fibroblast growth factor receptor signaling as a therapeutic target in tumor angiogenesis.

Review

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Tumor angiogenesis

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Pro-angiogenic functions of the FGF/FGFR system

3.

The FGF/FGFR system in tumor

The potential of fibroblast growth factor/fibroblast growth factor receptor signaling as a therapeutic target in tumor angiogenesis Roberto Ronca, Arianna Giacomini, Marco Rusnati & Marco Presta† University of Brescia, Department of Molecular and Translational Medicine, Brescia, Italy

angiogenesis

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4.

Inhibition of the FGF/FGFR system: experimental approaches

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FGF/FGFR inhibitors in cancer clinical trials

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Expert opinion

Introduction: Fibroblast growth factors (FGFs) are endowed with a potent pro-angiogenic activity. Activation of the FGF/FGF receptor (FGFR) system occurs in a variety of human tumors. This may lead to neovascularization, supporting tumor progression and metastatic dissemination. Thus, a compelling biologic rationale exists for the development of anti-FGF/FGFR agents for the inhibition of tumor angiogenesis in cancer therapy. Areas covered: A comprehensive search on PubMed was performed to identify studies on the role of the FGF/FGFR system in angiogenesis. Endothelial FGFR signaling, the pro-angiogenic function of canonical FGFs, and their role in human tumors are described. In addition, experimental approaches aimed at the identification and characterization of nonselective and selective FGF/FGFR inhibitors and their evaluation in clinical trials are summarized. Expert opinion: Different approaches can be envisaged to inhibit the FGF/FGFR system, a target for the development of ‘two-compartment’ antiangiogenic/anti-tumor agents, including FGFR selective and nonselective small-molecule tyrosine kinase inhibitors, anti-FGFR antibodies, and FGF ligand traps. Further studies are required to define the correlation between tumor vascularization and activation of the FGF/FGFR system and for the identification of cancer patients more likely to benefit from anti-FGF/FGFR treatments. In addition, advantages and disadvantages about the use of selective versus non-selective FGF inhibitors remain to be elucidated. Keywords: angiogenesis, cancer, fibroblast growth factor, fibroblast growth factor receptor, therapeutic target Expert Opin. Ther. Targets [Early Online]

1.

Tumor angiogenesis

Under physiological conditions, the angiogenic process that leads to the formation of new blood vessels from pre-existing ones occurs as a controlled series of molecular and cellular events supporting changes in tissue requirements [1]. However, under pathological situations such as cancer, these angiogenic signaling pathways are exploited in a deregulated manner. Angiogenesis is an essential process for tumor growth and progression, since the large-scale growth of a tumor ultimately requires an adequate blood supply [2]. Indeed, once a tumor lesion exceeds a few millimeters in diameter, hypoxia and nutrient deprivation triggers an ‘angiogenic switch’ to allow the tumor to progress [3]. Tumor cells can tilt the balance toward stimulatory angiogenic factors to drive vascular growth by releasing growth factors, chemokines and cytokines to attract and activate normal, quiescent cells within their microenvironment and initiate a cascade of events that quickly becomes deregulated. These signals create a concentration gradient that initiates the sprouting and proliferation of formerly 10.1517/14728222.2015.1062475 © 2015 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted

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Fibroblast growth factors (FGFs) are endowed with a potent pro-angiogenic activity. Activation of the FGF/FGF receptor (FGFR) system occurs in a variety of human tumors. This may lead to tumor neovascularization, supporting tumor progression and metastatic dissemination. Due to their pleiotropic effects, FGFs may also act on tumor cells and exert paracrine/autocrine functions on cancer epithelial/stromal cells thus providing druggable targets for the development of ‘two-compartment’ anti-FGF agents. Experimental and clinical evidences indicate that activation of the FGF/FGFR system may represent a mechanism of tumor escape from antiangiogenic vascular endothelial growth factor blockade. Several strategies have been experimentally attempted to inhibit the FGF/FGFR system in the search for efficacious antiangiogenic/antitumor approaches. At present, two major classes of inhibitors of the FGF/FGFR system are under clinical evaluation: FGFR selective and nonselective small-molecule TK inhibitors and extracellular anti-FGFR antibodies, a few studies focusing on FGFR decoy extracellular FGF ligand traps. Several challenges are being faced to further develop efficacious FGF/FGFR inhibitors for antiangiogenic therapies in cancer: i) the correlation between tumor vascularization and activation of the FGF/FGFR system in the different human cancer types remains uncertain; ii) identification of cancer patients more likely to benefit from a therapeutic anti-FGF/FGFR approach is required; iii) prognostic indicators, surrogate markers of angiogenesis and of response to antiangiogenic therapy in cancer patients need to be identified; iv) pros and cons about the use of selective versus nonselective FGF inhibitors are not fully elucidated; and v) the development of drugs specific for individual FGFs or FGFRs may reduce undesired systemic side-effects also related to alterations of hormonal FGFs.

This box summarizes key points contained in the article.

quiescent endothelial cells on nearby blood vessels and recruits fibroblasts that deposit a repertoire of extracellular matrix (ECM) proteins and enzymes in an attempt to remodel and repair the lesion. In addition, tumors may elicit an inflammatory response that attracts myeloid cells into the tumor microenvironment that release their stores of soluble factors to escalate the angiogenic response. Moreover, although tumor-associated angiogenesis has traditionally been defined as the sprouting of new vessels from pre-existing vessels, blood vessels supporting tumor growth or tumor rebound from therapy-induced trauma can also originate from cells recruited from the bone marrow or differentiate from tumor stem cells (vascular mimicry) [4]. During tumor angiogenesis, tumor microenvironment continually changes and evolves as the tumor grows, creating localized areas of hypoxia, inflammation, and ECM turnover that affect blood vessel growth, remodeling, and maturation [5]. 2

Although the induction of angiogenesis may initially provide the tumor with more oxygen and nutrients, the ultimate response is poor, as the continuously remodeled tumor vasculature is leaky and tortuous, causing irregular blood flow [6]. This environment makes the tumor cells more invasive, allowing them to intravasate into the systemic vasculature and metastasize to distant sites [7]. To date, numerous inducers of tumor angiogenesis have been identified, including the members of the VEGF family, angiopoietins, TGF-a and TGF-b, PDGF, TNF-a, interleukins, chemokines, and members of the fibroblast growth factor (FGF) family. Considering the variety of angiogenic modulators, cell types, and signals involved in tumor neovascularization, several aspects of this process can be targeted therapeutically. However, the identification of the steps and components most susceptible to drug treatment is a major concern. The most effective therapies will probably involve targeting combinations of factors, as well as improving the efficiency of drug delivery to the tumor microenvironment.

Pro-angiogenic functions of the FGF/FGFR system

2.

The 1980s saw for the first time the purification to homogeneity of angiogenic proteins, the breakthrough coming as a result of the observation that endothelial cell growth factors showed a marked affinity for heparin [8,9]. This led to the identification of the two prototypic heparin-binding angiogenic growth factors FGF1 and FGF2. Since then, 22 structurally related members of the FGF family have been identified able to interact with four high-affinity tyrosine kinase (TK) FGF receptors (FGFR1-4) [10]. FGFRs are composed of an extracellular portion consisting of three Ig-like domains (D1, D2, and D3), a hydrophobic transmembrane region, and a cytoplasmic TK tail. The ligand-binding site for FGFs is located in the Ig-like domains D2--D3 and the linker that connects them [11]. Each FGFR binds to and is activated by a unique subset of FGFs, its specificity being further regulated by alternative splicing events in the exons IIIb and IIIc that encode the carboxyl-terminal portion of the third Ig-like loop. Alternative splicing of FGFR1, FGFR2, and FGFR3 results in isoforms FGFR1-3IIIb and FGFR1-3IIIc with distinct FGF binding characteristics [11]. Phylogenetic analyses divide the human FGF gene family into seven subfamilies, each containing two to four members [12]. These subfamilies can be further classified into three different groups by their mechanism of action: canonical FGFs comprising the FGF1/2/5, FGF3/4/6, FGF7/10/22, FGF8/17/18, and FGF9/16/20 subfamilies; the intracellular FGF11/12/13/14 subfamily; and the hormone-like (endocrine) FGF19/21/23 subfamily [12]. Canonical FGFs mediate their biological responses as extracellular proteins by binding to and activating FGFRs with heparin/heparan sulfate proteoglycans (HSPGs) as cofactors. They act as local signaling

Expert Opin. Ther. Targets (2015) 19(11)

The potential of FGF/FGFR signaling as a therapeutic target in tumor angiogenesis


molecules in an autocrine/paracrine manner. In contrast, intracellular FGFs act as intracellular signaling molecules in a FGFR-independent manner; they play a major role in neuronal functions at postnatal stages by interacting with intracellular domains of voltage-gate sodium channels and with the neuronal MAPK scaffold protein islet-brain-2 [13]. Hormone-like FGFs show a reduced affinity for HSPGs and are mainly involved in metabolic homeostasis [14,15]. Similar to canonical FGFs, they mediate their biological responses in a FGFR-dependent manner, FGF19 and FGF23 also requiring b-Klotho and a-Klotho as cofactors, respectively [16,17]. FGFR signaling in endothelial cells Endothelial cells express different members of the FGF/FGFR family. Among them, FGFR1IIIc appears to be the most expressed isoform and the most relevant for angiogenesis. Also, FGFR3IIIc and to a lesser extent FGFR2IIIc isoforms are detectable in human umbilical vein endothelial (HUVE) cells together with the ligands FGF2, FGF5, FGF7, FGF8, FGF16, and FGF18 [18]. Even though the role in angiogenesis of the different FGFRs and of their splicing variants expressed by the various endothelial cell populations has not been fully elucidated, experiments performed on engineered mice deficient in FGFR1 and FGFR2 in both endothelial and hematopoietic cells indicate that these receptors are not required for vascular homeostasis or physiological functions. However, they represent a key requirement for cell-autonomous endothelial FGFR signaling in tissue repair and neovascularization following injury and validate endothelial cell FGFRs as a target for diseases associated with aberrant vascular proliferation [19]. In tumors, the FGFR1IIIb isoform was found to be specifically and differentially expressed by the intraductal vasculature in a murine model of prostate cancer progression [20] whereas the few studies focusing on the impact of FGFR2 or FGFR4 inhibition on tumor angiogenesis (see for instance, references [21] and [22]) do not distinguish between a direct effect on endothelial cells and an indirect effect on FGFR-expressing tumor cells. FGFs act on target endothelial cells by interacting with high-affinity FGFRs and low-affinity HSPGs. The formation of HSPG/FGF/FGFR ternary complexes [23] causes receptor dimerization and trans-phosphorylation of multiple residues in the intracellular TK domain. Downstream signaling occurs mainly through the intracellular specific adaptor protein FGFR-substrate-2 and phospholipase-Cg. Activated FGFRsubstrate-2 allows the recruitment of the adaptor proteins SOS and GRB2 to activate RAS and the downstream RAF/ mitogen-activated protein kinase pathway; phospholipase-Cg leads to the activation of protein kinase C and RASindependent AKT signaling [24]. An alternative signaling pathway like the JAK-signal transducers and activators of transcription (STAT) pathway may be activated by FGFRs in different contexts [24]. The pleiotropic and multifaceted functions exerted by the FGF/FGFR system are the consequence of the variety of 2.1

ligands and receptor isoforms as well as of the wide range of feedback/modulatory pathways that can fine tune FGF/FGFR effects. Indeed, FGFR signaling can be regulated/attenuated both at extra- and intra-cellular levels. At the plasma membrane, remodeling and modifications of the heparin-sulfate (HS) chains of transmembrane HSPGs (Syndecan-1, -2, and -4) may modulate FGF/FGFR interaction, deeply affecting FGFR signaling [25]. Also, cell-surface gangliosides [26] and avb3 integrin [27] may act as FGF2 co-receptors for a full angiogenic response in endothelial cells. Finally, cell membrane receptors like neural cell adhesion molecule and N-cadherin can associate with FGFR1 [28,29]. This interaction requires the presence of the acid box motif in the linker region between the D1 and D2 Ig-like loops of the receptor. As a consequence, neural cell adhesion molecule acts as a nonconventional FGFR1 ligand, promotes the stabilization of the receptor, and exerts a peculiar control on its intracellular trafficking [28,29]. Regulatory mechanisms of the FGF/FGFR system also include several intracellular mediators like the FGF-induced Sprouty proteins (SPRY1-4), a highly conserved group of negative feedback loop modulators of growth factor-mediated MAPK activation. All four SPRY isoforms are expressed by endothelial cells [30] and they mainly act by binding to GRB2 and RAF, thus disrupting the FGF activated downstream signaling cascade. Also, FGFs can induce the activation of phosphatases, such as MAPK-phosphatase 3 that specifically dephosphorylates and inactivates ERK1/2 and the transmembrane protein SEF that binds FGFR and prevents its activation [31]. Angiogenic activity of canonical FGFs FGFs are pleiotropic factors acting on different cell types, including endothelial cells. In the angiogenesis field, FGF1 and FGF2 represent the prototypical and best-studied members of the canonical FGF subfamily. In vitro they induce a complex ‘pro-angiogenic phenotype’ in endothelial cells that recapitulates several aspects of the in vivo angiogenesis process, including modulation of endothelial cell proliferation, migration, protease production, integrin and cadherin receptor expression, and intercellular gap-junction communication [32]. The angiogenic activity of FGF1 and FGF2 has also been demonstrated in various in vivo experimental models, including the chick embryo chorioallantoic membrane [33], rabbit/mouse cornea [34,35], and murine subcutaneous Matrigel plug [36] assays. However, FGF2 knockout and FGF1/ FGF2 double-knockout mice develop normally but show a poor wound-healing capacity when compared to control animals [37] whereas no abnormalities were found in mice lacking FGF1 only. The relatively mild phenotypic defects associated with FGF1/FGF2 deletion led to the hypothesis that the expression of other FGFs may exert a compensatory effect during development and under physiological conditions. To date, a comprehensive work comparing the effect of all canonical FGFs on endothelial cells has not been published. 2.2


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Nevertheless, as summarized below, scattered pieces of information indicate that, besides FGF1 and FGF2, other FGFs show clear pro-angiogenic properties (like FGF4 and FGF8) whereas few or controversial data have been reported for other members of the family. FGF4 Exogenous FGF4 exerts a response similar to FGF2 when administered to murine aortic endothelial cells, stimulating DNA synthesis and triggering ERK1/2 phosphorylation. In contrast, significant qualitative differences were observed in the biological behavior of these cells following the endogenous overexpression of FGF2 or FGF4. Indeed, at variance with FGF2-overexpressing cells, FGF4 transfectants show a limited capacity to growth under anchorage-independent conditions. Also, they lack the ability to invade 3D-fibrin gel, to undergo morphogenesis in vitro, and to induce hemangiomas when injected in vivo in the chorioallantoic membrane of the chick embryo [38]. Moreover, recombinant FGF4 did not induce any morphological change in HUVE cells cultured on collagen gel and the conditioned medium from bladder carcinoma cells expressing FGF4 failed to induce endothelial cell morphogenesis [39]. At variance, the conditioned medium from FGF4-overexpressing murine mammary cells shows a significant pro-angiogenic activity mediated by the upregulation of VEGF-A [40].


2.2.1

FGF5 Recombinant FGF5 and conditioned media from FGF5-overexpressing glioblastoma cell lines exerted a proliferative, migratory, and morphogenic effect on HUVE cells that was abolished by an anti-FGF5 antibody [41]. 2.2.2

2.2.3

FGF7

Also named keratinocyte growth factor, FGF7 is produced mainly by mesenchymal cells and acts on epithelial cells. Nevertheless FGF7 can act on microvascular endothelial cells by inducing cell proliferation, MAPK activation, and chemotaxis, while it is ineffective on macrovascular endothelial cells [42]. FGF7 also contributes to the maintenance of the barrier function of monolayers of capillary but not aortic endothelial cells, thus supporting its apparent selectivity for microvascular endothelium [42]. FGF8 It is actively produced by endothelial and tumor cells [18]. FGF8 acts on endothelial cells leading to the activation of the angiogenic process in vitro and in vivo [43,44] and its blockade has a deep impact on tumor angiogenesis [44]. For instance, S115 breast cancer cells transfected with the FGF8b isoform, but not with the FGF8a or FGF8e isoforms, originate highly vascularized tumors when injected in nude mice [43]. Also, the capacity of FGF8 to activate the STAT protein in murine microvascular endothelial cells has been described [45]. 2.2.4

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FGF9 It has been reported to play a unique role in bone healing, possibly by initiating angiogenesis via VEGF-A. Treatment with FGF9 protein promotes angiogenesis and successfully rescued the bone healing capacity in Fgf-9+/- mice where µCT angiography indicated dramatic impairment of neovascularization [46]. Also, FGF9 has been shown to orchestrate wrapping of vascular smooth muscle cells around newly formed microvessel [47]. 2.2.5

FGF16 and FGF18 They are both expressed in HUVE cells and in the rat aortic tissue. FGF16 and FGF18 exert a chemotactic response in HUVE cells and enhance cell migration in response to mechanical damage. However, both FGF16 and FGF18 failed to induce endothelial cell proliferation or sprouting in vitro in a 3D angiogenesis assay [48]. Nevertheless, FGF18 has been shown to affect skeletal vascularization and subsequent recruitment of osteoblasts/osteoclasts through the modulation of VEGF expression at the early stages of chondrogenesis [49]. FGF18 also controls the migration, invasion, and tumorigenic activity of ovarian cancer cells through NF-kB activation, thus creating a tumor microenvironment characterized by enhanced angiogenesis and augmented tumorassociated macrophage infiltration with M2 polarization. Accordingly FGF18 expression levels correlate with microvessel density (MVD) in tumors from ovarian cancer patients [50]. 2.2.6

The FGF/FGFR system in tumor angiogenesis

3.

Various tumor cell lines express FGF2 [51,52] and the appearance of an angiogenic phenotype correlates with the export of FGF2 during the development of fibrosarcoma in a transgenic mouse model [53]. Antisense cDNAs for FGF2 and FGFR1 inhibit neovascularization and growth of human melanomas in nude mice [54]. Also, the antiangiogenic activity of IFN-a/b appears to be related, at least in part, to the capacity to down-regulate FGF2 expression [55]. These data suggest that FGF2 production and release may occur in vivo and may influence the growth and neovascularization of tumor xenografts. Indeed, neutralizing anti-FGF2 antibodies and soluble FGFRs affect tumor growth under defined experimental conditions [56,57]. Of note, adenoviral expression of a soluble form of VEGFR-1 in spontaneous b-cell pancreatic tumors in Rip1-Tag2 mice affected the initial stages of tumor angiogenesis whereas soluble FGFR2 appeared to impair the maintenance of tumor angiogenesis. The combination of the two soluble receptors exerted a synergistic effect [57]. Accordingly, FGF2 and VEGF exert a synergistic effect on tumor blood vessel density even though they differently affect blood vessel maturation and functionality [58,59]. Given the pleiotropic activity of FGFs, it is not always possible to dissociate the effect of FGFs on tumor angiogenesis



Endothelial cell

Angiogenesis

Production FGFR

Production

Stromal cell

Production

FGF

FGFR

FGFR

FGF/FGFR inhibitor

Tumor cell

Tumor cell proliferation

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Tumorprogression

Figure 1. The FGF/FGFR system in cancer progression. The FGF/FGFR system may contribute to tumor growth by inducing neovascularization and by acting directly on tumor and stromal cells via autocrine and paracrine mechanisms of action, thus providing a druggable target for the development of ‘two-compartment’ anti-FGF/FGFR agents. FGF: Fibroblast growth factor; FGFR: Fibroblast growth factor receptor.

Table 1. FGFs and human cancers*. FGF

FGF upregulation in human tumors

FGF1 FGF2

Breast, pancreatic, prostate cancer [142-144] Melanoma, bladder, prostate, breast, pancreatic, small cell lung cancer [63,142,145-148] Bladder, mammary, gastric, and non-small cell lung cancer [149-152] Lymphoma, hepatocellular carcinoma, ovarian, breast cancer [149,152-155] Glioblastoma multiforme, melanoma, bladder, pancreatic, prostate, breast cancer [41,145,156-159] Colorectal, prostate, breast cancer [142,160,161] Gastric, lung, ovarian cancer [162-164] Hepatocellular carcinoma, breast, prostate, bladder, lung cancer [145,148,165,166] Glioblastoma, prostate, non-small cell lung cancer [41,167,168] Pancreatic, prostate cancer [169,170] Ovarian cancer [171] Hepatocellular carcinoma, prostate tumors [172-174] Melanoma, hepatocellular carcinoma, ovarian, colorectal, prostate, lung cancer

FGF3 FGF4 FGF5 FGF6 FGF7 FGF8 FGF9 FGF10 FGF16 FGF17 FGF18

[148,159,165,175,176]

FGF19 FGF20 FGF22 FGF23

Hepatocellular carcinoma, colon cancer, breast cancer, prostate cancer [177-182] Colon, gastric, lung, ovarian endometrioid adenocarcinomas [183-185] Non-melanoma skin cancer [186] Multiple myeloma [187]

*The table summarizes literature data pointing to a role of the upregulation of different FGFs in human tumors. For a more comprehensive description of the putative role(s) of the different members of the FGF family in human cancers, see also [31,64,124,136,188].

from those exerted directly on tumor cells (Figure 1). For instance, inhibition of the FGF/FGFR system in glioma cells by dominant negative FGFR transfection [60] or in prostate cancer cells by fgf2 gene knockout [61] results in the inhibition

of tumor growth by both angiogenesis-dependent and angiogenesis-independent mechanisms. Also, overexpression of the FGF-trap protein long pentraxin-3 (PTX3) suppressed the angiogenic and tumorigenic potential of TRAMP-C2 prostate cancer cells [62] and the metastatic potential of B16-F10 melanoma cells [63]. Accordingly, compelling evidence for deregulated FGF signaling in tumorigenesis continues to emerge [64]. Several studies performed with animal models have suggested that aberrant FGF and/or FGFR signaling has a wide range of effects and can involve both tumor cells and the surrounding stroma (Figure 1). These effects include cellular proliferation, resistance to cell death, increased motility and invasiveness, enhanced metastasis, and resistance to chemotherapy [64,65]. Deregulation of FGF signaling in tumorigenesis can result from FGFR activating mutations, gene amplifications, and chromosomal translocations [66]. Also, overexpression of different FGFs have been observed in various tumor types, including breast, pancreatic, prostate, bladder, gastric, colorectal, lung, and ovarian cancers as well as lymphoma, glioblastoma multiforme, melanoma, and hepatocellular carcinoma (Table 1). Of note, emerging evidence has suggested that upregulation of FGF and FGFR may serve as a mechanism of resistance to anti-VEGF therapy. FGFs not only directly promote endothelial cell proliferation, but also indirectly synergize with the VEGF and PDGF pathways, promoting tumor neoangiogenesis through complementary and overlapping functions [67,68]. In the preclinical setting of antiVEGF therapy, when U87MG glioblastoma cells were intracranially implanted as xenografts in mice who were then treated with the anti-VEGF antibody bevacizumab, a significant increase in FGF2 expression was observed at the time of tumor progression and at animal death. Interestingly, FGF2 was not elevated in tumors analyzed at early time points that had only received short-term VEGF


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E., F. Masking of co-receptors TK-FGFRs

FGF A. Inhibition of FGF production

FGF B. Sequestration of FGFs

Endothelial cell

Signal transduction


C., D. Inhibition of expression

FGF-producing tumor cell

Chemoterapeutics, antiblastics

G. Inhibition of TK activity

co-receptor TK-FGFR expression

H. Inhibition of effectors activity

Effectors (i.e., proteases)

Figure 2. Anti-FGF/FGFR strategies. Different approaches can be envisaged to neutralize the pro-angiogenic activity of the FGF/FGFR system: (A) inhibition of FGF production and release; (B) sequestration of FGFs in the extracellular environment; (C) inhibition of endothelial FGFR expression; (D) inhibition of the expression of endothelial FGF co-receptors (e.g., HSPGs, integrins, and gangliosides); (E) masking TK-FGFRs; (F) masking FGF co-receptors; (G) blockage of signal transduction pathway (s) triggered by FGF/FGFR interaction in endothelial cells; (H) neutralization of FGF-induced effectors whose activity is essential in mediating the angiogenic potential of FGFs. FGF: Fibroblast growth factor; FGFR: Fibroblast growth factor receptor; TK: Tyrosine kinase.

inhibition, thereby correlating FGF2 induction with tumor relapse and indicating the existence of a compensatory mechanism enhancing neovascularization. Moreover, MVD at the early time point remained significantly reduced compared with control tumors, whereas bevacizumab-treated tumors at the late stage exhibited restoration of MVD above that observed in controls, suggesting that tumors could indeed reactivate angiogenesis following long-term anti-VEGF therapy in part through FGF2 upregulation [69]. Similarly, in the Rip1 -- Tag2 model of pancreatic islet carcinogenesis, tumors with an initial response to VEGFR2 blockade expressed higher levels of FGF2 at the time of progression compared with stable tumors [70]. When the tumors were first treated with a VEGFR inhibitor alone and subsequently treated at the peak of response with a FGF trap, the combination attenuated revascularization and slowed tumor growth. Accordingly, combined blockade of VEGF and FGF2 with the dual inhibitor brivanib prolonged tumor stasis and angiogenic blockade when used as a first-line treatment or as a second-line therapy following previous antiangiogenic inhibition [71]. Interestingly, second-line brivanib treatment was more efficacious when initiated prior to first-line antiangiogenic failure and less beneficial when administered after tumors had already initiated revascularization. Clinical evidence also supports the role of FGF2 in resistance to antiVEGF-containing regimens. FGF2 levels were found to be higher in patients with colorectal cancer after the failure of 6

bevacizumab-containing regimens [72] and in glioblastoma patients after treatment with a VEGFR TK inhibitor [73]. Taken together, the bulk of experimental evidences indicate that the identification and development of new FGF pathway inhibitors as well as the possibility to target both the VEGF and FGF pathways by their combinatorial or sequential inhibition may translate into improvements in the clinical care of cancer patients.

Inhibition of the FGF/FGFR system: experimental approaches

4.

Different approaches can be envisaged to neutralize the proangiogenic activity of the FGF/FGFR system (Figure 2). They include: i) inhibition of FGF production and release; ii) sequestration of FGFs in the extracellular environment; iii) inhibition of endothelial FGFR expression; iv) inhibition of the expression of endothelial FGF co-receptors (e.g., HSPGs, integrins, and gangliosides); v) masking TK-FGFRs; vi) masking FGF co-receptors; vii) blockage of the signal transduction pathway(s) triggered by FGF/FGFR interaction in endothelial cells; and viii) neutralization of FGF-induced effectors whose activity is essential in mediating the angiogenic potential of FGFs. All these approaches have been challenged experimentally and will be described briefly below. Since the bulk of data refer to FGF2, we will focus on this prototypical FGF, even though many of the experimental



approaches herewith described may apply also to other members of the FGF family. Inhibition of FGF production Tumor and stromal cells may produce FGFs that trigger endothelial cell activation in a paracrine manner or release growth factors/cytokines that induce endothelial cells to produce FGFs, thus activating an autocrine mechanism of stimulation. In both cases, inhibition of FGF production will lead to inhibition of neovascularization and tumor growth. The capacity to inhibit the production of angiogenic growth factors (including FGFs) is a common feature of cytotoxic chemotherapeutics [74] that will decrease FGF production by exerting their antiblastic effect on tumor cells. More specifically, antisense-oriented FGF2 oligonucleotides have been developed that block the production of the growth factor by tumor or endothelial cells in vivo [54,75]. The inhibition of FGF production is also obtained by various inhibitors of second messengers involved in FGF production [74], selected endogenous cytokines [76] and natural products [74]. In particular, the antiangiogenic activity of IFN-a/b appears to be related, at least in part, to the capacity to down-regulate FGF2 expression [55].


4.1

Sequestration of FGFs in the extracellular environment

4.2

Classically, this approach is pursued by the production of antibodies targeting a specific FGF [75,77] or by soluble forms of the extracellular FGFR portion that can act as multi-FGF ligand traps [78]. Also, free heparin, a negatively charged glycosaminoglycan released in the blood stream during inflammation, can bind various members of the FGF family with different affinity. At variance with HSPGs that act as FGF co-receptors, free heparin sequesters FGFs in the extracellular environment, thus exerting an antagonist effect. However, due to its anticoagulant activity and its capacity to bind a wide array of growth factors, cytokines, enzymes, and proteases, unmodified heparin cannot be used as an antiangiogenic drug. This prompted a series of studies aimed at identifying heparin derivatives, biotechnological heparins, and polyanionic heparin-like molecules endowed with a more specific FGF antagonist activity and a more favorable therapeutic window [79]. A more recent approach consists in the identification of natural, proteinaceous extracellular FGF binders, the characterization of their FGF-binding domain, and the design of related peptidomimetics acting as low-molecular-weight extracellular FGF traps [80,81]. For instance, the ECM protein thrombospondin-1 (TSP-1) sequesters FGF2 in an inactive form, binds avb3 integrin and HSPGs (possibly preventing their interaction with FGF2), and inhibits FGF2 activity by a CD36-dependent mechanism of action [82]. TSP-1 is also endowed with a multitarget potential, being able to block simultaneously other angiogenic growth factors besides

FGF2, including VEGF, HGF, PDGF, and the HIV protein Tat [82,83]. At variance, the soluble pattern recognition receptor PTX3, a member of the pentraxin family produced locally in response to inflammatory signals [84], binds various FGFs via its N-terminal extension, including FGF2, FGF6, FGF8b, FGF10, and FGF17 [44,62,84,85], and inhibits FGF2and FGF8-dependent angiogenic responses without exerting any interaction with VEGF or other cytokines and growth factors [44,86]. Recently, computational analysis, nuclear magnetic resonance spectroscopy, and surface plasmon resonance analysis were utilized to map the amino acid residues at the TSP-1/FGF2 interface. The translation of this 3D information into pharmacophore models allowed the screening of small molecule databases and the identification of small molecule anti-FGF2 mimetics of TSP-1 [82,83]. Also, the 480 Da acetylated pentapeptide Ac-ARPCA-NH2 (in single letter code), corresponding to the N-terminal amino acid sequence PTX3(100--104) was identified as a minimal antiangiogenic FGF-binding peptide able to interfere with the angiogenic activity of FGF2 and FGF8b [87,88]. Inhibition of endothelial FGFR and FGF coreceptor expression

4.3

Transfection with FGFR1 antisense cDNAs inhibits FGF2-dependent endothelial cell proliferation and migration in vitro [89] and angiogenesis in vivo [60]. Also, the observation that IFN-g and IL-1 down-regulate FGFR expression may inspire new strategies to inhibit angiogenesis by FGFs [90]. As already mentioned, FGFs need to interact and activate different co-receptors to induce a full angiogenic response. Consequently, the blockage of endothelial cell surface expression of these co-receptors (i.e., HSPGs [91] or gangliosides [92]) has been exploited as an antiangiogenic strategy. A FGF antagonist effect can also be obtained by enzymatic digestion of endothelial HSPGs [89] or by enzymatic modifications of their sugar chain leading to a reduced affinity for FGFs [93-95]. Masking endothelial FGFRs and FGF co-receptors Neutralizing anti-FGFR antibodies [96-98], vaccination against FGFR1 [99] and synthetic peptides that bind and mask FGFRs [100] have been shown to block FGF2-mediated angiogenesis in vitro and in vivo. FGF-derived FGFR-binding peptides can also be exploited to deliver antiblastic drugs to FGFR-overexpressing tumor cells [101]. FGF2 contains two DGR amino-acid sequences (in single letter code) that are the inverse of the integrin-recognition sequence RGD. Consistently, DGR- or RGD-containing peptides and RGD-peptidomimetics inhibit avb3-mediated endothelial cell adhesion to immobilized FGF2, FGF2-dependent biological activities in vitro, and tumor growth and neovascularization in vivo [102-104]. Similarly, anti-avb3 antibodies and disintegrins, a class of naturally occurring integrin antagonists, inhibit FGF2/avb3 interaction and FGF2-dependent angiogenesis [102,105,106]. Besides integrins, also HSPGs [107-111] and 4.4


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gangliosides [92] can be masked by a wide array of natural or synthetic compounds to obtain an anti-FGF2 effect. Blockage of signal transduction pathway(s) triggered by FGFR activation


4.5

Each intracellular second messenger activated by FGFs in endothelium may represent a putative target for angiogenesis inhibitors. Accordingly, FGF activity can be inhibited in vitro and in vivo by a variety of synthetic compounds, antibodies, selective dominant negative mutants, and antisense cDNAs targeting various second messengers activated by FGFs [112]. Notably, the low molecular weight compound SSR128129E that binds the extracellular domain of all FGFRs without affecting orthosteric FGF interaction inhibits FGFR activation and signaling by an allosteric mechanism of action, thus eliciting antiangiogenic/antitumor effects [113]. Neutralization of FGF-induced effectors/biological activities induced by FGFs

4.6

FGFs upregulate the production of several proteases, including different metalloproteases and urokinase-type plasminogen activator. Natural inhibitors of these proteases, such as TIMPs and PAI-1, inhibit FGF2-induced neovascularization and may provide the rationale for the development of new antiangiogenic strategies. Accordingly, the citrus polymethoxyflavonoid Nobiletin inhibits FGF1/2-dependent angiogenesis in vitro by the simultaneous down-regulation of MMP2 and urokinasetype plasminogen activator expression and activity [114]. The properties of the neovasculature differ from those of the quiescent endothelium in terms of proliferation, permeability, maturation, and cytoskeleton organization. Vascular targeting agents exploit these differences to induce selective blood vessel occlusion and destruction [115]. Microtubule or stress fiber destabilizing agents [116-119] and microtubulestabilizing chemotherapeutics [120,121] disrupt immature, rapidly proliferating tumor endothelium exerting an antiangiogenic effect. In addition, 5’-O-tritylinosine (KIN59) inhibits angiogenesis, at least in part, by down-regulating the expression of laminin, a major component of endothelium basement membrane [122]. 5.

FGF/FGFR inhibitors in cancer clinical trials

As described above, a wide array of approaches might be theoretically pursued to develop anti-FGF strategies to be employed for the treatment of angiogenesis-dependent diseases, including cancer. For all these approaches, the demonstration of their antiangiogenic efficacy in vitro has been provided and their antiangiogenic/antitumor potential has been proved in vivo for many of them. Nevertheless, the search for anti-FGF drugs currently under evaluation in cancer clinical trials (https://clinicaltrials.gov) indicates that only two major classes of inhibitors of the FGF/FGFR system have so far been developed: FGFR 8

selective and nonselective small-molecule TK inhibitors and anti-FGFR antibodies, a few studies focusing on FGFR decoy extracellular FGF ligand traps (Table 2; see also [123-128] for a detailed description of FGF/FGFR-targeting agents in Phase I, Phase II, or Phase III clinical development). In addition, some compounds, including integrin antagonists, entered clinical trials for pharmacologic features different from their FGF antagonist activity that, nevertheless, may contribute to the observed therapeutic effects. This is the case also for heparin, originally included in anti-cancer regimens for its capacity to prevent thromboembolic diseases in tumor-bearing patients. Its ability to sequester different angiogenic growth factors, including FGFs (see above), encouraged the evaluation in clinical trials of modified heparin molecules endowed with low anticoagulant capacity but retaining their antiangiogenic, antitumor effect. Finally, it is worth noting that FGF/FGFR inhibitors are frequently evaluated in combination with classical chemotherapeutics, in agreement with the notion that, in respect to monotherapies, multidrug regimens may provide better therapeutic benefits in cancer patients. 6.

Expert opinion

The possibility that the FGF/FGFR system may play a role in human tumor vascularization represents an important issue in FGF biology and for the development of antiangiogenic therapies. Numerous studies attempted to establish a correlation between intratumoral levels of FGF2 mRNA or protein and intratumoral MVD in cancer patients. The results from 53 independent studies that investigated the correlation between intratumoral FGF2 levels and MVD and between these two parameters and cancer progression/prognosis highlighted a marked heterogeneity among different tumors and also among different studies within the same tumor type [68]. With a few exceptions (e.g., melanomas) FGF2 levels did not correlate persistently with MVD, in sharp contrast with VEGF levels that more systemically correlated with MVD. It is interesting to note that in some tumor types (e.g., breast and hepatocellular carcinomas) intratumoral levels of FGF2 correlated with the clinical outcome but not with MVD. As stated above, the pleiotropic activity of FGFs may affect both tumor vasculature and tumor parenchyma. Thus, FGF2, as well as other FGFs (Table 1), may contribute to cancer progression not only by inducing neovascularization, but also by acting directly on tumor cells, thus providing druggable targets for the development of ‘two-compartment’ anti-FGF/FGFR agents (Figure 1) [128]. Evaluation of MVD may have prognostic significance in solid tumors [129,130], lymphomas [131], and leukemia [132]. Quantification of the angiogenic proteins in body fluids may represent an indirect, non-invasive way to measure angiogenic activity in cancer patients. Serum concentration of angiogenic factors increases with tumor progression [133] and



Table 2. Mechanism of action of anti-FGF/FGFR drugs in clinic or in clinical trials. Compound

Specific target

Multitarget activity


Different FGFs/ FGFRs

Different angiogenic growth factors/pro-angiogenic receptors

Sequestering FGFs in the extracellular environment Anti-FGF5 vaccine FGF5 FP-1039 (decoy) FGF1, 2, 4 PI-88 (oligosaccharide) FGF1, 2 VEGF, IL-8 LMW 2--0, 3--0 desulfated * heparin Tinzaparin * (LMW heparin) Masking FGFRs PI-88 (oligosaccharide) FGFR1 IMCA1 (Ab) FGFR1 R3Mab (Ab) FGFR3 PRO001 (Ab) FGFR3 FP-1039 (decoy) FGFR1, 2, 4 BIIB022 (Ab) FGFRs VEGFRs AMG479 (Ab) FGFRs VEGFRs Cixutumumab (Ab) FGFRs VEGFRs Figitumumab (Ab) FGFRs VEGFRs MK0646 (Ab) FGFRs VEGFRs Masking FGF co-receptors † Etaracizumab (Ab) avb3 integrin † Intetumumab (Ab) av integrins † Cilengitide avb3, avb5 integrins Inhibition of TK-FGFRs RG7444 FGFR3 AZD4547 FGFR1--3 BGJ398 FGFR1--3 CH5183284 FGFR1--3 LY2374455 FGFR1--4 JNJ-42756493 FGFRs BAY1163877 FGFRs E3810 FGFR1 VEGFRs Lucitanib FGFR1, 2 PDGFRs, VEGFRs Brivanib FGFRs PDGFRs, VEGFRs TSU68 FGFRs PDGFRs, VEGFR2 Vargatef FGFRs PDGFRs, VEGFRs E7080 FGFRs PDGFRS, VEGFRs Lenvatinib FGFRs PDGFRS, VEGFRs Nintedanib FGFRs PDGFRS, VEGFRs Ponatinib FGFRs PDGFRS, VEGFRs Masitinib FGFRs VEGFRs Cediranib FGFRs VEGFRs Pazopanib FGFR1, 3 PDGFRs, VEGFRs AP24534 FGFRs VEGFRs AXL1717 FGFRs VEGFRs RG1507 FGFRs VEGFRs Regorafenib FGFRs PDGFRs, VEGFRs ENMD2076 FGFR1, 2 PDGFRs, VEGFR2, FLT3 Dovotinib FGFRs PDGFRs, VEGFRs, FLT3 Blockage of signal transduction pathway(s) triggered by FGFR activation PBI-05204 Endostar

Other receptors/second messengers involved in tumor growth

IGF-1R IGF-1R IGF-1R IGF-1R IGF-1R

c-KIT c-KIT c-KIT IGF-1R IGF-1R IGF-1R c-KIT, RET c-KIT, Aurora c-KIT, CSFR AKT, NF-kB p38, ERK1/2, AKT

Data referring to FGF/FGFR-targeting therapeutics in clinic or in clinical trials were obtained from https://clinicaltrials.gov. *They may sequester a variety of heparin-binding angiogenic mediators besides FGFs. † Integrin blockage may interfere with the activity of various angiogenic growth factor receptors besides FGFRs. All the compounds are synthetic small molecules unless otherwise specified. Ab, Antibody. Expert Opin. Ther. Targets (2015) 19(11)

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Undesired side effects Multitarget activity

Anti-FGF antibodies Anti-FGF peptides Selective TK inhibitors

Heparin-like compounds Nonselective TK inhibitors


Specificity of the inhibition

Figure 3. Relationship among specificity of antiangiogenic compounds, their multitarget potential and risk of undesired side-effects. FGF: Fibroblast growth factor; TK: Tyrosine kinase.

decreases in response to treatment and long-term disease control [134]. Thus, apart from providing prognostic information in early detection of primary tumors or to follow tumor progression, measurement of these circulating factors may be used for the identification of cancer patients more likely to benefit from a therapeutic anti-FGF/FGFR approach, to monitor tumor regression during therapy and for the selection of patients at high risk of recurrences after treatment [135]. To this respect, the observation that compensatory upregulation of the FGF/FGFR system may facilitate the escape from angiostatic anti-VEGF blockade [70,136] points to the possibility that the combinatorial or sequential inhibition of VEGF and FGF pathways may translate into improvements in the clinical care of cancer patients. At present, the prognostic significance of FGF levels in biological fluids of cancer patients is controversial and mostly limited to FGF2-related studies. Early observations had shown that elevated levels of FGF2 in urine samples collected from 950 patients having a wide variety of solid tumors, leukemia, or lymphoma were significantly correlated with the status and the extent of disease [137]. However, no association between increased serum levels of FGF2 and tumor type was observed in later studies on a large spectrum of metastatic carcinomas even though two-thirds of the patients showing progressive disease had increasing serum levels of the angiogenic factor compared with < 1 -- 10 of the patients showing response to therapy [138]. On the other hand, the analysis of the clinical significance of circulating FGF2 in individual types of cancer indicated that the levels of circulating FGF2 may have prognostic significance in head and neck cancer, lymphoma, leukemia, prostate carcinoma, and soft tissue sarcoma but they do not correlate with breast cancer progression and their significance in colorectal carcinoma is unclear [139]. Also, after an encouraging report about a positive correlation between MVD and cerebrospinal fluid FGF2 in children with brain tumors [140], further studies showed that FGF2 levels in body fluids do not always reflect tumor vascularity. Further studies assessing the correlation between the levels of different FGFs at 10

the tumor site and/or in body fluids and MVD are required before these growth factors, as well as other angiogenic factors, can be used as prognostic indicators, surrogate markers of angiogenesis, and of response to antiangiogenic therapy in cancer patients. As described above, both selective and nonselective inhibitors of the FGF/FGFR system have been described and are under clinical development. For instance, FGF family members and a variety of other angiogenic growth factors share the capacity to bind heparin/HSPGs. Thus, nonselective binding would confer to heparin-like drugs the capacity to sequester different angiogenic growth factors simultaneously, acting as ‘multitarget traps’ [79]. This may provide a more potent antiangiogenic response since tumor neovascularization is often the result of the synergic action of various angiogenic growth factors [141]. On the other hand, a too broad binding capacity may cause heparin-like compounds to interfere with physiological cytokines and related biological processes with consequent undesired side-effects and/or toxicity (Figure 3). The same considerations may apply when considering the pros and cons of selective versus nonselective TKFGFR inhibitors. Indeed, the latter ones may show a more potent antitumor activity in patients affected by FGF/ FGFR-driven tumors but are potentially endowed with a broader nonspecific toxicity profile (see [128] for a further discussion about this point). Frequently, adverse effects related to anti-FGF/FGFR therapy are represented by tissue calcification and abnormally high serum phosphate levels, resulting from the loss of FGF23 systemic signals, and potentially manageable through diet, phosphate-lowering therapy, or drug interruptions. Sub-type-specific FGF or FGFR inhibitors should significantly reduce these adverse effects. In particular, this could be achieved by an optimal titration of neutralizing specific antibodies or ligand traps in order to reduce FGF/FGFR signals to physiological levels rather than to a complete inhibition [128]. Relevant to this point is the recent characterization of an allosteric pan-FGFR inhibitor that did not show signs of toxicity in animal models, possibly due to its ceiling effect that cannot completely wipe out TK signaling [113]. In conclusion, experimental and clinical evidences point to a role for the FGF/FGFR system in tumor neovascularization, growth and metastatic dissemination. However, several challenges are being faced to further develop efficacious FGF/ FGFR inhibitors for antiangiogenic therapies in cancer: i) the correlation between tumor vascularization and activation of the FGF/FGFR system in the different human cancer types remains uncertain; ii) identification of cancer patients more likely to benefit from a therapeutic anti-FGF/FGFR approach is required; iii) prognostic indicators, surrogate markers of angiogenesis and of response to antiangiogenic therapy in cancer patients need to be identified; iv) pros and cons about the use of selective versus nonselective FGF inhibitors are not fully elucidated; v) the development of drugs



specific for individual FGFs or FGFRs may reduce undesired systemic side-effects related also to alterations of hormone-like FGFs.

Declaration of interests This work was supported in part by grants from Ministero dell’Istruzione, Universita e Ricerca (FIRB project RBAP11H2R9 2011), and Associazione Italiana per la


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Affiliation Roberto Ronca, Arianna Giacomini, Marco Rusnati & Marco Presta† † Author for correspondence University of Brescia, Department of Molecular and Translational Medicine, Brescia, Italy Tel: +39 030 371 7311; E-mail: [email protected]

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Fibroblast growth factor receptor 2: a therapeutic target in gastric cancer.

Fibroblast growth factor receptor 1 as a putative therapy target in colorectal cancer.

Insulin-like growth factor 1 receptor signaling via Akt: a general therapeutic target in neurocutaneous melanocytosis?

Fibroblast growth factor receptor 4 as a potential prognostic and therapeutic marker in colorectal cancer.

Fibroblast growth factor receptor 2 fusions as a target for treating cholangiocarcinoma.

Angiogenesis and basic fibroblast growth factor.

Fibroblast growth factor receptor (FGFR) alterations in squamous differentiated bladder cancer: a putative therapeutic target for a small subgroup.

Cell-permeable iron inhibits vascular endothelial growth factor receptor-2 signaling and tumor angiogenesis.

The hepatocyte growth factor receptor as a potential therapeutic target for dedifferentiated liposarcoma.

Fibroblast growth factor 21: a promising therapeutic target in obesity-related diseases.

Fibroblast Growth Factor Receptor 3 (FGF-R3): A Promising Therapeutic Target for the Treatment of Bladder Cancer.

Fibroblast growth factor signaling in the vasculature.

Cross-talk between endothelial and tumor cells via basic fibroblast growth factor and vascular endothelial growth factor signaling promotes lung cancer growth and angiogenesis.

The nerve growth factor signaling and its potential as therapeutic target for glaucoma.

Ferulic Acid Exerts Anti-Angiogenic and Anti-Tumor Activity by Targeting Fibroblast Growth Factor Receptor 1-Mediated Angiogenesis.

The Fibroblast Growth Factor signaling pathway.

Angiogenesis as a therapeutic target for obesity and metabolic diseases.

Response to "Insulin-like growth factor 1 receptor signaling via Akt: a general therapeutic target in neurocutaneous melanocytosis?".

Factor XI as a Therapeutic Target.

Fibroblast growth factor receptor 1 is a key inhibitor of TGFβ signaling in the endothelium.

Prostanoid receptor EP2 as a therapeutic target.

Azithromycin effectively inhibits tumor angiogenesis by suppressing vascular endothelial growth factor receptor 2-mediated signaling pathways in lung cancer.

Mutational identification of fibroblast growth factor receptor 1 and fibroblast growth factor receptor 2 genes in craniosynostosis in Indian population.

The basic fibroblast growth factor-saporin mitotoxin acts through the basic fibroblast growth factor receptor.