**2. FGFs and FGFRs**

The human *Fgf* gene family consists of 22 members, and they are classified into seven subfamilies based on phylogenetic analysis (Figure 1) [3]. FGFs are also classified into three subfamilies (intracrine, paracrine, and endocrine FGFs) based on their mechanism of action. Intracrine FGFs are not typical and does not bind FGFR. Their function is not mediated by a receptor. Therefore, we herein focused two

subfamilies, paracrine FGFs and endocrine FGFs. Paracrine FGFs—which consist of 15 members—exert their biological function through binding to an extracellular domain of FGFRs with heparin/ heparan sulfate and activating tyrosine kinase signaling of FGFRs.

**Figure 1.** *Fgf* genes consisting of 7 subfamilies and *Fgfr* genes. Asterisks indicates fibroblast growth factor (FGF)/ fibroblast growth factor receptor (FGFR) whose expression are enhanced in prostate cancer cells and/or tissues.

Endocrine FGFs consist of FGF19, FGF21, and FGF23. These molecules have a low affinity to heparin/heparan sulfate in contrast to paracrine FGFs. They have to form complexes with co-receptors, α/β-Klotho, to bind to the extracellular domain of FGFR. Because the endocrine FGFs' affinity to FGFRs changes depending on the existence of α/β-Klotho, they have hormone-like activity beyond functioning as a growth factor (Figure 2). Several studies have demonstrated the physiological function of endocrine FGFs (Table 1) [4]. Among endocrine FGFs, FGF15 (the mouse orthologue of human FGF19) was the first molecule to be identified. The physiological activity of FGF19, including the regulation of glucose and bile acid metabolism, is exerted through the formation of a complex with FGFR4 and beta-klotho and follows the activation of a signaling cascade by recruiting adaptor proteins in cytoplasm [5–7]. FGF21 mainly acts as a metabolic regulator in the liver, adipose tissue, and the pancreas. The tissue-specific metabolic action of FGF21 depends on its specificity to the receptor. FGF21 binds with beta-klotho and FGFR1c [6,8,9]. FGF23 is a bone-derived endocrine hormone. Expression of FGF23 is induced by activation of the vitamin D receptor (VDR) with 1,25 dihydroxyvitamin D (1,25D), and FGF23 is a suppressor of 1,25D. So FGF23 and 1,25D are linked by mutual regulation. A signaling cascade of FGF23 activates through formation of a complex with alfa-klotho and FGFR1 for the kidney and FGFR3c for the parathyroid gland. FGF23 regulates

phosphate and vitamin D metabolism in the kidney and inhibits parathyroid hormone secretion and vitamin D synthesis in the parathyroid gland [10,11].

**Figure 2.** Difference in the mechanism in signal transduction between paracrine FGFs and endocrine FGFs.



ADHR, autosomal dominant hypophosphataemic rickets; FGF, fibroblast growth factor; IBD, irritable bowel disease; NAFLD, non-alcoholic fatty liver disease; PTH, parathyroid hormone; TIO, tumor-induced osteomalacia; WAT, white adipose tissue; and XLH, X-linked hypophosphataemic.

Four FGFRs, FGFR1-4, contain an extracellular ligand binding domain with three immunoglobulin (Ig)-like domains (I–III), a transmembrane domain, and a split intracellular tyrosine kinase domain. FGFR1-3 have two kinds of Ig-like III domains, IIIb and IIIc, which are generated by alternative splicing. The Ig-like domain is crucial for determining ligand-binding specificity, and as a result, seven FGFR with different ligand-binding specificities are derived from four *Fgfr* genes [12].

### **3. FGF-FGFR Signaling in Epithelial-Stromal Interaction in Prostate Tissue**

Epithelial-stromal interaction plays an important role in maintaining the homeostasis in normal prostatic tissue [13,14]. Stromal tissues secrete paracrine factors that include FGF ligands, and they lead to stimulation of epithelial maintenance and growth. Huang et al. reported the significance of FGFR2 signaling for preserving stemness and preventing differentiation of prostate stem cells [15]. FGFR2IIIb, a splicing variant of the FGFR2, is a resident form of FGFRs expressed in normal prostate epithelial cells. FGFR2IIIb is specific to FGF7, and FGF7-FGFR2IIIb contributes epithelial-stromal interaction [16]. The loss of FGFR2 isoforms is shown in human PCa tissues, and the loss of FGFR2IIIb is associated with the characteristics of castration-resistant prostate cancer (CRPC) in particular [17].

The expression pattern of FGFR is different in each PCa cell line. For instance, FGFR1 expression is detectable and FGFR2IIIb expression is undetectable in PC3 cells that show androgen-independent growth and high potential of cell proliferation. On the other hand, FGFR2IIIb expression is detectable in LNCap cells that show expression of androgen receptor (AR), androgen-dependent growth, and relatively low potential of cell proliferation [18]. In addition to FGFs and FGFRs, FGF receptor substrate 2alpha (FRS2alpha), an FGFR interactive adaptor protein, involves multiple signaling pathways to the activated FGFR kinase. FRS2 alfa is required for prostate development and tumorigenesis [19], as well as in angiogenesis [20].

### **4. Effects of the Restoration of FGFR2IIIb in Prostate Cancer Cells**

Many studies have demonstrated the association of aberrant FGFR signaling with the development and progression of PCa [21,22]. Binding ligands, FGFRs form functional dimerization and lead transphosphorylation and activation of downstream signaling pathways such as Ras, Src, PKC γ, MAPK, PI3K-AKT, and STAT [23–25]. The involvement of FGF signaling in various molecular mechanisms has been reported in PCa. Shao et al. reported that FGF-FGFR signaling plays an important role in transformation induced by the loss of a PTEN tumor suppressor when combined with the expression of the TMPRSS2/ERG fusion gene [26], and activation of FGF-FGFR signaling by FGF8b overexpression in PTEN deficiency is reported to be associated with prostate tumorigenesis [27]. FGF-FGFR signaling is also related to the induction of an inflammatory response in PCa tissues [28]. The involvement of aberrant FGFR1 signaling in the progression of PCa in particular was demonstrated in several studies. FGFR1 signaling promotes the reprogramming of energy metabolism from oxidative phosphorylation to aerobic glycolysis by regulating the expression of an LDH isoenzyme [29]. It also promotes an inflammatory response through activation of NF-κB signaling [30]. Furthermore, activation of FGFR1 signaling promotes epithelial to mesenchymal transition and androgen independency in PCa cells [31,32]. Loss of FGFR2IIIb and enhancement of ectopic expression of FGFR1 PCa progression have been reported as common events in the progression of PCa [21]. As FGFR2IIIb plays an important role in the maintenance and its disorder is found in PCa cell lines and tissues, several investigators have reported the effects of restoring FGFR2IIIb. In animal models and PCa cell lines, FGFR2IIIb's restoration also restored responsiveness to stroma and significantly reduced in vivo tumorigenesis. In castration-resistant human PCa cells, restoration of FGFR2IIIb showed the inhibition of cell proliferation, the induction of differentiation, and the enhancement of apoptosis in a ligand-independent manner [21,33,34]. In addition, in PCa cells overexpressing FGFR2IIIb, clonogenic cell death increased in concurrence with enhanced apoptosis and cell cycle arrest in the G2/M-phase and radiosensitivity by gamma-irradiation [35]. Another study reported the effect FGFR2IIIb's restoration had on the chemosensitivity in PCa cells. Restoration of FGFR2IIIb led to the enhanced chemosensitivity of several agents, especially docetaxel. The expression of N-cadherin, vimentin, survivin, and XIAP were induced by restoring FGFR2IIIb [36]. This data indicates that PCa cell lines are induced to a more differentiated phenotype when changing the pattern of gene expression that became sensitive to radiation and chemotherapy when FGFR2IIIb was restored.

### **5. Involvement of FGFs in the Development and Progression of Prostate Cancer**
