To study the key roles of TLR4 in LPS-induced EMT of HCC cells, we selected four different HCC cells (HepG2, Huh7, Hep3B, and SMMC-7721) and carefully evaluated the direct effect of LPS on the invasive potential of these cells. We demonstrated that LPS could significantly enhance the invasive potential and induce EMT of HCC cells with higher expression of TLR4, which was confirmed to be in a TLR4 dependent manner. Further studies showed that LPS could directly activate NF-κB signaling through TLR4 in HCC cells. Importantly, blocking NF-κB signaling significantly inhibited Snail expression and inhibited EMT occurrence. Finally, we revealed that high expression of TLR4 in HCC tissues was strongly associated with both poor cancer-free survival and overall survival. Our results indicate that TLR4 is necessary for LPS-induced EMT and metastasis in HCC cells, which increases our understanding of the pathogenesis and provides clues for developing new strategies against LPS-related metastasis of HCC.
This study provides the first evidence that LPS-induced metastasis and EMT of HCC cells is TLR4 dependent. EMT is a key event in the metastasis of tumors from epithelial cells. An imaging study in vivo
has shown that carcinoma cells migrate from mouse primary tumors through a process of EMT and that this process is dependent on an inflammatory microenvironment [26
]. Recently, the new finding that TNF-α induces EMT in MCF-7 breast cancer cells reinforced the connection between inflammation and EMT [27
]. In HCC patients, the level of endotoxin in the portal and peripheral veins has been determined to be higher, which results in a high level of LPS in the portal vein. In this study, four different human HCC cell lines (HepG2, Huh7, Hep3B, and SMMC-7721) were tested in the scratch wound-healing and Matrigel invasion assay. Our results showed that not all tumor cell lines responded in vitro
to LPS, the motility and invasion abilities of SMMC-7721 cells were enhanced significantly, and the data of the splenic vein metastasis assay in vivo
were consistent with this. However, Liu et al.
have inferred that LPS may be ignored by immune systems and may directly promote the invasion abilities of HepG2 and HepG2.2.15 cells [9
]. Our experiment showed that LPS had little effect on HepG2 cells; the different experimental methods may have led to the inconsistent results.
Administration of LPS can induce EMT of human IBECs, which makes tumor cells undergo a phenotypic change to produce bipolar cells with a fibroblastic morphology. In these four HCC cells, LPS induced EMT in SMMC-7721 cells but not the others, and SMMC-7721 cells constitutively expressed a high level of TLR4, which might be associated with LPS-induced metastasis and EMT of HCC cells. When we used adeno-associated virus to express TLR4 in HepG2 cells or knocked down TLR4 in SMMC-7721 cells with siRNA, the metastasis and EMT of HCC cells were changed by being treated with LPS. All of the above results highlight a direct link between LPS and tumor cell metastasis. The effect of LPS is possible because SMMC-7721 cells constitutively express TLR-4. Not all tumor cells are positive for TLR-4, and this could depend on the tissue from which the tumor cells originated. Molteni et al.
analyzed the responses of tumor cell lines from different origins (melanoma, ovarian carcinoma, neuroblastoma) to LPS in vitro;
the results showed that only melanoma cells significantly increased cell adhesion, when triggered with LPS and these effects were associated with the constitutive expression of TLR-4 [28
]. Different HCC cell lines also have different expression of TLR4, and LPS-induced EMT in cells is in a TLR4 dependent manner as well. However, notably, HCC development is a multifactorial and complicated process, which has a close association with various risk factors, such as viral infection, alcohol consumption, steatosis, toxic substances and inflammation. In this process, LPS is an important EMT inducer but not the only one. Many gene alterations and cytokines also could induce EMT and promote metastasis [29
]. Therefore, although HCC cells with low expression or even a lack of TLR4 are not susceptible to LPS, they might perform EMT induced by other TLR4-independent mechanisms.
NF-κB activation is well known to play an important role in responses to LPS via TLR-4 [8
]. Previous studies have shown that LPS may be able to induce NF-κB activation in colon and pancreatic cancer cells [30
], which strongly suggests that LPS can act not only on immune cells but also on some cancer cells from epithelial cells. In the current study, we found that ligation of LPS and TLR4 could activate the NF-κB pathway in SMMC-7721 cells, but not in HepG2 cells. Furthermore, activation of NF-κB is involved in the invasion and metastasis of HCC. Many studies have shown that NF-κB is a key regulator of Snail expression in cancer cell lines and metastatic tumor samples [32
]. In our study, activation of NF-κB in SMMC-7721 cells up-regulated Snail expression, which was a prerequisite for these cells to undergo an EMT toward an invasive, metastatic tumor phenotype. Snail is identified as a transcription factor in the control of EMT, and expression of Snail represses expression of E-cadherin and induces EMT in breast cancer cells, indicating that Snail plays a fundamental role in EMT [36
]. To confirm the contribution of NF-κB activation to the enhanced invasive ability and EMT, we used BAY-11-7082 to inhibit NF-κB activation in SMMC-7721 cells, and the inhibitor not only reduced NF-κB activation, but also down-regulated Snail expression. Our data strongly suggest that activation of the NF-κB pathway by LPS is TLR4-dependent and increases Snail expression to induce EMT.
TLR4 expression has been described in different human tumors [10
]. Gonzalez-Reyes et al.
have showed that TLR4 expression in breast carcinomas was associated with an increased incidence of metastasis and has prognostic significance [14
]. Cammarota et al.
analyzed 116 tissue samples from patients with different stages of colorectal disease and found that adenocarcinoma patients with higher TLR-4 expression in the stromal compartment had a significantly increased risk of disease progression and relapsed significantly earlier than those with lower expression levels [38
]. Interestingly, analysis of the association of TLR4 expression and the clinicopathological characteristics of 106 HCC patients reveals that TLR4 expression is significantly correlated with margin, vascular invasion, portal vein thrombosis, and so on, which are widely accepted markers for metastasis and poor prognosis of HCC [39
]. The Kaplan-Meier analysis shows that the HCC patients with high TLR4 expression in general had a shorter cancer-free interval and a worse overall survival than those with low expression, suggesting that TLR4 may be a useful biomarker of HCC metastasis and recurrence.
In conclusion, our study has shown for the first time that LPS-induced EMT and the invasive potential of HCC cells is in a TLR4 dependent manner and that activation of the NF-κB-Snail regulatory axis may function as a therapeutic target. Furthermore, we have demonstrated that high expression of TLR4 in HCC tissues significantly correlates with metastasis and recurrence of HCC. Our data suggest TLR4 as a novel prognostic marker and a potential therapeutic target for LPS-induced EMT and metastasis of HCC.