Deciphering role of FGFR signalling pathway in pancreatic cancer

Abstract Recently, fibroblast growth factors are identified to play a vital role in the development and progression of human pancreatic cancer. FGF pathway is critical involved in numerous cellular processes through regulation of its downstream targets, including proliferation, apoptosis, migration, invasion, angiogenesis and metastasis. In this review article, we describe recent advances of FGFR signalling pathway in pancreatic carcinogenesis and progression. Moreover, we highlight the available chemical inhibitors of FGFR pathway for potential treatment of pancreatic cancer. Furthermore, we discuss whether targeting FGFR pathway is a novel therapeutic strategy for pancreatic cancer clinical management.


| INTRODUC TI ON
Pancreatic cancer is one of the common malignancies in human worldwide. In fact, 56 770 new cases of pancreatic cancer and 45 750 deaths have been expected this year in the United States. 1 More than 400 000 deaths annually due to pancreatic cancer are observed in the worldwide. 2 Pancreatic cancer is the third leading cause of cancer death behind lung cancer and colon cancer in the United States in 2018. However, deaths from pancreatic cancer are predicted to be the second leading cause of mortality in the United States by 2030. 3 The causes of pancreatic cancer are still unclear, although accumulating evidence has suggested that pancreatic cancer occurrence is associated with several factors such as smoking, drinking, coffee consumption, high fat and high protein diet, and genetic background. In addition, the patients with diabetes and chronic pancreatitis have high risk for developing pancreatic cancer. 4,5 In contrast to the increase in survival for most cancer types, the 5-year relative survival rate for pancreatic cancer is about 8% in the United States. One of the reasons is that pancreatic cancer is often diagnosed at a distant stage, which has 3% for the 5-year survival rate. 6 Because the early symptoms of pancreatic cancer are same as gastric disease such as upper abdominal discomfort and loss of appetite, most of the patients with pancreatic cancer often exhibit locally invasion or metastatic tumour when they are diagnosed. 7 About 95% of pancreatic cancer cases are adenocarcinoma, known as PDAC (pancreatic ductal adenocarcinoma), which arises from the epithelium of a duct. [8][9][10] In recent years, emerging evidence has demonstrated that vital genes and signalling pathways are critically involved in the tumorigenesis and progression of pancreatic cancer, such as K-ras related proteins, 11 Notch, 12 Hedeghog, 13 Wnt, 14 F-box proteins, 15 PI3K (phosphatidylinositol 3-Kinase)/Akt 16 and mTOR (mammalian target of rapamycin). 17 Several lines of evidence has revealed that various growth factor signalling pathways are participated in pancreatic tumorigenesis and progression, including TGF (transforming growth factor), 18 EGF (epidermal growth factor), 19 HGF (hepatocyte growth factor), 20 IGF (insulin-like growth factor), 21 PDGF (platelet-derived growth factor) 22 and FGF (fibroblast growth factor). 23,24 Recently, FGF has been paid attention to pancreatic cancer development and progression. In fact, these pathways could have interplays. For example, Notch signalling activation increases FGF1-mediated invasion in oral squamous cell carcinoma. 25 FGF activates Ras-MAPK pathway, leading to skin tumour induced by Pten deficient. 26 Similarly, FGFR1 promotes activation of MAPK and mTOR pathway in palbociclib resistant non-small-cell lung cancer. 27 Another study identified that FGF2 exerts tumour lymphangiogenesis via activating the Akt/mTOR/p70S6K. 28 FGF signalling activates the expression of the sonic hedgehog receptor and Ptch2. 29 In this review article, we will describe recent advances of FGF signalling pathway in pancreatic cancer. Moreover, we will dissect the available chemical inhibitors of FGF pathway for potential treatment of pancreatic cancer. Furthermore, we will discuss whether targeting FGF pathway is a novel therapeutic strategy for pancreatic cancer clinical management.

| FG F/FG FR S I G NALLING PATHWAY
FGF, a kind of peptide molecule, has been identified to bind to its specific receptors of cell membrane and to govern cell growth. FGF is named due to its promotion of fibroblast proliferation and is located in various tissues. FGF is also called heparin conjugate growth factor because of its high affinity for heparin. At present, more than 20 members of the FGF family are identified, which are encoded by various genes. 30 34 Interestingly, FGFR5 (also named as FGFRL1) lacks tyrosine kinase domain, which is different from the other four types. 35 Clearly, FGFs as ligands bind to FGFRs and activate tyrosine kinase domain of FGFRs, leading to activation of FGF/FGFR signalling pathway. Interestingly, FGF1 also binds to heparin sulphate proteoglycans (HSPG), suggesting that HSPG could be a co-receptor of FGF1. In addition, FGF1 co-localizes with both proteoglycans CD44 and CSPG4 at the cell surface, indicating that these receptors could be storage molecular to create a reservoir of FGF1. 36 Heparin and heparin sulphate glycosaminoglycans (HSGAGs) can stabilize FGFs against degradation. 37 The activation of FGF/FGFR pathway regulates several downstream targets such as PI3K/Akt, MAPK (mitogenactivated protein kinase) or PLCγ. 38 FGF signalling pathway plays a role in a myriad of cellular biological and physiological processes such as proliferation, differentiation, survival, migration, invasion, metastasis, wound repair and angiogenesis. 30 FGF signalling pathway has been identified in tumorigenesis and progression in a variety of human cancers including pancreatic cancer. In the following sections, we will decipher the role of FGF/FGFR signalling pathway in pancreatic carcinogenesis.

| FGF in pancreatic cancer
FGF-1 and FGF-2 are overexpressed in pancreatic carcinoma cells, which are associated with advanced tumour stage and shorter survival. 40 In line with this finding, one study has demonstrated that the expression of FGF-1, FGF-2 and their receptors were highly increased in pancreatic adenocarcinomas compared with normal pancreatic tissue. 41 Moreover, increased FGF and FGFR were associated with upregulation of iNOS (inducible nitric oxide synthase) and protein tyrosine nitration in pancreatic cancer tissues, predicting the potential involvement of oxidant stress in FGF pathway-mediated pancreatic cancer development. 41 Subsequently, this group identified that FGF-1 signalling inhibited peroxynitrite-induced cell death in pancreatic cancer, suggesting that FGF-1 plays a vital role in pancreatic adenocarcinoma. 42 Another study reported that FGF-1 and FGF-2 treatment led to induction of phosphorylation of E-cadherin and beta-catenin on tyrosine residues, resulting in an increase in cell adhesion, tubular differentiation and reduction of invasion in pancreatic cancer cells. 43,44 Twenty-eight years ago, one study has shown that FGF-2 at picomolar concentrations promoted cell proliferation via regulation of ornithine decarboxylase in AR4-2J rat pancreatic cancer cell line. 45 Moroever, more evidence has emerged to validate the role of FGF-2 in pancreatic cancer. For exmaple, high expression of FGF-2 was observed in PDAC, and patients with high level of FGF-2 and VEGF (vascular endothelial growth factor) had shorter survival times. 46 Consistently, tumour cell proliferative indices were significantly higher in pancreatic cancer cells with FGF-2-positive, indicating that the expression of FGF-2 is associated with cell proliferation in pancreatic cancer. 47 Similarly, a specific neutralizing antibody against FGF-2 led to a 50% inhibition in cell proliferation in pancreatic cancer cells. 48 Further, the high gradient of FGF-2 enhanced cell invasiveness in pancreatic cancer cells, whereas inhibition of FGF pathway by anti-FGF receptor antibody retarded cell invasion, demonstrating that FGF-2 is involved in cell invasiveness in pancreatic cancer. 49 Additionally, Pim-3, a proto-oncogene with serine/threonine kinase activity, promoted tumour neovascularization and tumour growth via upregulation of FGF-2 in pancreatic cancer. 50 Klotho, a transmembrane protein, suppressed cell growth in vitro and in vivo through inactivation of FGF-2 pathway in pancreatic cancer. 51 Interestingly, secretory FGF-2 upregulation was exhibited to have the potential to inhibit spreading of pancreatic cancer cells. 52 FGF-5 has been reported to be involved in various biological processes including development, tissue growth, repair and morphogenesis. 53 FGF-5 was initially identified to be an oncogene in human cancers. 54  suggesting that FGF-10 could be a predictive biomarker for chemotherapeutic treatment response in pancreatic cancer patients. 63 FGF-13 was found to be significantly associated with the shorter survival and occurrence of liver metastasis in pancreatic cancer. 64 This investigation identifies FGF-13 as a novel prognostic biomarker in pancreatic cancer. Overexpression of FGF-19 did not affect the cell proliferation, but inhibited cell migration, invasion and attachment via stimulation of FGFR4 in pancreatic cancer cells. 65 Several knockout mouse phenotypes have demonstrated the role of FGFs in tumorigenesis. Ffg15 (human homolog, FGF19) deficiency impairs liver regeneration in mice. 66 Moreover, fibrosis-induced hepatocellular carcinoma development is retarded in Fgf15 knockout mice. 67 Inducible Fgf13 ablation in cardiomyocytes enhances caveolae-induced cardioprotection during cardiac pressure overload. 68 Loss of

| FGF-binding proteins in pancreatic cancer
FGF-binding proteins (FGF-BP) release FGFs from the extracellular matrix storage, leading to increased FGF activity. Therefore, FGF-BP plays a critical role as an extracellular chaperone in FGF-mediated signalling pathway and mitogenesis. [75][76][77] Moreover, FGF-BP expression is remarkably increased in a variety of human cancer tissues. 78 FGF-BP1 expression is highly elevated in pancreatic adenocarcinoma compared with normal pancreas, suggesting that FGF-BP1 might a biomarker for high-risk premalignant lesions. 79 In consistent, FGF-BP1 was found to be induced early during the pancreatic cancer initiation. 80 These reports clearly indicate that FGF-BP could become an indicator of early diagnosis for pancreatic cancer. The results from Fgfbp3 knockout mice showed that FGF-BP3 impacts carbohydrate and lipid metabolism. 81 To further investigate the role of FGF-BP in tumorigenesis, Fgf-bp engineering mice are required.

| FGFR in pancreatic cancer
Twenty-five years ago, aberrant expression of FGFR1 was observed in pancreatic cancer. 82 Moreover, the 2-immunoglobulin-like form of FGFR1 was reported to involve in aberrant autocrine and paracrine pathways in pancreatic cancer. 82 One study showed that inhibition  84 However, overexpression of FGFR-1β led to formation of tumour xenograft and exhibited resistance to chemotherapy. 84 Liu et al found that   93 One study showed that targeting the CYP2B1/cyclophosphamide suicide system to FGFRs led to tumour suppressive response and an increased survival rate in pancreatic cancer. 94 FGFR4 was expressed in a majority of pancreatic cancer patients, and its expression was related to longer overall survival. FGFR4 stimulation led to increased cell adhesion to laminin and fibronection, and inhibited cell migration, suggesting that FGFR4 could contribute to tumour suppressive function via enhanced cell adhesion to extracellular matrix. 65 Consistently, dominant-negative FGFR-4 and inhibitors of FGFR signalling inhibited matrix adhesion induced by N-CAM (neural cell-adhesion molecule) in pancreatic cancer.
Moreover, N-CAM promoted β1-integrin-involved cell-matrix adhesion via activation of FGFR signalling pathway. 95 Additionally, FGFR4 knockout mice bred with FGF19 transgenic mice fail to develop liver tumours. 71 The engineering mice are necessary to explore the function of FGFR in tumorigenesis.

| FG FR INHIB ITOR S FOR PAN CRE ATI C C AN CER TRE ATMENT
Several FGFR inhibitors have been discovered for potential treatment of human cancers including pancreatic cancer 96 (Table 1). For example, SSR128129E is an orally effective allosteric FGFR inhibitor, which has no effect on other related RTKs. Chemical SSR128129E Moreover, TKI258 inhibited tumour growth and lymph node metastases in mouse model, suggesting that TKI258 could be an effective agent for human pancreatic cancer. 98 Dovitinib treatment exhibited pro-apoptotic effect in pancreatic cancer cells with heightened FGFR signalling activation via regulation of Akt/Mcl-1 axis. 92 Recently, a phase 1b study showed that dovitinib with gemcitabine and capecitabine achieved efficacy signals in advanced pancreatic cancer. 99 Lenvatinib, an oral inhibitor of multiple RTKs targeting FGFR1-4, VEGFR1-3, PDGFRα, RET and KIT. One study has shown that lenvatinib suppressed in vivo angiogenesis induced by overexpressed FGF in pancreatic cancer. Notably, lenvatinib also inhibited tumour growth in tumour xenograft models. This report indicates that lenvatinib inhibited FGF-and VEGF-driven angiogenesis in pancreatic cancer. 100 Masitinib, a tyrosine kinase inhibitor of several targets, inhibits c-Kit, FGFR and PDGFR. Masitinib could decrease inflammation in pancreatic cancer patients with increased pain scores. 101 Masitinib and gemcitabine combination exhibited synergy in vitro on proliferation of pancreatic cancer cells. 102 The efficacy and safety of masitinib/gemcitabine have been evaluated and shown to extend survival and median time-to-progression in pancreatic cancer. 103,104 PD173074, an effective inhibitor of FGFR1, inhibited neoangiogenesis and mitogenesis, induced apoptosis, leading to inhibition of orthotopic tumour growth in pancreatic cancer mouse model. 105 In addition, PD173074 inhibited cell proliferation and self-renewal of pancreatic cancer stem cells via suppression of Oct4, Sox-2, Nanog, c-Myc, XIAP (X-linked inhibitor of apoptosis protein), Bcl-2 and survivin. However, it has no direct evidence to show the role of FGF/ FGFR in pancreatic cancer stem cells. Two papers suggest that FGF signalling and FGF10 were involved in enhancing differentiation of pluripotent stem cells into pancreatic progenitors. 106,107 Moreover, PD173074 induced cell apoptosis via upregulation of caspase-3 and cleaved PARP (poly-ADP ribose polymerase) in pancreatic cancer cells. PD173074 also inhibited the activation of c-Met, Src, ERK1/2 and NF-κB in pancreatic cancer cells. 108 BGJ398 is an effective, bioactive FGFR1/2/3 inhibitor with low inhibitory effect on FGFR4, which inhibited cell proliferation of pancreatic cancer. 109 Nintedanib (BIBF 1120), a triple tyrosine kinase inhibitor that targets VEGFR1/2/3, FGFR1/2/3 and PDGFRα/β signalling, inhibited tumour growth, enhanced the activity of gemcitabine and decreased metastatic burden in orthotopic pancreatic xenografts, suggesting that nintedanib could be a potent anti-angiogenesis agent for pancreatic cancer. 110 Moreover, nintedanib inhibited cell proliferation, induced apoptosis via blocking PI3K/MAPK activity and enhanced gemcitabine inhibitory effects in pancreatic cancer. 111 Furthermore, nintedanib was identified as a highly effective therapeutic for neuroendocrine carcinoma of the pancreas using transgenic mouse model. 112 Notably, nintedanib plus afatinib exhibit anti-tumour activity with a manageable safety in pancreatic cancer. 113 Ponatinib (AP24534) is an effective multitargeted inhibitor that act on FGFRs, Bcr-Abl, Src kinase, PDGFRα, VEGFR2, Akt, ERK1/2 and other kinases. 114 Ponatinib plus an MEK inhibitor were effective in inhibition of pancreatic cancer cell growth. 115 BGJ398 is an effective, bioactive FGFR1/2/3 inhibitor with low inhibitory effect on FGFR4, which inhibited cell proliferation of pancreatic cancer. 109 We believe that more FGFR inhibitors will be discovered for the treatment of pancreatic cancer. It is noteworthy that using these FGFR inhibitors could cause side effects on cancer patients. For instance, TKIs could lead to adverse effects on viral organs, including the cardiovascular system and liver. 116 Hypertension is associated with the treatment of nintedanib, lenvatinib, ponatinib, cabozantinib and trametinib. 116 Moreover, ponatinib treatment for chronic myeloid leukaemia results in cardiovascular adverse effects, such as vascular occlusive event. 117 Due to inhibition of VEGFR by these TKIs, these inhibitors' application could lead to bleeding and thrombosis. 118 Hence, it is required to reduce adverse effects of FGFR inhibitors.

| CON CLUS I ON AND PER S PEC TIVE
In summary, FGF plays an important role in the development and progression of human pancreatic cancer because FGF pathway is critical involved in numerous cellular processes including proliferation, apoptosis, migration, invasion, angiogenesis and metastasis ( Figure 1). FGF/FGFR has been revealed to participate in its  For example, miR-214 inhibits the expression of FGFR-1, leading to suppression of hepatocellular carcinoma metastasis. 119 One study showed that miR-99a targets FGFR3 in epithelial ovarian cancer cells. 120 Another study validated the miRNA panel, including let-7c, miR-155 and miR-218, could be useful for prediction of response to ponatinib in lung cancer cells. 121

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R CO NTR I B UTI O N S
All authors are involved in writing this manuscript and approved this article.