocellular Carcinoma

Abstract

Overexpression of the tumor necrosis factor receptor-associated factor 4 (TRAF4) has been detected in many cancer types and is considered to foster tumor progression. However, the role of TRAF4 in hepatocellular carcinoma (HCC) remains elusive. In this study, we found that TRAF4 was highly expressed in HCC cell lines and HCC tissues compared with normal liver cell lines and adjacent noncancerous tissues. TRAF4 overexpression in HCC tissues was correlated with tumor quantity and vascular invasion. In vitro studies showed that TRAF4 was associated with HCC cell migration and invasion. An in vivo study verified that TRAF4 overexpression facilitated metastasis in nude mice. In addition, overexpressed TRAF4 promoted the phosphorylation of Akt and induced Slug overexpression, leading to downregulated E-cadherin and upregulated vimentin, while silencing TRAF4 moderated the phosphorylation of Akt and repressed the expression of Slug, which resulted in upregulated E-cadherin and downregulated vimentin. These effects were inversed after pretreatment of the PI3K/Akt inhibitor LY294002 or overexpression of constitutively active Akt1. Our study demonstrated that TRAF4 was involved in promoting HCC cell migration and invasion. The process was induced by the EMT through activation of the PI3K/Akt signaling pathway.

Figure 1

Tumor necrosis factor receptor-associated factor 4 (TRAF4) is overexpressed in hepatocellular carcinoma (HCC) cell lines and HCC tissues. (A) TRAF4 is overexpressed in HCC cell lines at the mRNA level. Data are presented as the mean ± standard deviation (SD); n = 9. *p < 0.05. (B) TRAF4 is highly expressed in HCC cells at the protein level. (C) Representative immunohistochemistry images show that TRAF4 is more highly expressed in HCC tissues than in adjacent tumor tissues. Stronger stain intensity and higher stain density exist in HCC tissues with vascular invasion than in those without vascular invasion. (D) Quantitative charts of TRAF4 expression in tumor-adjacent tissues and HCC tissues are shown. The expression of TRAF4 is correlated with the incidence of HCC and vascular invasion. Data are presented as the percentage. *p < 0.05.

Figure 2

TRAF4 regulates HCC cell mobility. (A) The upregulation and downregulation of TRAF4 in HepG2 cells and Sk-Hep-1 cells are confirmed through qRT-PCR and Western blotting. Data are presented as the mean ± SD; n = 9. *p < 0.05. (B) Cell mobility is tested using a Transwell assay. Representative views shows that upregulated TRAF4 promotes cell migration and invasion, while downregulated TRAF4 attenuates these effects. (C) Quantitation of cell migration and invasion. Data are presented as the mean ± SD; n = 6. *p < 0.05. (D) TRAF4 overexpression promotes HCC metastasis in vivo. Representative views of lung tissues from each group and pulmonary metastasis rates are shown. Data are presented as the number.

Figure 3

TRAF4 regulates the epithelial–mesenchymal transition (EMT) in HCC cells. (A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) shows changes in EMT marker expression following stable overexpression or knockdown of TRAF4. (B) EMT marker expression is regulated at the protein level following the expression of TRAF4. Data are presented as the mean ± SD; n = 9. *p < 0.05.

Figure 4

TRAF4 induces the EMT via PI3K/Akt signaling activation. (A) TRAF4 knockdown mitigates the phosphorylation of Akt, while forced TRAF4 expression enhances the phosphorylation of Akt. (B) Constitutive activation of Akt1 rescues TRAF4 knockdown-impeded EMT in Sk-Hep-1 and Hep2 cells. (C) PI3K/Akt inhibitor LY294002 restricts the TRAF4-induced EMT in Sk-Hep-1 and Hep2 cells. Data are presented as the mean ± SD; n = 3. *p < 0.05.

Figure 5

Regulatory effect of PI3K/Akt signaling pathways on TRAF4-induced cell mobility. (A) Constitutive activation of Akt1 recovered cell migration and invasion impaired by TRAF4 downregulation (p < 0.05). (B) PI3K/Akt inhibitor LY294002 hampered the TRAF4-induced cell migration and invasion (p < 0.05). Data are presented as the mean ± SD; n = 6. *p < 0.05. All experiments were repeated independently three times; significant difference between groups: *p < 0.05.