s2fieldsofstudy: Biology Chemistry

text:
Identification of ABC Transporter Interaction of a Novel Cyanoquinoline Radiotracer and Implications for Tumour Imaging by Positron Emission Tomography

Background The epidermal growth factor receptor (EGFR) is overexpressed in many cancers including lung, ovarian, breast, head and neck and brain. Mutation of this receptor has been shown to play a crucial role in the response of non-small cell lung carcinoma (NSCLC) to EGFR-targeted therapies. It is envisaged that imaging of EGFR using positron emission tomography (PET) could aid in selection of patients for treatment with novel inhibitors. We recognised multi-drug resistant phenotype as a threat to development of successful imaging agents. In this report, we describe discovery of a novel cyanoquinoline radiotracer that lacks ABC transporter activity. Methods Cellular retention of the prototype cyanoquinoline [18F](2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-({[1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl]methyl}amino)-but-2-enamide ([18F]FED6) and [18F](2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-[({1-[(2R,5S)-3-fluoro-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]-1H-1,2,3-triazol-4-yl}methyl)amino]but-2-enamide ([18F]FED20) were evaluated to establish potential for imaging specificity. The substrate specificity of a number of cyanoquinolines towards ABC transporters was investigated in cell lines proficient or deficient in ABCB1 or ABCG2. Results FED6 demonstrated substrate specificity for both ABCG2 and ABCB1, a property that was not observed for all cyanoquinolines tested, suggesting scope for designing novel probes. ABC transporter activity was confirmed by attenuating the activity of transporters with drug inhibitors or siRNA. We synthesized a more hydrophilic compound [18F]FED20 to overcome ABC transporter activity. FED20 lacked substrate specificity for both ABCB1 and ABCG2, and maintained a strong affinity for EGFR. Furthermore, FED20 showed higher inhibitory affinity for active mutant EGFR versus wild-type or resistant mutant EGFR; this property resulted in higher [18F]FED20 cellular retention in active mutant EGFR expressing NSCLC. Conclusion [18F]FED20 binds EGFR but is devoid of ABC transporter activity, thus, has potential for EGFR imaging.

Introduction
The epidermal growth factor receptor (EGFR) is a 170 kDA transmembrane tyrosine kinase receptor member of the ErbB family of receptors, which includes HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4) [1]. Pathological expression of EGFR has been observed in many cancers including head and neck, breast, pancreatic, non-small cell lung cancer (NSCLC) and colorectal cancers [2]. Currently clinically approved therapies which target EGFR include monoclonal antibodies such as cetuximab or panitumumab [3,4], and small molecular tyrosine kinase inhibitors (TKI) such as gefitinib and erlotinib [4]. In the context of NSCLC, the efficacy of TKI's was found to be significantly higher in patients who express active mutant EGFR of which the L588R and Del E746-A750 mutations are the most common. The frequency of mutations in EGFR recorded in NSCLC patients has been found to vary [5][6][7], but can be as high as 44% in female Asian patients with adenocarcinoma and no previous history of smoking [5,8,9]. The Iressa Pan-Asia-Study (IPASS), which compared gefitinib to a standard carboplatin and paclitaxel chemotherapy in a first line treatment setting in NSCLC Asian patients [10] was the first study that demonstrated a benefit of TKI therapy in selected NSCLC patients. The most prevalent mechanism of acquired resistance is a secondary mutation in EGFR (T790M); this mutation occurs in half of patients administered reversible TKIs and results from an increase in affinity for the receptor for ATP [11,12]. The issue of acquired resistance is complex; more recently C797S mutations were reported as a new mechanism to third generation TKIs [13]. Despite advances in EGFR targeted therapies and the increasing number of EGFR inhibitors undergoing preclinical evaluation, there are currently no non-invasive probes for the in vivo imaging of EGFR used routinely in the clinical setting. Such probes could be used to aid patient stratification in a setting where multiple biopsies are difficult to obtain.
A number of positron emission tomography (PET) probes targeting EGFR have been developed and preclinically evaluated in the last decade. Both gefitinib and erlotinib have been radiolabelled with carbon-11 and/or fluorine-18. [ 18 F]Gefitinib gave disappointing results as no differences were detected in vitro or in vivo in models that expressed different levels of EGFR or mutant forms of EGFR [14]. On the other hand [ 11 C]erlotinib showed more promising results in vivo, with higher uptake in active mutant EGFR compared to wild type EGFR expressing xenografts [15]. Furthermore, a clinical trial evaluating the use of [ 11 C]erlotinib for the in vivo assessment of EGFR mutational status in NSCLC patients have been published [16]. This small trial of five patients with del E746-A750 and five patients with WT EGFR showed that the volume of distribution of the radiotracer was on average two times greater in the del E746-A750 EGFR than in the wild-type EGFR expressing tumours [16].
Other EGFR targeting radiotracers have been developed based on irreversible TKIs, which covalently bind cysteine-773 of the EGFR tyrosine kinase-binding site. Irreversible tracers were believed to be more appropriate for in vivo imaging as they would compete less with the high intracellular ATP concentration for domain occupancy [17]. One of the first compounds of this series was [ 18 F]ML04, which although retained selectivity for EGFR in vitro was poorly specific in vivo, with high uptake in EGFR negative tumour models and no decrease in tumour radioactivity upon pre-treatment with a blocking dose of cold ML04 [17]. Our group developed fluorine-18 labelled cyanoquinoline irreversible probes including FED6 ([ 18 [18]. FED6 showed high uptake in EGFR overexpressing tumours compared to low EGFR expressing tumours and was metabolically stable. There were however high levels of uptake in the intestines. This was partially attributed to less favourable pharmacokinetics and/or non-specific interactions. In the context of PET imaging of mutant forms of EGFR, one of the first examples of irreversible TKI reported in the literature is [ 18 F]PEG6-IPQA; [ 18 F]PEG6-IPQA was shown to bind with higher selectivity and specificity to L858R activating mutant EGFR expressing tumour xenografts compared to resistant EGFR L858R/T790M mutant xenografts [19]. More recently, Slobbe and co-workers have demonstrated potential usefulness of [ 18 F]afatinib as an irreversible TKI for imaging treatment-sensitive xenografts harboring exon-19 deletion mutations in EGFR [20]. One of the threats to use of quinoline or quinazoline-based molecules as imaging agents is susceptibility to efflux by members of the ATP binding cassette transporter proteins (ABC transporters) overexpressed at the cell surface of most tumours [21]. The key ABC transporter family members of relevance to the multi-drug resistance (MDR) phenotype in oncology include permeability glycoprotein (P-gp also known as ABCB1), breast cancer resistance protein (BCRP or ABCG2) and multidrug resistance protein 1 (MRP1or ABCC1) [21,22]. In relation to probe design, compounds that are substrates for ABCB1 despite having different structures share certain physicochemical properties such as a molecular weight (MW) > 400 Da, logP > 3 and polar surface area (PSA) > 85 Å 2 [23]. Both gefitinib and erlotinib have been found to be substrates of ABCB1 and ABCG2 [24,25]; furthermore, gefitinib has also been shown to be an inhibitor of ABCG2 [22]. Second generation irreversible inhibitors including EKB-569 (pelitinib; Fig 1) from which the cyanoquinoline series of compound was based, also interact with ABCG2 [24]. Regarding EGFR targeted radiotracers, [ 18 F]ML04 was found to be a substrate for ABCB1 [17]; more recently [ 18 F]afatinib was also shown to be responsive to ABCB1 modulation [20]. The threat posed by ABC substrate specificity for imaging probes results from the low mass of injected compound resulting in efficient efflux and consequently low tumour localisation. Against this background, we investigated the interaction of a series of cyanoquinolines EGFR antagonists previously evaluated in our laboratory for their substrate specificity for key ABC transporters, ABCB1 and ABCG2. Based on the findings, we tested a new probe and found it to be devoid of ABC transporter substrate specificity.

Inhibitors and Cell Lines
Unless otherwise stated, all compounds were purchased from Sigma (St Louis, Missouri, USA). For brevity, cell lines used to investigate ABC transporter specificity, together with their respective growth media are summarised in Table 1. 3T3 and 3T3 transfected with cDNA expressing P-gp (pHamdr1) were kindly provided by Dr E Schuetz from St Jude's Children Research Hospital (Memphis, TN, USA) [26], and MCF7 and mitoxantrone (MX)-resistant subclones MCF7MX by Dr E Schneider from the University of Maryland (Baltimore, MD, USA) [27]. Generation of the isogenic PC9ER from PC9 lung cancer cells has been reported [28]. Media were supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) (all purchased from Invitrogen, Carlsbad, California, USA). All cells were grown in a humidified atmosphere at 37°C and 5% CO 2 .

Inhibition of EGFR Autophosphorylation
Affinity of the cyanoquinolines for EGFR was established by measuring their inhibition of EGFR autophosphorylation via western blot. Cells were seeded in 6 well plates and following 24 h of growth, were subject to overnight serum starvation. The cells were then treated with increasing concentrations of cyanoquinolines prepared in serum free growth media from a DMSO stock (final DMSO concentration < 1%) for 3 h at 37°C, and stimulated for receptor autophosphorylation by addition of 100 ng/mL of EGF during the last 15 minutes of drug incubation. After 3 h the cells were placed on ice, media removed and then the cells were washed two times with ice cold phosphate buffered saline (PBS). Lysates were analysed by western blot to measure changes to p-EGFR Y1068.

Cell Viability Assay
Cell viability by the sulforhodamine B (SRB) assay was used to assess cell growth and cytotoxicity of test compounds [29]. Cells were seeded at densities of 3,500 to 5,000 cells per well in 96 well plates overnight and pre-treated for 1 h with ABC transporter modulators: 10 μM fumitremorgin C (FTC; in MCF7MX and MCF7 cells), or 0.3 μM of zosuquidar (in 3T3 and 3T3-MDR1). Cells were then treated with test compounds for 72 h, at 37°C. After drug treatment, cells were fixed by addition of 50% trichloroacetic acid (TCA), and the plates washed and dried to allow staining with 0.4% solution of sulforhodamine B (Sigma) in 1% acetic acid for 30 minutes. Excess dye was removed upon washing with 1% acetic acid. Plates were dried and bound protein was solubilised upon addition of 150 μL of 10 mM Tris base buffer. Absorbance was measured at 540 nm using Thermo Multiskan-Ex plate reader. Cell viability versus compound concentration curves were plotted as a percentage of control cells. The concentration of drug that gave 50% inhibition in cell growth (GI50) was extrapolated from these curves using the sigmoid curve fit with variable slope in Prism (GraphPad Prism version 5.01, GraphPad Software, San Diego California USA). Three to six replicate wells were used for each drug concentration and experiments were carried out on three separate occasions for each compound.

Caco2 Transwell Assay
The caco2 transwell assay was performed to assess permeability. Cells were seeded at a density of 60,000 cells per well in 24 well transwell plates (Millipore, Billerica, Massachutsetts, USA), and maintained in DMEM supplemented with 4.5 g/mL glucose (20% FBS, 1% penicillin/streptomycin, 1% glutamine) for 21 days at 37°C in 5% CO 2 . After 21 days the cells differentiate into enterocytes and express all three MDR proteins, ABCB1 and ABCG2 at the apical side and ABCC1 on the basal side. On day 21, cells were washed three times with Hank's Balanced Salt Solution (HBSS) (Fisher scientific, Loughborough, UK) and the trans-epithelial electric resistance (TERR) was measured using an Epithelial Voltohmmeter (EVOM) (World Precision Instruments, Sarasota, USA); only transwells with TERR of 400 Ω/cm 2 or above were used for the experiment. Cells were treated either at the apical (n = 3) or basal (n = 3) side of the transwell by addition of 10 μM of cyanoquinoline compound or 50 μM vinblastine prepared in a volume of 400 μL HBSS (compound added to apical well) or 800 μL of HBSS (added to basal well). When transport inhibitors FTC (10 μM) or zosuquidar (0.3 μM) were used, they were added to both the apical and basal wells 1 h prior to drug treatment. After 1 h pre-treatment the medium was removed and cells were treated with the compounds prepared in HBSS containing FTC or Zosuquidar. Plates were incubated in shaker incubator at 37°C and 60 rpm for 120 min. After incubation, 200 μL aliquots from the apical and the basal wells were collected and analysed using high performance liquid chromatography (HPLC). Samples were run on a Millipore Waters HPLC system which included a 717 plus autosampler and 2487 dual wavelength absorbance detector system. The stationary phase comprised of a Phenomenex Luna C 18 reverse phase column (150 X 4.6 mm; 5 μm particle size). The mobile phase comprised water (0.1% TFA) and acetonitrile (0.1% TFA) (80:20 v/v) at a flow rate of 1 mL/min for all compounds except vinblastine. For vinblastine, the stationary phase comprised a Supelcosil LC-ABZ reverse phase column (5 × 4.6 mm; 5 μm particle size). The mobile phase comprised water (0.1% formic acid) and methanol (90:10 to 10:90 v/v gradient over 20 min). Peak areas corresponding to the compounds of interest were recorded. Using standard curves the areas under the curve were converted into concentrations of compound. The permeability coefficient (Papp) was calculated using the following equation [30]: Papp = (dQ/dt)/A C0, where A is the surface area of the transwell membrane in cm 2 (= 0.33 cm 2 ), C0 is the drug concentration in the donor chamber at time t = 0 and dQ/dt is the rate of transfer of the compound to the receiver chamber, determined from the slope of the graph concentration (dQ) versus time (dt). Papp values corresponding to absorption and secretion were assessed and used to calculate efflux ratio.
Where Papp A-B = Papp absorption and Papp B-A = Papp secretion. The integrity of the caco2 cell membrane was assessed for each well following the experiment by addition of a 100 μM solution of luciferase yellow (Sigma) in HBSS to the apical side of the plate. The plates were placed in the shaker incubator for 1 h at 37°C and 60 rpm. Samples from the basal wells were transferred into a 96 well plate and the levels of luciferase yellow were assessed by measuring with an excitation of 450 nm and an emission of 540 nm using the Per-kinElmer Wallac 1420 counter. The membrane was considered intact if measured values from the basal side were inferior to 1% that of the measured values on the apical side.

siRNA Transfection
SiRNA transfections were carried out using ON TARGET plus-SMARTpool siRNA or nontargeting siRNA (Dharmacon, Lafayette, Louisianna, USA); ABCG2, L-009924-00-0005 and scramble (non-targeting) D-001810-10-05. Lipofectamine RNAiMAX (Invitrogen UK) was used as the transfection reagent and was prepared as indicated on the product protocol. Cells were seeded in black 96 well plates in antibiotic free media. On the day of transfection, siRNA and Lipofectamine were separately diluted in opti-MEM media, mixed in a 1 to 1 ratio and incubated at room temperature for 20 min; the siRNA-Lipofectamine complexes were then added to the cells. Fresh media containing 1% penicillin streptomycin was added to the cells after 24 h followed by Hoechst fluorescent assays at 72 h.

Cell Uptake Assay
[ 18 F]FED6 and [ 18 F]FED20 were synthesised as previously reported [18,31]. To increase specific radioactivity of [ 18 F]FED20, we modified the synthesis by decreasing the amount of alkyne precursor from 1 mg down to 0.5 mg. While this had the desired effect of increasing the specific activity from 7.3 GBq/μmol up to 52 GBq/μmol, the conversion of the alkyne to the triazole product dropped from > 95% down to 45%. Cells were seeded in 6 well plates and [ 18 F]FED6 (0.22 MBq) or [ 18 F]FED20 (0.55 MBq or 0.74 MBq) added to each well and incubated at 37°C for 1 h. Media were then removed and cells were washed twice with PBS. The cells were trypsinised and centrifuged for 3 minutes at 2000 rpm to obtain a pellet. The cell pellet was washed once with PBS and resuspended in 200 μL of RIPA buffer. Cell lysates were transferred to gamma counting tubes and the radioactivity in the pellet was measured on a Packard Cobra II gamma counter, (Perkin-Elmer, Waltham, Massachusetts, USA). Protein concentrations of samples were evaluated using Pierce 1 BCA Protein Assay Kit (Thermo Scientific, Waltham, Massachusetts, USA). Data were expressed as background-and decay-corrected counts per minute (CCPMA) per μg of cellular protein.

Statistics
Unless otherwise specified, data were expressed as mean ± standard error (SE). Data were compared by unpaired two tailed Student's t-test using GraphPad Prism. Statistical significance was assigned to values 0.05. Ã corresponded to p value < 0.05 and ÃÃ corresponded to p < 0.01.
We investigated the substrate specificity of the cyanoquinoline series to the two ABC transporters previously reported to interact with TKI compounds: ABCB1 and ABCG2 by a number of approaches. First, we used two paired cell models -3T3 cells (transfected with an empty vector) and 3T3-MDR1 cells (transfected with the human MDR1 gene which encodes ABCB1) or MCF7 cells and a mitoxantrone resistant, MCF7MX cell line, which overexpresses ABCG2and investigated the impact of FED6 treatment on cell viability. The GI50 (causing 50% growth inhibition) values obtained from these cell viability assays are summarised in Table 2. EKB-569, FED2 and FED6 were associated with GI50s of 5.75, 36, and > 100 μM, respectively, in the 3T3-MDR1 cells; corresponding values in the 3T3 cells were 1.06, 7 and 10.7 μM. Compounds associated with ABC transporter proficient to isogenic ABC transporter deficient cell line GI50 ratio of > 2 are considered to be actively effluxed by the transporters. There was a 5.4, 5.1 and > 10 fold increase in GI50 values for EKB-569, FED2 and FED6, respectively, in the ABCB1 overexpressing cell line (Fig 2A) suggesting that all three compounds were effluxed via ABCB1. Pre-treatment with specific ABCB1 inhibitor, zosuquidar [32], led to a decrease in the GI50 of EKB-569, FED2 and FED6 in 3T3-MDR1 but not 3T3 cells (Table 2 ; Fig 2A), confirming that the lower cytotoxicity of these compounds in 3T3-MDR1 cells was a result of efflux of the compounds via ABCB1. Similarly, there was a 1.7, 3.2 and 6.3 fold increase in GI50 values, for EKB-569, FED2 and FED6, respectively, in the ABCG2 overexpressing cell line (Fig 2B), suggesting that both FED2 and FED6 were effluxed via ABCG2. Pre-treatment with specific ABCG2 inhibitor, FTC, led to a decrease in the GI50 of EKB-569, FED2 and FED6 in  Fig 2B) cells confirming that the differential cytotoxicity of these compounds in the two cell lines were related to efflux via ABCG2.
Finally we examined the efflux of cyanoquinoline compounds in the universally exploited caco2 transwell assay. EKB-569, FED2, and FED6 showed efflux ratios >3 (Fig 4A), the accepted threshold for active efflux, indicating active efflux via the ABC transporters [33]. Interestingly, FED14 had an efflux ratio < 1 in keeping with its physicochemical properties (Fig 1). The efflux ratio of FED6 decreased from 6.9 to 1.1 in the presence of ABC transporter inhibitor verapamil further confirming the active efflux of this molecule via the ABC transporters (Fig 4B).

Higher Affinity of FED6 for Active Mutant EGFR Did Not Translate into Higher [ 18 F]FED6 Uptake in Active Mutant EGFR Expressing Cells
The cyanoquinoline compound FED6 had previously been investigated in cells expressing wildtype EGFR only. We investigated FED6 in the context of mutant EGFR. Inhibition of EGFR phosphorylation (p-EGFR Y1068) by FED6 was measured. The IC50 value for FED6 The highest cell uptake of [ 18 F]FED6 was seen in H3255 cells and lowest in H1650 cells ( Fig  5B). Wildtype expressing H358 cells also showed high uptake of [ 18 F]FED6 (86 CCPMA/μg of protein). The high affinity of FED6 in H1650 cells (low IC50 value) was discordant with the low [ 18 F]FED6 uptake in these cells; similarly, the low affinity of FED6 for wildtype EGFR (high IC50 value) in H358 cells was discordant with high uptake of [ 18 F]FED6 in these cells.
EGFR, ABCB1 and ABCG2 protein expression in these cells (Fig 5C) indicated that a lack of correlation between the affinity of FED6 for the different mutant forms of EGFR and the degree of cell uptake of [ 18 F]FED6 could be a result of differential expression of EGFR and ABCG2. Indeed, H3255 cells express significantly higher levels of EGFR than the other cell lines which could explain the highest uptake of [ 18 F]FED6 in these cells. The lack of ABCG2 in H3255 and H358 could also explain why the uptake of [ 18 F]FED6 was highest in these two cell lines regardless of the EGFR mutational status. As ABCB1 protein expression was not detected in any of the cell lines studied we focused on the possibility of overcoming the efflux of [ 18 F]FED6 via ABCG2.

Can ABCG2 Knockdown or Inhibition Overcome Reduced Cell Uptake of Radiolabelled [ 18 F]FED6?
One way of simplifying EGFR-specific tracer uptake interpretation is to remove the contribution of ABCG2 activity. Thus, we examined the specific role of ABCG2 in the efflux of [ 18 F] FED6 in MCF7MX cells (+++ ABCG2; + EGFR and p-EGFR expression) following ABCG2 siRNA transfection or pre-treatment with ABCG2 inhibitors, FTC and gefitinib. The effect of FTC (positive control) and gefitinib were examined in isogenic ABCG2 negative MCF7 cells. [ 18 F]FED6 uptake increased from 0.6 CCPMA/μg protein in scramble siRNA transfected MCF7MX cells to 3.5, 6 and 6.3 following pre-treatment with ABCG2 siRNA, FTC (positive control) and gefitinib, respectively (Fig 6A). The lower [ 18 F]FED6 uptake following ABCG2 siRNA pre-treatment compared to FTC or gefitinib could be due to incomplete knockdown of ABCG2. The cell uptake of [ 18 F]FED6 was unchanged following pre-treatment with FTC or gefitinib in ABCG2 negative MCF7 cells (Fig 6B). To further scrutinize the specificity of this approach, we repeated the study in an independent cell line, A431 (+ ABCG2; +++ EGFR and p-EGFR expression). The high unmodulated [ 18 F]FED6 uptake at baseline could be explained in part by high EGFR expression (Fig 6C). ABCG2 knockdown was near complete and was associated with increased [ 18 F]FED6 uptake. FTC and gefitinib again increased [ 18 F]FED6 uptake in this cell line.

Evaluation of a Hydrophilic Cyanoquinoline, FED20
While it is feasible to enhance radiotracer uptake via inhibition of ABCG2, an ultimate goal for this class of radiotracers is to design out substrate specificity for ABC transporters. As well as being a substrate for ABCB1 and ABCG2, FED6 is also associated with high non-specific binding. The high lipophilicity (LogP) of FED6 (3.85) (Fig 1) is thought to contribute at least in part to this property. We recently reported the synthesis of a glycosylated cyanoquinoline derivative, FED20, designed to reduce the lipophilicity [31]. The LogP of FED20 was significantly lower than that of FED6 at 2.51, while retaining affinity for EGFR; IC50 for inhibition of cell-free EGFR phosphorylation was 4.5 nM compared to 1.8 nM for FED6 [18,31]. While features such as MW, PSA, and n(N+O) were still suggested of active efflux, we predicted that FED20 would have improved uptake selectivity profile due to the lower LogP [34].
A number of observations indicated that FED20 was not a substrate for ABCB1 or ABCG2 transporters: a) In the MCF7/MCFMX and 3T3/3T3-MDR isogenic cell lines, no biologically significant differences in uptake of radiolabelled FED20 was seen (Fig 7A and 7B). Furthermore, pre-treatment with specific transporter inhibitors, FTC in the MCF7/MCF7MX cells and zosuquidar in the 3T3/3T3-MDR1 cells, did not lead to an increase in the uptake of [ 18 F] FED20 in the ABCG2 or ABCB1 expressing cell lines. b) FED20 was not actively effluxed by caco-2 monolayers, with efflux ratio close to unity (1.3) (Fig 7C). In addition, pre-treatment with zosuquidar did not have any effect on the permeability ratio. In contrast, the permeability ratios of vinblastine (positive control), a substrate of ABC transporters, and FED6 (Fig 4) were greater than 3, the accepted threshold for active efflux; pre-treatment with zosuquidar reduced the ratio for vinblastine from 6.7 to 1.7 (Fig 4). Expectedly, [ 18 F]FED20 uptake was higher in A431 cells compared to EGFR low MCF7 (Fig 7D and 7E). Pre-treatment with unlabelled FED20 decreased [ 18 F]FED20 uptake in the former but not in the latter cell line. Notably, the lower overall uptake selectivity of [ 18 F]FED20 in cells could be explained by the hydrophilic nature of the radiotracer.
Finally, we examined the utility of [ 18 F]FED20 for discriminating isogenic mutant EGFR cell lines that expressed del E746-A750 EGFR (PC9) or del E746-A750/T790M (PC9ER) for which [ 18 F]FED6 was not discriminatory. Both cell lines expressed similar levels of EGFR, however, the IC50 for inhibition of EGFR phosphorylation was 2-fold higher in PC9ER (0.7μM) compared to PC9 cells (0.3μM) (Fig 8A-8C). The same trend of higher potency in PC9 versus PC9ER was seen with gefitinib and FED6 (positive controls) although the magnitude of reduction was higher for gefitinib and FED6. In keeping with this observation, the uptake of [ 18 F]FED20 was higher in the PC9 compared to PC9ER cells (Fig 8D). The overall lower potency for inhibition of p-EGFR inhibition is also likely due to the hydrophilic character of the compound. [ 18 F]FED20 uptake was also low in cells harboring EGFR wildtype or T790M mutation. The H1650 cell line was unique in that while it harbors del E746-A750 active mutant EGFR, it showed low [ 18 F]FED20 uptake. The absolute uptake of [ 18 F]FED20 was improved at higher specific radioactivity (Fig 8E). Discussion EGFR remains an important transmembrane receptor target in cancer and molecular imaging of EGFR status-expression or mutation-using PET could aid patient selection for treatment with novel reversible or irreversible inhibitors. A key threat to developing successful imaging agents is existence and evolution of multidrug resistance phenotype. Starting from the ABC transporter substrate [ 18 F]FED6, we describe a novel cyanoquinoline radiotracer [ 18 F]FED20 that is devoid of ABC transporter activity.
One of the key components of MDR is the increase in expression of ABC transporters [35,36]. In the context of PET imaging, ABC transporter substrate specificity is also an important mechanism of radioprobe clearance. Indeed, as only tracer amounts of the radiolabelled probe are injected into the patients, efflux via such transporters will significantly decrease the potential accumulation of the radiotracer in the tumour. Several TKIs, including EKB-569 from which the FED series of compounds are derived have been shown to be substrates for members of the ABC transporter family [24]. The initial in silico characterisation of the FED series of compounds, indicated that these compounds had several characteristics which make them potential substrates for both ABCB1 and ABCG2 transporters: MW above 400 Da, LogP > 3 (ABCB1 substrate) and PSA>85Å 2 (ABCG2 substrate). [37,38]. We therefore predicted that FED6, FED2, and FED9 would be substrates for ABCB1 and ABCG2, whereas FED14 that had a lower MW would not. The cell viability assays performed in the paired cells lines that are proficient or deficient for specific ABC transporters, allowed us to indirectly verify this prediction. The higher GI50 values of FED6 in both the ABCB1 expressing 3T3-MDR1 cells and the ABCG2 expressing MCF7MX cells compared to the paired matched cells that expressed no transporters, as well as decreases in GI50 when the transporters were inhibited with zosuquidar or FTC indicated substrate specificity of FED6 for both transporters. By measuring efflux ratios, corresponding to the ratio between the rate of secretion over the rate of absorption, we determined that FED6, together with EKB-569, and FED2 were indeed effluxed by the physiologically relevant differentiated caco2 cell model; efflux ratios above 1 were indicative of active ABC transporter efflux [33]. The efflux of FED6 was reduced in the presence of ABC transporter inhibitor verapamil further confirming the active efflux of FED6. The indirect measurement of cytotoxicity in paired cells, compound efflux in caco2 and modulation by pharmacological inhibitors translated into predicted differences in [ 18 F]FED6 uptake in the ABC transporter proficient versus deficient cells.
EGFR is overexpressed in several cancers including, breast, ovarian, brain and colorectal [39], and this has led to development of specific TKIs directed against EGFR. However, efficacy of gefitinib and erlotinib against wildtype EGFR in patients was disappointing, with only a small fraction of unselected patients responding to TKI treatment [40]. The subgroup of patients, shown to express active mutant EGFR, had significantly higher response rates to TKI treatment [10]. Identifying patients that express mutant EGFR, non-invasively via PET imaging could assist in therapeutic selection. To date no PET tracers have been approved for the imaging of either wildtype or mutant EGFR in the clinic. The discovery of [ 18 F]PEG6-IPQA, an L858R mutant EGFR specific [ 18 F] PET radiotracer that showed a 7.6 fold higher uptake in H3255 (L858REGFR) compared to H1975 (L858R/T790M EGFR) xenografts was an important breakthrough in the imaging of EGFR [19]. This radiotracer was only studied in L858R mutant EGFR and further investigation in other common active mutant EGFR, including del E746-A750 EGFR would be important. Furthermore, tumour uptake of the most recent iteration of irreversible EGFR imaging agents, [ 18 F] afatinib, was sensitive to modulation by the ABCB1-specific inhibitor, tariquidar.
We investigated [ 18 F]FED6 for the imaging of mutant EGFR. FED6 was more potent against active mutant EGFR expressing cells than wildtype or resistant mutant EGFR expressing cells with respect to inhibition of p-EGFR. The uptake of [ 18 F]FED6 was high in H3255 (L858R EGFR) and the PC9 (del E746-A750) cells, and lowest in gefitinib resistant H1650 (del 746-750) and H1975 cells (L858R/T790M) cells. The higher uptake of [ 18 F]FED6 in H3255 cells as opposed to the low uptake in H1975 cells correlated with the differential affinity of FED6 in these two cells lines, assessed by western blot. However, it is important to note that expression levels of EGFR as well as mutant status could impact on the uptake of [ 18 F]FED6. H3255 cells had the highest expression of EGFR compared to the other NSCLC cell lines investigated. We cannot claim that the higher uptake of [ 18 F]FED6 in H3255 cells is a consequence of the higher affinity of FED6 for L858R mutant EGFR since it could also be due to higher expression of EGFR or a combination of these. We addressed this conundrum by assessing uptake of [ 18 F]FED6 in isogenic PC9 and PC9ER cell lines that have the same levels of EGFR protein expression but differ in mutational status; no statistical difference in the uptake of [ 18 F]FED6 was seen. Furthermore, the low uptake of [ 18 F]FED6 in H1650 cells was at odds the potent reduction of p-EGFR by FED6 in these cells. Notably, ABCB1 was not expressed in any of the cell lines studied, whereas ABCG2 was expressed in all the cell lines with the exception of the H3255 and H358 cells. Therefore the higher uptake of [ 18 F]FED6 in H3255 and H358 compared to the low uptake in A549 and H1650 could also be related to expression of ABCG2.
As only ABCG2 was expressed in the cell lines used, we wondered whether it would be feasible to improve EGFR detection properties of [ 18 F]FED6 by blocking this transporter using siRNA or pharmacological inhibitors FTC and gefitinib [41][42][43][44]. Indeed, both siRNA and pharmacological inhibition of ABCG2 increased [ 18 F]FED6 uptake with magnitude depending on ABCG2 expression/knockdown and EGFR expression. The uptake of [ 18 F]FED6 following inhibitor pre-treatment was consistently higher than following ABCG2 siRNA transfection which could be due to more efficient inhibition by the compounds. Rather than having to block the effect of the ABC transporters to which [ 18 F]FED6 is a substrate, designing a radiotracer than lacks the ABC transporter substrate specificity is preferred.
We have recently reported a hydrophilic cyanoquinoline compound FED20 [31]. The addition of a glucose moiety to form FED20 reduced LogP (to 2.1) compared to that of FED6 (3.85). The higher uptake of [ 18 F]FED20 in the A431 versus MCF7 cells was indicative of the specificity of the tracer for EGFR. Furthermore, the uptake of [ 18 F]FED20 in isogenic ABCB1 and ABCG2 proficient or deficient cell lines, supported by the unremarkable efflux characteristics of unlabelled FED20 by differentiated caco2 cells indicated that the compound lacked substrate specificity for ABCB1 and ABCG2. We have not tested the impact of all known ABC transporters in this study. For example, we did not directly test the impact of ABCC1 (MRP1) expressed in NSCLC as a potential risk for interaction with FED6. Even though no isogenic cell line information was generated for MRP1, the caco-2 data are broadly suggestive of a lack of interaction. Furthermore, verapamil is a dual ABCB1-MRP1 inhibitor, thus, a lack of interaction could be inferred from the verapamil inhibitor data [45,46]. The magnitude of [ 18 F]FED20 uptake was > 5-fold lower than that seen for [ 18 F]FED6. Despite its lower lipophilicity, this result is counterintuitive given the lack of transporter activity. The lower potency of FED20 on p-EGFR compared to FED6 suggests that the lower lipophilicity of FED20 may somewhat compromise its cellular delivery and engagement of target. An additional potential reason for the low [ 18 F]FED20 uptake is lower specific activity (7.3 GBq/μM) of the reported radiosynthetic method [31] compared to [ 18 F]FED6 (48 GBq/μmol) with some self-blocking by cold compound being a possibility. In keeping with this hypothesis, absolute uptake of [ 18 F]FED20 increased at higher specific radioactivity (52 GBq/μmol) although the difference in uptake between PC9 and PC9ER was less (albeit significant) than seen under low specific activity conditions.
In view of the lack of ABC transporter substrate specificity, we investigated the potential of this tracer for the imaging of mutant versus wild type EGFR. We initially focused on two cell lines with nearly equivalent EGFR expression and for which [ 18 F]FED6 uptake was non-discriminatory; PC9 cells expressing active mutant EGFR had a 2-fold higher uptake of [ 18 F] FED20 compared to PC9ER cells. [ 18 F]FED20 uptake was also low in other cells harboring EGFR wildtype or T790M mutation except the H1650 cell line, which while harboring a del E746-A750 active mutant EGFR, showed low [ 18 F]FED20 uptake. Despite harboring active mutant EGFR, this cell line is known to be resistant to gefitinib. PTEN loss has been proposed as a key genomic resistance mechanism in the cell line [47], but it is unclear if this alteration affects cellular ATP levels or ATP affinity of the EGFR receptor in addition to the known activity of PTEN loss/AKT activation in cell survival.
So far we have demonstrated that [ 18 F]FED20 has useful properties for discriminating EGFR inhibitor resistance (genomic or functional) in NSCLC cell lines. Further work-in particular metabolism, biodistribution and imaging contrast-is required to advance [ 18 F]FED20 towards imaging in humans for upfront detection of mutational status, monitoring response to treatment, and assessing the development of drug resistance [16,48]. The current recommendations are that, NSCLC patients undergo screening for mutations to EGFR before the start of therapy [49]. This allows the selection of patients with active mutant to be given TKI gefitinib or erlotinib. Indeed gefitinib was approved in the UK by NICE in 2009 for first line treatment for advanced metastatic NSCLC with active mutant EGFR [50]. PET imaging of active mutant EGFR could complement this screening as biopsies are not always possible and different nodules may present different mutational expression that would not be picked up if one site was biopsied [51]. PET may also be useful in detecting secondary resistance.

Conclusion
We demonstrated the role of the substrate specificity of the cyanoquinoline compound FED6 in the low accumulation of the radiotracers in the tumour. Blocking ABCG2 with gefitinib led to an increase in [ 18 F]FED6 uptake. FED20, which lacked substrate specificity for ABCB1 and ABCG2 was found to have favourable properties for detection of EGFR resistance.


s2fieldsofstudy: Biology Medicine

text:
Fibroblast Growth Factors and their Emerging Cancer-Related Aspects

Fibroblast growth factors (FGF) are proteins that are involved in several biological and developmental processes. Cell proliferation, cell motility, cell survival, and angiogenesis are the fundamental mechanisms that are carried out by these growth factors. FGF ligands when to interact with their receptors-FGFRs, elicit certain signaling pathways that ultimately lead to precise regulated cellular responses. Alterations in FGF/FGFR signaling results in various types of abnormalities. The transition of normal signaling mode of FGFs/FGFRs to an unusual functional pattern causes neurological disorders, skeletal disorders, tumor progression and tumor neovascularization. Overexpression of FGFs and FGFRs has been correlated with advanced tumor forms, mainly prostate, mammary gland, bladder and renal cell carcinomas. These GFs and their receptors are among the fundamental mediators of tumor angiogenesis, others being VEGF, Ang-1, etc. Many pharmaceutical therapies have evolved in the past years to minimize the effects of FGF/FGFR and VEGF signaling, individually as well as simultaneously. Tyrosine kinase inhibitors are the most prominent compounds that have been investigated exclusively in phase I and II clinical trials. However, the applicability of developing therapeutic techniques needs a further illustration better to comprehend the complexity of the underlying mechanism and to provide patients with a better outcome. 22 members of the fibroblast growth factor family have been classified in humans (Table 1). Hese22 genes of the FGF family encode proteins that share about 25 to 55 percent sequence similarity within the core region (120-130 aa) [4]. Among the 22 members of the FGF family, 4 of them (Fibroblast growth factor -11, -12, -13, -14) although show high sequence homology with prototypic FGFs, however, are unable to activate the FGFRs and are therefore not employed as FGFs. Instead, they have been entitled as Fibroblast homologous factors-FHFs. Whereas FGF-19 (human) is orthologous to FGF-15 (mouse) [5]. The members of the fibroblast growth factor family are involved in diverse biological activities such as wound healing, MAPK cascade, angiogenesis, cell differentiation, cell growth and proliferation, axon guidance, neuron development, etc. FGFs are localized within the nucleus and cytoplasm (cytosol) whereas some of them function in the extracellular space. Other members, for instance, bFGF, get accumulated in the extracellular matrix and are activated by other factors [6-9]. The FGF ligands interact with their specific receptors, i.e. fibroblast growth factor receptors (FGFRs) leading to the activation of the intracellular signaling cascade [10]. The FGFRs are tyrosine kinase receptors, encoded by four genes i.e. fgfr-1, fgfr-2, fgfr-3 and fgfr-4 [11]. These receptors, upon interaction with FGFs, form complexes with their ligands and Heparin sulfate proteoglycans (HSPGs), undergo dimerization to initiate pathways that ultimately promotes the stimulation physiological and developmental processes. Genetic modifications within the FGF family or irregularities in the processes carried out by FGFs provoke several disorders of nervous system, Citation: Khalid A, Javaid MA (2016) Fibroblast Growth Factors and their Emerging Cancer-Related Aspects. J Cancer Sci Ther 8: 190-205. doi:10.4172/1948-5956.1000413

Introduction
For years, it was known that the brain and the pituitary glands enclosed some mitogenic agents [1]. In 1973, an entity dissimilar from pituitary hormones was isolated from the bovine pituitary 3T3 fibroblasts. The identified substance was fibroblast growth factor that induced the growth of 3T3 cells [2]. Two different forms of proteins were involved in the growth of 3T3 cells. The first one was aFGF (acidic Fibroblast Growth Factor) and the second one was bFGF (basic fibroblast growth factor). aFGF had a 5.6 isoelectric point (pI) (acidic); bFGF had >9 pI (basic). bFGF and aFGF share 55% homology within their sequences. Fibroblast growth factor-1 and Fibroblast growth factor-2 were previously known as aFGF and bFGF, respectively [3]. With the advancing technology and usage of emerging capacities, up to Abstract Fibroblast growth factors (FGF) are proteins that are involved in several biological and developmental processes. Cell proliferation, cell motility, cell survival, and angiogenesis are the fundamental mechanisms that are carried out by these growth factors. FGF ligands when to interact with their receptors-FGFRs, elicit certain signaling pathways that ultimately lead to precise regulated cellular responses. Alterations in FGF/FGFR signaling results in various types of abnormalities. The transition of normal signaling mode of FGFs/FGFRs to an unusual functional pattern causes neurological disorders, skeletal disorders, tumor progression and tumor neovascularization. Overexpression of FGFs and FGFRs has been correlated with advanced tumor forms, mainly prostate, mammary gland, bladder and renal cell carcinomas. These GFs and their receptors are among the fundamental mediators of tumor angiogenesis, others being VEGF, Ang-1, etc. Many pharmaceutical therapies have evolved in the past years to minimize the effects of FGF/FGFR and VEGF signaling, individually as well as simultaneously. Tyrosine kinase inhibitors are the most prominent compounds that have been investigated exclusively in phase I and II clinical trials. However, the applicability of developing therapeutic techniques needs a further illustration better to comprehend the complexity of the underlying mechanism and to provide patients with a better outcome.
22 members of the fibroblast growth factor family have been classified in humans (Table 1). Hese22 genes of the FGF family encode proteins that share about 25 to 55 percent sequence similarity within the core region (120-130 aa) [4]. Among the 22 members of the FGF family, 4 of them (Fibroblast growth factor -11, -12, -13, -14) although show high sequence homology with prototypic FGFs, however, are unable to activate the FGFRs and are therefore not employed as FGFs. Instead, they have been entitled as Fibroblast homologous factors-FHFs. Whereas FGF-19 (human) is orthologous to FGF-15 (mouse) [5].
The members of the fibroblast growth factor family are involved in diverse biological activities such as wound healing, MAPK cascade, angiogenesis, cell differentiation, cell growth and proliferation, axon guidance, neuron development, etc. FGFs are localized within the nucleus and cytoplasm (cytosol) whereas some of them function in the extracellular space. Other members, for instance, bFGF, get accumulated in the extracellular matrix and are activated by other factors [6][7][8][9]. The FGF ligands interact with their specific receptors, i.e. fibroblast growth factor receptors (FGFRs) leading to the activation of the intracellular signaling cascade [10]. The FGFRs are tyrosine kinase receptors, encoded by four genes i.e. fgfr-1, fgfr-2, fgfr-3 and fgfr-4 [11]. These receptors, upon interaction with FGFs, form complexes with their ligands and Heparin sulfate proteoglycans (HSPGs), undergo dimerization to initiate pathways that ultimately promotes the stimulation physiological and developmental processes. Genetic modifications within the FGF family or irregularities in the processes carried out by FGFs provoke several disorders of nervous system, Basic fibroblast growth factor; bFGF, Heparin-binding growth factor 2; HBGF-2. Phosphatonin, Tumor-derived hypophosphatemia-inducing factor Table 1: Members of Fibroblast Growth Factor Family (Homo sapiens). The information mentioned above is to the widely expressed fibroblast growth factors, their genes, chromosomal locations along with their sequence lengths, cellular localization. FGFs are concerned with several biological activities and diseases [18].
factors. Distinct amino acid residues that reside within the receptor binding site of FHFs are responsible for their inability to activate FGFRs. Assembly of FHFs excludes many amino acid residues that are critical for the binding of FHFs to the receptors [25].
An FGF gene is typically composed of three exons, where exon 1 starts with a Met residue. However, there are some exceptions; for instance, FGF-2 and FGF-3 have particular sequences transcribed at their 5' end [26][27]. In some FGFs, sub-exons are formed when exon 1 passes through the splicing process. Genes of the FGF family are dispersed or clustered (in some cases) throughout the genome. FGF-3, -4 and -19 genes are present on the 11q13 chromosome, 12p13 chromosome contains FGF-6 and -23 genes whereas 8p21-p22 chromosome profiles FGF-17 and -20 genes chromosome.

Structure
The domain structure of FGFs is composed of a core region of about 120 aa -140 aa, containing 28 conserved residues as well as six identical amino acid residues [28]. Among the 28 conserved residues, 10 of them are involved in interactions with the fibroblast growth factor receptors [29]. The homologous core region in most of the FGF members constitutes a cylindrical barrel formed by the precise folding of 12 antiparallel beta strands. Variable amino-and carboxy-terminals that border the core region of FGFs [30]. Exceptions exist in the structure of FGF-1 and FGF-2. These two fibroblast growth factors own a triangular array (allocated within the beta-trefoil structure) formed renal system, defects in embryonic development, tumor proliferation and cancer metastasis (Table 1). FGFs have been related to several types of tumors and cancers in association with vascular endothelial growth factor (VEGF) where these growth factors operate to carry out angiogenesis [12][13][14]. Some of the FGFs are secreted into the extracellular environment while others function intracellularly. All fibroblast growth factors were known to function through paracrine signaling, but advancements in the recent years lead to the affirmation of FGF-19, 21 and -23 as endocrine signaling molecules [15][16][17][18].
This article reviews data about the fibroblast growth factors, their receptors, and signaling pathways, structure and their biological roles, participation in cancer as well therapeutic and pharmacological implications of FGFs. Several studies have been conducted in the past to elucidate the various features of the FGF family. Using X-ray crystallography, NMR and other peculiar techniques, characteristic like 3D structure, domain structure, sequences, cell surface receptor complexes, expression, pathology, etc. have been exquisitely outlined in the literature.

Fibroblast growth factor-FGF family
The fibroblast growth factor family consists of 22 genes that encode particular proteins, FGFs. The fibroblast growth factor family is composed of related molecules ranging from 17 kDa to 34 kDa.The first FGFs to be discovered were aFGF (FGF-1) and the bFGF (FGF-2) from the brain and pituitary extracts of the bovine in the 1970s. In the upcoming years, various other FGFs were identified. Genes of FGF-3, FGF-4, FGF-5 and FGF-6 were isolated as oncogenes [19][20][21]. FGF-7 is also known as Keratinocytes growth factor and FGF-8 as a growth factor from the carcinoma cells (androgen-induced). FGF-9 was described to be associated with the nervous system and was isolated from glioma cells [22]. Fibroblast growth factors 1 to 9 share a 30-70 percent sequence homology, though these FGFs are of different sizes ranging from 150 to 250 aa [23]. Almost all the members of the FGF family experience ca. 120 similar amino acids i.e. about 16% to 65% sequence similarity [24].
The fibroblast growth factor family embodies only 18 FGFR ligands i.e. FGF1-FGF10, FGF16-FGF23. FGF11-FGF14, although share high structural as well as sequence similarity with the other FGF family members but they lack the capacity to activate the FGF receptors and are, thus, more correctly designated as FHFs, i.e. fibroblast homologous by a peculiar arrangement of four beta strands [31]. In FGF-2, among the 12 beta strands, two of them i.e. β10 and β11 possess essential aa residues that are involved in the formation of the primary binding site for heparin. This site is different from the site that interacts and binds with FGFRs [32].
155 aa protein (17-18 kDa) forms FGF-1, which is a nonglycosylated polypeptide that does not possess a distinct signal peptide and is in fact, secreted in the shape of a dimer linked by a disulfide bond and by an ATP-dependent pathway that features synaptotagmin-1 rather than the classical pathway [33]. The domain structure of FGF-1 characterizes an NLS-nuclear localization sequence that depicts the involvement of FGF-1 in DNA synthesis. The 18kDa non-glycosylated protein, FGF-2, is secreted as a monomer and forms four isoforms (22 kDa, 22.5 kDa, 24 kDa, and 34 kDa) based on the presence of different start codons. This growth factor is thought to perform its activities, both intracellularly and extracellularly. Apart from similar domains that FGF-3 shares with other members of the FGF family, it has an NLS motif located at the C-terminal [34]. FGF-4 has two heparin binding sites within its 176 glycoprotein domain structure. FGF-5 is a precursor polypeptide that contains a signal peptide and a mature peptide, 17 aa and 251 aa, respectively. Its unique feature is that it has both types of glycosylation's, i.e. N-and O-linked. FGF-6 could be secreted in three precursor isoforms (208,198 and 175 amino acids) because it contains three different AUG (methionine) start codons. The keratinocyte growth factor, FGF-7, is synthesized as a glycosylated precursor protein that later matures to form the 163 amino acid activated form. The activated form of FGF-7 activity is thought to be hindered by heparin molecules [35]. Three regular exons encode the members of the FGF family. However, in the case of FGF-8, exon one has been split up into four small exons followed by the typical exon 2 and 3. FGF-9 do not own a signal peptide but is secreted adequately, unlike FGF-1 and FGF-2. Known as the keratinocyte growth factor-2, FGF-10 contains a serine-rich motif located at the amino terminal and retains an oddly long signal peptide [36].
FGF-11, also known as FHF-3 (FGF homologous factor-3), maintains an NLS whereas its domain structure has no signal peptide. FGF-12 (FHF-1) possess two NLS sequences (designated as bipartite NLS) near the amino terminal as well as a typical NLS. FGF-13 (FHF-2) features an amino-terminal alternate splicing. FGF-14 has a secondary signal motif along with a bipartite NLS. FHF-4 lacks the typical signal peptide. FGF-16 and FGF-20 do not have the typical signal sequence, like FGF-9, and are, therefore, secreted like that of FGF-9. Like FGF-8, FGF-17s exon one is also divided into mini exons, leading to several amino terminal isoforms and the other exons i.e. two and three are the same as others. FGF-18 is a secreted glycosylated polypeptide that interacts with heparin molecules whereas FGF-19, FGF-21 and FGF-23 lack the heparin binding site within their domain structures [37] ( Figure 3).

Fibroblast growth factor receptors
As a result of either of the two possible mechanisms, several isoforms of the FGF receptors are expressed in humans. A) Expression of various FGF receptor genes or B) expression of particular splice variants that result from the alternative splicing of the same FGF receptor gene. Four genes (fgfr1, fgfr2, fgfr3 and fgfr4) encode these receptors. Seven isoforms of FGF receptors exist due to alternative splicing that occurs within the mRNA of fgfr1, fgfr2, and fgfr3 [38,39]. Isoforms include FGFR1b, 1c, FGFR2b, 2c, FGFR3b, 3c, and FGFR4. In some of them, splicing results in the removal of the Ig-I domain and acid box whereas Ig-III alternative splicing results in the formation of type b and c isoforms. Alternative splicing in Ig-III (carboxyl-terminal half), retains any one of the exons that lie within the domain region. Isoform IIIb (Ig-IIIb) and isoform IIIB results when the splice variant retains exon 8 or exon 9, respectively ( Figure 4). Particular tissue types i.e. epithelial clans express isoform IIIb whereas mesenchymal clans express the isoform IIIc. The various isoforms of the FGF receptors display peculiar specificity and selectivity for different FGF ligands as well as the signal transduction pathway they activate, depending on the type of tissue and the targeted biological activity. The FGF members show increased affinity for particular isoforms. FGF-3, -7, -10, and -22 interacts with isoforms IIIb. Acidic FGF binds with both the isoforms, whereas the other 13 members of the FGF family bind to isoform IIIc [40]. The gene of FGFR4 is exclusive in a sense since no isoforms for this gene exist.

Structure and signaling
The general arrangement of an FGF receptor features an extracellular domain, a transmembrane domain, and a tyrosine kinase cytoplasmic domain. The specific portions of the extracellular domain are a signal peptide followed by three Immunoglobulin-like (Ig-like) domains, designated as Ig-I, Ig-II, and Ig-III. An acid box (serine-rich acidic sequence) is present between Ig-I and Ig-II domains, within the linker region ( Figure 4). Ig-I and the acid box might participate in autoinhibition of the receptor itself [41]. Ig-II and Ig-III domains are important for determining ligand specificity and its binding. A heparin molecule that serves as a co-factor assists the functioning of the receptor and increases the efficacy of the receptor-ligand complex. Ig-II domain (166-177 amino acids) contains the receptor interaction site. Alternative splicing within the Ig-III area determines the isoform of FGFR. A juxtamembrane region is present downward the transmembrane portion of the receptor which then leads to the tyrosine kinase-TK domain. A kinase insert of around 14 amino acids divides the TK region into two functional kinases i.e. kinase 1 and kinase 2 or TKI-I and TKI-II. The carboxy-terminal of FGFR1 contains a PLC γ interaction site that binds with PLC γ within the cytoplasmic region. The CAM homology domain-CHD is involved in the formation of an endosome and nuclear transport of the receptor, FGFR1 [42].
When the FGF ligand binds to the FGF receptor through its secondary receptor binding site, the activated receptor undergoes dimerization. As the ligand approaches its receptor, it interacts with the Ig-II and Ig-III domains of the receptor and forms a complex. The complex is, indeed, a ternary complex since the heparin molecules are also involved in the formation of the compound. A fully operating complex is composed of FGF, FGFR, and Heparin molecules in the ratio of 2:2:2 to form a symmetrically positioned dimer. After the binding of the FGF ligand and the co-factor (heparin molecule), the receptor is activated i.e. dimerized, and the tyrosine kinase domains are now trans-phosphorylated mainly within the A-loop region. Phosphorylation of the cytoplasmic domain region is then extended to the carboxy terminal region, juxtamembrane region and kinase insert region. The PLC gamma site is also phosphorylated, which in turns activates the effector molecules within the cytoplasmic region. The most important signaling cascades that are triggered by the activation of FGFR are the RAS/MAPK pathway, PLC γ pathway, and PI3K/ Akt pathway. Heparin molecules or heparin sulfate proteoglycans participate in the formation and arrangement of the FGFR dimer. It functions to stabilize the interactions within the protein structures. In this case, it works to secure the attachment of FGF with the FGF receptor in a 1:1 ratio.
Considering the vast range of activities that FGFs perform within the extracellular environment, many efforts have been made to investigate their structure as well as their particular role in various malignancies and diseases. Due to the rapid evolvement of many cancer types, FGFs have provoked concern as the frontline players of cancer development due to their well-studied roles in directing cell proliferation, angiogenesis, and cell migration. Hence, the modern day research is more focused on finding out the likely job of FGFs and FGFRs in the advancement of individual tumors. In the upcoming section, the role of FGFs and FGFRs in various biological activities, tumor development, and cancer metastases will be discussed. Also, the inhibitors of FGFs/FGFRs used as therapeutic drugs for clinical trials will be reviewed (Figures 4 and 5).

FGFs and FGFRs: Roles in biological processes
FGFs and FGFRs function to promote cell proliferation, angiogenesis, cell survival, and cell migration particularly at inflammatory, tumor and other hypoxic sites surrounding the primary tumor region as well as within the normal extracellular environment to carry out normal physiological activities. The signal transduction pathways activated by FGFs are well-characterized and follow specific orders from the guiding proteins within the cellular environment. Eventually, the outcome observed is standardized according to the needs of the surrounding cells and tissues. FGFs carry out cell proliferation in epithelial cells, adipocytes, and endothelial cells  [57]. FGF-2 promotes the chondrogenic ability of hMSCs [58], whereas FGF-7 and FGF-20 are responsible for the differentiation that occurs in keratinocytes and the conversion of stem cell to dopamine-producing neurons within monkeys, respectively. FGFs have been reported to be responsible for making angiogenesis liable within the standard as well as many cancerous settings. FGF1 and FGF2 are concerned with the occurrence of angiogenesis via endothelial cells [59]. FGF-1 is a known angiogenic factor that triggers angiogenesis within the chorioallantoic membrane-CAM of chickens whereas FGF-9 has been described to stimulate angiogenesis during bone repair [60][61][62].
The functioning of FGFs in the process of wound healing is an intriguing phenomenon and marks another biological activity that is carried out in the presence of these GFs [63]. The conduct of FGFs, particularly that of FGF-1 and -2, is well-defined in angiogenesis. Angiogenesis leads to the successful accomplishment of the wound healing process. Wound healing requires four phases (inflammation, contraction, repair and regeneration) to reach an outcome [64,65]. Evidently, FGFs have been proven to take part in three out of four phases of wound repairing [66]. These GFs are found in the ECM as well as in the fibroblasts and endothelial cells [67]. Usually, FGFs are thought to be expressed at a particular site as it encounters a wound [68]. Inflammation, the first phase of wound healing, features various members of the FGF family i.e. FGF-1, FGF-4, and FGF-2 [69][70][71]. These FGFs attract other immune cells to the site of inflammation, control the proliferation of platelets since they have significant activity at inflammation sites [72]. FGFs regulate the division of megakaryocyte progenitor cells at the point of inflammation. Endothelial cells and fibroblasts are present in the second phase of wound healing. These cells express certain FGFs that display their activity in the cellular proliferation of these cells [73]. FGF-2 stimulates the migration of endothelial cells by the activation of uPA (urokinase-type plasminogen activator) [74]. uPA when interacts with its receptor on the cell surface, in turn, converts plasminogen to plasmin that cleaves fibrin, causing fibrinolysis and promoting the movement of endothelial cells [75]. ΑVβ3 cell-surface adhesion molecule characterizes another mechanism adopted by FGF-2 to facilitate the movement of endothelial cells. FGF-7 plays a role in the final phase, regeneration, of wound healing [76,77]. FGFs induce the limb buds to evolve and grow gradually [78,79].

FGF and FGFRs: Roles in cancer
Various cancers express FGFs-FGFRs at an increased level. The signaling scheme of these growth factors reinforces the survival, growth, and dissemination of tumors. In cancer state, FGFs facilitates the tumors to propagate to secondary locations either by promoting angiogenesis or by guiding other angiogenic factors to bring about their activity. As a result of FGF-FGFR interactions, various cellular elements like transcription factors and secondary proteins are triggered. An orderly working of all these elements is crucial at every step of the cascade and loss of check at any point may lead to uncontrolled cell division and neoplasia [80,81]. Tumor cell proliferation marks overexpression of FGFs. These GFs may probably be expressed either by tumor cells or by the stromal cells found in the surrounding environment, in response to a signal received from tumor cells. They operate in a paracrine [82] or an autocrine manner [83,84], thereby targeting tumor or the stromal cells. Another possible undercover mechanism behind FGF participation in cancer is their release from the extracellular matrix -ECM [85]. ECM, upon its degradation by MMPs, releases FGFs that are stored or trapped within by MMPs. Otherwise, FGFs may associate with a carrier protein that delivers these FGFs to their respective receptors and in turn activate them [86].
FGFs when overexpressed in the tumor microenvironment, invasive and metastatic properties are gained by the cancer cells. However, the expression of FGF receptors is more discriminatory when it comes to the type of tumor [87]. FGFR1 signaling outlines prostate cancer progression. It's possible due to the overexpression of FGFR1 in basal cells, prostate epithelial cells and prostate progenitor cells that are embodied by the basal prostate gland-benign [88]. The ligands of FGFR1 i.e. FGF-1, 2, 8b and 17, also show increased expression in prostate cancer [89]. Studies concerned with the role of FGFR1 in the progression of prostate cancer have reported that hyperplasia or neoplasia within the prostate epithelium is caused by a continuously activated form of FGFR1 [90]. Another study revealed the possible cause of transition of non-malignant prostate cancer cells to the malignant phenotype. The shift of FGFR2 splice forms, IIIb to IIIc is concerned with the underlying mechanism behind the proliferation of prostate epithelial cells. As mentioned earlier, the expression of IIIb and IIIc isoforms are characterized by epithelial and mesenchymal cells, respectively. Furthermore, FGF-2, IIIc ligand, was also overexpressed indicating its role along with that of IIIc isoform in the epithelial cell division. Hence, the conversion of non-invasive epithelial cells to the malignant phenotype [91] is due to the switch that ensures the FGFR2 signaling through the IIIc isoform (expressed by mesenchymal cells and hence has the potency to invade surrounding tissues) rather than the IIIb isoform. FGFR2-IIIb isoform has also been described to possess repressive tumor properties. Therefore, the fgfr2-IIIb gene might be viewed as a tumor suppressor gene [92]. However, further research is required to enlighten the mechanism that underlies the functioning of FGFR2-IIIb isoform [93].
Fibroblast growth factors are related to mammary gland carcinogenesis. Studies have confirmed that mammary oncogene families sometimes operate in cooperation with each other for the successful tumorigenesis of the mammary gland. Wnt1 was the first oncogene to be identified that was activated by MMTV [94]. Shortly after its discovery, fgf3 oncogene was located at a separate locus. Subsequent selection of oncogenes for induction of mammary tumor development within the MMTV infected mice, correlated wnt1 and fgf3 genes [95].The link between FGFs and breast cancer aroused the probability to look for the particularities of the oncogenes mentioned above. During the RNA analysis of fgf3 and fgf4 oncogenes, their particular role in the mammary tumor DNA amplification was not establishment on an authentic ground. However, the CCND1 oncogene that lies close to the fgf3/4 genes on chromosome 11q was later found to be the active oncogene within the region. Indeed, fgf3/4 genes were being transcribed as mere "riders" of the amplicon containing the oncogene concerned with the disease [96,97]. Moreover, roles of FGF-1, -2, and -8 have also been described in assorted types of breast cancers [98][99][100]. FGF receptors, on the other hand, have been associated with breast carcinogenesis. For instance, FGFR1, FGFR4 have been detected to be overexpressed in some types of BCs, studies report [101,102]. FGFs serve as special players of certain carcinogenesis process due to their cell proliferating, anti-apoptotic and angiogenic properties. FGFs and FGFRs participate in skin [103] and urothelial carcinogenesis [104]. The MAPK pathway which is triggered by activation of FGFR1 has been related to the proliferation of tumor cells [105]. This path indicates the role of Ras that is can be activated by two separate proteins, in the activation of MAPK. FRS2 [106] and Shc-src homology 2 containing protein [107]. Proliferating cells express both of these proteins. However, the mechanisms by which these proteins work is entirely different from one another, Point mutations, genetic alterations, alternative splicing that results in various splice variants and abnormal expression of FGFs and FGFRs have the potential to form cancer [108]. Point mutations have the least connectivity with the disease advancement in humans. However, a point mutation in three out of four FGFRs i.e. FGFR1, 2 and 3, has been linked to several skeletal disorders. Genetic alterations that modify the arrangement of the genes leads to the synthesis of a different form of protein that does not function properly. For instance, the FGFR2 receptor in osteosarcoma cell line of rats is continuously activated, even in the absence of a ligand [109]. Continuous activation of FGFR2 is due to a rearrangement that takes places within the carboxy-terminal region of the receptor. Other than this, overexpression of FGFR1 and FGFR2 has been reported in renal cell carcinoma [110]. FGFR2 is overexpressed in breast cancer and gastric cancer [111,112]. Human myeloid cells, bladder carcinoma as well as urinary tract carcinomas overexpress FGFR3 [113][114][115]. Alternative splicing, on the other end, creates such variants that do not hold back their ability to help survive the tumor environment, facilitating its cells through various signal transduction pathways by devising desired results. Splice variants of FGFR2 are linked to the type of breast cancer. FGFRs variants have also been associated with ovarian cancer, prostate cancer, etc. [116,117].
Low-grade urothelial bladder carcinomas are reported to have FGFR3 mutations [118,119]. Glioblastoma is a type of brain tumor in adults. A subgroup of this tumor type has been described to be caused due to a fusion protein, mainly involving the tyrosine kinase domain of FGFR1 or 3 that has been fused with the TACC1 or 3 encoding regions [120,121]. FGFR3-TACC3 fusion proteins have been associated with bladder cancer, cervical cancer [122,123]. Other examples include overexpression of FGFR2-TACC3 in cholangiocarcinomas (CCAa). FGF receptor 2, on the other hand, has been illustrated to undergo mutation in cervical, endometrial and squamous cell lung cancer [124][125][126]. Missense mutations of FGFR2 can also occur in gastric and ovarian cancer [127,128]. Polymorphism of FGFR2 can be related to breast carcinoma, and the isoform FGFR2-IIIc polymorphism has been outlined in breast cancer (BC) caused by a BRCA2 mutation. An aggressive form of breast cancer has been correlated with FGFR3 overexpression [129] and another form of breast cancer malignancy has been associated with FGFR1. Squamous cell lung cancer and NSCLC is related to FGFR1 amplification or other less defined mechanisms [130,131]. A study featuring Caucasian males demonstrated FGFR4 allele Arg-388 mutation with an aggressive form of prostate carcinoma [132].
Fibroblast growth factors act as mitogenic agents thus compelling the cells to undergo division. They may also act as anti-apoptotic agents thereby supporting cell survival pathways. FGFs carry out their roles in cancer by acting as any of the agents mentioned above. FGF-1 and FGF-2 are known potent mitogenic agents [133]. The other FGFs such as FGF-3, -4 and -5 have also been related to maintaining the proliferative properties of individual cancer cells lines [134,135]. Cell survival by anti-apoptotic mechanism is, indeed, an alluring process that assists the tumor cells to escape the body defense attacks and in turn, show resistance to different therapeutic agents. FGF-2 has been depicted to govern various anti-apoptotic proteins, such as Bcl-2 and XIAP, within the cellular environment [136,137]. On the other hand, FGF-2 has been reported actually to make the cancer cells undergo apoptosis, such as MCF-7 breast cancer cell lines by the down-regulation of Bcl-2 structure [138]. Lastly, FGFs have been resolutely linked with angiogenesis, discussed in the upcoming section.

FGFs and FGFRs: Roles in angiogenesis
Angiogenesis is the formation of new blood vessels into an organized vasculature system that operates to transport nutrients and supplements to the target cells and tissues that reside within a particular environment, either in the close surroundings or at other distant locations [139]. Many pathological anomalies, as well as developmental mechanisms, have been characterized by angiogenesis that is carried out by several pro-angiogenic factors [140]. Various stimulatory and inhibitory factors maintain an equilibrium which, when disturbed, can result in the generation of several disorganized vessels and therefore, owns the ability to agitate the non-malignant cancer setup and convert it into a malignant form of cancer [141]. With the passage of time, the number of tumor cells within a local tumor microenvironment keeps increasing. These tumor cells tend to escape such surrounding due to the hypoxic conditions created by a large number of cells [142]. Angiogenesis is, in fact, responsible for metastasizing various tumors since it facilitates the movement of tumor cells to other locations. Thus aiding in tumor progression and growth [143].
The generalized concept about angiogenesis involves the induction of endothelial cells by various growth factors like FGF [144] and VEGF [145], etc. Stimulation of ECs allows the endothelial cells (EC) to release various MMPs into the extracellular matrix [146]. Numerous studies have been directed to investigate the role of fibroblast growth factors and their receptors in angiogenesis. In fact, these GFs are among the angiogenic factors that were first specified and are capable of triggering a pro-angiogenic environment that is suitable for carrying the process of angiogenesis. FGFs when to interact with FGFRs on the cell surface of EC, they directly promote tumor angiogenesis. FGFR1 and FGFR2 (in some cases) are only expressed by endothelial cells [147,148]. In the group of 22 FGF members, the role of FGF-1 (aFGF) and FGF-2 (bFGF) has been extensively demonstrated as angiogenesis promoting growth factors.
Members of the FGF family are involved in the favorable implementation of angiogenesis by actively maintaining the endothelial cell division, migration, ECM degradation processes and interactions between the cell receptors and effector molecules. When FGF-1, -2 and -4 activate FGF receptor 1 and 2, discrete cellular pathways are triggered that facilitate the maintenance of the proliferative environment of endothelial cells [149,150]. FGF-8b is also involved in carrying out the same kind of activity [151]. When FGF-2 stimulate a proliferative response in ECs, PKC is upregulated [152]. When down-regulated, PKC, in turn, diminishes the effect of FGF-2 and as a result, the mitogenic activity is lost. FGFs increase the expression of MMPs and uPA from endothelial cells. In the case of uPA, its receptor expression is, indeed, harmonized on the cell surface by FGF-1, -2 and -4 to increase the efficacy of ECM-degrading mechanism FGF-1, -2 and -4 [153]. Gelatinase A (MMP-2) and gelatinase B (MMP-9) have the potency to carry out angiogenic activities [154]. Their release from the endothelial cells is promoted by FGFs [155,156]. Angiogenesis requires the migration of ECs to form the new vessels. FGF-1,-2, -10 and 8b induce migratory features in endothelial cells. FGF-2 binds to its receptor and activates MAPK pathway thereby supporting the migration of endothelial cells [157][158][159][160]. Certain integrins, cell adhesion molecules, and cadherins mediate endothelial cells interaction with surrounding extracellular components. FGF-2 governs the expression of these molecules on the cell surface of endothelial cells, assisting in the cell distribution, cell-cell communication, and maturation [161,162]. Finally, endothelial cell organization and maturation to form the vessel lumen is simplified by the effect of FGF-2 and FGF-8b [163]. This action of FGFs requires backing from several other elements like the expression of TIMPs, MMP-2, MMP-9, uPA, several integrin receptors, etc. Under the effect of FGF-2, signals from VEFG receptor 1 are also significant in the development of mature endothelial cells [164].
FGF-BP (FGF binding protein) is present in various tissues where FGF-2 or other members of FGF family are expressed. This protein competes with heparin sulfate molecules and interacts with FGF-2 that is trapped in the extracellular matrix and facilitates its release [165]. When bound to FGF-2, FGF-BP reduces the availability of bFGF to heparin sulfate molecules, stimulating the growth of prostate and adrenal cancer cells in an FGF-2 dependent manner [166,167]. Reduction in the levels of FGF-BP has been correlated with the inhibition of angiogenic activity within the xenograft models of squamous cell cancer, colon cancer as well as prostate cancer [168,169].
In certain inflammatory conditions, angiogenesis could be induced in response to an injury that may be caused by any external stress. Cells found in inflammatory conditions include mast cells, cytotoxic and helper T cells as well as phagocytic cells [170]. Inflammation provokes the release of bFGF from these cells [171]. Moreover, other traumatic conditions may result in the expression of FGF-2 from endothelial cells [172]. Other factors that are responsible for the expedition of FGF-2 release from endothelial cells may include cytokines like IFNα also with IL-2, nitric oxide-NO, and IL-1β. NO, produced by the effect of various inflammatory molecules and cytokines, is concerned with the up-regulation of FGF-2 that ultimately leads to the progression of angiogenic activities [173,174]. In addition to the data-mentioned above, excessive inflammation may also result in cell damage and hypoxic conditions. Hypoxia stimulates the synthesis of heparin sulfate proteoglycans, thus increasing the impartiality of endothelial cells to FGF-2 [175]. On the other hand, damaged endothelial cells express increased levels of FGF-2, facilitating the synthesis of new vesselsangiogenesis [176,177].
A crosstalk mechanism exists within the tumor microenvironment where various factors coordinate with each other to make sure the survival of tumor cells. As the tumor progresses, the expression profile of tumors also evolves where particular pro-or anti-angiogenic factors may be expressed at one point but might be absent at another [178,179]. VEGF is known to regulate and manage the angiogenic process. Upon integration, FGF-2 and VEGF bring about complex mechanisms that are essential for carrying out angiogenic activities [180]. However, the effect of these two GFs is evident at different stages of the process. Almost every step of angiogenesis expresses FGF whereas VEGF is usually observed at the initial phases of tumor development [181,182]. FGF-2 signaling triggers VEGF expression in endothelial cells. A decrease in the expression of VEGF reduces the levels of FGF-2 consequently. Blocking FGFR1 and FGFR2 reduces the levels of VEGF and neovascularization [183]. A study reported an increase in the levels of VEGF and VEGFR1/2 along with the levels of an angiogenic factor Hif-1α, observed in epithelial cells of the prostate tumor, upon induction of FGFR1 [184,185]. FGFR1 functions to promote the paracrine signaling of VEGF on endothelial cells to execute angiogenesis. VEGF and VEGFR signaling pathway may be induced by FGFs, resulting in the indirect stimulation of angiogenesis. FGF-2 acts to enhance the expression of NRP-1, which serves as a co-factor in VEGF-VEGFR interactions. The expression of NRP-1, when observed in VSMCs, in turn, facilitates the cell migratory properties induced by VEGF [186], thus endorsing the angiogenic process. On the other hand, FGF-2 reinforces the secretion of HIF-1α that consequently releases VEGF [187]. A reciprocal interaction exists among the VSMCs bearing FGFR1 and endothelial cells that retain PDGFR α and β. This peculiar communication is carried out by FGF-2, where it operates to make endothelial cells more responsive to PDGF-BB that in turn responses to VSMCs and promotes their reactivity towards FGF-2 by increasing FGFR1 expression. Angiogenesis carried out under the effect of FGF-2 is not entirely dependent on VEGF signaling. According to one report, even in the presence of high levels of VEGF, FGF-2 overexpression can induce angiogenesis [188]. Apart from mediating the VEGF signaling, the FGFR1 is involved in reducing the levels of Angiopoietin 1 while augmenting the expression profile of Angiopoietin 2 [189]. These are angiogenic factors that interact with their respective receptors (RTKs) on endothelial cells. Ang-1 is responsible for maintaining balanced and stabilized embryonic blood vasculature system [190]. Paradoxically, Ang-2 is involved in the disruption of new blood vessels [191]. The presence of Ang-2 and VEGF at the point of angiogenesis suggests the undermining effect of Ang-2 on endothelial cells makes these cells more responsive to VEGF-regulated angiogenesis.
Participation of fibroblast growth factors in commencing and implementing various angiogenic activities is quite evident. Numerous research studies conducted in the recent years have proved the role of FGFs in promoting tumor angiogenesis. However, most of the research administered was based either on animal models or in vitro studies. The link of tumor progression and tumor angiogenesis with FGFs in human cancer requires further affirmatory data. Respective therapeutic techniques have been investigated in various clinical trials to limit the operation of FGFs in the tumoral environment.

Therapeutic inhibition of FGFs-FGFRs
The association between cancer metastases and angiogenesis is genuinely acceptable. Efforts are being made to develop such kind of drugs that could ultimately limit the effect of FGFs on tumor angiogenesis and the crosstalk mechanism they undergo with various molecules within the tumor microenvironment [192]. FGF-FGFR signaling, apart from directly participating in angiogenesis and tumor growth, is linked with resistance to various VEGF inhibitory therapies, confirming the actuality of cross talk system between FGF and VEGF [193]. Upregulation of interactions among FGF and FGFR may result in resistance to an anti-VEGF therapy [194]. In certain malignancies, FGFs have been reported to hinder the inhibitory action of therapies targeted against VEGF-mediated signaling activities. When studied in transgenic mice models of pancreatic tumor, Rip1-Tag2, blocking of VEGFR elevated the levels of FGF-2. Upon treatment of the same tumor with VEGFR inhibitor alone and later on with an FGF-trap, the result recorded was a decrease in vessel density and tumor progression [195].
Various types of therapeutic drugs have been manufactured and tested for their inhibitory actions against FGFs and VEGF since these two GFs show a highly integrated response mechanism that leads to tumor angiogenesis [196]. Inhibitors developed against VEGF or FGF usually aims the growth factor itself, its receptor or any downstream signaling molecule angiogenic activities [197][198][199].
Bevacizumab, rhumAb VEGF, is a recombinant monoclonal antibody that binds to VEGF-A growth factor and hinders its interaction with its receptor thus down-regulating angiogenesis, investigated in various studies. However, FGFs' show resistance to its mode of action [200,201]. A treatment study of bevacizumab along with 5-fluorouracil (5-FU), leucovorin, and irinotecan (FOLFIRI) was carried out on the patients of colon cancer to demonstrate the role of FGF-2 against bevacizumab. At various levels of disease progression, FGF-2 levels were assessed. The results displayed an increase in the levels of FGF-2 just before cancer progression [202,203]. Another study concerned with renal cancer neoadjuvant therapy included bevacizumab along with other radiations. Here too, FGF-2 showed no response to this treatment, suggesting resistance to bevacizumab [204,205]. An antibody directed against VEGF receptor 2 KDR/Flk-1 obstructs the binding of VEGF with VEGFR2, thus attenuating the signaling pathways directed by VEGF-VEGFR2 interaction [206]. SU6668 is an inhibitor that aims to interrupt the kinase activity of FGFR, VEGF, and PDGFR. Interferon is correlated with inhibition of angiogenesis [207]. Interferon α reduces angiogenic activity that is being maintained by increased levels of FGF-2. It also decreases the expression of FGF-2 from tumor cells. Another inhibitor of FGF-2 induced angiogenesis is thalidomide, which is an immunomodulatory drug [208]. Platelet factor 4 is a CXC chemokine that possesses anti-angiogenic properties. It interacts with FGF-2 and heparin sulfate simultaneously to form a ternary complex and inhibits angiogenesis by binding with FGF-2/HS at the cell surface. It also obstructs the dimerization of bFGF [209,210].
In the past years, advancements in pharmaceutical therapies against FGFs have been made to limit their effects on the cancerous environment. Though, tumor angiogenesis is mainly regulated by VEGF and passively by FGF. However, fibroblast growth factors resist the effects of anti-VEGF therapies, as mentioned earlier. Recently developed techniques against growth factors target FGF receptors to limit angiogenesis. Individual tyrosine kinase inhibitors are being developed, known as "small molecule TKIs" that may act against FGFs either specifically or as a part of the larger target group depending on their mode of action [211]. The primary strategy applied here is to block the ATP binding site of the FGFR receptor. The intracellular tyrosine kinase domain of FGFR contains the ATP site. The "non-specific" TKIs class usually, targets VEGFRs as well as FGFRs since these two have almost identical kinase domain structure. However, nonspecific TKIs have a higher potency to select VEGFR as compared to FGFR [212,213]. Dovitinib (TK1258) is a tyrosine kinase inhibitor that works against several factors like VEGFR1, 2, 3; FGFR1; PDGFR-β; CSf-1, etc. This compound possesses antitumor and anti-angiogenic properties when it targets receptors like FGFR, PDGFR or VEGFR, FGFR, and PDGFR, respectively. Dovitinib has been investigated in gastrointestinal stromal tumors, endometrial, breast, hepatocellular carcinomas, etc. [214][215][216][217][218][219]. Side effects recorded in different studies mainly included diarrhea, hypertension, abdominal pain, nausea, dehydration, etc. [220]. E3810 (may or may not be labeled as lucitanib) acts as a dual inhibitor of FGFR1/2 and VEGFR1/2/3 that can be administered orally and is currently in phase II clinical trials. Side effects reported were hypertension, hyperthyroidism, etc. [221][222][223]. Nintedanib (BIBF1120) is another non-specific TKI that targets VEGFR, FGFR as well as PDGFR. Phase II clinical trials of nintedanib are under way for endometrial cancer, hepatocellular carcinoma, glioblastoma multiform and prostate cancer, etc. This inhibitor mainly causes gastrointestinal side effects [224]. Used as a leading therapy for chronic myelogenous leukemia, Ponatinib (AP24534) is a BCR-ABL tyrosine kinase inhibitor. It also shows anti-angiogenic properties against FGFR1, VEGFR2, PDGFRα, etc. [225,226]. Apart from the above mentioned prominent non-selective TKIs other compounds, like brivanib (BMS582664) a dual FGFR/VEGFR inhibitor, ENMD-2076, lenvatinib (E7080), orantinib (TSU-68), etc., though have shown anti-FGFR properties. However, their effects against VEGFR or other kinases are more relevant [227][228][229][230].
"Selective" TKIs are the other class of inhibitors that specifically target FGFRs. BGJ398 is a selective TKI that targets FGFRs1-3. This compound, either exclusively or in combination with other compounds, is being investigated in various phase II clinical trials of certain tumors [231,232]. The other two selective TKIs are pan (Greek meaning all) inhibitors i.e. they block all FGFRs indiscriminately. These inhibitors are AZD4547 and LY287445 [233,234]. The former is being tested in phase I and II clinical trials whereas the latter has been tested in breast cancer patients during a phase I clinical trial. TAS120, Debio1347, JNJ-42756493, and ARQ087 are other specific tyrosine kinase inhibitors that have been developed to hinder the activity of FGFRs [235]. Besides tyrosine kinase inhibitors, other forms of therapies have evolved in the recent years to hinder the activity of FGFRs. These types include monoclonal antibodies directed towards FGFRs [236,237] and FGFligand traps. The novel strategy for blocking FGF/FGFR interaction is the ligand trap. It has the potency to hinder the binding of FGF ligand with its respective receptor. MGFR1877S is a monoclonal antibody that targets FGFR3. FP-1039 is an FGF ligand trap that has been developed by combining the regions of human IgG and extracellular domain region of FGFR1-IIIc isoform [238].
The developmental steps of FGF/FGFR inhibitors have come across various challenges. When it comes to limiting angiogenic process within a cancerous setting, FGFs or FGFRs alone are not solely responsible for carrying out this particular process. FGF/FGFR targeted remedies need to be combined with other therapies to attain the desired result. Further evaluation of tumor microenvironment and various factors that reside within is required. The timing for the application of a certain therapy is another hurdle. The tumor environment is a dynamic environment that keeps on modifying its expression profile. Hence, any tumor would have unique FGF/FGFR expression levels at certain points, and these levels may decrease or increase as the tumor advances. On the other hand, in such a complex environment administration of a certain therapy usually, faces resistance from some other factor. Identification of such factors makes the developmental process even complicated and time-consuming. Furthermore, selection of patients for conducting a clinical trial, cost effectiveness of therapy, possible adverse outcomes, recurrence time, etc. are some other factors that need to be figured out before launching a treatment against the FGF/FGFR activity.

Drug resistance
FGFs' expression shows resistance to anti-cancer therapies in certain tumors. In the excess of ligands i.e. GFs, the response of cancer cells to tyrosine kinase inhibitors is impaired. FGF is expressed by stromal cells and resists the anti-cancer effects of imatinib in gastrointestinal stromal cancer. FGFs decrease the anti-cancer effects of imatinib in a mice model of cervical carcinogenesis. FGF-2, when administered on a cell line of human bladder cancer, shows resistance to cisplatin therapy [239]. According to one study, FGF-1 and FGF-2 also resist the response of paclitaxel, doxorubicin, and 5-fluorouracil in metastatic tumors [240]. FGFs and FGFRs resist individual therapies that target cancer cells. In a breast cancer study, FGFR1 overexpression has been correlated with the induction of resistance to 4-hydroxytamoxifen. Certain novel inhibitory techniques have been developed that lower the expression of EGFR along with that of FGFR3 and FGFR2 in the cell lines of non-small cell lung cancer-NSCLC [241]. In a particular type of melanoma, the activation of MAPK pathway by FGFR3 results in the resistance to vemurafenib, an anti-cancer drug [242]. A crosstalk mechanism that exists between FGFR3 and KIT gene induces drug resistance in gastrointestinal stromal cancer [243]. FGFR3 hinders the effects of dexamethasone in multiple myeloma cells [244]. FGFR4 has been linked with drug resistance in colorectal cancer [245]. Selective mutations in FGFR2 can lead to resistance to dovitinib.
An enormous amount of data is available to validate the role of FGF and FGFR in drug resistance. Development of FGF/FGFR combined resistant drugs and exclusively designed clinical trials that implement a combined treatment of specific anti-cancer drugs would help achieve the standardized outcome in the future.

Summary
Several biological and developmental processes that take place during the lifespan are brought about by Fibroblast growth factors. These growth factors act as ligands and upon binding to their receptors-FGFRs and cooperating with co-factors, i.e., heparin sulfate molecules/ HSPG, trigger a signal transduction cascade that ultimately lead to cell proliferation, cell survival, angiogenesis and other mechanism carried out by this class of GFs. FGFs may act in an endocrine, paracrine or an intracrine manner depending upon their domain structure and secretion.
FGF/FGFRs play a distinct role in facilitating tumor progression and angiogenesis. With the assistance of other elements, particularly VEGF in the case of angiogenesis, FGFs can implement the angiogenic process. Since their discovery in 1970's and revelation of their structure and mode of action in cancer, attempts have been made to restrict their activity. Clinical trials have evaluated several pharmaceutical remedies. Advanced schematic analysis of tumor microenvironment, for isolating determinants that coordinate with the surrounding components to form an alliance and operate as a compact unit to favorably actuate the mechanism of angiogenesis and tumor survival is required. Detailed studies on cancer types specified by FGFs and FGFRs would provide a lead in the future for the better development of medicinal therapeutics.