Wnt signaling

The Wnt signaling pathway plays a crucial role not only in cell-cell communication during development and normal tissue homeostasis, but its deregulation is also often associated with tumorigenesis and cancer progression. The more than 19 identified Wnt ligands belong to a family of highly conserved secreted glycoproteins and are capable of activating Wnt signaling (Gordon & Nusse, 2006). During ß-catenin-dependent, canonical Wnt signaling binding of Wnt proteins to a receptor complex consisting of a Frizzled receptor and the co-receptor LRP5/6 (Tamai et al, 2000; Wehrli et al, 2000) induces stabilization and translocation of cytoplasmic ß-catenin to the nucleus where it binds to LEF/TCF transcription factors and activates target gene expression (van de Wetering et al, 1997). Apart from the Wnt inhibitory factor-1 (WIF-1) and secreted Frizzled-Related Proteins (sFRP), the family of Dickkopf (DKK) proteins is known to inhibit Wnt signaling by binding to the co-receptor LRP6 (Semenov et al, 2008).

In addition to the canonical pathway, two ß-catenin-independent non-canonical pathways have been described: The planar cell polarity (PCP) pathway and the Wnt/Ca²⁺ pathway.

Activation of the latter has been associated with an increase in intracellular calcium levels

which leads to activation of PKC and CamK II (Kuhl et al, 2000) which, in turn, activate several nuclear transcription factors including e.g. NFκB or CREB (De, 2011). The Wnt/Ca²⁺

pathway was shown to play a pivotal role in cardiac development (Rao & Kuhl, 2010) and was suggested to exert tumor-suppressing effects in human basal cell carcinoma (Nitzki et al, 2010).

In contrast, PCP signaling was described to enhance tumor cell migration and invasion in several human cancer models due to its regulatory influence on cytoskeletal organization and cell motility (Ripka et al, 2007; Weeraratna et al, 2002). Activation of the PCP pathway induces a signaling cascade that results in activation of JNK-signaling and ROCK-mediated actin reorganization (Wallingford & Habas, 2005). Both non-canonical pathways, Wnt/Ca²⁺

and PCP, can be activated by the Wnt ligand Wnt5a (De, 2011; Yamanaka et al, 2002).

1.4.1 Wnt5a – a controversially discussed Wnt ligand

Wnt5a was originally described as a non-canonical ligand although recent data show that it is also capable of activating ß-catenin-dependent canonical Wnt signaling (Mikels & Nusse, 2006). In this context, its influence on canonical signaling seems to depend on the receptor context (Mikels & Nusse, 2006).

The Wnt5a protein was found to be glycosylated and palmitoylated, the latter being essential for its function (Kurayoshi et al, 2007). Due to its high hydrophobicity, it can bind to the ECM which was suggested to be involved in formation of local gradients (Ai et al, 2003). The role of Wnt5a in cancer is controversially discussed (McDonald & Silver, 2009; Pukrop &

Binder, 2008). On the one hand, it was shown to have no transforming activity (Olson &

Papkoff, 1994; Wong et al, 1994), was found to be downregulated on the mRNA level in neuroblastoma, leukemia or endometrial carcinoma compared to corresponding benign tissues (Blanc et al, 2005; Bui et al, 1997; Liang et al, 2003) and even inhibited cell migration and invasion in several cancer models (Dejmek et al, 2005; Jiang et al, 2013; Kremenevskaja et al, 2005). On the other hand, Wnt5a is overexpressed in many human cancers (Pukrop & Binder, 2008) and was shown to induce expression of several pro-invasive factors including MMP (Prieve & Moon, 2003), CD44 and vimentin (Dissanayake et al, 2007) or laminin γ2 (Hanaki et al, 2012). Wnt5a was not only expressed in the tumor cells themselves, but especially in tumor-associated Mϕ (Pukrop et al, 2006; Smith et al, 1999), which were predominantly localized at the invasive front of the tumor (Pukrop et al, 2006).

1.4.2 Secretion of Wnt proteins

Although the first Wnt gene was already isolated in 1982 (Nusse & Varmus, 1982), the mechanism of Wnt protein synthesis and secretion is still not completely understood. Wnt proteins are generally modified by a series of posttranslational modifications which are important for their secretion and function. Especially palmitoylation was found to be crucial for the activity of Wnt proteins (Willert et al, 2003). After translation most Wnt proteins are lipid-modified by the acyltransferase Porcupine which is located in the membrane of the endoplasmic reticulum (Kadowaki et al, 1996; Willert et al, 2003). It was shown that Porcupine-mediated acetylation requires correct previous glycosylation of Wnt3a (Komekado et al, 2007) or Wnt5a (Kurayoshi et al, 2007) and missing glycosylation significantly impaired secretion of both Wnt proteins.

Fig. 3: Evi is essential for secretion of Wnt proteins

After lipid modification by the acyltransferase Porcupine in the endoplasmic reticulum, Wnt proteins are transported to the Golgi complex where they bind to Evi and are escorted to the cell surface in secretory vesicles for secretion (left). In contrast, loss of Evi results in an accumulation of synthesized Wnt proteins inside the cell (right). Taken from (Ching & Nusse, 2006) with permission from Elsevier.

Lipid-modified Wnt proteins then bind to p24 proteins and are transported to the Golgi complex (Buechling et al, 2011; Port et al, 2011) where they associate with Evi, also known as WLS or Gpr177. Evi is a seven-pass transmembrane protein that is specifically required for escorting Wnt proteins from the Golgi complex to the plasma membrane (Banziger et al, 2006; Bartscherer et al, 2006). Loss of Evi causes an accumulation of Wnt proteins in the Wnt-producing cell (Bartscherer et al, 2006) as depicted in Fig. 3. Furthermore, Evi has often been proposed to exert a chaperone function for the correct folding of synthesized Wnt proteins, although this has not been confirmed yet. After secretion of Wnt proteins Evi is either targeted to lysosomal degradation or can be recycled via endosomes and the retromer

complex to the Golgi where it can associate with new Wnt proteins and participate in a second round of Wnt secretion (Bartscherer & Boutros, 2008; Port et al, 2008). Following secretion, Wnt proteins are found in the supernatant of the Wnt-producing cell, but were also predicted to be tethered to the cellular plasma membrane due to their hydrophobic lipid moieties (Janda et al, 2012).