Wnt/β-catenin signaling pathway

1.4.1. Wnt/β-catenin signaling pathway

Wnt/β-catenin pathway plays vital roles in the development of various organs by directing cell proliferation, cell fate decision and cell polarity (Logan and Nusse, 2004). During tissue homeostasis, Wnt signaling was also shown to be critical to stem cell maintenance (Lowry et al., 2005; Reya et al., 2003). Therefore, dysregulation of Wnt signaling may cause cancers or other diseases. For instance, mutations of Wnt pathway were often observed in colon cancer (Lammi et al., 2004).

β-catenin is the major effector of Wnt signaling. In the absence of Wnt ligand, β-catenin forms a complex with GSK-3β, Axin and APC in the cytoplasm, leading to its ubiquitination and proteasomal degradation. When Wnt ligand binds onto its receptor, Frizzled (Fzd), β-catenin is released from the complex and then translocates into the nucleus. By binding with DNA-binding factors, T-cell factor/lymphoid enhancer factor (TCF/LEF), β-catenin initiates transcription of downstream target genes involved in various biological functions (Clevers, 2006) (Figure 1.6).

Figure 1.6. Scheme of canonical Wnt/β-catenin signaling.

1.4.2. Wnt/β-catenin signaling in liver diseases

Growing evidences have shown that Wnt signaling is also crucial to liver diseases as well as liver regeneration (Huch et al., 2013). Recent studies indicated β-catenin could stimulate cell-cycle entry of hepatocytes during liver regeneration after PHx treatment (Sekine et al., 2007). Likewise, accumulated β-catenin were shown to be involved in HCC progression by promoting cell proliferation (Thompson and Monga, 2007). Other researches further examined the role of Wnt signaling in the regeneration of chronic liver injury since it could regulate cell proliferation after PHx. A study using DDC diet to induce LPCs emergence and proliferation depicted that Wnt/β-catenin signaling was a part of niche signals that regulated LPCs behaviors (Itoh et al., 2009). Also, resection specimens from patients of liver injuries revealed an important role of Wnt signaling in human LPCs proliferation (Spee et al., 2010). As mentioned above, Wnt signaling could maintain stem cell properties, and it was proved to drive LPCs specification into hepatocytes (Boulter et al., 2012). These evidences implied the significance of Wnt signaling in liver regeneration, especially LPCs activation.

1.4.3. HIF-1α and Wnt/β-catenin signaling

In previous studies, HIF-1α has been proved to promote the expression of

β-regeneration. For example, hypoxia led to β-catenin accumulation in HCC and further stimulated EMT of cancer cells (Liu et al., 2010). Moreover, hypoxia could promote self-renewal in ESCs through Wnt signaling (Mazumdar et al., 2010).

Though, whether similar mechanisms work in other stem cell populations are still unknown.

Some recent researches further examined the direct interaction between 1α and β-catenin. A study showed that β-catenin would directly interact with HIF-1α in HCC, increasing the transcription activity of HIF-HIF-1α and facilitating EMT (Zhang et al., 2013). Another study found out that β-catenin could serve as coactivator of HIF-1α and improve hepatocyte survival using ischemia and reperfusion to induce liver injury (Lehwald et al., 2011). However, the relationship between HIF-1α and β-catenin during LPC activation has not been explored.

1.5. Epithelial cell adhesion molecule (EpCAM)

1.5.1. Epithelial cell adhesion molecule (EpCAM)

EpCAM is a transmembrane glycoprotein, containing an extracellular domain (EpEx), a transmembrane domain and a small intracellular domain (EpICD) (Schnell et al., 2013) (Figure 1.7). It was first characterized to function as an adhesive factor, which located on cell membrane and mediate cell-cell

adhesion (Litvinov et al., 1997). Later, EpCAM was found overexpressed in various carcinoma, but the mechanisms and its function in cancer cells are still not well known (Maetzel et al., 2009). Also, EpCAM was frequently detected in stem/progenitor cells and was lost in differentiated cells (Schmelzer et al., 2006).

Recent studies revealed that EpCAM can engage in signal transduction, which is associated with cell proliferation, migration, de-differentiation, maintenance of stem cell population (Gonzalez et al., 2009; Munz et al., 2004). The signal transduction begins by proteolysis of EpCAM, releasing EpICD into cytoplasm.

EpICD would form a complex with FHL2, β-catenin and Lef-1, translocate into nucleus, and then stimulate the transcription of downstream target genes (Munz et al., 2009) (Figure 1.8).

1.5.2. EpCAM in liver disease

As mentioned above, EpCAM may serve as an oncogenic signaling protein and was highly expressed in carcinomas. Some studies indicated that EpCAM could be a biomarker of HCC, and that the expression level of EpCAM was correlated with poor prognosis (Yamashita et al., 2008). EpCAM is also a marker of LPCs, thereby suggesting that EpCAM may be an essential factor to maintain cancer stem cells and promote their growth. As mentioned above, EpCAM is able

Figure 1.7. EpCAM is a transmembrane glycoprotein, containing an extracellular domain

(EpEx), a transmembrane domain (TM) and an intracellular domain (EpICD).

Figure 1.8. EpICD releases from plasma membrane and form a complex with FHL2,

β-catenin and Lef. The complex then stimulates the transcription of downstream target genes in the nucleus.

showed that EpCAM⁺ cells increased during liver injury (Okabe et al., 2009;

Yoon et al., 2011). Based on these results, it is reasonable to suspect that EpCAM might mediate LPCs proliferation during liver injury and promote regeneration.

1.5.3. HIF-1α and EpCAM

The relationship between HIF-1α and EpCAM has not been confirmed yet.

However, a recent study discovered that EpCAM increased under hypoxia condition and contributed to renal regeneration (Trzpis et al., 2008). Additionally, by examining specimens from HCC patients, HIF-1α and EpCAM were significantly highly expressed during recurrence after radiotherapy (Yamada et al., 2014). The results suggested that HIF-1α increment may subsequently induce the expression of EpCAM, a cancer stem cell marker, leading to tumor aggressiveness.

In silico analysis of EpCAM promoter discovered several regulatory enhancers

including hypoxia responsive element (HRE). Thus, HIF-1α may probably bind onto the HRE of EpCAM and regulate its expression.

1.5.4. Crosstalk between Wnt/β-catenin and EpCAM

EpCAM was characterized as a hepatic stem cell marker and Wnt/β-catenin signaling is critical to stem cell maintenance. A study found that EpCAM is a

EpCAM induction would be important to maintain cancer stem cell growth (Yamashita et al., 2007). Later, a nuclear complex formed by EpICD, FHL2, β-catenin and Lef-1 was detected. The complex could induce gene transcription by binding to Lef-1 consensus sites and promote cell proliferation (Maetzel et al., 2009). Other researches revealed the crosstalk between Wnt signaling and EpCAM in a variety of carcinomas, including HCC, colon cancer, thyroid cancer and ovarian cancer (Ralhan, 2010; van der Gun et al., 2011; Yamashita et al., 2009;

Zhou et al., 2015). However, whether Wnt signaling and EpCAM could synergistically mediate liver regeneration and LPCs activation is still unknown.