Role of FGF System in Neuroendocrine Neoplasms: Potential Therapeutic Applications

Neuroendocrine neoplasms (NENs) are a heterogeneous group of tumors originating from neuroendocrine cells dispersed in different organs. Receptor tyrosine kinases are a subclass of tyrosine kinases with a relevant role in several cellular processes including proliferation, differentiation, motility and metabolism. Dysregulation of these receptors is involved in neoplastic development and progression for several tumors, including NENs. In this review, we provide an overview concerning the role of the fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) system in the development and progression of NENs, the occurrence of fibrotic complications and the onset of drug-resistance. Although no specific FGFR kinase inhibitors have been evaluated in NENs, several clinical trials on multitarget tyrosine kinase inhibitors, acting also on FGF system, showed promising anti-tumor activity with an acceptable and manageable safety profile in patients with advanced NENs. Future studies will need to confirm these issues, particularly with the development of new tyrosine kinase inhibitors highly selective for FGFR.

Receptor tyrosine kinases are a subclass of tyrosine kinases with a relevant role in several cellular processes including proliferation, differentiation, motility and metabolism. Dysregulation of these receptors plays a relevant role in neoplastic development and progression for several tumors, including NENs (6,7).
In this review, we provide an overview concerning the role of the fibroblast growth factor (FGF)/ fibroblast growth factor receptor (FGFR) system in NENs.

FGF SYSTEM IN HEALTH AND CANCER
FGFs and related receptors are members of a large family with a wide range of effects. This system is involved in organogenesis (during development), homeostasis and repair of adult tissues. Moreover, FGF family promotes angiogenesis, growth, differentiation and migration of cells mainly through the activation of RAS-MAPK, PI3K-AKT and PLCg pathways, with a relevant role in the development and progression of several tumors (8). These effects are mediated by the interaction of FGFs with four tyrosine kinase receptors: FGFR1, FGFR2, FGFR3 and FGFR4, which are composed by an extracellular domain, a transmembrane domain and an intracellular domain. The binding of ligands induces conformational changes that lead to a dimerization of these receptors. This event activates the intracellular tyrosine kinase domain, which in turn triggers the signalling cascade (8). FGFs, based on their biochemical functions, sequence similarity and evolutionary relationships, are classified into different subfamilies: FGF1, FGF4, FGF7, FGF8, FGF9, FGF15/19 and FGF11.
A deregulation of the FGF/FGFR system can be involved in cancer development and progression through modulation of cell proliferation, migration and angiogenesis (22). Besides its role in physiological angiogenesis, FGF2 is implied in tumor-induced angiogenesis and metastatic process and appears to direct tumor-associated macrophages toward a protumorigenic state (23)(24)(25). FGF4 promotes cancer cell proliferation, invasion and migration by causing a switch of the receptor FGFR2-IIIb, a splice variant expressed in epithelial cells, into FGFR2-IIIc, expressed in mesenchymal cells and able to induce epithelial-mesenchymal transition (26). FGF5 can promote osteosarcoma proliferation by activating the MAPK signaling pathway (27) and the FGF5/FGFR1 axis contributes to melanoma progression (28). FGF6 can stimulate proliferation of prostate cancer cells through the activation of FGFR4 (29). Among the FGF7 subfamily, FGF3 and FGF7 have been reported to be highly expressed in breast cancer (30,31) and gastric adenocarcinoma (32), respectively. In addition, the FGF10/FGFR-IIb signaling appears to have a role in breast and pancreatic tumors (15,33). Although the mechanism is unclear, Jarosz et al. observed a potential role of FGF22 in skin tumorigenesis (34). In a recent study, FGF22 and its receptor FGFR-IIb appear to be associated with the development of lung adenocarcinoma through the MAPK and Rap I signaling pathways (35). A deregulation of FGF18, caused by an altered expression of its negative regulator miR-590-5p, is able to stimulate proliferation and epithelial-mesenchymal transition, with enhanced invasion abilities, in gastric cancer cells (36). In HER + breast cancer cell lines, overexpression of FGF18 stimulates the expression of genes involved in migration and cancer metastasis through Akt/GSK3b pathway (37). By the interaction with FGFR2 and FGFR3 and the activation of the ERK/Akt pathway, FGF18 is able to induce proliferation and invasion in endometrial carcinoma (38).
The FGF/FGFR pathway has also a key role in the onset of drugresistance (39). FGF/FGFR pathway is the first compensatory mechanism in tumors resistant to drugs targeting the vascular endothelial growth factor (VEGF) system (40)(41)(42). Indeed, VEGFdependent vessels are suppressed during prolonged anti-VEGF therapy, while the expression of FGF2 is increased, leading to a novel angiogenesis dependent on FGF2 signaling pathway. This condition drives the tumor toward drug-resistance (42). Boichuk et al. (43) showed that FGF signaling is activated in gastrointestinal stromal tumors after the acquisition of imatinib resistance. Interestingly, the use of a potent FGF inhibitor markedly reduced cell growth in resistant cells compared to imatinib-sensitive cells. This effect increased when the two molecules were combined in resistant cells, showing also that the FGF-inhibitor can restore sensitivity to imatinib.

FGF SYSTEM IN NEUROENDOCRINE NEOPLASMS
The role of the FGF/FGFR system has been analyzed also in NENs and several lines of evidence support its function in the modulation of tumor fibrosis, proliferation, angiogenesis and drug resistance, through a dynamic cross talk between NEN cells, fibroblasts, endothelial cells and inflammatory cells (44). Bordi et al. identified FGF2 by immunohistochemistry in endocrine cells of the gastric oxyntic mucosa and mRNA of FGF2 in enterochromaffin-like carcinoid tumors (45). Immunohistochemical studies demonstrated the staining for FGF-2 in 100% of NEN cells from the midgut and the pancreas, while FGF2 receptors were observed only in the stromal component (46). La Rosa et al. found cytoplasmic immunoreactivity for FGF1 in 26 (43%) out of 60 GEP-NENs and FGFR1-4 were found in 68-88% of tumors with tumor microenvironment components also expressing FGFRs (47). The authors observed also that normal endocrine cells of the gut rarely expressed FGFRs thus hypothesizing that in normal mucosa the FGF/FGFR system has not an autocrine role on modulating endocrine cells functions. Therefore, de novo expression of FGFRs by NEN cells may play a role in the autocrine/paracrine signaling responsible of tumorigenesis, stromal fibrosis and tumor-induced angiogenesis.
NEN are often characterized by the development of fibrosis, local or distant. The best-known fibrotic complications are carcinoid heart disease, which develops in about 20% of patients with carcinoid syndrome (48), and mesenteric fibrosis, which affects up to 40-50% of small bowel NENs (49,50). Less known complications are represented by retroperitoneal fibrosis (50), scleroderma (51), infiltration of the pleura (52) or alveoli (53) and fibrosis of the bladder (54). Although the pathogenesis of fibrotic complications is unclear, serotonin, with a relevant mitogenic power on fibroblasts, mesangial cells, smooth muscle cells, endothelial cells and NEN cells, may have a role in these events (55). The FGF system appears to be also implicated in the mechanism of gastrointestinal NEN fibrosis (56). In fact, Bordi (45) showed that among the 10 patients suffering from type 3 gastric NEN and with positive immunohistochemistry for FGF2, some had diffuse stromal fibrosis. Another study (57), which analyzed a pool of 41 gastrointestinal NENs, showed a positive correlation between FGF1 and the amount of fibrous stroma in tumors. The FGF is responsible of cell proliferation and stroma formation and its action is potentiated by serotonin (58). Moreover, FGF may activate also the expression of the connective tissue growth factor genes that regulate myofibroblast differentiation, collagen synthesis and fibroblast proliferation (59).
The mRNA expression of FGF receptor was found more frequently in functioning NENs (including gastrinomas and insulinomas) than in functionally inactive NENs (53.6% vs. 22.2%) (60). Although this difference was not statistically significant (p=0.10), speculating on the association between FGFR expression and hormone production may be not totally irrational, but further evidence is required to corroborate these findings.
The FGFR4-G388R single-nucleotide polymorphism was investigated in 71 patients with pancreatic NEN (61). The authors observed that FGFR4-R388 allele was independently associated with liver metastases. To further analyze the impact of the FGFR4 SNP, the same authors transfected BON1 cells with either FGFR4-G388 or FGFR4-R388 and injected them in SCID mice. They found that xenografts expressing FGFR4-R388 displayed a more aggressive biological behavior and were resistant to everolimus treatment. This latter aspect was investigated also among 17 patients previously treated with everolimus in a clinical trial. Patients harboring FGFR4-R388 allele achieved a worse tumor response (9% vs. 25%) and a reduced median PFS (4.8 vs. 16.6 months) and OS (9.3vs 40 months) compared to patients homozygous for FGFR4-G388. Although decreased drug response was related to persistently high mTOR and STAT3 phosphorylation despite of everolimus treatment, these data were not confirmed by Cros et al., who reported no modification of the mTOR pathway in patients with pancreatic or ileal NENs harboring FGFR4-R388 allele (62). This apparent inconsistency corroborates the need for further studies validating the identification of molecular parameters useful to predict drug efficacy and resistance (63).
The FGF/FGFR system collaborates with the VEGF signaling pathway in the initiation and maintenance of tumor angiogenesis. These mechanisms have been demonstrated in allograft transplantation experiments and in mouse model of pancreatic NEN (the Rip1Tag2 transgenic mice), where interfering with the FGF function by a soluble form of the FGFR2 IIIb significantly inhibited tumor-induced angiogenesis and tumor growth (64). The FGF system acts as a second proangiogenic circuit, indeed VEGF is the main regulator of angiogenesis but, as reported by Casanovas et al., experiments in the Rip1Tag2 model of pancreatic islet carcinoma documented that initial inhibition of the angiogenesis achieved by VEGF signaling blockade was restored by the upregulation of the FGF system (65). Therefore, blocking both VEGF and FGF signaling pathways may reveal synergic antiangiogenic effects and inhibit tumor progression secondary to compensatory feedback loops driving tumor revascularization. For instance, Allen et al. investigated the effect of brivanib, a selective inhibitor targeting both VEGF and FGF receptors, in a mouse model of pancreatic NEN. Brivanib was effective not only as first-line therapy, but also as second-line treatment after failure of two agents inhibiting VEGF receptors (DC101 and sorafenib) (66).

FGFRs AS THERAPEUTIC TARGET IN NENs
In the last few years, the therapeutic approach for NENs has changed following the approval of several innovative targeted treatments such as tyrosine kinase inhibitors (TKIs). Although no specific FGFR kinase inhibitors have been evaluated in NENs, several clinical trials on multitarget TKIs, acting also on FGF, are ongoing and few published studies have demonstrated their efficacy in NENs (44). The interest in FGF pathway inhibitors relies also in the possibility to overcome resistance to VEGF inhibition that may arise after long term use of these drugs or could be intrinsic in tumor expressing FGF2 (67)(68)(69). The results of clinical trials in NENs evaluating multitarget TKI, acting also on FGF, are described below ( Table 1).
Another study evaluated the effect of surufatinib dose escalation/expansion in 32 patients with heavily pre-treated progressive NENs, 16 patients with pancreatic NENs and 16 with extra-pancreatic NENs. Nineteen patients remained on active treatment (13 extra-pancreatic and 6 pancreatic), 9 patients discontinued due to disease progression, 2 withdrew consent and 2 discontinued due to AE. An ORR of 9.4% was observed (72).
An open label phase II study evaluated efficacy and tolerability of surufatinib (300 mg/day) in 27 patients with progressive medullary thyroid cancer (MTC). Objective response was observed in 22.2% of patients with MTC, and the majority (88.9%) achieved disease control. The therapy was well tolerated (73).
Therefore, surufatinib demonstrated promising anti-tumor activity with an acceptable and manageable safety profile in advanced NENs.
Recently, a prospective, post-marketing observational study evaluated, in daily clinical practice, the safety and effectiveness of lenvatinib in 28 patients with MTC. Hypertension, proteinuria and palmar-plantar erythrodysesthesia syndrome were the most frequently reported AE. The 12-months OS rate was 83%. ORR was 45% (77).
Nintedanib is a dual inhibitor of VEGFR1, -2, and -3 as well as FGFR2 and showed both antiangiogenic and antitumor activity in the RIP1-Tag2 transgenic mouse model of tumorigenesis for pancreatic NEN (44). A multicenter phase II study evaluated efficacy, safety and tolerability of nintedanib in 30 patients with unresectable/metastatic carcinoids on stable dose of SSA for ≥3 months. PFS at 16 weeks was 86.7% in 26 patients. PR was observed in 4%, SD in 83%, disease progression in 8% of patients. Quality of life was maintained or improved in at least 50% of subjects. The most common grade 3 AE were hypertension and decreased appetite (78).
A prospective randomized double-blind phase II study evaluated the efficacy and tolerability of nintedanib in progressing MTC after prior TKI treatment. The study was stopped due to slow accrual with 32/67 patients enrolled, without reaching the targeted statistical power. The most common AE were diarrhea (18%), nausea (9%), GGT increase (18%) and lymphopenia (18%) (83).
These data have been confirmed in a phase IIb study (ALTER01031), enrolling a larger cohort of patients (80). Ninety-one patients with advanced MTC were randomized: 62 to anlotinib arm and 29 to placebo arm (12 mg/die from day 1 to 14 of a 21-day cycle). Median PFS was 20.7 months in anlotinib arm vs. 11.1 months in placebo arm. The most common AE after anlotinib arm were hand-foot syndrome, hypertension, hypertriglyceridemia and diarrhea (80).
Several clinical trials on the use of multi target TKI, with an action also on FGFR, in patients with NENs are currently ongoing.

CONCLUSIONS AND FUTURE PERSPECTIVES
In the last years there is mounting evidence supporting the role of FGF/FGFR system in the development and progression of NENs and probably in the occurrence of fibrotic complications (mesenteric and/or retroperitoneal fibrosis). In addition, the FGF/FGFR pathway could also have a key role in the onset of drug-resistance. Indeed, FGF/FGFR pathway is a main compensatory mechanism in anti-VEGF-therapy-resistant tumors. Currently no specific FGFR kinase inhibitors have been evaluated in patients affected by advanced NENs. Although recent clinical trials have reported a significant antitumor activity and manageable safety profile of several multitarget TKIs, which are able to block many molecular pathways including FGFR, it is not possible to isolate the efficacy of FGFR inhibition alone. Future studies should better confirm these issues and clarify the role of FGF/FGFR pathway in promoting drug-resistance in NENs. The development of new TKIs, highly selective for FGFR and with less toxicity, may open an innovative therapeutic strategy to be integrated into a personalized approach for this heterogeneous class of tumors. In addition, recent preclinical studies showed a potent inhibition in tumor growth both in hepatocellular carcinoma (85) and in ovarian cancer (86), through the simultaneous blockade of mTOR and FGFR pathways. Considering the pivotal role of deregulated mTOR signaling activation in the proliferation of NENs, particularly in pancreatic tumors, combining mTOR inhibitors and TKIs targeting FGFRs could represent a future therapeutic approach in NENs.

AUTHOR CONTRIBUTIONS
GV, AlC, PM, RM, GP, and DS conceptualized and wrote the manuscript. AF and AnC contributed to draft the manuscript. All authors contributed to the article and approved the submitted version.