Primary liver cancer is the fifth most common cancer worldwide and the third most common cause of cancer mortality. Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancers and usually occurs within an established background of chronic liver disease. It has been well documented that HCC originates from a series of genetic and epigenetic alterations, including gene mutations and aberrant DNA methylation patterns (1, 2). During HCC progression, hepatocytes gradually adopt a fibroblast-like phenotype, a process that is reminiscent of the epithelial-to-mesenchymal transition (EMT) that normally occurs during embryogenesis. A key event in EMT includes the disruption of cellular architecture, polarity and connectivity. This enables the cancer cells to become migratory and invasive, which in turn triggers metastasis (1–5).
The drastic changes in the cellular microenvironment that take place during EMT are accompanied by the disassembly of junctional structures (3–6). Epithelial cells typically display four types of intercellular junctions, namely adherens junctions, desmosomes, gap junctions and tight junctions. Tight junctions, also referred to as occluding junctions, are composed of a wide variety of proteins, and the list of newly discovered members is still growing. Tight junction components are grouped as transmembrane and cytoplasmic members (see Fig. 1). The former are all integral membrane proteins and include occludin, claudins, junctional adhesion molecules (JAM), the coxsackie-adenovirus receptor (CAR) and the recently discovered tricellulin. Cytoplasmic tight junction building stones are subdivided into two classes according to the presence of a so-called post-synaptic density protein-95/disc large protein/zonula occludens-1 (PDZ) domain within their primary protein structure. PDZ-containing tight junction proteins include members such as zonula occludens (ZO) proteins and partitioning-defective proteins (PAR), whereas symplekin and cingulin belong to the group of non-PDZ-containing cytoplasmic tight junction proteins (7–11). Tight junctions are specialized in sealing cells together. Basically, occluding junctions perform two functions, namely the control of paracellular diffusion of solutes (barrier function or gate function) and the restriction of intramembrane diffusion of proteins and lipids between the membrane poles (fence function). The latter, as well as the mechanical link between the tight junction and microfilamentous cytoskeleton (F-actin), are major determinants of cell polarity. Besides these basic activities that play essential roles in virtually all aspects of the cell's life cycle, tight junctions also control the homeostatic balance at another level. Thus, a number of tight junction components, such as the ZO-1-associated nucleic acid-binding protein (ZONAB), shuttle between the cell membrane surface and the nucleus, and are directly involved in the regulation of gene expression (10, 11).
In the last decade, many efforts have been focussed on the elucidation of the molecular mechanisms that underlie the abrogation of cellular contacts in EMT. In fact, EMT is governed by the interplay between a number of prominent signal transduction pathways, including Smad signalling, receptor tyrosine kinase signalling, Notch signalling and phosphatidyl-3′-kinase/mitogen-activated protein kinase (PI3K/MAPK) signalling. The central role of transforming growth factor β (TGF-β) within the EMT signalling circuit has been well acknowledged. Upon ligand binding, type II TGF-β receptor subunits phosphorylate their type I counterparts, which trigger signalling effectors such as PI3K and MAPK. In a more straight forward pathway, stimulated TGF-β receptors phosphorylate particular members of the cytoplasmic Smad protein family. Activated Smad proteins form complexes among each other and then travel to the nucleus where they interfere with the transcription of genes that assist in cell proliferation, differentiation, apoptosis and migration. The prototypic adherens junction building stone E-cadherin is a well-studied target in this regard. A complex network of Smad-regulated transcription factors, including Snail, Slug and Smad interacting protein 1 (SIP1), are activated downstream of TGF-β, and they all repress E-cadherin expression. A similar scenario seems to hold for other junctional proteins, because these transcription factors also downregulate the expression of connexin26, plakophilin 2, desmoplakin, ZO proteins, claudins and occludin (3–6).
In this issue of Liver International, Kojima et al. (12) explored the biological impact of EMT on hepatic tight junctions. To this end, the authors used primary cultures of adult rat hepatocytes that were exposed to TGF-β. The EMT-elicited response was evidenced by downregulated E-cadherin expression and concomitant increased SIP1 and Snail mRNA contents. The fence function, inherent to tight junctions, was disrupted in this experimental setting. This was associated with decreased and increased expressions of claudin-1 and claudin-2, respectively, whereas occludin was not affected. As such, this information is not new, because EMT-associated modifications in hepatocellular tight junction production have already been reported (13, 14). However, the Kojima study is innovative in the sense that new light is shed on the molecular mechanisms that drive EMT-linked alterations in hepatocellular tight junctions. The authors demonstrate that the downregulation of the fence function and claudin-1 expression during TGF-β-induced EMT in primary rat hepatocytes occurred through a Smad-independent mechanism that involves protein kinase C, p38MAPK, PI3K but not MAPK. Of course, a crucial question that remains to be answered is how these individual signalling effectors exactly perform their actions on tight junction expression and functionality, and to what extent this occurs. Based on the findings of Kojima et al., it seems tempting to speculate that TGF-β could also interfere with alternative tight junction activities, including the barrier function as well as its involvement in the control of gene expression. These issues certainly deserve further investigation. Another interesting topic to be addressed in follow-up studies is the impact of EMT on other junctional proteins in hepatocytes, whether or not belonging to the tight junction complex.
The work of Kojima et al. is a mechanistic study in the first instance. It is clear that the molecular scenario that is proposed by the authors should be confirmed in human hepatocytes and may actually be an oversimplification of the in vivo situation. Nevertheless, the data that are provided by the authors could have important clinical implications. Tight junction proteins have recently been proposed as potential biomarkers in HCC (15, 16). Attenuated expression of claudin-1, for example, was found to correlate with dedifferentiation and invasion of HCC and may therefore serve as a marker for poor HCC prognosis (15). The research of Kojima et al. further underscores the diagnostic value of claudins. In addition, boosting of tight junctions could be an interesting strategy in HCC therapy. A number of compounds are known to upregulate tight junction expression in hepatoma cells, including retinoic acid (17) and chenodeoxycholic acid (18). A most promising class of tight junction inducers are epigenetic modulators of gene expression, which are typically represented by histone deacetylase (HDAC) inhibitors. HDAC inhibitors display pleiotropic antitumour properties, including the induction of cell cycle arrests, differentiation and apoptosis, as well as the inhibition of angiogenesis and metastasis (8). Tight junction proteins, like occludin, CAR, cingulin and ZO proteins have been repeatedly found as positive targets for HDAC inhibitors (8, 19). Further exploration of such targeted anticancer strategies should be strongly encouraged as they provide an important contribution to the way forward of successful management of HCC.