In this study, we demonstrated that co-culture of Bel-7402 cells with HL-7702 or RF/6A cells led to initiation of MET and an attenuation of in vitro invasion/migration, characterized by increased expression of the E-cadherin/catenin complex. However, the mesenchymal properties were not markedly affected. E-cadherin repressors including Snail, Slug, Twist1 and ZEB-2 were moderately-to-strongly induced, and mesenchymal markers including vimentin, MMP-3 and MMP-7 were significantly induced. Accordingly, co-cultured Bel-7402 underwent only a partial reversion of EMT. These results correlate with previous reports [24, 25] demonstrating that the induction or silencing of one or some of the most potent pro-EMT agents did not necessarily impinge on the expression of all of the other EMT markers. This phenomenon could be explained by the complexity of the EMT regulation network.
It is important to keep in mind that EMT is probably a transient and reversible event during tumor progression and could occur at several stages of the metastatic process, such as during the intra- or extravasation . Additionally, since EMT could be initiated by many different signaling effectors in a paracrine fashion, only a minimal number of tumor cells might be responsive to EMT-inducing cues . Furthermore, the classical EMT event is considered as the exception, and the induction of all EMT characteristics within tumors is difficult to observe . To further understand the complex biological processes, such as cell motility and reversion of EMT, we analyzed the expression of vascular endothelial growth factor (VEGF)-c, hepatocyte nuclear factor 4α (HNF4α), CD147 and Sonic hedgehog-Gli pathway components (SHH, Patch-1 and SMO) in co-cultured Bel-7402 cells, all of which are associated with tumor cell invasion and migration [29–32], as shown in (Additional file 5: Figure S3). HNF4α, which could suppress the development of HCC via inhibiting activation of β-catenin, was increased in co-cultured Bel-7402 cells. However, CD147, which could promote cell motility through regulating annexin II-activated RhoA and Rac1 signaling pathways, was also up-regulated. Moreover, Sonic hedgehog-Gli pathway components changed in opposite trends. The expression of VEGF-c did not change. Therefore, the relevance of EMT/MET in human tumors remains to be resolved.
Our studies verified that MRC-5 induced Bel-7402 cells to undergo an EMT-like transformation. These were the only cells that produced a large quantity of hepatocyte growth factor (HGF) and co-expressed the HGF receptor c-Met and HGF activator (HGFA). The HGF/MET signaling pathway is associated with cancer cell migration and invasion . However, the mechanism underlying the induction of Bel-7402 cells to undergo EMT-like transformation remains to be elucidated.
Recently, significant insight had been obtained linking EMT and the acquisition of epithelial stem cell properties . In the present study, we demonstrated for the first time that HL-7702 cells could significantly inhibit the tumorigenic ability and viability of Bel-7402 cells. Contradictorily, RF/6A cells enhanced the colony formation ability of Bel-7402 cells. Intriguingly, CM of MRC-5 did not promote the tumorigenicity of Bel-7402 cells; rather, MRC-5-CM inhibited the colony-forming efficiency of Bel-7402 cells. It appears that microvessels maintain the survival of tumor cells in the blood and could operate as a “seed repertory” for tumor metastasis. This is in contrast to parenchymal cells in tumor-host tissue that might suppress tumor development. Also, MRC-5 fibroblasts might preferentially generate HCC cells with enhance motility. However, whether MRC-5 cells could generate HCC cells with stem cell potential remains a matter for debate.
Laminins and integrins have recently been identified as EMT biomarkers in head and neck squamous cell carcinomas progression , suggesting them to be related to invasion and migration of cancer cells. In the present study, we confirmed that the expression of laminin α1 and integrin α4, α11, AL, AV, β1, β6, β7, β8 was significantly associated with the malignant potential of HCC cells. In addition, we evaluated the expression of integrin β1, β3, β4, β7, laminin β3, E-cadherin and Snail in 42 paired HCC surgical tissues. The results showed that down-regulation of integrin β1 was correlated with capsular formation, and that integrin β4 was negatively correlated with CK19 expression. The role of integrin β1 has been well documented in breast cancer  and non-small cell lung cancer  during the few last years, but to date, its role in HCC progression has received less attention. Moreover, the integrin β1 expression in breast cancer remains controversial. Some studies reported that down-regulated expression of integrin β1 was correlated with more aggressive disease , while others demonstrated that up-regulated expression of integrin β1 was correlated with decreased survival . One possible explanation is that the alteration of integrin β1 expression is associated with different stages of tumor progression. Positive expression of CK19 indicated more aggressive HCC and was a valuable predictor of early recurrence and poor prognosis . Expression of integrin β4 showed a significant negative correlation with CK19, suggesting that integrin β4 inhibits the progression of HCC.
The expression of integrin β7, laminin β3, Snail and E-cadherin was frequently decreased in HCC tissues. Expression levels of these factors did not differ significantly according to age, gender, liver cirrhosis, tumor size, CK19, CK34, capsular formation, vascular invasion and hitological differention, likely due to the limited number of cases.