Review

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Targeting fibroblast growth factor receptor in breast cancer: a promise or a pitfall? 1.

Introduction

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Fibroblast growth factor ligands and receptors

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FGF/FGFR signal in BC

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FGFRs role in BC treatment

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Conclusion

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Expert opinion

Francesca Bedussi, Alberto Bottini, Maurizio Memo, Stephen B Fox, Sandra Sigala & Daniele Generali† †

UOM di Patologia Mammaria/US Terapia Molecolare, Azienda Istituti Ospitalieri di Cremona, Cremona, Italy

Introduction: Fibroblast growth factors (FGFs) along with their receptors (FGFRs) are involved in several cellular functions, from embryogenesis to metabolism. Because of the ability of FGFR signalling to induce cell proliferation, migration and survival in cancer, these have been found to become overactivated by several mechanisms, including gene amplification, chromosomal translocation and mutations. New evidences indicate that FGFs and FGFRs may act in an oncogenic fashion to promote multiple steps of cancer progression by inducing mitogenic and survival signals, as well as promoting epithelialto-mesenchymal transition, invasion and tumour angiogenesis. This review focuses on the predictive and prognostic role of FGFRs, the role of FGFR signalling and how it may be most appropriately therapeutically targeted in breast cancer. Areas covered: Activation of the FGFR pathway is a common event in many cancer types and for this reason FGFR is an important potential target in cancer treatment. Relevant literature was reviewed to identify current and future role of FGFR family as a possible guide for selecting those patients who would be poor or good responders to the available or the upcoming target therapies for breast cancer treatment. Expert opinion: The success of a personalised medicine approach using targeted therapies ultimately depends on being capable of identifying the patients who will benefit the most from any given drug. Outlining the molecular mechanisms of FGFR signalling and discussing the role of this pathway in breast cancer, we would like to endorse the incorporation of specific patient selection biomakers with the rationale for therapeutic intervention with FGFR-targeted therapy in breast cancer. Keywords: breast cancer, breast cancer biological target, breast cancer therapy, fibroblast growth factor receptor Expert Opin. Ther. Targets (2014) 18(6):665-678

1.

Introduction

In breast carcinoma (BC), the response to chemotherapy or targeted therapies varies according to histology and/or molecular subtypes [1-5]. Although effective regimens are currently established for invasive ductal carcinoma, the most common invasive breast cancer, the treatment efficacy and the prognosis of other minor types of BC are not adequately developed. Thus, the lobular histotype, the second most common subtype of BCs (15%), actually shows poor responsiveness to available chemotherapies [6,7]. BC is has been conventionally subclassified phenotypically depending on the expression of different receptors, such as oestrogen receptor (ER), progesterone receptor and human epidermal growth factor receptor 2 (HER2) [8], but this traditional classification [1-5,9] has been augmented by 10.1517/14728222.2014.898064 © 2014 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted

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The fibroblast growth factors and their receptors (FGFRs) play an important role in a wide range of biological functions (cell proliferation, migration, motility, survival, tissue repair, hematopoiesis and angiogenesis). The aberrant regulation of this pathway has been implicated in many forms of human malignancies as a mediator of proliferation, anti-apoptosis, drug-resistance, epithelial-to-mesenchymal transition and invasion. FGFR-targeted drugs exert direct as well as indirect anticancer effects through processes such as angiogenesis. Based on the genetic aberrations in FGFRs identified in breast carcinoma patients and their consequences on a molecular level, several approaches can be used to target FGFR signalling: inhibitors of FGFR tyrosine kinases activities, therapeutic antibodies and upstream intervention. FGFRs amplification status could be either predictive or prognostic markers. FGFR pathway must be further clarified to develop drugs more selective and targeted. Randomised Clinical Trials will establish the subpopulation in which these drugs should be used.

This box summarises key points contained in the article.

expression profiling into at least five molecular subtypes namely luminal A and luminal B (ER-positive tumours), HER2 (HER2-positive tumours), basal and normal-like tumours [10,11]. More recently, Next-Generation Sequencing (NGS) technology have identified gene alterations in fibroblast growth factor receptor (FGFR)-1/2 that may be suitable for selecting patients for targeted therapies, such as the combination of everolimus with exemestane for treatment of ER-positive metastatic BC patients [12]. In light of this, we would emphasise that even within the above-defined phenotypic subtypes defined by classical markers, there is a wide spectrum of tumour behaviour requiring the identification of additional novel prognostic markers [13]. In a context of suboptimal medical therapies, new promising predictive biomarkers that provide potential for selecting appropriate patients suitable for receiving new effective regimens are needed [14,15]. Genetic variations, particularly gene amplification, chromosomal translocation and point mutation, have been shown to be excellent biomarkers for selection of targeted drugs. Because a spectrum of aberrant FGFR gene changes has been associated with prostate and breast tumorigenesis [16], we focus our attention on FGFR as new therapeutic target for treatment of BC [17-22]. 2. Fibroblast growth factor ligands and receptors

The FGFR family consists of four members, FGFR1, FGFR2, FGFR3 and FGFR4, which bind to their high-affinity ligands, 666

the fibroblast growth factors (FGFs) [23,24]. The FGF/FGFR signalling pathway has been shown to mediate cell proliferation, migration, motility, survival and other biological processes, including tissue repair, hematopoiesis and angiogenesis [25]. The aberrant regulation of this pathway has been implicated in many forms of human malignancies [26] and affects proliferation, anti-apoptosis, drug-resistance, epithelialto-mesenchymal transition (EMT) and invasion [23,27-31]. It has also been determined that activation of the FGF/FGFR pathway leads to an increase in tumour angiogenesis and may play a role in tumour resistance to anti-angiogenic and other chemotherapies [21,26]. The FGF family consists of 18 secreted glycoproteins: FGF1 (aFGF), FGF2 (bFGF), FGF3(INT2), FGF4, FGF5, FGF6, FGF7(KGF), FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22 and FGF23 [17]. The different FGFs and their corresponding receptors are expressed in a tissue-specific manner, which contribute to the specificity of the ligand--receptor interaction [21,24]. FGFs are secreted glycoproteins that are generally readily sequestered into the extracellular matrix, as well as in the cell surface, by heparan sulphate proteoglycans (HPSGs). To signal, FGFs are released from the extracellular matrix by heparinases, proteases or specific FGF-binding proteins, and the liberated FGFs subsequently bind to cell surface through the HPSGs. HPSGs also stabilise the FGF ligand--receptor interaction, forming a ternary complex with FGFR [32,33]. The specificity of the FGF--FGFR interaction is established partly not only by the differing ligand-binding capacities of the receptor paralogues [34,35] but also by alternative splicing of FGFR, which substantially alters ligand specificity. Other secreted proteins facilitate the FGF--FGFR interaction [36], such as the Klotho family [37], for hormonal FGFs, which further increases ligand specificity [21]. There are four FGFR genes (FGFR1, FGFR2, FGFR3 and FGFR4), located on chromosomes 8p12, 10q26, 4p16.3 and 5q35.1-qter, respectively [21,38,39]. A fifth related receptor, FGFR5 (also known as FGFRL1), is capable of binding FGFs [21]. FGFR genes are proto-oncogenes activated in human cancers as a result of gene amplification, chromosomal translocation and point mutation [17-22]. Encoded receptors consist of an extracellular ligand-binding domain, a singlepass transmembrane domain and an intracellular tyrosine kinase (TK) domain [24]. The extracellular domain has three Ig-like domains (IgI -- IgIII) with an acidic, serine-rich region between domains I and II (termed the acid box): the first Ig-like domain, together with the acid box, plays a role in receptor auto-inhibition; the second and third Ig-like domains are responsible for binding the FGF ligand [38,40,41]. In FGFR1 -- 3, alternative splicing in Ig-like domain III creates isoforms with different ligand-binding specificities (FGFR1 IIIb, FGFR2 IIIb, FGFR3 IIIb and FGFR1 IIIc, FGFR2 IIIc, FGFR3 IIIc) [38]. The FGFR IIIb isoforms are predominantly epithelial and the IIIc isoforms are

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Targeting fibroblast growth factor receptor in breast cancer

predominantly mesenchymal, with their corresponding ligands only activating either the epithelial or mesenchymal isoforms, except FGF1, which binds all receptor isoforms [35]. Thus, paracrine signalling is achieved by, for instance, epithelial cells producing ligands that only activate the corresponding mesenchymal FGFR IIIc isoforms and vice versa [40]. The intracellular kinase domain is similar to the VEGF receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) kinases in that it has an insert, resulting in a split kinase domain [40]. FGF ligand binding to the FGFR causes receptor dimerisation, trans-phosphorylation and activation of an intracellular TK domain that is separated into two contiguous active regions [21]. Several docking proteins (molecules containing Src homology 2 domains) bind to these phosphorylated residues, resulting in their phosphorylation and subsequent activation. Major pathways downstream of activated FGFRs include the rat sarcoma MAPK (RAS--MAPK) pathway and the phosphoinositide-3 kinase v-akt murine thymoma viral oncogene (PI3K [PIK3CA]--AKT [AKT1]) pathway whose activation is mediated via FGFR substrate 2a (FRS2a [FRS2]) and several other adaptor molecules, such as GRb2 [21,39,42]. FRS2 is a key adaptor protein that is largely specific to FGFRs, although it can also bind other TK receptors, such as neurotrophic TK receptor type 1, ReT and anaplastic lymphoma kinase 12. FRS2 binds to the juxtamembrane region of FGFRs through its phosphotyrosine-binding domains. The activated FGFR phosphorylates FRS2 on several sites, allowing the recruitment of the adaptor proteins son of sevenless and growth factor receptor-bound 2 (GRb2) to activate RAS and the downstream RAF and MAPK pathways [21,24]. A separate complex involving GRb2-associated binding protein 1 recruits a complex, which includes PI3K, and this activates an AKTdependent anti-apoptotic pathway [21,43]. Downstream signalling can be attenuated through the induction of MAPK phosphatases (MKPs), such as MKP3, Sprouty (Spry) proteins and SeF family members that modulate receptor signalling at several points in the signal transduction cascade [21]. Another prominent example is phospholipase Cg (PLCg), which binds to a phosphotyrosine in the C-terminal tail of the activated receptors. PLCg hydrolyses phosphatidylinositol 4,5-bisphosphate to produce diacylglycerol and inositol 1,4,5-triphosphate, which trigger the release of calcium and subsequent activation of PKC [21,40]. PKC partly reinforces the activation of the MAPK pathway by phosphorylating RAF [21]. Several other pathways are also activated by FGFRs, depending on the cellular context, including the p38 MAPK and Jun N-terminal kinase pathways, signal transducer and activator of transcription signalling [44] and ribosomal protein S6 kinase 2 [45] as described in Figure 1. In this way, FGF/ FGFR signals trigger a variety of responses in target cells, such as proliferation, anti-apoptosis, drug resistance, angiogenesis, EMT and invasion, that are aspects strictly implicated in cancer biology [17].

A major deactivation pathway for RTKs, termed receptor downregulation, involves their ligand-induced internalisation by means of endocytosis, followed by degradation in lysosomes [46]. Once activated, receptors can be removed from the cell surface by endocytosis [47]. Internalisation of activated RTK by endocytosis followed by sorting to lysosomes and subsequent degradation of the receptors is one of the ways cells achieve signal attenuation. After internalisation, endocytosed FGF/FGFR complexes reach early/sorting endosomes. From here, FGFR4 is sorted mainly to the recycling compartment, whereas FGFR1 -- 3 are sorted mostly to degradation in the lysosomes [48]. In vertebrates, other modulators of RTK signalling include the members of the Spry family of proteins, comprising various Spry and Spred (Spry-related proteins with enabled/ vasodilator-stimulated phosphoprotein homology 1 domain) isoforms. Depending on the cell type and physiological conditions, Sprys and Spreds modulate RTK signalling (and sometimes also modulate signalling by G-protein-coupled receptors) by mainly repressing the MAPK pathway [49]. RTK activity is tightly controlled also through the coordinated action of many other negative protein regulators that function at multiple levels of the signalling cascade, and at different time points after receptor engagement, and through microRNAs that have emerged as an abundant class of small (~ 22 nucleotides) non-protein-coding RNAs that play an important role in the negative regulation of gene expression, controlling the translational efficiency of target mRNAs [50]. Suppression pathways for RTKs are important to be considered because the failure of RTKs to be appropriately deactivated may be a cause of neoplastic growth [50,51].

3.

FGF/FGFR signal in BC

Fibroblast growth factor receptor 1 Several studies have identified amplifications of FGFR1 in BC [39,52]. The FGFR1 is one of the TK receptors for the pro-angiogenic FGF2, secreted by endothelial and tumour cells. Recent lines of evidence indicate that FGFR1 may play a significant role in the biology of BCs, in particular for the hormonal receptor positive and/or low grade BCs [21]. A variety of FGFR abnormalities have been identified in BC. FGFR1 amplification (8p11.2 -- p12 amplicon) is found in 8 -- 15% of all BCs [9,53,54]; it is correlated with FGFR1 protein overexpression [53,55,56] and it has been found as well in 16 -- 27% of luminal type B BC [55]. In addition, both cytoplasmic and nuclear expression of FGFR1 are elevated in invasive ductal carcinoma compared to normal tissue, thus predicting worse outcomes in terms of overall survival (OS) and disease-free survival (DFS). Further, using FISH probes on tissue microarray (TMA), FGFR1 amplification was shown to be more frequent in invasive BC than ductal in situ carcinoma, and these amplifications were more commonly located in the invasive components of tumours [57]. 3.1

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Figure 1. FGF-FGFR pathway is shown. FGF ligand binding to the FGFR causes receptor dimerisation, trans-phosphorylation and activation of an intracellular TK domain that is separated into two contiguous active regions. Major pathways downstream of activated FGFRs include RAS--MAPK pathway and PI3K--AKT pathway. FGF/FGFR signals trigger a variety of responses in target cells, such as proliferation, anti-apoptosis, drug resistance, angiogenesis, EMT and invasion, that are aspects strictly implicated in cancer biology. DAG: Diacylglycerol; EMT: Epithelial-to-mesenchymal transition; FGF: Fibroblast growth factor; FGFR: Fibroblast growth factor receptor; FRS2: FGFR substrate 2; GRb2: Growth factor receptor-bound 2; IP3: Inositol 1,4,5-triphosphate; PI3K: Phosphoinositide-3 kinase; PLCg: Phospholipase Cg; STAT: Signal transducer and activator of transcription; TK: Tyrosine kinase.

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Targeting fibroblast growth factor receptor in breast cancer

As such, it is proposed the activation of FGFR1 may drive the transition from the in situ to the invasive disease [57]. The amplification/overexpression of FGFR1 has been recently shown to be associated with a poor prognosis, early relapse and hormone resistance [55,56]. FGFR1 amplification remained a significant independent risk factor for poor DFS and OS in ER-positive but not in ER-negative cases [54]. The effects of FGFR1 amplification on prognosis were confirmed by chromogenic in situ hybridisation analysis of TMAs and subsequent correlation of FGFR1 overexpression to survival [54]. FGFR1 overexpression was shown to be an independent predictor of poor OS in ER-positive tumours [54]. These FGFR1-amplified ER-positive tumours are commonly endocrine therapy-resistant -- a result of increased ligand dependent and independent signalling, with enhanced MAPK activation promoting upregulation of the gene encoding cyclin D 1 [55]. The role of FGFR1 amplification in lobular carcinoma has also been investigated. Lobular BC usually shows poor responsiveness to chemotherapies and often lacks targeted for therapies. FGFR1 amplification and overexpression has been detected in 43% of classic lobular carcinomas, a subtype accounting for 5 -- 15% of invasive BCs [9]. Recently, Brunello et al. confirmed that a subset of metastatic lobular BC harbours FGFR1 gene amplification [58], thus suggesting that patients affected by the lobular BC positive for FGFR1 could be treated by the inhibitors of FGFR signalling [56,59]. Amplification of FGFR1 is uncommon in HER2-amplified tumours, thus suggesting that amplification of FGFR1 and HER2 may be mutually exclusive ways of activating similar downstream pathways [54].

Fibroblast growth factor receptor 2 A further link between FGFR signalling and BC has been provided by recent genome-wide association studies that identified FGFR2 as a BC susceptibility gene [21]. Using a network derived from 2000 transcriptional profiles, it was identified that SPDEF, ERa, FOXA1, GATA3 and PTTG1 are master regulators of FGFR2 signalling and was shown that ERa occupancy responds to FGFR2 signalling. Their results indicate that ERa, FOXA1 and GATA3 contribute to the regulation of BC susceptibility genes, which is consistent with the effects of anti-oestrogen treatment in BC prevention and suggest that FGFR2-related signalling has an important role in mediating BC risk [60]. A locus within an intron of the FGFR2 gene is consistently most strongly associated with BC risk [61-63]. FGFR2 amplification and enrichment was also detected in approximately 4% of triple-negative breast tumours [21]. In two triple-negative FGFR2-amplified cell lines, constitutive signalling appeared to confer a survival advantage over nonamplified cell lines [21]. The role of FGFR2 in these cancers has been confirmed by in vitro studies using a FGFR-targeted small molecule TK inhibitor (TKI) (PD173074) or RNAi 3.2

treatment, which reduced cell survival, blocked PI3K/AKT signalling and induced apoptosis [21]. Fibroblast growth factor receptor 3 FGFR3 gene mutations are common in certain cancers and thus this gene has been considered an oncogene. However, in some normal tissues, FGFR3 has been shown to limit cell growth and promote cell differentiation. Lafitte et al.’s data raise the possibility that FGFR3 has biphasic effects during multistage carcinogenesis in carcinomas, acting initially as a tumour suppressor through oncogene-induced senescence via signal transducer and activator of transcriptions (STATs) activation and apoptosis enhancement [64]. In this context, FGFR3 loss would help in tumour progression. Alternatively, after additional mutations have occurred in the developing tumour, decoupling FGFR3 from its canonical inhibitory pathway (STATs), FGFR3 signal might be redirected to other intracellular factors, promoting tumour progression. In the same way, epithelial-to-mesenchymal phenotype transition would reveal FGFR3 as an oncogene by coupling the receptor to MAPKs pathways. This model supports a new aspect of FGFR3 function, explaining why in epithelial cancers FGFR3-activating mutations in epithelial cancers were associated with good prognosis tumours, whereas in soft tissue cancers, FGFR3 promoted tumour progression [64]. Nevertheless, no FGFR3 mutations have been found in BC [39,65-67]. From the clinical point of view, the presence of elevated levels of FGFR3 in malignant breast tissues has been demonstrated in a number of studies [68], and a significant correlation between elevated levels and poor survival has been observed [69]. The function of nuclear FGFR3 is uncertain, but proteolytically cleaved FGFR3 has been reported to traffic to the nucleus [70] and nuclear FGFR1 has recently been reported to drive invasive behaviour of BC cells [71]. Also testing FGFR3 on TMAs from patients treated with tamoxifen, it was shown that the differential expression of FGFR3 was able to select those patients who responded from those who did not, based on the intensity of the staining [72]. 3.3

Fibroblast growth factor receptor 4 A single-nucleotide polymorphism in exon 9 results in an amino acid change (substitution of a glycine residue for an arginine -- Gly388Arg) within the FGFR4 transmembrane domain and results in a positive correlation with poor prognostic parameters in several human cancers, including breast, colon, lung, prostate and head and neck cancers [73-79]. However, its role in cancer is not yet clear [79-83]. Although all four FGFRs signal through a similar network of pathways in general, the kinase domain of FGFR1 drives stronger downstream pathway activation than FGFR4 [84]. There is also some evidence that differential responses to signalling are initiated by the FGFR1 and FGFR2 kinase domains, with more rapid attenuation of FGFR2 signalling mediated by receptor internalisation and degradation [21,85-87]. 3.4

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Meijer et al. showed that FGFR4 may play a role in the biological response of the tumour to tamoxifen treatment. The end points of their study were the clinical benefit of the therapy and the progression-free survival in patients with recurrent breast cancer. Gene mRNA levels were measured by real-time quantitative reverse transcriptase polymerase chain reaction in 285 ER-positive frozen primary breast tumours from patients who developed recurrent disease that was treated with tamoxifen as first-line therapy. The study showed that increasing levels of FGFR4 were related with a higher probability of tamoxifen failure [88]. Even if the complexity of intracellular pathways and of the cancer pathology and physiology suggested that FGFR4 mRNA levels are not merely associated with prognosis, FGFR4 could play a role in the biological response of the tumour to tamoxifen treatment. 4.

FGFRs role in BC treatment

FGFR-targeted drugs exert direct as well as indirect anticancer effects, as FGFRs are also expressed on endothelial cells and thereby effect angiogenesis/vasculogenesis, as well as tumorigenesis [89], based on the genetic aberrations in FGFRs identified and their consequences on a molecular level in BC patients. In this setting, several pharmaceutical companies have developed FGFR TKIs targeting FGFR signalling [39] that are in the early phases of clinical trials [21]. Inhibitors of FGFR TKs activities Dual inhibition with VEGFRs has the obvious potential benefit of targeting two pro-angiogenic growth factors or of simultaneously targeting angiogenesis and tumour cell proliferation. However, many of these TKIs with multiple targets are less potent against the FGFRs and it is uncertain if this will be a disadvantage in clinical response. Targeting multiple kinases may also increase the side effects of these compounds, limiting the ability to deliver drugs at doses required for FGFR inhibition. Consequently, several pharmaceutical companies are developing highly potent and specific FGFR TKIs, which are selective over VEGFRs [21]. AZD4547 (N-[5-[2-(3,5-dimethoxyphenyl)ethyl]-2Hpyrazol-3-yl]-4-(3,5 diemthylpiperazin-1-yl)benzamide) is a selective inhibitor of recombinant FGFR1, 2 and 3 TKs activities in vitro (IC50 values of 0.2, 2.5 and 1.8 nM, respectively) with significantly weaker activity against FGFR4 (IC50 165 nM). In vitro drug selectivity has been examined against a diverse panel of representative human kinases and AZD4547 has been shown to inhibit recombinant VEGFR2 (KDR) kinase activity with an IC50 of 24 nmol/l. However, when compared with FGFR1, this represents a selectivity of approximately 120-fold. Excellent selectivity for FGFR was observed across a range of unrelated TK and serine/threonine kinases, including IGFR (> 2900-fold), CDK2 (> 50,000-fold) and p38 (> 50,000-fold). AZD4547 inhibited recombinant FGFR kinase activity in vitro and suppressed 4.1

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FGFR signalling and growth in tumour cell lines with deregulated FGFR expression [25]. To date, seven trials are ongoing as displayed in Supplementary Table 1. PD173074, a selective FGFR1 TKI, was evaluated for its anti-angiogenic activity and anti-tumour efficacy in combination with photodynamic therapy (PDT). PD173074 displayed selective inhibitory activity toward FGFR1 TK at 26 nM. PD173074 demonstrated (> 100-fold) selective growth inhibitory action toward human umbilical vein endothelial cells (HUVECs) compared with a panel of tumour cell lines. PD173074 (at 10 nM) inhibited the formation of microcapillaries on Matrigel-coated plastic. In vivo antiangiogenesis studies in mice revealed that oral administration (p.o.) of PD173074 (25 -- 100 mg/kg) generated dosedependent inhibition of angiogenesis against a murine mammary 16c tumour, and significantly prolonged tumour regression was achieved with daily p.o. doses of PD173074 (30 -- 60 mg/kg) following PDT compared with PDT alone (p < 0.001) [90]. Ye et al. demonstrated that PD173074 could also inhibit the proliferation, migration and invasion and promote the apoptosis of murine mammary tumour cell line 4T1 [91]. Sharpe et al. showed that triplenegative breast cancer cell line (CAL51) in xenografts are sensitive to PD173074, thus suggesting that it is active based on FGFR2 expression in this subgroup in regard to the basal-like breast cancer [92]. However, PD173074 has been abandoned for clinical development due to toxicity as it has a narrow therapeutic window with relatively high toxicity, and future studies on the impact of pharmacological blockade of FGFR4 will require alternative approaches [93]. Dovitinib (TKI258) is an oral multi-targeted TKI with potent activity against receptors for VEGF, PDGF and basic FGF (bFGF). In contrast to other TKIs, it inhibits not only VEGFR1 -- 3 (IC50: 8 -- 13 nM) and PDGFRb (IC50: 12 nM) but also FGFR1 (IC50: 8 nM), FGFR3 (IC50: 9 nM) and FGFR2 (IC50: 40 nM). It, therefore, has the potential to have anti-tumour activity through inhibition of both FGFR and PDGFR, as well as anti-angiogenic activity through inhibition of FGFR, VEGFR and PDGFR [94]. Chase et al. demonstrated that, in the Ba/ F3-ZNF198-FGFR1 and BCR-FGFR1 cell lines, treatment with TKI258 resulted in the inhibition of cell proliferation and survival, accompanied by an inhibition of phosphorylation of the respective fusion proteins, ERK and STAT5 [95]. These results are similar to those demonstrated for the inhibitors SU5402, PD173074 and PKC412 in similar Ba/F3 cell line experiments [96-98]. Chase et al. have also demonstrated activity of TKI258 on proliferation, survival and apoptosis in both cell lines with cellular IC50 values similar to those obtained in the Ba/F3-ZNF198-FGFR1 cell line [95]. Andre´ et al. showed that dovitinib monotherapy inhibited proliferation in FGFR1- and FGFR2-amplified, but not FGFR normal, BC cell lines and also inhibited tumour growth in FGFR1-amplified BC xenografts. Andre´ et al. also clinically suggested that dovitinib showed anti-tumour

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activity in FGFR-amplified BCs and may have activity in BCs with FGF pathway amplification [99]. The 4T1 and 67NR cell line models of basal-like breast cancer have been used by Issa et al. for examining the impact of FGFR inhibition on tumour growth and metastatic spread [100]. They previously showed that blocking FGFR in vitro was sufficient to inhibit Erk and PI3K signalling and to induce cell death via blockade of the latter pathway. In vivo targeting of FGFR significantly slowed tumour growth, but neither tumour stasis nor strong inhibition of PI3K/Akt signalling was observed [101]. The PI3K/mammalian target of rapamycin (mTOR) inhibitor NVP-BEZ235 robustly blocks this pathway and the combination of dovitinib + NVP-BEZ235 had significantly better antitumour and anti-metastatic activities than treatment with single inhibitors. It is becoming clear that in vivo response to TKIs is optimal only when tumours show high levels of apoptosis. Indeed, the most durable tumour responses and the highest levels of apoptosis were observed in mice treated with the FGFR inhibitor in combination with either the PI3K/mTOR inhibitor or the pan ErbB inhibitor [100]. For both treatments, a strong inhibition of the FGFR/FRS2/Erk pathway and the PI3K/Akt/mTOR pathway was observed. Thus, it has been suggested that that combinatorial inhibition of FGFR and ErbB receptors may have a particularly significant impact on the in vivo tumour growth and metastatic spread of BC models [100]. New strategies based on dovitinib are now under development in clinical trials (Supplementary Table 1). Brivanib alaninate is an example of a TKI targeting both FGFRs and VEGFRs. Its effect in BC has so far only been tested in vitro: cell lines with FGFR1 amplification/ overexpression are more sensitive than non-amplified ones [102]. Nintedanib (BIBF-1120) targets additional pro-angiogenic intracellular signalling pathways beyond VEGF signalling and have also the potential to contribute to anticancer therapies. It targets not only VEGFRs but also FGFR and PDGFR [103]. NVP-BGJ398 (N-aryl-N¢-pyrimidin-4-yl ureas) has been optimised to afford potent and selective inhibitors of the FGFR1, FGFR2 and FGFR3 by rationally designing the substitution pattern of the aryl ring. Based on the in vitro data, NVP-BGJ398 was selected for in vivo evaluation and showed significant anti-tumour activity in RT112 bladder cancer xenograft models that overexpress wild-type FGFR3, supporting the potential therapeutic use of NVP-BGJ398 as a new anticancer agent [104]. Guagnano et al. showed that somatic mutations of FGFR family members predict sensitivity to NVP-BGJ398. NVPBGJ398 inhibits FGFR1, FGFR2 and FGFR3 with single digit nmol/l IC50 in biochemical and cellular autophosphorylation assays and FGFR4 with 38- to 60-fold lower potency. In cellular assays, the most potently inhibited kinase, in addition to the FGFRs, was found to be VEGFR2, displaying 70- to 100-fold reduced potency as compared with FGFR1,

FGFR2 and FGFR3. Therefore, NVP-BGJ398 is a selective, pan-FGFR kinase inhibitor, with predominant activity on FGFR1, FGFR2 and FGFR3 [105]. Lucitanib (E-3810) is a dual inhibitor of VEGFR and FGFR TKs inhibiting the kinase activity of VEGFR1, 2 and 3 and FGFR1 and 2 at nM concentrations [106]. In vitro E-3810 inhibits the VEGF- and bFGF-dependent proliferation and the signalling transduction pathways elicited by VEGF and bFGF receptor binding to their receptors in HUVECs in the nM range. Much higher concentrations (µM) were needed to interfere in vitro with the growth of different cell lines in non-ligand stimulated conditions, suggesting that the drug effect on tumour cells occurs at quite high concentrations and that primary effect of the drug is inhibition of VEGF and FGF signalling pathways, which are pivotal in the proliferation and survival of endothelial and stromal cells. In vivo 7 days of treatment with E-3810 completely inhibited the FGF-induced angiogenesis in an implanted Matrigel plug in mice; E-3810 treatment significantly reduced tumour vessel density in treated tumours (as assessed by the decrease in CD31 staining) by increasing the percentage of tumour necrosis and changing the composition of tumour stroma (with a decrease in collagen IV content) [107]. Bello et al. also showed a striking activity of the E-3810--paclitaxel combination with complete, lasting tumour regressions; the anti-tumour activity of the combination was also confirmed in another triple-negative breast xenograft, MX-1. The activity was superior to that of the combinations paclitaxel + brivanib and paclitaxel + sunitinib [108]. This drug is now in a Phase I clinical trials (Supplementary Table 1). Ponatinib (AP24534) is an oral multi-targeted TKI and has been explored as a pan-FGFR inhibitor in vitro and in vivo using a broad panel of engineered cell lines and cell lines derived from a variety of cancer types, including breast models. In all the 14 cell lines examined, ponatinib had the most potent inhibitory effect on cell growth. Ponatinib displayed greater potency compared with BIBF 1120 and brivanib and dovitinib across all models, with the greatest differences (2- to 13-fold increased potency) observed in cell lines containing dysregulated FGFR1 or FGFR2 [109]. FGFRs are expressed on various different cell types to regulate key cell behaviours and play an important role in tumour cell proliferation, differentiation, survival, cellmigration and angiogenesis [91]. On this assumption, a number of potent inhibitors of the FGFRs are in early phase clinical trials (Phase I or II) [110]. No drug is currently under Phase III trials. Currently there are insufficient data in breast cancer to state any one of the above therapies is better than another. Although there some proven targeted therapies that have radically changed the outcome of breast cancer, such as endocrine therapy for luminal-type breast cancer or trastuzumab for HER2-positive breast cancer, many cancer have de novo or develop resistances to targeted treatment or do not have a target such as triple-negative breast cancer (ER,

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progesteron receptor and HER2 negative). For these patients, it is important to evaluate alternative pathway to circumvent the mechanisms of resistance. For example, exemestane with mTOR inhibitor Everolimus was used in hormone-receptorpositive, HER2-negative, advanced breast cancer, following progression/recurrence after endocrine therapy with aromatase inhibitors to improve patients’ outcome. The disadvantage of this approach is that many of the TKIs are multi-targeted drugs blocking not only the FGFRs but also PDGFR family and VEGFR family at similar concentrations and lead to increased frequency of treatment-related adverse events.

Therapeutic antibodies To minimise the side effects of targeting FGFRs, therapeutic antibodies could be used which may have substantial benefits, as they can be used in treating cancer cells that are reliant on a particular FGFR and thereby reduce the potential toxicity of pan-FGFR inhibition. Antibodies targeting FGFR3 have been shown to have an anti-proliferative effect on bladder cancer cells and myeloma. A single chain Fv antibody that targeted FGFR1IIIc could not be pursued further as it was found to potentially block FGF signalling in the hypothalamus, resulting in severe anorexia in rodents and monkey models, but it still remains to be ascertained whether this would be a class effect for all FGFR1IIIc antibodies [21]. No therapeutical antibodies against FGFR or FGF are in clinical trials in BC currently, but there are many efforts to develop mAbs against this attractive therapeutic target. To address the role of FGFR2 in tumorigenesis and to explore FGFR2 as a potential therapeutic target, Bai et al. generated a mAb against the extracellular ligand-binding domain of FGFR2: GP369, an FGFR2-IIIb isoform-specific mAb. GP369 inhibited the ligand-induced phosphorylation of FGFR2 and its downstream signalling, as well as the proliferation driven by FGFR2 overexpression. Administration of GP369 in mice inhibited the in vivo growth of human cancer xenografts harbouring FGFR2 amplification [111]. Zhao et al. also generated and characterised several anti-FGFR2 mAbs and showed that they block the functional activity of FGFR2 in vitro and inhibit growth of FGFR2 overexpressing gastric tumour xenografts in vivo. Among the three mAbs developed, GAL-FR21, GAL-FR22 and GAL-FR23, the ability of the GAL-FR21 and GAL-FR22 mAbs, which are highly specific for FGFR2, is almost to completely inhibit the growth of SNU-16 and OCUM-2M in xenografts providing decisive evidence for the central role of FGFR2 in tumour growth [112]. These findings provide a rationale for clinically testing their therapeutic potential in human cancers with activated/amplified FGFR2 signalling [113]. As all FGFR TKI drugs inhibit several TK receptors in addition to FGFR2, mAbs may compensate this loss of selectivity in decreasing adverse events. Another avenue that interferes with ligand binding is the use of antagonistic peptide mimics, which have been designed 4.2

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for FGFR1-IIIc and FGFR2-IIIb and have shown apparent therapeutic potential [114]. Upstream intervention A further approach is to develop FGF ligand traps. FP-1039, a fusion protein, comprises the extracellular domain of FGFR1 and the Fc region of IgG [21]. FP-1039 has been shown to have anti-angiogenic effects in vivo. Moreover, FP-1039 was able to block tumour formation of BC cell line xenografts, depending on their expression of FGFs and FGFRs. As the FGFRs have several ligands in common, not only FGFR1 activation but also activation of FGFR3 and FGFR4 is blocked by binding of FP-1039 to FGFs, making FP-1039 a rather universal blocker of FGFR signalling [39,115]. To achieve blockade of the mitogenic FGFs while avoiding inhibition of hormonal FGFs, Harding et al. developed this soluble decoy receptor. The FGFR1c isoform (FGFR1 containing the c-splice region in domain III) was chosen because it has a broad FGF ligand-binding profile and does not bind the hormonal FGFs with high affinity in the absence of klotho. Consistent with these findings, FP-1039 binds to and inhibits the mitogenic members of the FGF family but exhibits little or no affinity for the hormonal FGFs. FP-1039 inhibits angiogenesis and the FGF/FGFR autocrine growth loops that drive tumour cell proliferation [116]. 4.3

5.

Conclusion

Despite improvements in BC detection and development of new therapeutic approaches, there are many breast cancers for which efficacious therapies are unavailable. Kinases inhibitors have become one of the most intensively pursued classes of drug target with many kinase targets being developed to the level of a Phase I clinical trial [117]. Because of the multiple mechanisms of action for FGFR inhibitors to overcome drug resistance, FGFR-targeted therapy is a promising strategy for the treatment of refractory cancer [118,119]. With regard to the BC, several studies have identified amplification of FGFRs such as FGFR1 and 2 [39,52] that play a significant role in the biology of BCs, in particular hormonal receptorpositive and/or low-grade BCs [21]. Indeed, amplification of FGFR’s signal is reported in up to one-third of BCs and is correlated with concomitant proteins’ overexpression [53,55,56]. The amplification/overexpression of FGFR1 has recently been shown to be associated with a poor prognosis, early relapse and hormone resistance [55,56], supporting the rationale for targeting the FGF/FGFR family members. The use of these changes as biomarkers for patient selection is seen in the study of Shiang et al. which demonstrated growth inhibition by brivanib, an FGFR1 inhibitor correlated with FGFR1 DNA copy number, mRNA and its protein expression measured in 21 cell lines [102]. Among non-amplified cells, there was no correlation between FGFR1 mRNA or protein expression levels and brivanib sensitivity. Two of three FGFR1 amplified cells were sensitive to bFGF-induced growth stimulation,

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Targeting fibroblast growth factor receptor in breast cancer

which was blocked by brivanib. In cells with amplified FGFR1, brivanib decreased receptor autophosphorylation, inhibited bFGF-induced TK activity and reduced phosphorylation of ERK and AKT. These finding suggest that FGFR family amplification or protein overexpression in breast cancers may be an indicator of anti-FGFR inhibitor-based treatment. The most advanced approaches for targeting the FGFR network are small molecule-like inhibitors of FGFR TK activities and blocking antibodies for specific receptors or ligands. Nevertheless, two important points that need to be considered when considering these drugs are, in particular, the FGFR inhibitor activity and the potential side effects of blocking FGFRs. First, the selection of patients by physicians is through molecular testing to determine tumour-specific gene amplification and/or protein overexpression as indicators for sensitivity. Second, as many of TKIs are multi-targeted drugs because they block at similar concentrations not only the FGFRs but also PDGFR family and VEGFR family, the side-effects profile need to be managed. In conclusion, although there are still some clinical problems to be overcome, there is good evidence suggesting that targeting FGFRs in certain subtypes of BC would be a valuable approach in the future. 6.

Expert opinion

The FGFs and FGFRs play an important role in a wide range of biological functions, controlling developmental events such as brain patterning, morphogenesis and limb development with multiple physiological functions in the adult, including angiogenesis, wound repair and endocrine functions. The majority of FGFs bind receptor in a trimeric complex with heparins, triggering a conformational change in the receptor that leads to activation of the FGFR that results in phosphorylation of multiple sites on the intracellular domain, adapter protein binding and intracellular signalling. The deregulation of FGF signalling in cancer results in activation of the pathway without appropriate regulation leading to/contributing to development of cancer, promoting cancer cell proliferation, survival and migration. Activation of the FGFR pathway is a common event in many cancer types, and for this reason FGFR is an interesting potential target in cancer treatment. Up to now several therapeutic approaches have been in use in clinical trials. Multiple different therapeutics are under development, so it is important to consider whether different therapeutic approaches lend themselves to specific oncogenic aberrations. Possible way to target FGFR pathway are mAbs binding FGFR, ligand traps or downstream blockage, but they are still in a very premature development phase. However, the most advanced in clinical development are TKIs. Different FGFR TKIs vary substantially in potency against FGFRs. Kinases with constitutive ligand-independent activation, through mutation or amplification, are generally more sensitive to TKIs than wild-type receptors. The first generation of inhibitors, represented by multi-targeting ATP

competitive inhibitors or the second generation of inhibitors which selectively target FGFRs with an undoubted higher potency are the most tested in clinical trials. The most advanced first-generation small molecules inhibiting FGFR are TKI258 (dovitinib) or/and BMS540215 (brivanib). Dovitinib targets FGFR, PDGFR and VEGFR. In a Phase II trial, treatment with dovitinib induced stable disease for > 6 months in the 25% of patients with FGFR1-amplified ER-positive and HER2-negative metastatic breast cancer [120]. Also recent data showed that the overexpression of FGFR1 in association with the mutational status of the PI3K could help in understanding those patients who will have the benefit from the combination therapies such as the exemestane and everolimus. The multiple different mechanisms through which FGF signalling can be activated necessitate a complex approach to clinical development. Only a subset of breast cancers is likely to be sensitive to FGFR inhibitors, and screening will be required to specifically identify cancers with amplification. One approach is to screen a very large number of patients; another approach is to potentially combine different cancer types with the same genetic aberration into a single trial but this requires the target and its downstream effect to be the same in different cancer subtypes. This has shown not to be the case with BRAF inhibition in melanoma and colorectal cancer, for example, the current knowledge suggests that FGFRs amplification status could be not only a predictive and prognostic marker, but it could be also a potential antitumour target and that FGFRs inhibition could be a valid approach for a selected subpopulation of breast cancer patient, probably in association with conventional therapies. Importantly, concrete progresses are being made in understanding how FGF signalling may impact breast cancer pathogenesis and progression, but we are only at the beginning of understanding how, and in which cancers, FGF signalling might be targeted for therapeutic benefit. Several questions arise about combination of FGFR inhibitors and standard chemotherapy or how FGFR signalling affects the response to chemotherapy. While we are waiting for further scientific and clinical research to clarify the potential role of FGFR targeting in breast cancer treatment, we believe the proper questions, in order to be ready when the drug will be available for the oncologist, are related to ‘how could we select the proper patient for the appropriate anti-FGFR drug?’. The real success will depend on understanding additional pharmacokinetic and pharmacodynamic factors that influence anti-tumour efficacy of these drugs targeting FGFRs, which are in development. Another practical issue that faces the clinical development of these targeted therapies is the validation, in tandem, of predictive markers for treatment sensitivity and resistance. Moreover the assay for such biomarkers should have sufficient sensitivity to detect low-frequency aberrations, in routine paraffin-embedded specimen, particularly as tumour cells can be heterogeneous and background material of normal tissue cannot be avoided. Ideally, this should be

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incorporated into a platform that is capable of multiplex testing of a large panel of candidate genes to fully utilise the typically small quantity of tumour genetic material available, such as a mass-array system or NGS, with some assays allowing copy number to be determined. However, this requires dedicated technological, human and financial resources that may not be readily available for the community practitioner, thereby requiring sending to a reference laboratory. Although immunohistochemical and TMA methods are readily available in pathology laboratories, this might be a feasible approach, although this may be hampered by availability of relevant antibodies, optimisation required and the general poor concordance in community laboratories with reference laboratories in quantitative testing. There is also a need to understand both intrinsic and acquired resistance to serial biopsies along each patient’s treatment course that will enable Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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Declaration of interest The authors state no conflict of interest and have received no payment in preparation of this manuscript.

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Targeting fibroblast growth factor receptor in breast cancer

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dimethoxy-phenyl)-1-{6-[4-(4-ethylpiperazin-1-yl)-phenylamino]-pyrimidin4-yl}-1-methyl-urea (NVP-BGJ398), a potent and selective inhibitor of the fibroblast growth factor receptor family of receptor tyrosine kinase. J Med Chem 2011;54(20):7066-83 105. Guagnano V, Kauffmann A, W€ohrle S, et al. FGFR genetic alterations predict for sensitivity to NVP-BGJ398, a selective pan-FGFR inhibitor. Cancer Discov 2012;2(12):1118-33 . In the era of personalised medicine, the paper showed data about the molecular profiles, useful in clinic, of potential predictors with sensitivity to FGFR inhibitors. 106. Chen TW, Bedard PL. Personalized medicine for metastatic breast cancer. Curr Opin Oncol 2013;25(6):615-24 107. Bello E, Colella G, Scarlato V, et al. E-3810 is a potent dual inhibitor of VEGFR and FGFR that exerts antitumor activity in multiple preclinical models. Cancer Res 2011;71(4):1396-405 108. Bello E, Taraboletti G, Colella G, et al. The tyrosine kinase inhibitor E-3810 combined with paclitaxel inhibits the growth of advanced-stage triple-negative breast cancer xenografts. Mol Cancer Ther 2013;12(2):131-40 109. Gozgit JM, Wong MJ, Moran L, et al. Ponatinib (AP24534), a multitargeted pan-FGFR inhibitor with activity in multiple FGFR-amplified or mutated cancer models. Mol Cancer Ther 2012;11(3):690-9 110. Cortes JE, Kim DW, Pinilla-Ibarz J, et al. A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias. N Engl J Med 2013;369(19):1783-96 111. Bai A, Meetze K, Vo NY, et al. GP369, an FGFR2-IIIb-specific antibody, exhibits potent antitumor activity against human cancers driven by activated FGFR2 signaling. Cancer Res 2010;70(19):7630-9 112. Zhao WM, Wang L, Park H, et al. Monoclonal antibodies to fibroblast growth factor receptor 2 effectively inhibit growth of gastric tumor xenografts. Clin Cancer Res 2010;16(23):5750-8 113. Ceccarelli S, Romano F, Angeloni A, et al. Potential dual role of KGF/KGFR as a target option in novel therapeutic strategies for the treatment of cancers

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115. Zhang H, Masuoka LB, Sadra AB, et al. 2007 LT FP-1039 (FGFR1:Fc), A soluble FGFR1 receptor antagonist, inhibits tumor growth and angiogenesis. AACR-NCI-ERTC International Conference, San Francisco 2007. p. B55 116. Harding TC, Long L, Palencia S, et al. Blockade of nonhormonal fibroblast growth factors by FP-1039 inhibits growth of multiple types of cancer. Sci Transl Med 2013;5(178):178ra39 117. Zhang J, Yang PL, Gray NS. Targeting cancer with small molecule kinase

inhibitors. Nat Rev Cancer 2009;9(1):28-39 118. Liang G, Liu Z, Wu J, et al. Anticancer molecules targeting fibroblast growth factor receptors. Trends Pharmacol Sci 2012;33(10):531-41 119. Hynes NE, Dey JH. Potential for targeting the fibroblast growth factor receptors in breast cancer. Cancer Res 2010;70(13):5199-202 .. The paper is a clear overview of FGFR signal transduction inhibitors focused on breast cancer. 120. Andre F, Bachelot T, Campone M, et al. Targeting FGFR with dovitinib (TKI258): preclinical and clinical data in breast cancer. Clin Cancer Res 2013;19:3693-702 .. The translational approach is fundamental in understanding the new drugs, which are available, as it helps

Supplementary material available online Supplementary Table 1.

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the clinicians to treat their patients. The paper is a very interesting translational study moving from the bench to the bedside on a FGFR inhibitor in breast cancer.

Affiliation Francesca Bedussi1, Alberto Bottini2, Maurizio Memo1, Stephen B Fox3, Sandra Sigala1 & Daniele Generali†2 † Author for correspondence 1 University of Brescia Medical School, Department of Molecular and Translational Medicine, Section of Pharmacology, Brescia, Italy 2 UOM di Patologia Mammaria/US Terapia Molecolare, Azienda Istituti Ospitalieri di Cremona, Viale Concordia 1, 26100, Cremona, Italy E-mail: [email protected] 3 Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, 3002, Australia