Fibroblast Growth Factor Signaling in Non–Small-Cell Lung Cancer

Review

Fibroblast Growth Factor Signaling in Non–Small-Cell Lung Cancer Thomas J. Semrad,1,2 Philip C. Mack1 Abstract Despite recent progress in the treatment on non–small cell lung cancer (NSCLC), outcomes remain suboptimal. Treatment advances that target the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) signaling pathways highlight the need to understand the multiple convergent growth factor signaling pathways involved in the pathogenesis of NSCLC. Signaling through fibroblast growth factors (FGF), long recognized for its pro-angiogenic activity, has recently emerged as a contributing factor in the pathogenesis and progression of NSCLC through an autocrine signaling loop. In addition, this pathway may function as a mechanism of resistance to anti-EGFR and anti-VEGF treatment. Clinical experience with FGF receptor (FGFR) inhibitors is mounting, and more specific inhibitors of this signaling pathway are in development. This review describes the structure of the FGF signaling pathway, delineates its dual roles in angiogenesis and proliferation in NSCLC, evaluates FGF ligand and receptor expression as prognostic biomarkers in NSCLC, and discusses the development of FGF pathway inhibitors for the treatment of lung malignancies. Clinical Lung Cancer, Vol. 13, No. 2, 90-5 Published by Elsevier Inc. Keywords: Angiogenesis, Cellular proliferation, Fibroblast growth factors, Fibroblast growth factor receptor, Non–small-cell lung

Introduction In the United States, lung cancer is the leading cause of cancerrelated death for both men and women, and accounted for 157,300 deaths in 2010.1 The majority of lung cancer cases (approximately 85%) are classified as non–small cell lung cancer (NSCLC) for which survival has minimally improved in the last several decades.2 Recently, novel treatments that target the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) have emerged from an improved understanding of growth factor signaling in NSCLC. Although EGFR inhibitors exhibit some benefit in unselected patients, activating mutations in the EGFR identify a subset of tumors that are exquisitely sensitive to EGFR tyrosine kinase inhibitors.3,4 Similarly, anti-VEGF treatment produces an improved outcome for certain patients with NSCLC,5 although defining pre-

1 Division of Hematology/Oncology, Department of Internal Medicine, University of California, Davis, Sacramento, CA 2 Section of Hematology/Oncology, The Department of Veterans Affairs Northern California Health Care System, Sacramento, CA

Submitted: May 12, 2011; Revised: Jul 27, 2011; Accepted: Aug 01, 2011 Address for correspondence: Thomas J. Semrad, MD, Division of Hematology/ Oncology, Department of Internal Medicine, University of California, Davis, 4501 X Street, Suite 3016, Sacramento, CA 95817 Tel: 916-734-3771; fax: 916-734-7946; e-mail contact: thomas.semrad@ ucdmc.ucdavis.edu

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dictive biomarkers for anti-VEGF therapeutic efficacy has been less successful.6 The experience with EGFR and VEGF inhibitors in NSCLC highlights the need to define other signaling pathways involved in tumor progression, angiogenesis, and resistance to currently available treatments. Fibroblast growth factors (FGF) comprise a complex family of signaling molecules that have been implicated in angiogenesis and inflammation in a wide variety of human disorders.7 Signaling of FGFs through the FGF receptors (FGFR) has been implicated as an autocrine signaling loop that leads to tumor proliferation and angiogenesis in a variety of NSCLC cell lines8 and is potentially a mechanism of resistance to both anti-VEGF and anti-EGFR therapies.9,10 In this review, we provide an overview of FGF signaling, consider its role in NSCLC, and describe potential therapeutic strategies for the ongoing development of FGF pathway inhibitors.

FGF Signaling The mammalian FGF family of growth factors consists of 18 distinct members in 6 subfamilies involved in multiple physiologic processes, including angiogenesis, organogenesis, tissue development, and endocrine signaling.7 The FGFR tyrosine kinases are coded by 4 genes (FGFR1, FGFR2, FGFR3, and FGFR4) but exist in numerous isoforms due to alternative messenger RNA splicing.7,11 The extracellular domain of FGFRs is composed of 2 or 3 immunoglobulin (Ig)-like binding loops.12 Importantly, tissue-specific alternative

1525-7304/$ - see frontmatter Published by Elsevier Inc. doi: 10.1016/j.cllc.2011.08.001

Figure 1 Overview of Fibroblast Growth Factor Signaling. Schematic Representation of Signal Transduction Pathways Activated by Signaling of FGFs Through FGFRs, HSPGs, and Integrins

FGF FGFRs

Integrins

HSPGs Transmembrane FRS2

PIP2 PLCγ1

GRB2 FAK

c-Src

SOS

PIP2 Raf

Ras

DAG+IP3 Ca2+

MAPKK PKC MAPK

Activation of Target Genes

Nucleus

Abbreviations: DAG ⫽ diacylglycerol; FAK ⫽ focal adhesion kinase; FGF ⫽ fibroblast growth factor; FGFR ⫽ fibroblast growth factor receptor; FRS2 ⫽ fibroblast growth factor receptor substrate 2; GRB2 ⫽ growth factor receptor bound protein 2; HSPG ⫽ heparan sulfate proteoglycans; IP3 ⫽ inositol 1,4,5-trisphosphate; MAPK ⫽ mitogen-activated protein kinase; MAPKK ⫽ mitogen-activated protein kinase kinase; PIP2 ⫽ phosphatidylinositol 4,5-bisphosphate; PKC ⫽ protein kinase C; PLC␥1 ⫽ phospholipase C-gamma 1; Raf ⫽ v-raf 1 murine leukemia viral oncogene homolog 1; Ras ⫽ retrovirus-associated DNA sequences; Src ⫽ v-src sarcoma viral oncogene homolog; SOS ⫽ son of sevenless.

splicing in FGFR1-3 of the invariant exon IIIa with exon IIIb tends to occur in epithelial cells, whereas splicing of exon IIIa with exon IIIc is preferentially seen in mesenchymal cells.13 Local paracrine loops can be generated by the epithelial-specific expression of ligands cognate for the mesenchymal isoform or, conversely, by expression of ligands for the epithelial isoform in adjacent mesenchymal cells. The binding of heparin sulfate glycosaminoglycan (HSGAG) to the FGF/ FGFR complex is required for dimerization, autophosphorylation, and activating of intracellular signaling.7 In addition, the binding of FGF to HSGAG traps FGF at the cell surface and protects the ligand from degradation (Figure 1).14 Signaling through FGFRs is mediated by direct recruitment of signaling intermediates to autophosphorylation sites on the activated receptor and by phosphorylation of fibroblast growth factor receptor substrate (FRS) docking proteins.15 Autophosphorylation of the receptor leads to binding and activation of phospholipase C-gamma, which results in the generation of the second messengers diacylglycerol and inositol triphosphate, that ultimately leads to activation of protein kinase Cs. In addition, activated FGFR phosphorylates the FRS2␣ and FRS2␤ docking proteins that recruit a multiprotein adaptor complex, including growth factor receptor bound protein 2 and son of sevenless. This complex ultimately activates both the

phosphatidylinositol-3-kinase and Ras/mitogen-activated protein kinase (MAPK) signaling cascades. Signaling through FGF/FGFR pathways is regulated by multiple feedback systems. Members of the Sprouty, Sprouty-related protein with EVH-1 domain, and similar expression to FGF families negatively regulate FGFR-induced MAPK signaling by binding to growth factor receptor bound protein 2, son of sevenless, and Raf1.16 MAPK phosphorylation of FRS2␣ reduces its binding to the FGFRs and thus reduces FGFR signaling.17 Moreover, interactions of FGF and FGFR with syndecans, integrins (especially ␣v␤3), Ncadherin, and neural cell adhesion molecule may modulate the intensity of signaling in different biologic contexts.18 Finally, there is emerging evidence that FGFs can induce signaling independent of the FGFR, although these signals largely seem to converge on the same intracellular pathways.18

Implications of FGF Signaling in NSCLC Signaling through FGFs has been implicated in cell proliferation, motility, and angiogenesis in a variety of human malignancies, including NSCLC.12,19 Mutations in FGFRs have only been identified in a small minority of NSCLCs.20,21 However, a case-control study of 274 Italian patients with surgically treated lung adenocarcinoma

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FGF Signaling in NSCLC compared with 401 controls suggested that a Gly388Arg polymorphism in the transmembrane domain of FGFR4 may impart a higher risk of recurrence.22 This polymorphism is associated with altered tumor cell motility in a breast cancer cell line.23 Further investigation in an expanded cohort of Italian patients with NSCLC associated the Gly388Arg polymorphism with an increased risk of node positivity (hazard ratio, 1.8 [95% confidence interval, 1.3-2.6]) and poorer survival (hazard ratio, 1.5 [95% confidence interval, 1.1-1.9]); however, the same associations were not seen in a smaller cohort of Norwegian patients in the same analysis.24 In addition, no impact of the Gly388Arg polymorphism was observed in 619 patients with lung cancer from the United Kingdom , although only 164 of these patients had adenocarcinoma.25 In a retrospective study of 387 Japanese patients with surgically treated NSCLC, poorer survival with the FGFR4 Gly388Arg polymorphism was not evident in the entire cohort (P ⫽ .4889).26 However, in the subset of patients who were node positive (n ⫽ 118), the Gly388Arg polymorphism was associated with a significantly (P ⫽ .0397) inferior overall survival. Current experimental evidence suggests that FGF signaling may play a major role in neoangiogenesis.27 FGFs induce endothelial cell proliferation in vitro28 and facilitate the degradation and reorganization of the extracellular matrix through upregulation of the urokinase-type plasminogen activator and matrix metalloproteinase systems in endothelial cells.29,30 In addition, activation of FGFR2 stimulates chemotaxis, which leads to capillary-like structures and endothelial cell reorganization when cultured on 3-dimensional matrices.31–33 Furthermore, FGFR2 regulates the expression of certain cadherins and integrins that contribute to the organization and maturation of new blood vessels.34 –36 There is emerging evidence that a subset of NSCLC relies on the FGF pathway for cellular proliferation through autocrine or paracrine signaling loops. Results of several studies have demonstrated the coexpression of specific FGFs, particularly FGF2 and FGF9, along with FGFR1 and FGFR2 in human lung cancers.37 FGF2 and FGF9 specifically bind the FGFR-IIIc splice variants of FGFR1 and FGFR2, which are expressed in NSCLC cell lines.8,9,38 Furthermore, inhibition of FGFR signaling through antisense RNA, RNA interference, naturalizing FGF2 antibodies, or FGFR tyrosine kinase inhibitors leads to inhibition of cellular proliferation and tumor growth in vitro.8 In addition, NSCLC cell line data suggest that transcriptional derepression of FGFR2 and FGFR3 expression is induced by EGFR inhibitors and that these reactivated receptors provide proliferation signals through the extracellular signal-regulated kinase (ERK) pathway.39 Moreover, FGFR1 amplification has recently been observed in approximately 20% of squamous cell NSCLC. These FGFR1 amplified tumors are exquisitely sensitive to FGFR inhibition in vitro, which suggests that FGFR1 may be a critical target in this subset of NSCLC.40 Together, these studies demonstrate multiple potential mechanisms for the induction of autocrine and paracrine signaling loops through FGFs and FGFRs that are necessary for tumor proliferation and survival in a subset of NSCLC, and suggest a subset of tumors that may have heightened sensitivity to FGFR inhibitors. Finally, the FGFR signaling pathway also has been implicated in the epithelial to mesenchymal transition (EMT), which is necessary for invasion and metastases of tumor cells, and has been associated

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with resistance to EGFR agents.41 FGF2 stimulates an EMT phenotype in vitro.42 In addition, the expression of both the plateletderived growth factor receptor (PDGFR) and FGFR1 are elevated in a NSCLC cell line (H358) induced into EMT by exogenous transforming growth factor ␤.43 Furthermore, FGFR1 expression is higher in NSCLC cell lines with a mesenchymal phenotype compared with those with an epithelial phenotype.43 Inhibition of FGFR reduces proliferation in these cell lines, which are relatively resistant to EGFR inhibition. Thus, FGFR signaling may stimulate the development of EMT and maintain cellular proliferation in the mesenchymal state.

FGF2 and FGFR1 Expression As Prognostic Biomarkers in NSCLC There are conflicting data on the prognostic impact of FGF2 expression in NSCLC. In a recent study of 335 patients with resected NSCLC, high tumoral FGF2 expression levels by immunohistochemistry (IHC) were associated with a poorer 5-year survival (59% vs. 37%; P ⫽ .015); however, no such impact was seen for a high expression of FGFR1 (P ⫽ .15).44 Interestingly, high stromal expression of FGF2 was associated with an improved 5-year survival (70% vs. 53%; P ⫽ .024), an effect that remained significant in multivariate analyses.44 Separate analysis of FGF2 expression by IHC in 111 resected stage I to III NSCLCs associated higher expression with poor prognosis (P ⫽ .0173),45 and a similar analysis of FGF2 IHC expression in 143 lung adenocarcinoma corroborated these findings (P ⫽ .0089).46 In addition, a poor prognosis was associated with higher levels of FGF2 measured by enzyme-linked immunosorbent assay (ELISA) on frozen tumor specimens in 71 patients with surgically resected NSCLC (P ⫽ .0059).47 These results were not corroborated by 2 separate studies that evaluated tumoral IHC expression levels and that did not find a significant prognostic value of FGF2 in 132 patients with resected stage I NSCLC48 or 206 patients with stage I to III NSCLC.49 However, the latter study did find a significant association with high levels of FGFR1 expression and poor prognosis (P ⫽ .025). Similarly discrepant results have been observed in studies that evaluated the prognostic impact of serum FGF2 levels in patients with NSCLC.50 FGF2 levels measured by ELISA have been associated with a better prognosis in 2 studies,51,52 and a poor prognosis in at least 3 others.53–55

FGF Signaling Inhibitors A variety of FGFR tyrosine kinase inhibitors and FGFR-targeted monoclonal antibodies are in development. Because of the considerable similarity among the tyrosine kinase domains of FGFRs, PDGFRs, and VEGFRs, a number of small molecules that developed primarily as VEGFR antagonists have anti-FGFR activity as well.9 The considerable overlap in activity of these agents for VEGFR-2 and PDGFR␤ clouds the ascertainment of activity that is dependent on FGFR inhibition. Indeed, it is quite likely that the toxicity induced by inhibition of alternative kinases such as VEGFR-2 precludes dosing of many of these agents to sufficient levels for therapeutic inhibition of FGFR. Published half maximal inhibitory concentration (IC50) values of selected multikinase inhibitors for FGFR1, VEGFR-2, and PDGFR are listed in Table 1. Cediranib (Recentin; AstraZeneca, London, U.K.) is an oral small molecule tyrosine inhibitor that is in advanced stages of clinical de-

Thomas J. Semrad, Philip C. Mack Table 1 Selected Tyrosine Kinase Inhibitors With FGFR1, VEGFR-2, and PDGFR␤ Activity IC50 (nM) FGFR1

VEGFR-2

PDGFR␤

8

13

27

XL999

8.2

2.6

1.5

E-381066

17.5

25

525

PD17307467

22

100-200

18

Cediranib (AZD2171)68

26

⬍1

5

Dovitinib (TKI258)64 65

57

BIBF 1120

69

21

65

Pazopanib69

140

30

84

Brivanib (BMS-540215)70

148

25

⬎6,000

Abbreviations: FGFR ⫽ fibroblast growth factor receptor; IC50 ⫽ half maximal inhibitory concentration; PDGFR ⫽ platelet-derived growth factor receptor; VEGFR ⫽ vascular endothelial growth factor receptor.

velopment. Although it principally inhibits VEGFRs, PDGFRs, and stem cell factor receptor (c-kit), FGFR1 is also a putative target. Interim analysis of a phase II-III study of cediranib vs. placebo in combination with carboplatin and paclitaxel (n ⫽ 251) as initial therapy for NSCLC suggested improved responses in the cediranib arm (38% vs. 16%; P ⬍ .001), although this study was halted due to excess toxicities, including higher rates of hypertension, hypothyroidism, hand-foot syndrome, and gastrointestinal toxicity on the cediranib arm.56 A similar study has been initiated with a lower dose of cediranib (NCT00795340). BIBF 1120 (Boehringer Ingelheim, Ingelheim am Rhein, Germany) is an oral small molecule inhibitor of FGFRs, PDGFRs, and VEGFRs as well as fms-like tyrosine kinase 3 and v-src sarcoma viral oncogene homolog.57,58 In a randomized phase II trial conducted in advanced NSCLC (n ⫽ 73) after failure of platinum-based chemotherapy, 48% of patients treated with BIBF 1120 had stable disease as the best tumor response and the median progression-free survival was 6.9 weeks.59 Toxicities were manageable, with grade 3/4 enzyme elevations, diarrhea, nausea, vomiting, and abdominal pain being the most common. In addition, 2 phase III trials that compared BIBF 1120 to placebo in addition to either docetaxel (LUME-Lung 1; NCT00805194) or pemetrexed (LUME-Lung 2; NCT00806819) as second-line treatments are underway. Pazopanib (Votrient; GlaxoSmithKline, Middlesex, U.K.) is an oral small molecule inhibitor of the VEGFRs, PDGFRs, and c-kit that has activity against FGFRs. Pazopanib is approved by the U.S. Food and Drug Administration for the treatment of advanced renal cell carcinoma. In a multicenter, open-label, phase II “window of opportunity” trial of 2 to 6 weeks of treatment with pazopanib in clinical stage I-II NSCLC (n ⫽ 26), 86% of patients had some tumor reduction, with 3 response evaluation criteria in solid tumors partial responses.60 Pazopanib was well tolerated in this study, with the most common adverse events consisting of grade 2 hypertension, diarrhea, and fatigue. Phase II/III clinical trials of pazopanib in NSCLC are ongoing (NCT01208064, NCT00775307). Additional kinase inhibitors of FGFR that are in various phases of clinical development include brivanib (BMS-540215; Bristol-Myers

Squibb, New York, NY), XL999 (Symphony Evolution, Inc, Rockville, MD), dovitinib (TKI-258; Novartis, East Hanover, NJ), E-3810 (Ethical Oncology Science, Milano, Italy), AZD4547 (AstraZeneca), and BGJ398 (Novartis). Brivanib is being tested in NSCLC as part of a randomized discontinuation trial in multiple tumor types (NCT00633789). A phase I trial of XL999 in NSCLC was terminated due to safety concerns (NCT00491699). Dovitinib is being studied in metastatic breast, urothelial, and prostate cancers, although no trials in NSCLC are currently ongoing. Single-agent phase I trials of E-3810 (NCT01283945), AZD4547 (NCT00979134 and NCT01213160), and BGJ398 (NCT01004224) are in process. Interestingly, the BGJ398 trial is limited to those patients with amplification of FGFR1 or FGFR2 or mutation of FGFR3. The development of monoclonal antibodies that inhibit FGFR has lagged somewhat behind the development of tyrosine kinase inhibitors, although the potential advantage of this approach is the ability to selectively inhibit specific FGFR isoforms.9 Fully human antibodies for FGFR1-IIIb and FGFR1-IIIc have been developed.61 In addition, soluble FGFR1 ligand traps have been developed that exhibit potent antitumor activity in human NSCLC xenografts.62 These agents remain in early clinical development, although a phase I trial of a FGF ligand trap (FP-1039; Five Prime Therapeutics, Inc, South San Francisco, CA) has completed enrollment (NCT00687505).

Discussion The FGF signaling pathway is aberrantly activated in at least a subset of NSCLC, which leads to tumor proliferation and/or angiogenesis. Emerging data suggest that some NSCLCs may rely on FGF signaling through autocrine or paracrine loops for proliferation and survival.8,40 In addition, the FGF pathway may serve as an angiogenic growth factor pathway that allows tumor escape from VEGF inhibition.10 Furthermore, the FGF signaling pathway has been implicated as a mechanism of resistance to anti-EGFR treatment.9 To date, the predominant evidence for a proliferative dependency on FGFR signaling in NSCLC is derived from squamous and large cell lung cancer, subtypes that are frequently intrinsically resistant to EGFR inhibitors.8,39,40 Because the elucidation of driver mutations in NSCLC (eg, EGFR and EML4-ALK) has been largely confined to the adenocarcinoma subtype, the discovery of FGFR1 amplification may represent a major breakthrough in the search for a therapeutic target in squamous NSCLC. Inhibitors of the FGF signaling pathway should be further explored in tumors with evidence for FGFR signaling dependence as well as in those that are resistant to antiVEGF and anti-EGFR treatment. An improved understanding of the role of FGF signaling in angiogenesis, proliferation, and resistance is required to optimize investigations into FGF pathway inhibition in NSCLC. Subsets of NSCLC addicted to FGFR mutations in an analogous manner to those with activating EGFR mutations have not been identified. The relative binding affinities of multitargeted agents for FGFR vs. other targets, for example, VEGFR, must also be taken into consideration. Because currently available agents have lower specificity for FGFR, it is difficult to ascertain the relative importance of FGFR inhibition to their therapeutic effect or whether FGFR is even being inhibited at doses used. Data derived from agents with increased relative potency

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FGF Signaling in NSCLC toward FGFR compared with other tyrosine kinases will be instructive. It will be important to identify the level of cross-talk and redundancy between these different signaling pathways to design combination strategies that tip the therapeutic balance toward cell death.63 Current preclinical evidence suggests that FGFR signaling may be an important proliferative pathway for squamous and large cell NSCLC, which are generally refractory to EGFR inhibition. In addition, cell line studies imply a potential for synergism of FGFR and EGFR inhibition in tumors that are susceptible to EGFR inhibition.38 These preclinical observations need to be prospectively validated in clinical trials.

Conclusions The FGF signaling pathway is an alternative growth factor signaling pathway associated with normal development and tumor angiogenesis. Preclinical evidence suggests that it may form an autocrine loop in a subset of NSCLC. Further evidence suggests that activation of the FGF signaling pathway may provide a mechanism of resistance to anti-VEGF and anti-EGFR therapy. FGFR inhibitors may have multiple potential therapeutic roles in NSCLC, including (1) a direct effect on tumor growth and survival through interference with FGFR autocrine signaling dependency, (2) an anti-angiogenic effect on tumors when using FGF as a pro-angiogenic stimulant, and (3) a means of overcoming FGF-mediated acquired resistance to angiogenic and tyrosine kinase inhibitors. A growing number of small molecule inhibitors of the FGFRs are in development, with varying degrees of selectivity and specificity. Further research is needed to clarify the contribution of FGF signaling to NSCLC pathogenesis and resistance to therapy to define a role of FGF pathway inhibitors in the clinical management of NSCLC.

Acknowledgments This work was supported by Boehringer Ingelheim Pharmaceuticals, Inc (BIPI). Editorial assistance was provided by Alyssa Tippens, PhD, of MedErgy, who was contracted by BIPI for these services. The authors met criteria for authorship as recommended by the International Committee of Medical Journal Editors and were fully responsible for all content and editorial decisions, and were involved at all stages of manuscript development. The authors received no compensation related to the development of the manuscript.

Disclosure All authors have no conflicts of interest.

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57. 58.

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