Molecular and clinical significance of fibroblast growth factor 2 (FGF2 /bFGF) in malignancies of solid and hematological cancers for personalized therapies

Fibroblast growth factor (FGF) signaling is essential for normal and cancer biology. Mammalian FGF family members participate in multiple signaling pathways by binding to heparan sulfate and FGF receptors (FGFR) with varying affinities. FGF2 is the prototype member of the FGF family and interacts with its receptor to mediate receptor dimerization, phosphorylation, and activation of signaling pathways, such as Ras-MAPK and PI3K pathways. Excessive mitogenic signaling through the FGF/FGFR axis may induce carcinogenic effects by promoting cancer progression and increasing the angiogenic potential, which can lead to metastatic tumor phenotypes. Dysregulated FGF/FGFR signaling is associated with aggressive cancer phenotypes, enhanced chemotherapy resistance and poor clinical outcomes. In vitro experimental settings have indicated that extracellular FGF2 affects proliferation, drug sensitivity, and apoptosis of cancer cells. Therapeutically targeting FGF2 and FGFR has been extensively assessed in multiple preclinical studies and numerous drugs and treatment options have been tested in clinical trials. Diagnostic assays are used to quantify FGF2, FGFRs, and downstream signaling molecules to better select a target patient population for higher efficacy of cancer therapies. This review focuses on the prognostic significance of FGF2 in cancer with emphasis on therapeutic intervention strategies for solid and hematological malignancies.


INTRODUCTION
The fibroblast growth factor (FGF) family consists of 23 FGF signaling polypeptides that function as potent mitogens [1][2][3]. FGFs exert broad mitogenic activity by stimulating the growth of fibroblasts, endothelial, and cancer cells. The family plays important roles in embryonic development, maintenance of adult organ systems, tissue regeneration, wound repair, and hematopoiesis. FGF2, also known as basic FGF (bFGF), is the prototypical and most studied member of the FGF superfamily. FGF2 is an important regulator of cell growth and differentiation under physiological and pathological conditions [1][2][3]. Previous studies have suggested a role of FGF2 as a prognostic marker for different types of malignancies. This review summarizes the biology of FGF signaling by demonstrating biological roles of FGF2 in regards to pathogenesis and prognosis of solid and hematological tumors with a special focus on clinical development of FGF2 inhibitors in the era of personalized medicine.

FGF2 isoforms, receptors, binding partners, and signaling
FGF2 exists in low and high molecular weight isoforms, which are translated from a single common Review Oncotarget 44736 www.impactjournals.com/oncotarget mRNA through alternative translation-initiation codons [4]. Low molecular weight (LMW) FGF2 is an 18 kDa protein translated from a conventional AUG start codon [5]. LMW FGF2 is found in the cytoplasm and nucleus and can be also secreted by target cells [6]. In order to initiate signaling, LMW FGF2 interacts with cell surface heparan sulfate proteoglycans (HSPGs) and fibroblast growth factor receptor (FGFR) in a ternary complex consisting of FGF2, FGFR, and HSPG resulting in activation of downstream signaling pathways, including Ras, Raf, MAPK and ERK ( Figure 1A) [7]. High molecular weight (HMW) FGF2 isoforms  are produced through translation initiation at CUG sites upstream and in frame of the AUG codon. HMW FGF2 localizes to the nucleus and signals independently of FGFRs [8]. Similar to HMW FGF2, LMW FGF2 can also function in the cytosol and nucleus of cells through endocytosis of activated FGF-FGFR complexes [9]. FGFR1 and FGF2 have been shown to co-localize in the nuclear matrix, where together they may co-activate transcription and thus control cell proliferation ( Figure  1A) [10,11].
Five FGFRs have been identified, four of which (1)(2)(3)(4) are highly conserved single-pass transmembrane   [12,101] HSPG FGF2 binds with low affinity to heparan sulfate chains of HSPG. This interaction can activate intracellular signaling, promote FGF2 internalization, or by presenting FGF2 to FGFR in proper conformation. HSPG also act as reservoirs for FGF2 which protect them from degradation Cell membrane Cancer, endothelial cells FGFR [102][103][104] αvβ3 integrin FGF2 binds to αvβ3 integrin leads to assembly of focal adhesion plaques Cell membrane Endothelial cells FAK [105] Gangliosides Gangliosides bind FGF2 via Neu-Ac residues and acts as coreceptors Cell membrane Endothelial cells FGFR [106] Free gangliosides     [12]. The extracellular regions of these receptors contain three immunoglobulin (Ig)-like domains (designated as IgI, IgII, and IgIII) linked to the cytoplasmic domain via a transmembrane α-helix ( Figure  1A). FGFRs 1-3 can undergo alternative splicing during gene expression, and the IgIII domain is composed of an invariant IgIIIa exon alternatively spliced to IgIIIb or IgIIIc. The expression of IgIIIb and IgIIIc is important in defining FGF signaling specificity. While FGF1 binds to all FGFRs, FGF2 binds to FGFR1 (IIIb), FGFR1 (IIIc), FGFR2 (IIIc), and FGFR4 [2]. It has been reported that LMW FGF2 predominantly binds to FGFR1 (IIIc) and weakly to the other FGFRs [5,13]. The cytoplasmic domain of FGFRs 1-4 contains a juxtamembrane split kinase domain, which contains tyrosine kinase motifs and a C-terminal tail [12]. Although FGFR5 lacks intracellular tyrosine kinase domain, this receptor can bind to multiple FGF ligands acting as a negative regulator of signaling [14].
FGF2 utilizes HSPGs, such as syndecans (SDC), as binding partners to stabilize the FGF-FGFR interaction and enhance resistance to proteolysis [15,16]. FGF2 cannot activate FGFRs in cells lacking heparan sulfate [17]. After the binding of FGF and HSPG to FGFR to form a ternary FGF:FGFR:HSPG complex, FGFRs dimerize leading to conformational changes in FGFR structure and subsequent intermolecular transphosphorylation of multiple cytoplasmic tyrosine residues ( Figure 1A) [12,18]. FGFR transmits extracellular signals to two main intracellular substrates, which are phospholipase C-γ1 (PLC-γ1) (also known as FRS1) and FGFR substrate 2 (also known as FRS2) ( Figure 1A). The phosphorylation of FGFR1 tyrosine residues creates binding sites for SH2 domain of PLC-γ required for phosphorylation and activation of PLC-γ. Conversely, FRS2 constitutively associates with the juxtamembrane region of the FGFR. The phosphorylation of FRS2 is essential for activation of the Ras-mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase-Akt (PI3K-Akt) signaling pathways in cancer and endothelial cells ( Figure 1A) [12,19].
FGF2 also interacts with immobilized molecules bound to extracellular matrix (ECM), including cell membrane receptors and soluble molecules (Table 1, Figure 1B). The complex interactions between FGF2 and these molecules control bioavailability, stability, and concentration of FGF2 in the microenvironment [20]. FGF2 can tightly bind HSPG in ECM and is only released through the action of FGF-binding protein (FGF-BP), which is a critical controller of FGF bioavailability (Table 1, Figure 1B). In addition, the binding of FGF to heparin, released HSPG, or cell surface-bound HSPG also regulate FGF bioavailability and the interactions with FGFRs (Table 1, Figure 1B). Conversely, thrombospondin-1 (TSP-1) and pentraxin 3 (PTX3) prevent the interaction of FGF2 with cell surface HSPGs and FGFRs. Similarly; xcFGFR1 (a soluble form of the extracellular portion of FGFR1) binds FGF2 and prevents FGF2/FGFR interaction (Table 1, Figure 1B).
Gangliosides are glycosphingolipids bound to cell membranes regulating growth of a wide variety of normal cells by binding to FGFs. Gangliosides are highly synthesized in metastatic tumors and are known to shed into the ECM. Integrins are transmembrane receptors that regulate the response to soluble FGF2 in endothelial cells. The interaction between αvβ3 integrin and FGF2 promotes endothelial cell proliferation by activating the MAPK pathway [21]. In addition, the binding of fibrinogen to FGF2 requires αvβ3 integrin to promote endothelial cell proliferation (Table 1, Figure 1B). These findings indicate that FGF2 bioactivity and interaction with FGFR is highly regulated by a complex network of interactions with various FGF2 binding partners.

ROLES OF FGF2 IN TUMOR PROGRESSION
Accumulating evidence suggests that FGF2/FGFR signaling is involved in several biological functions, such as embryonic development, tissue regeneration, wound repair, and normal hematopoiesis [1][2][3]. Expression of FGF2 and FGFRs in normal cells is highly regulated, and termination of FGF2 signaling is achieved through receptor internalization (Figure 2A) [1][2][3]. However, FGF2/FGFR signaling in cancer cells is dysregulated, which may contribute to the pathogenesis of many types of cancer (Figure 2A). Several studies have shown that FGF2 is a key tumor-promoting factor in the tumor microenvironment. The following section reviews current knowledge of the molecular pathways associated with FGF2 signaling in cancer, which represents a critical step for the implementation of strategies toward the development of personalized cancer therapy.

Deregulation of FGFR signaling
FGFR amplification and/or upregulation occur in cancer due to deregulated transcription or chromosomal amplification (Figure 2A) [22]. The upregulation of FGFR1 induces cellular transformation of nontransformed cells [23]. FGFR2 upregulation is associated with poor prognosis in patients of multiple cancer types [24]. Furthermore, FGFR2 amplification has been shown to be accompanied with C-terminal exon abrogation, which regulates receptor internalization [25]. Impaired termination of FGFR signaling leads to continuous receptor activation [22]. In addition, mutation of FGFR can also render it insensitive to endocytosis by maintaining its expression at the cell surface [22]. A number of germline activating point mutations of FGFRs have been identified in human cancers and are associated with poor Oncotarget 44743 www.impactjournals.com/oncotarget survival and chemoresistance [26,27]. Mutations in the extracellular domain of FGFRs facilitate ligand binding, while mutations in transmembrane and kinase domains lead to constitutive activation of receptors (Figure 2A) [22]. Furthermore, alternative splicing of the third Ig-like domain could promote tumorigenesis. Paracrine signaling typically occurs through FGFR-IIIb that is expressed on mesenchymal cells and -IIIc expressed on epithelial cells [22]. In cancer models, however, the switch from FGFR IIIb to FGFR IIIc by alternative splicing results in autocrine activation of the receptor (Figure 2A) [28]. For example, FGFR2-IIIb to IIIc switch is related to increased invasiveness in bladder and prostate cancers [29,30]. In addition, FGFR1-IIIc has been upregulated in pancreatic cancer where it is regarded as a strong oncogene [31].

FGF2 as pro-angiogenic agent
FGF2 is an extremely potent pro-angiogenic growth factor. FGF2 exerts its effects on endothelial cells via a paracrine mode after being released by tumor and stromal cells or through mobilization from ECM ( Figure 2B) [32]. In addition, FGF2 plays autocrine roles in endothelial cells [32]. It has been reported that endothelial cells predominantly express FGFR1 and to some extent FGFR2 [33,34]. Activation of these receptors by FGF2 leads to endothelial cell proliferation, migration, protease production, and angiogenesis. Furthermore, the full mitogenic and chemotactic responses of FGF2 in endothelial cells require activation of ERK1/2 and protein kinase C (PKC) signaling pathways [35]. FGF2 upregulates plasmin-plasminogen activator (uPA) and matrix metalloproteinase (MMP) production in endothelial cells eventually leading to ECM degradation and angiogenesis [36]. In addition, the response of endothelial cells to FGF2 can be regulated by integrins [21]. Immobilized FGF2 binds to αvβ3 integrin causing endothelial cell adhesion, migration, proliferation, and morphogenesis ( Figure 2B) [37]. There is also considerable cross-talk between FGF and vascular endothelial growth factor (VEGF) signaling, whereby FGF-induced signaling promotes resistance to VEGF receptor signaling for blocking of the VEGF [38]. Moreover, transient expression of FGF2 in endothelial cells control the expression of genes implicated in cell cycle, differentiation, adhesion, and cell survival [39]. Taken together, these data suggest an important role of FGF2 in promoting endothelial cell angiogenesis ( Figure  2B).

FGF2 as mitogen for tumor cells
Although FGF2 levels are elevated in several human cancers, FGF2 levels do not always correlate with microvessel density [40]. For example, in a study  [41]. A significant correlation was observed between VEGF expression and MVD but not between FGF2 and MVD [41]. However, in a study conducted by Garcia de la Torre et al, FGF2 expression was high in primary parathyroid hyperplasia (PPH) and FGF2 scores and MVD were significantly correlated [42]. Therefore, FGF2 may contribute to cancer progression through alternative mechanisms involve acting directly on tumor cells [19]. Mutations in genes encoding FGFs and FGFRs deregulate FGFR signaling [43,44]. However, no activating mutations have been reported as yet for FGF2 [44]. FGF2-induced activation of FGFR signaling and subsequent activation of PI3K/Akt and ERK1/2 signaling pathways in cancer cells [19,45]. FGF2 contributes to tumor progression through enhanced expression and/or release from tumor, endothelial, or stromal cells as well as release from local reservoirs in the ECM ( Figure 2B) [43]. Secretion of proteases leads to release of the sequestered FGF2 [43]. Therefore, FGF2 functions in an autocrine or paracrine manner in cancer cells ( Figure 2B).

Dysregulated downstream FGF2/FGFR signaling pathway in hematological tumors
Growing evidence supports a role of FGF2 in hematopoiesis starting at early stages of development through adulthood. In early stages of development, FGF2 has an important role in the proliferation of hemangioblasts, which are common progenitors of hematopoietic and endothelial cells [51] that play a central role in hematopoietic and angiogenic differentiation [52]. In addition, FGF2 plays a role in self-renewal, cell survival, and cell adhesion of human embryonic stem cells [53]. In adult hematopoiesis, FGF2 induces proliferation of stromal cells of bone marrow [54]. FGF2 also induces the production of interleukin-6 (IL-6) [55] and counteracts the suppressive effect of transforming growth factor beta (TGF-β) on myeloid progenitor cells [56]. Myeloid precursor cells can be induced by FGF2 to give rise to erythroid progenitors [57]. In the absence of FGF2, myeloid progenitors generate cells of the neutrophilgranulocyte lineage upon FGF2 induction [58].
Neoplastic cells that define each hematological tumor are descendants of a specific lineage of the hematopoietic process. The involvement of FGF2 in various stages of hematopoiesis suggests that its dysregulation can result in hematological cancers [59]. FGFRs are expressed on all cell types of haematopoietic origin, and deregulation of FGFR gene expression or mutation has been observed in haematologic malignancies [59]. The ability of FGF2 to induce stem cell proliferation and differentiation implies that FGF2 is involved in very early stages of hematopoiesis. It has been reported that lymphoma cell lines express FGF2 and FGFRs and release FGF2 into culture media [45].

Putative mechanisms of FGF2 in Hodgkin Lymphoma
Hodgkin lymphoma (HL) is a rare B-cell malignant neoplasm characterized by a paucity of malignant Hodgkin and Reed-Sternberg cells (HRS cells) embedded within an inflammatory infiltrate [60]. FGF2 causes aberrant signaling activities in HRS cells involving SDC1, NF-κB, IL-6, Ras/ERK, and JAK/STAT as shown below. SDC1 regulates bioavailability, dimerization, and interaction of FGF2 with FGFRs [20]. Witzig and colleagues reported that SDC1 was expressed in the bone marrow of patients with plasma cell proliferative disorders [61]. In line with this, increased expression of FGF2 and SDC1 was also reported in HL cell lines [68]. Overall, findings suggest that increased expression of FGF2, FGFR, and SCD1 is associated with poor prognosis and chemoresistance [62].
Activation of the NF-κB pathway is a wellestablished mechanism for protection of tumor cells from apoptosis [63,64]. In HL, NF-κB is constitutively activated and represents an important step for the proliferation of neoplastic HRS cells (Figure 3) [65]. Epstein-Barr Virus (EBV) is a risk factor for HL [74]. In EBV-positive (EBV+) HL, the EBV oncoprotein, latent membrane protein-1 (LMP1), has been implicated in the activation of NF-κB signaling leading to enhanced B-cell survival [65]. Alternatively, NF-κB may serve as a transcription factor for the FGF2 gene regulating expression and release of FGF2 by LMP1 [66,67]. This process could be of clinical importance for determining the relationship between EBV status and FGF2 levels in HL patients (Figure 3). IL-1β, which is expressed in subsets of cells in the HL tumor microenvironment, activates PI3K signaling pathway to upregulate FGF2 production through NF-κB [67].
The interleukin-6 (IL-6) signaling pathway has also been implicated in tumor progression [68,69]. In multiple myeloma, stromal-derived IL-6 stimulates FGF2 expression in tumor cells, which in turn stimulates the secretion of IL-6 [70]. In addition, IL-6 and FGF2 together can enhance proliferation of myeloma cells [71]. In HL, IL-6 and its receptors are expressed by HRS cells ( Figure  3) [72,73]. Moreover, IL-6 is upregulated in serum of HL patients resulting in poor prognosis [74]. FGF2 upregulates IL-6 gene expression in the fibroblast NIH-3T3 cell line [75]. Moreover, in a basal cell carcinoma cell line, IL-6 mediates upregulation of FGF2 through activation of JAK/STAT3 and PI3K/Akt pathways which are aberrantly activated in HL [65]. Therefore, IL-6 and FGF2 may be involved in paracrine and autocrine interactions to promote chemoresistance in relapsed/ Components of the Ras/ERK pathway are aberrantly expressed in malignancies and associated with chemoresistance [76]. MEK/ERK signaling pathway is essential for proliferation and survival of neoplastic HRS cells (Figure 3) [77]. FGF2 induces MEK signaling to upregulate anti-apoptotic proteins and enhance chemoresistance [78]. In addition, FGF2 mediates chemoresistance to doxorubicin in endothelial cells by inhibiting the pro-apoptotic protein ASK1, which is a member of the MEKK family [79].
The Janus kinase-signal transducer and activator of transcription (JAK/STAT) is a frequently altered pathway in the pathogenesis of HL [80]. The JAK/STAT pathway has been implicated in FGF2-induced chemoresistance in human cancer cells [81]. The JAK2 inhibitor lestaurtinib can overcome JAK/STAT-induced drug resistance in refractory HL cell lines [82]. Therefore, FGF2 may promote chemoresistance by deregulation of JAK/STAT signaling in HRS cells of relapsed and refractory HL patients ( Figure 3).
The above studies demonstrated key downstream signaling pathways in HL, including cell growth, survival, and angiogenesis. It seems clear that the main pathways contributing to HL are regulated, at least in part, by FGF2 ( Figure 3). Therefore, clinical trials using agents that target FGF pathway may be promising for treatment of HL.

CLINICAL PROGNOSTIC VALUE AND FUNCTIONAL SIGNIFICANCE OF FGF2 IN SOLID AND HEMATOLOGICAL TUMORS
Several studies have compared FGF2 serum levels in cancer patients to those in healthy volunteers (Tables 2  and 3). FGF2 expression in sera quantified by ELISA was strongly increased in cancer patients compared to healthy donors. Significant correlations between serum FGF2 levels and tumor stage, size, and metastasis were reported in endometrial, colorectal, esophageal, head and neck, liver, renal, and testicular cancers. However, no significant correlation was observed between increased serum FGF2 levels and tumor grade in bladder, breast, lung, and prostate cancers. In all leukemia and lymphoma studies, there was no correlation between increased serum levels of FGF2 and microvessel density or stage of the disease. However, high serum FGF2 levels were significantly correlated with tumor bulkiness in Non-Hodgkin's lymphoma (NHL). Therefore, the levels of serum FGF2 may have prognostic significance in these cancers, and quantification of FGF2 may provide an indirect, noninvasive way to monitor patients with high risk of relapse from solid and hematological tumors (Tables 2 and 3). FGF2 expression in cancer surgical sections has been evaluated using immunohistochemical, Western blot, and qRT-PCR techniques (Tables 2 and 3). Upto-date diagnostics and antibodies that allow detection and precise quantification of FGF2 are listed in Table 4. Immunohistochemical and immunofluorescence studies have shown that FGF2 staining is heterogeneous and significantly increased in malignant tissues compared to benign or normal tissues (Tables 2 and 3). The differential expression and localization of FGF2 was also studied in different cancers. For example, FGF2 expression is generally limited to the cytoplasm of breast cancer tissues, while it is exclusively expressed in the nuclei of normal mammary tissues. Similarly, FGF2 is strongly expressed in the cytoplasm of malignant melanocytes and prostate cancer tissues, while it is almost entirely restricted to the nuclei of benign cells. In pancreatic cancer cells, FGF2 staining is intense in both the cytoplasm and nucleus, while it is weak in normal control tissues. Different expression and localization of FGF2 suggests that FGF2 and different members of FGFR may have different functions and signaling in various cancers. Moreover, FGF2 expression was elevated in tumor stroma, including inflammatory cells, myofibroblasts, and endothelial cells in colorectal, pancreatic, and prostate carcinomas, respectively ( Table 2). These findings suggest that FGF2 can modulate tumor progression by activating signaling pathways in cancer-associated fibroblasts, endothelial cells, and cancer cells. In addition, fibroblasts that are abundant in the stroma of carcinomas at advanced stages of disease can mediate resistance to treatment via FGF2 secretion [83]. Additional studies on tissue sections have revealed that high FGF2 intratumoral levels are associated with advanced tumor stages of bladder, glioma, head and neck, liver, and prostate cancers ( Table 2).
There is an urgent need for the identification of novel prognostic biomarkers to improve treatment of poor outcome cancer patients. It is worth noting that in the vast majority of studies, high serum and intratumoral FGF2 levels were associated with reduced cancer patient survival. In addition, high intratumoral and serum levels of FGF2 were associated with relapse and/or recurrence in various cancers such as bladder, breast, esophageal cancers and HL (Tables 2 and 3). In spite of a few contradictory finding, FGF2 is considered a significant tumor biomarker and a potential therapeutic target. Ongoing and future clinical trials are warranted to determine whether FGF2 could be incorporated in cancer prognosis and whether FGF targeting therapies have a favorable effect on cancer recurrence and mortality.

TARGETING THE FGF2/FGFR PATHWAY IN CANCER
The trend in cancer personalized medicine is to search for biomarkers to predict a patient's response to the targeted therapy and the emergence of secondary resistance [84]. High expression of FGF2 correlates with Oncotarget 44751 www.impactjournals.com/oncotarget a worse survival for relapsed/refractory cancer patients (Tables 2 and 3). Therefore, targeting FGF signaling may provide opportunities for personalized therapy in those patients. Several FGF2/FGFR inhibitors have shown promising anticancer and antiangiogenic efficacy in several in vitro assays and in vivo preclinical animal models (Table 5, Figure 4). Clinical studies on these compounds have been conducted in the last decade to evaluate their safety, efficacy, and tolerability (Table  6). Ongoing clinical trials are recruiting patients with metastatic, advanced, or relapsed/ refractory cancers to evaluate the importance of blocking the FGF2/FGFR signaling in progressive and poor outcome cancer patients ( Table 6).
It has been reported that various molecules can inhibit FGF2 (ligand) activity, binding, or expression in endothelial and tumor cells (Table 5, Figure 4). FGF ligand traps, such as FP-1039, block FGF2 interaction with FGFR (Tables 5 and 6, Figure 4) [85]. A phase I clinical trial was conducted to investigate the safety and tolerability of FP-1039 in advanced solid tumors (Table 6) [86]. FP-1039 is a soluble FGFR1 Fc fusion protein that was engineered to strongly bind all mitogenic FGF ligands. This compound showed promising results and inhibited FGF-mediated cell proliferation and angiogenesis in lung and endometrial cancer models [86]. AGM-1470, miRNA 646, and interferon alpha downregulate FGF2 expression in cancer cells (Table 5, Figure 4). A phase II clinical study was conducted to evaluate the efficacy of pegintron (peginterferon alpha-2b) in patients with stage IV metastatic melanoma overexpressing FGF2 (Table  6) [87]. The results showed that peginterferon alpha-2b suppressed FGF2 levels in 97% of patients with metastatic melanoma to reference range with a median progression free survival (PFS) and overall survival (OS) of 2.0 and 9.7 months, respectively (Table 6) [87].
Other FGF2 antagonists are under investigation for cell line and animal preclinical cancer models. Small molecules, such as sm27, pentosan, and PI-88 as well as proteins such as pentraxin-3 inhibit FGF2 binding to FGFRs (Table 5, Figure 4) [3]. The capability of FGF2 to bind heparin/heparan sulfate indicates that molecules able to interfere with this interaction may act as angiogenesis inhibitors. On this basis, compounds such as suramin, which mimic heparin, can interfere with FGF2 signaling (Table 5). Thalidomide, PAMPS, sirolimus, suramin, and platelet factor 4 inhibit FGF2-induced angiogenesis, while anvirzel inhibits FGF2 export via Na + /K + pumps (Table 5).
It has been reported that direct inhibition of FGFRs using small molecule inhibitors may be effective in cancer treatment. Several FGFR tyrosine kinase inhibitors (TKIs) are currently in early clinical development (Tables 5 and  6, Figure 4), and many of them exhibit dual specificity for FGFR and VEGFR due to similarity in the ATP binding pocket structure [85]. The first generation of inhibitors were developed as VEGFR and PDGFR inhibitors, but also can inhibit FGFR [88]. These compounds were successful in clinical trials and some of these drugs and drug combinations were subsequently approved by regulatory administrations worldwide for the treatment of different cancers (Tables 5 and 6).
Recently, the U.S. Food and Drug Administration (FDA) granted approval to Lenvima (lenvatinib, developed by Eisai) based on the results of a study on 392 patients with locally recurrent or metastatic, progressive, radioactive iodine-refractory differentiated thyroid cancer (DTC) who were randomly assigned to receive either Lenvima or a placebo. Study results showed that Lenvimatreated participants lived a median of 18.3 months (PFS) compared to a median of 3.6 months for participants who received placebo. Moreover, approximately two thirds of participants treated with Lenvima showed a reduction in tumor size compared to only two percent of participants who received placebo. Similarly, FDA recently approved Iclusig (ponatinib, developed by ARIAD Pharmaceuticals), which is a multi-tyrosine kinase inhibitor for the treatment of adult patients with chronic myeloid leukemia or acute lymphoblastic leukemia (Table  5) [89]. Moreover, the multi-tyrosine kinase inhibitor Votrient (pazopanib, developed by GlaxoSmithKline) was approved by FDA to treat patients with advanced renal cell cancer and patients with advanced soft tissue sarcoma who have received chemotherapy in the past (Tables 5 and  6) [90].
Dovitinib (TKI-258, CHIR-258, developed by Novartis) is a potent VEGFR, PDGFR and FGFR inhibitor. Dovitinib showed promising anticancer and antiangiogenic activity against multiple myeloma and colon cancer models [91,92]. A phase II trial was conducted to evaluate the safety and tolerability of Dovitinib in relapsed or refractory multiple myeloma patients, who are with or without t(4;14) chromosomal translocation ( Table 6). The t(4;14) translocation is observed in approximately 15-20% of multiple myeloma patients, and it is associated with upregulation of FGFR3 and poor prognosis [93,94]. Upregulation of FGFR3 occurs in nearly 70% of patients with the t(4;14) translocation. Therefore, development of inhibitors such as Dovitinib may show promise in t(4;14) multiple myeloma patients [94]. Further phase II clinical trials were conducted on Dovitinib for treatment of other types of cancers including lung, bladder and gastric cancers ( Table 6).
Other investigational multi-kinase inhibitors, such as Brivanib, Orantinib, and Lucitanib, are currently progressing into phases II and III of clinical trials (Tables 5 and 6). Recent clinical trials have assessed the combination of a tyrosine kinase inhibitor with cytotoxic agents (Table 6). Combination strategies that involve the blockade of FGFR signaling with cytotoxic agents have the best clinical outcome for cancer treatment [85]. Recently, Vargatef (nintedanib, developed by Boehringer Ingelheim) was approved in the EU based on the results Oncotarget 44752 www.impactjournals.com/oncotarget of a phase III study comparing Vargatef plus docetaxel to placebo plus docetaxel in patients with locally advanced/ metastatic non-small-cell lung carcinoma (NSCLC) after first-line therapy in over 1,300 patients in 27 countries [95]. Vargatef is an oral triple angiokinase inhibitor that simultaneously inhibits the VEGFR, PDGFR, and FGFR signaling pathways. The results indicated that treatment with Vargatef and docetaxel significantly extended the median overall survival from 10.3 to 12.6 months for patients with adenocarcinoma compared to placebo and docetaxel, with a quarter of patients surviving for two years or more with minimum side effects [95]. Several clinical trials are ongoing currently with compounds targeted for FGF2; a phase III study was designed to compare the safety and efficacy of a multi-tyrosine kinase inhibitor, masitinib, in combination with bortezomib and dexamethasone to placebo in combination with bortezomib and dexamethasone in patients with relapsing multiple myeloma. Masitinib is an orally active and bioavailable compound that is a weak inhibitor of FGFR3 [96].
More recently, second-generation inhibitors targeting FGFRs with high selectivity have been developed (Table 5, Figure 4). For example, Debio 1347, developed by Debiopharm Group, a Swiss-based global biopharmaceutical company, is an orally bioavailable inhibitor of FGFRs 1-3 that inhibits FGFR-mediated signal transduction pathways and consequently tumor cell proliferation and angiogenesis [97]. Debio 1347 will be used for personalized cancer treatment through the development of a companion diagnostic. It is currently being evaluated in Europe and the USA in a phase I trial to evaluate its safety and tolerability in patients with advanced solid tumors displaying alterations in FGFRs 1, 2, or 3 genes (Tables 5 and 6) [97]. Similarly, AZD4547, developed by AstraZeneca, is an orally tolerated inhibitor of FGFRs 1-3 [98]. AZD4547 inhibits FGFR kinase activity and tumor growth in vitro and in vivo [98]. This compound is currently being tested for safety and efficacy in different clinical trials against advanced tumors (Table 6). Other small molecule FGFR inhibitors, such as BGJ398, JNJ-42756493, ARQ 087, LY2874455, and TAS-120, are currently under clinical investigation for advanced, relapsed or refractory tumors and most of these trials are still recruiting patients (Tables 5 and 6). Other selective small molecule tyrosine kinase inhibitors, such as PD 161570, PD 173074, PD 166285 dihydrochloride, PD 166866, FIIN hydrochloride, SU 5402, and SSR128129E are currently being tested for their antitumor activity on cell lines and in vivo preclinical models (Table 5). Monoclonal antibodies specifically targeting particular FGFR isoforms are also being developed. MFGR1877S (developed by Genentech) is a human anti-FGFR3 monoclonal antibody that inhibits tumor progression of bladder carcinoma and multiple myeloma xenografts in mice by antagonizing FGFR3 signaling [99]. A phase I clinical trial was conducted to evaluate the response to MFGR1877S in patients with relapsed or refractory multiple myeloma (Tables 5 and 6). Similarly, BAY 1179470 (developed by Bayer) is a human anti-FGFR2 monoclonal antibody. BAY 1179470 showed antitumor activity in gastric cancer xenograft models with high FGFR2 expression [100]. The anti-FGFR2 antibody BAY 1179470 is currently in Phase I testing in subjects with advanced, refractory solid tumors (Tables 5 and 6) CONCLUSIONS FGF2 is frequently dysregulated in cancer, especially in advanced stages of disease. The upregulation of FGF2 or FGFRs can promote resistance to chemotherapy. FGF2 is currently being evaluated in clinical studies as a potential predictive biomarker for hematological and solid tumors. In addition, FGF2/FGFR inhibitors are being developed and evaluated as monotherapy or as part of a combination therapy for the treatment of different types of cancer. Identifying patients with advanced, relapsed or refractory cancers that would benefit from FGF2/FGFR signaling inhibition will allow for better treatment options of those patients in the era of personalized medicine.  55. Berardi AC, Wang A, Abraham J and Scadden DT. Basic fibroblast growth factor mediates its effects on committed myeloid progenitors by direct action and has no effect on hematopoietic stem cells. Blood. 1995; 86:2123-2129.