Planteamiento de hipótesis e identificación de variables

The components of the Hh signalling pathway include the secreted glycoproteins; Sonic (Shh), Desert (Dhh) and Indian (Ihh) (Ingham and McMahon, 2001; Pasca di Magliano and Hebrok, 2003). These ligands bind to one of the receptors including; Hedgehog-interacting protein 1 (Hip1), Patched1 (Ptch1) and Patched2 (Ptch2) (Marigo et al., 1996; Chuang and McMahon, 1999). The receptors then release the blocking effect on another transmembrane protein called Smoothened, Smo (Figure 0-6-B, and Figure 0-7-C)(Pasca di Magliano and Hebrok, 2003; Flemban and Qualtrough, 2015). As a consequence of this, Smo activates intracellular signalling via the Glioma-associated oncogene (Gli1, Gli2, and Gli3) transcription factors (Figure 0-7-D) (Ruiz i Altaba et al., 2002; Pasca di Magliano and Hebrok, 2003). These transcription factors are sequestered in the cytoplasm through connection to the cellular cytoskeleton by interaction with multiprotein complex Fused (Fu) and suppressor of Fused (SuFu) (Figure 0-6-D) (Murone et al., 2000; Pasca di Magliano and Hebrok, 2003).

Figure 0-6, The resting status of the Hh signalling pathway.

The resting (inactivated) hedgehog (Hh) signalling pathway. (A) In the absence of Hh ligand, Patched (Ptch) protein form an inactive complex with downstream receptor Smoothened (Smo) (Corbit et al., 2005). It works as inhibitor of Smo protein expression (Denef et al., 2000) and a suppressor of Smo (B) (Ding and Wang, 2017). (C) Several Hh pathway components transduce the signal intracellularly including suppressor of Fused (SUFU) protein that function to inhibit the Gli transcription factors by sequestering them to cytoplasm in absence of Hh signalling (Nozawa et al., 2013). Thus, blocking Hh target genes by localisation of repressor form of Gli (Gli3) in nucleus of inactive cells (Rubin and de Sauvage, 2006). The figure was drawn by the author from the mentioned references.

Figure 0-7, The active status of the Hh signalling pathway.

The active status of Hedgehog (Hh) signalling. (A) Hh ligands are synthesised as precursor proteins in the Golgi apparatus (Huangfu et al., 2003; Ramsbottom and Pownall, 2016) that is cleaved in endoplasmic reticulum before secretion (Ramsbottom and Pownall, 2016). The ligand presence and binding to Ptch receptor results in release of inhibiting function of Ptch on the expression (B) and function of Smo receptor (C) (Ramsbottom and Pownall, 2016), which leads to the activation of pathway (D). This result in induction of activated form of Gli that translocate to nucleus and act as a transcription factor and result in transcription of Hh target genes (F) (Rubin and de Sauvage, 2006; Ramsbottom and Pownall, 2016). The figure was drawn by the author from the mentioned references.

The Gli proteins are large zinc-finger transcription factors that are composed of more than a thousand amino acids (Ruiz i Altaba et al., 2002; Ruiz i Altaba et al., 2007). There are three Gli proteins in humans, and they have a context-dependent transcriptional regulation function (Stecca and Ruiz, 2010). These Gli proteins have both activator and repressor forms (Figure 0-8) which is referred to as the Gli code (Ruiz i Altaba et al., 2007; Aberger and Ruiz, 2014).

Figure 0-8, The gli code.

The repressor and activator forms of Gli proteins. During resting status of Hh signalling, Gli1 protein is not expressed while Gli2 and Gli3 are expressed in full-length form that is cleaved into repressor forms (Ruiz i Altaba, 1998; Stamataki et al., 2005). With activation of Hh signalling, Gli1 is transcribed as an active form (full-length) while cleavage of both Gli2 and Gli3 halts. Thus, Gli2 remain in full-length activator form, and Gli3 is no longer active (full-length) (Stamataki et al., 2005; Ruiz i Altaba et al., 2007). The figure was drawn by the author from the mentioned references.

The nuclear localisation of these Gli transcription factors is essential for protein function (Ruiz i Altaba et al., 2007). Negative modulators of the Hh signalling pathway (including Ptch) sequester Gli1 in cytoplasm of cells (Ruiz i Altaba et al., 2007). Thus, studying of localisation of Gli transcription factors is essential for the determination of the activation status of the pathway.

Other factors can activate Hh signalling in a non-canonical way including dysregulation of the signalling pathway and activation by other signalling cascades such as Wnt signalling. Several mechanisms were indicated to dysregulate Hh signalling including both ligand-independent and ligand-dependent mechanisms (Hui et al., 2013). The ligand-dependent mechanisms are a result of increased expression of SMO and

Gli1 or inactivation of PTCH (Hui et al., 2013).

1.5.1.1 Crosstalk between the hedgehog signalling and Wnt signalling pathways One of the pathways that can activate the Hh signalling pathway non- canonically is the Wnt pathway (Figure 0-9 and Figure 0-10) (Ding and Wang, 2017). In fact, interaction between the Wnt and Hh signalling pathway is seen during embryogenesis and cellular differentiation (Price, 2006; Moussaif and Sze, 2009; Ding and Wang, 2017).

Several studies showed that there was cross reaction between the two signalling pathways. The integration between Hh and Wnt signalling was revealed by pharmacological studies using inhibitory drugs. Singh et al. (2012) found that the Hh signalling inhibits the activation of Wnt signalling during embryogenesis (Figure 0-9- A). Additionally, Wnt activation rescues the suppression of the Hh signalling. Kim et

al. (2010a) found that increased Gli1 expression resulted in a decreased level of nuclear

b-catenin localisation (Figure 0-9-B). The Wnt/b-catenin signalling inhibits Hh activity via transcriptional regulation of Gli3 (Figure 0-10-A) (Alvarez-Medina et al., 2008). Gli3 expression was directly regulated by the b-catenin/TCF/LEF complex (Figure 0-10-B) (Alvarez-Medina et al., 2008).

The overlap between these signalling pathways is not well understood, but the regulation of both signalling pathways is disturbed in human cancer. Co-activation of both Wnt and Hh pathways has been observed in several types of human cancers (Yang

et al., 2008b). Qualtrough et al. (2015) found that inhibition of Hh signalling using

cyclopamine resulted in a reduction in b-catenin-related transcription (CRT) in colorectal cancer cell lines that was rescued by addition of Shh protein. Furthermore, a significant association between the nuclear expression of Gli1 and nuclear expression of b-catenin was observed in human breast cancer samples (Arnold et al., 2017). Thus, indicating that the crosstalk between the Hh and Wnt signalling pathways in breast cancer is context-dependent and requires further study.

Figure 0-9, Hh signalling mediated inhibition of the Wnt signalling pathway.

The inhibitory effect of Hh signalling on the Wnt signalling pathway. (A) Active Gli1 expression directly inhibit Wnt receptor Frizzled. (B) Gli1 protein directly inhibits localisation of b-catenin in nucleus of cells. The figure was drawn by the author from the mentioned references (Ding and Wang, 2017).

Figure 0-10, Wnt signalling mediated inhibition on the Hh signalling pathway.

The inhibitory effect of the Wnt pathway on Hh signalling. (A) APC protein directly regulates expression of Gli3 protein. B- b-catenin/TCF/LEF complex control expression of Gli3 protein which inhibit Gli1. The figure was drawn by the author from (Ding and Wang, 2017).

1.5.2 The Hedgehog signalling pathway is essential for mammary gland