Fibroblast growth factors (FGFs) consist of a family of 22 structurally related polypeptides that share a common core of 140 amino acids. In developmental processes, they are responsible for mesoderm induction, anterior-posterior patterning, limb development, neural induction and neural development. In mature tissues, FGFs are involved in angiogenesis, keratinocyte organisation and wound healing processes that play a key role in proliferation
71 | P a g e
and differentiation of a wide variety of cells and tissues (Ornitz & Itoh, 2015). They interact with one of four receptor tyrosine kinases (FGFR1-4) and one kinase deficient receptor, FGFR5 (Ornitz & Itoh, 2001; Sleeman et al., 2001; Eswarakumar et al., 2005). Binding of FGFs to its receptor results in receptor homodimerisation, autophosphorylation and recruitment of cytosolic adaptors such as fibroblast growth factor substrate 2 (FSR2) which initiates multiple signalling pathways (Eswarakumar et al., 2005; Beenken & Mohammadi, 2009) (see figure 1.8.1.below). Dysregulation of this signalling axis has been shown to play a significant role in tumour development and progression of several cancers (Sugiyama et al., 2010; Frullanti et al., 2011; Miura et al., 2012).
Figure 1.8.1. Signalling cascades involved in FGF-FGFR interaction
PLCγ Activates DAG-PKC and IP3 pathways FRS2 SHP2 GRB2 FGF Receptor GAB1 PI3K AKT PIP2 PIP3 GSK-3β SNAIL FOXO EMT Cell survival SOS RAS RAF MAP3K MAPK Cell different- iation
72 | P a g e
Fibroblast growth factor receptors (FGFR) 1-4 consist of three extracellular immunoglobulin-type domains (D1-D3), a single span transmembrane domain and an intracellular split tyrosine kinase domain. Alternative mRNA splicing gives rise to b and c variants of FGFR1-3 therefore there are 8 types of receptor that can be expressed at the cell surface (Eswarakumar et al., 2005; Turner & Grose, 2010). FGFs bind to D2/D3domains with D3 interaction being responsible for ligand binding specificity. Heparan sulfate (HS) binding is conferred through the D2 domain and it is likely that FGFRs are permanently complexed with heparan sulfate on the cell surface (Powell et al., 2002). Each FGF binds different FGFRs with differing affinity (Ornitz & Itoh, 2015). The specificity of FGFs binding to cellular targets will be influenced not only by the type of FGFRs expressed by the cell but also by the pattern of heparan sulfate binding that is generated by the cellular enzymes.
Most FGFs are only capable of a paracrine action as they are secreted proteins that bind heparan sulfates and therefore are usually caught up in the extracellular matrix of tissues that contain heparan sulfate proteoglycans (Itoh & Ornitz, 2011). In comparison, members of the FGF19 subfamily (FGF15, FGF19, FGF21, and FGF23) have poor affinity with heparan sulphate and therefore are able to diffuse through the HS-rich extracellular matrix to enter into the bloodstream (Goetz et al., 2007). This enables them to have an endocrine action such as is seen with FGF15/19 which is produced by intestinal cells but acts upon FGFR4 expressed in the liver to downregulate CYP7A1 in the bile acid synthesis pathway (Jones, 2012). Similarly, FGF23 is produced in the bone but acts upon FGFR1 expressing kidney cells to regulate vitamin D synthesis and maintain phosphate homeostasis (Razzaque, 2009).
73 | P a g e
Furthermore, the FGF19 subfamily members have poor affinity for their cognate FGF receptors and cannot bind to them and activate them in a solely HS-dependent fashion. Therefore, they require members of the Klotho family of proteins for high affinity receptor binding (Kurosu & Kuro-O, 2009). There are different types of Klotho proteins that interact with FGFs: α-klotho is the cofactor for FGF23, β-klotho for FGF15/19 and FGF21 (Kurosu
et al., 2006; Kurosu et al., 2007; Urakawa et al., 2006; Ogawa et al., 2007; Wu et al., 2007b). Despite having widespread tissue distribution, sites of action of the endocrine FGFs are limited by the distribution of klotho proteins (Fon Tacer et al., 2010).
Aberrant FGF signalling is found in many different cancers including cancer of the prostate, breast, lung, bladder and colon (Brooks et al., 2012; Turner & Grose, 2010). The most widely studied FGF in CRC is FGF2 which has a synergistic action with VEGF on angiogenesis and has been shown to have prognostic relevance (Elagoz et al., 2006). Other fibroblast growth factors that have been implicated in CRC include FGF19 and FGF7. As alluded to earlier, FGF19 belongs to the endocrine family of FGFs and usually plays a role in bile acid, protein and glucose metabolism (see figure 1.8.2.).
74 | P a g e
Figure 1.8.2. The physiological roles of FGF19
FGF19 has been implicated in a number of cancers, particularly hepatocellular carcinoma (French et al., 2012). FGF19 transgenic mice develop HCC and liver dysplasia by 10-12 months of age (Nicholes et al., 2002). FGF19 appears to interact with FGFR4 in driving HCC formation. Blocking the action of FGFR4 using an antibody (LD1) prevented interaction of FGF19 with FGFR4. This reduced colony formation and proliferation in vitro
and was shown to decrease tumour growth in a preclinical model of liver cancer in vivo
(French et al., 2012). β-klotho has also been shown to play a role in HCC carcinogenesis. Beta klotho expression is increased in HCC tumour tissue compared to non-tumour tissue and a greater than 2 fold increase in its expression is associated with the development of multiple tumours (Poh et al., 2012). This suggests that it is not only the action of FGF19 that
Bile acids FXR Terminal ileum FGF19 secreted in blood stream GB Increases cAMP levels which increase gallbladder filling Liver
Inhibits CYP7A1 reducing bile acid synthesis
Blocks CREB phosphorylation which reduces gluconeogenesis
Stimulates glycogen and protein synthesis through Erk pathway
75 | P a g e
is important but that there is concurrent increase in the expression of its receptor and cofactor that contribute to HCC formation.
Previous analysis suggests that FGF19 may also play a role in colorectal cancer by acting on the wnt signalling pathway. Ectopic expression of FGF19 in transgenic mice results in development of HCC and these liver tumours contain neoplastic cells with nuclear localisation of beta-catenin indicative of activation. Co-activation of Wnt and FGF signalling pathways in tumours leads to a more malignant phenotype and inhibition of beta catenin using siRNA completely abolishes FGF19 expression in HCT116 cells suggesting that beta-catenin influences FGF19 expression (Katoh, 2006).
Besides the direct action of FGF19 expression on tumour growth, modifications of its receptor, FGFR4 have also been linked to carcinogenesis. Reduced expression of FGFR4 leads to upregulation of E-cadherin and downregulation of other epithelial-mesenchymal transition (EMT) mediators such as SNAIL, TWIST and ZEB. E-cadherin loss has been recognised as one of the central events in EMT as the study noted that such a change led to an accompanied concurrent reduction in tumour growth both in vitro and in vivo models. The in vivo tumours that were produced from FGFR4 silenced cells were not only smaller, but consisted of a whitish aspect indicative of deficiency in angiogenesis (Peláez-García et al., 2013). FGFR4 may also play a role in crosstalk between tumour associated fibroblasts (TAF) that govern EMT and the tumour tissue itself. Upregulation of FGFR4 was observed in CRC cells that were co-cultured with TAFs (Liu et al., 2013b). This study demonstrated that inhibition of FGFR4 reduced TAF-induced signalling cascades including FRS2 and ERK phosphorylation. Using both in vitro and in vivo models, suppression of FGFR4 was able to reverse TAF-induced migration and invasion of CRC cells. The concept that was proposed suggested that TAFs produce CCL2 which induces FGFR4 expression. FGFR4 overexpression leads to phosphorylation of beta catenin which translocates to the nucleus
76 | P a g e
and initiates expression of SNAIL. This represses expression of E-cadherin, which leads to induction of EMT in CRC cells.
In contrast to FGF19, the studies investigating FGF7 and FGFR2b expression in cancer have yielded conflicting results. Fibroblast growth factor 7 (FGF7), also known as keratinocyte growth factor, is produced by cells of mesenchymal origin (Finch et al., 1989; Rubin et al., 1989) and acts on epithelial cells through its interaction with a specific isoform of the FGF receptor, FGFR2b (Miki et al., 1991). Generally, KGF is not expressed in epithelial cancer cell lines (Dahiya et al., 1996; Iida et al., 1994; Knerer et al., 1998) except in a few cell lines from pancreatic and breast cancer (Bansal et al., 1997; Siddiqi et al., 1995). In comparison, tumour tissue usually expresses KGF. However, there is disagreement across the literature with regards to whether KGF expression levels are higher (Siddiqi et al., 1995; Watanabe et al., 2000) or lower (Knerer et al., 1998) in tumour tissue compared with paired normal mucosa. Despite these differences, studies where in situ hybridisation is used to locate cells expressing KGF have shown that it is usually expressed by stromal cells lying in close proximity to cancer cells. Hence, cancer cells probably induce KGF production which stimulates further epithelial cell proliferation.
FGFR2b is expressed in cancer cell lines including those of the breast, colon, stomach, oesophagus, stomach, pancreas, prostate and oral mucosa. There is similar disagreement in the literature with regards to whether expression levels are higher or lower in cancer tissue. This is further complicated by the possibility of class switch. Reduced FGFR2b expression has been associated with increased FGFR2IIIc expression, which confers a more malignant phenotype and has been related to progression of prostate cancer and EMT in bladder cancer cells (Carstens et al., 1997; Chaffer et al., 2006; Oltean et al., 2006). Conversely, FGFR2IIIb and FGF7 have been shown to increase VEGF-A expression and are associated with a poor prognosis in pancreatic cancer (Cho et al., 2007). Therefore, in certain
77 | P a g e
situations, FGFR2b actually helps to maintain differentiation, inhibit tumour growth and tumour invasion and in others, it promotes venous invasion and tumour angiogenesis leading to a more malignant form of cancer.