Emergent targets in metastatic breast cancer








Filippo DE BRAUD, MD

Giulia Valeria BIANCHI, MD
Elisa ZANARDI, MD
Department of Medical
Oncology 1
Fondazione IRCCS
Istituto Nazionale dei Tumori, Milan
ITALY

Emergent targets in metastatic breast cancer


by E. Zanardi, G. V. Bianchi, and F. De Braud,
Italy



Despite the important results of research to date, metastatic breast cancer (MBC) remains an incurable disease, therefore the research of new targets and targeted therapies is needed. Hormone receptors and human epidermal growth factor 2 (HER2) hyperexpression represent current drug targets in the management of MBC. Specific treatments against these targets enable a greater disease control rate and an increase in overall survival. Furthermore, recent evidence demonstrated that a multitarget approach is more effective than monotherapy. For example, in hormone receptor–positive MBC the association of hormone therapy with mTOR (mammalian target of rapamycin) inhibitors is superior to hormone therapy alone. In HER2-positive breast cancer the association of pertuzumab and trastuzumab is superior to trastuzumab alone. Fibroblast growth factor (FGF) and its pathway are involved in tumor growth, proliferation, invasion, and angiogenesis. It therefore represents an interesting new target for cancer treatment, particularly fibroblast growth factor receptor 1 (FGFR1), which is amplified in about 10% of cases of MBC expressing estrogen receptors (ER).This aberration is associated with poor prognosis. Several molecules targeting the FGF-FGFR pathway are under investigation, and two phase 1/2 studies have already demonstrated the efficacy of tyrosine kinase inhibitors in luminal B tumors. Further studies are needed to confirm these preliminary results, identify which molecules inhibit the FGFR pathway most effectively, and determine if these molecules should be administered in association with other targeted therapies. The results of ongoing studies will help to define the importance of the FGF-FGFR pathway as a new target in MBC.

Medicographia. 2015;37:248-255 (see French abstract on page 255)



Metastatic breast cancer (MBC) accounts for about 1 million new cases of cancer each year worldwide.1 Many systemic treatment options are available for advanced breast cancer, including endocrine therapy, chemotherapy, and anti–human epidermal growth factor 2 (HER2) molecules, but it remains the most common cause of cancer and cancer death in women. In recent years, the median survival of patients affected by breast cancer has dramatically improved, in particular in HER2-positive breast cancer and estrogen receptor (ER)–positive tumors, due to new, approved targeted therapies. In 1998, trastuzumab was approved by the US Food and Drug Administration (FDA) for the treatment of MBC, completely changing the prognosis and survival of patients in the metastatic setting. In a monoinstitutional retrospective analysis in 2010, Dawood et al observed that HER2-positive MBC patients treated with trastuzumab have a survival rate similar to that of MBC patients with HER2-negative disease, independent of the expression of hormone receptor.2 Recently, the results of the CLEOPATRA (CLinical Evaluation Of Pertuzumab And TRAstuzumab) and trial have further improved the prognosis of these breast cancer subtypes, demonstrating that the combination of two HER2 monoclonal antibodies (trastuzumab and pertuzumab) with docetaxel prolongs progression-free survival (PFS) and overall survival (OS) in comparison with the combination of trastuzumab and doce-taxel. In fact, median PFS was 18.5 months in the pertuzumab arm versus 12.4 months in the control arm (hazard ratio [HR], 0.62; 95% confidence interval [CI], 0.51-0.75; P<0.001); median OS was 56.5 months in the pertuzumab group versus 40.8 months in the control group (HR, 0.68; 95% CI, 0.56-0.84; P<0.001).3,4 In ER-positive HER2-negative MBC, everolimus was the first targeted therapy associated with hormonal treatment demonstrating a better PFS than endocrine therapy alone.5 Unfortunately, in triple-negative breast cancer (TNBC) no targeted therapies have been identified to date; chemotherapeutic options remain the standard of care, with dismal impact on survival for these patients. The expression of receptors of endothelial growth factor receptor and androgen are under investigation as possible targets in TNBC.6,7 Despite targeted therapies changing the scenario of breast cancer treatment, this disease remains incurable in the metastatic setting. The treatment of metastatic disease, therefore, remains a clinically meaningful unmet need, and many preclinical and clinical studies are in progress in order to identify new molecular targets, their relevance in tumor progression, and their potential role as anticancer drugs.





Figure 1
Figure 1. Interaction between cyclin D1 and CDK (cyclin-dependent kinase) 4/6 represents the key passage from the G1 to the S phase in the cell cycle.

Several mitogenic signals converge at the level of this complex. The interaction of CDK 4/6 with cyclin D1 is responsible for hyperphosphorylation of retinoblastoma (RB) tumor suppressor proteins; this results in pRB inhibition and release of E2F transcription factors and transcriptional regulation of genes determinant in G1/S transition and cell cycle progression through the restriction point (lightning bolt).
Abbreviations: Akt, protein kinase B; AR, androgen receptor; ER, estrogen receptor; G0, gap 0 (resting); G1, gap 1; G2, gap 2; M, mitosis; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; PI3K, phosphatidylinositol-4,5-bisphosphonate 3-kinase; PR, progesterone receptor; S, synthesis; STAT, signal transducer and activator of transcription; Wnt, Wingless-related integration site.
Copied from reference 8: Lange and Yee. Endocr Relat Cancer. 2011;18(4):C19-C24. © 2011,Society for Endocrinology.



Currently emerging new targets such as cyclin-dependent kinases (CDK) and phosphoinositide 3–kinase (PI3K) pathways are the most widely studied mechanisms implicated in both primary and secondary endocrine resistance. Cell cycle–related genes and proteins are frequently deregulated in breast cancer. The progression from theG1to Sphase is a key checkpoint during cellular replication, and the fundamental step in this process is the interaction between CDK and cyclin proteins. The primary target of CDK action is the retinoblastoma protein (pRb) in a phosphorylation process that leads to the release of transcription factors of the E2 promoter-binding– protein-dimerization partners (E2F-DP) family, permitting phase S entry (Figure 1).8


Figure 2
Figure 2. Fibroblast growth factor receptor (FGFR) structure and pathway.

FGFR is composed of three parts: the extracellular immunoglobulin (Ig)-like domain that binds fibroblast growth factor (FGF) ligands, the binding of which is stabilized by heparan sulfate proteoglycans (HPSG); the single-pass transmembrane domain; and the intracellular domain with
several tyrosine residues. The ligand-receptor binding determines activation of intracellular pathways, such as Ras-Raf-MAPK, STAT, PLCg, and PI3K. Signaling can be negatively regulated at several levels by inhibitors such as SPRY and SEF. The hyper-expression of FGF ligands or the downregulation of FGFR inhibitors determine the transcription of genes related to cell proliferation, survival, metastasis, and angiogenesis.
Abbreviations: AKT, protein kinase B; FGFRL1, FGFR-like 1 protein; FRS2α, FGFR substrate 2α; GAB1, GRB2-associated–binding protein 1; GRB2, growth factor receptor-bound protein 2; IP3, inositol triphosphate; MAPK, mitogen-activated protein kinase; CBL, E3 ubiquitin-protein ligase; DAG,
diacylglycerol; MAPK, mitogen-activated protein kinase; MEK, mitogen/extracellular signal-regulated kinase; MKP1, MAPK phosphatase 1; MKP3, MAPK phosphatase 3; P, phosphorylation; PI3K, phosphatidylinositol-4,5-bisphosphonate 3-kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; PKC, protein kinase C; PLCϒ, phospholipase Cϒ; SEF, similar expression to FGF genes; Sos, son of sevenless; SPRY, Sprouty; STAT, signal transducer and activatorof transcription.
Copied from reference 15: Turner and Grose. Nat Rev Cancer. 2010;10(2):116-129. © 2010, Macmillan Publishers
Limited.



Approximately 15% to 20% of human breast cancers exhibit amplification of the cyclin D1 gene (CCND1), and a higher proportion of tumors overexpress CCND1 protein. Many CDK4-6 inhibitors are under evaluation in clinical trials.9 The FDA recently approved palbociclib (Ibrance®, Pfizer), an inhibitor of CDK 4 and 6, in combination with letrozole for the treatment of postmenopausal women with ER-positive, HER2- negative advanced breast cancer, as a first-line endocrine therapy in metastatic disease. This accelerated approval was based on the results of a randomized multicenter phase 2 trial, in which the combination of palbociclib and letrozole improved PFS versus letrozole alone, in postmenopausal women with ER-positive, HER2-negative MBC who had not received previous systemic treatment for advanced disease.10

The PI3K/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) pathway is an intracellular pathway that leads to cell growth and tumor proliferation. This pathway is associated with resistance to endocrine therapy, HER2-directed therapy, and cytotoxic therapy in breast cancer.11,12 Multiple inhibitors of the PI3K/Akt/mTOR pathway are under investigation in preclinical or clinical trials, mostly in association with hormone therapies. Everolimus is a rapamycin analog that inhibits the mTORC1 protein complex of the mTOR complex. It is currently the only compound approved for the treatment of hormone receptor–positive, HER2-negative metastatic or locally advanced breast cancer, as previously described.5

Buparlisib is an investigational oral pan-PI3K inhibitor that targets the four isoforms of class I PI3K (α β ϒ δ).13 It has been widely studied in phase 2 and 3 trials in combination with endocrine therapies or with chemotherapy. Other compounds, such as GDC-0941 and BEZ235, are being evaluated in phase 1 and 2 clinical trials. The next promising target in breast cancer is the fibroblast growth factor (FGF) pathway, which will be discussed in the subsequent paragraphs.

FGF-FGFR signaling pathway

FGFs and their receptors, known as fibroblast growth factor receptors (FGFRs), play an important role in cancer pathogenesis. The effects of activation of this pathway not only concern tumor cells, but also surrounding stroma, and are involved in cancer cell growth, survival, migration, and angiogenesis.14 The FGF family comprises 18 small molecules residing mainly in the extracellular matrix. These ligands exert their effect by binding with four tyrosine kinase receptors (FGFR1, FGFR2, FGFR3, FGFR4); a fifth receptor (FGFR5) has no tyrosine kinase domain, but FGFs can still bind to it. The FGF-FGFR complex is stabilized by heparan sulfate proteoglycan (HPSG) on the cell surface or Klotho proteins, forming a ternary complex that increases the stability of the interaction.15

FGFRs are transmembrane receptors composed of three parts: an extracellular domain containing three immunoglobulin (Ig)–like fragments, a single-pass transmembrane domain, and an intracellular portion with a tyrosine kinase domain and a carboxylic acid tail.15 The binding between FGFs with the receptor determines FGFR dimerization and the activation of intracellular signaling through the process of tyrosine phosphorylation. This involves multiple pathways, which include the mitogen-activated protein kinases (MAPKs), PI3K, phospholipase Cϒ (PLCϒ), protein kinase C (PKC), and signal transducers and activators of transcription (STATs) pathways (Figure 2). Several feedback inhibitors of the FGF pathway, including members of the Sprouty (Spry) family and Sef (similar expression to FGF), have been identified. These inhibitors interfere at different steps of the pathway to block intracellular signaling. The down regulation of feedback inhibitors in association with an increased expression of FGF ligands through autocrine or paracrine production, and mutations/ amplification in FGFR, results in tumor cell proliferation, progression, meta-stasis, and angiogenesis.14,15

FGFR’s aberration in breast cancer
All four FGFRs have been studied in breast cancer to evaluate a possible correlation between breast cancer and deregulation of FGFR activity. Several studies report amplification of FGFR1 in about 10% of breast cancers. Evidence of a correlation between this amplification and biological markers, such as hormone receptors or HER2, is inconsistent, but evidence indicates that a correlation between FGFR1 amplification and worsening of breast cancer prognosis exists. ERpositive patients with FGFR1 amplification are more likely to develop metastases and have significantly shorter OS, independent of other prognostic factors such as tumor size, lymph node invasion, and grading. The prognostic correlation was observed only in ER-positive tumors, suggesting a negative interaction between FGFR1 and ER signaling that results in a poor prognosis.16 Moreover, Turner et al observed that FGFR1 amplification drives resistance to endocrine therapy in vitro and that FGFR1 amplification occurs more frequently in progesterone receptor (PR)–negative tumors. Loss of PR expression could reflect activation of FGFR signaling, and it is thought to be a biomarker of FGFR1 activity in breast cancer proliferation. This evidence suggests that FGFR1 amplification is one of the major drivers of luminal B breast cancer, which is associated with poor prognosis.17 Amplification of FGFR2 is observed in about 4% of TNBCs, while no amplification was found in the other subtypes.

No breast cancer–related amplification was observed with FGFR3, although FGFR3 could have a role in resistance to tamoxifen. Increased levels of FGFR3 protein were found in a group of patients that did not respond to treatment with tamoxifen.18 FGFR4 amplification was found in 10% of breast cancers in a small study, and they were associated with positive ER and PR status. In a retrospective analysis of ER-positive breast cancers treated with tamoxifen in a metastatic setting, high levels of FGFR4 were associated with poor clinical benefit and shorter PFS, suggesting a relationship between FGFR4 expression and tamoxifen failure.19 It is noteworthy that FGFR4 is one of the HER2-enriched specific genes included in the 50-gene intrinsic subtype predictor (PAM50),20 but the significance of FGFR4 activation in HER2-positive breast cancer is still unknown.21 The genomic aberrations described above lead to constitutive receptor activation responsible for cancer growth.15

FGF pathway and angiogenesis
FGFs are among the first angiogenic factors described. They regulate endothelial cells inducing proliferation, migration, and differentiation of endothelial cells, and creating a favorable environment for vasculature growth. Angiogenesis is a crucial point for tumor proliferation and metastatic diffusion, and FGFs play a central role by promoting cell growth in endothelial cells expressing FGFR. The main FGFR expressed by endothelial cells is FGFR1, but FGFR2 is also present in small amounts. The binding of FGF to FGFR stimulates new vessel formation and maturation by inducing endothelial cell proliferation, favoring extracellular matrix degradation, and altering intracellular adhesion.22


Figure 3
Figure 3. Schematic representation of fibroblast growth factor (FGF) effects triggered in endothelial cells to induce neoangiogenesis.
From reference 22: Presta et al.2005;16:159-178. © 2005, Elsevier Ltd.



Other important regulators of angiogenesis are the vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). Intimate crosstalk is thought to occur between VEGF/VEGFR and FGF pathways. In fact, FGF and VEGF synergistically induce vascularization, but each has distinct effects on tumor survival and vessel functionality; VEGF acts at the beginning of angiogenesis, whereas FGF stimulates vessel growth in both early and late angiogenesis (Figure 3, page 251).22

Since angiogenesis is one of the milestones for the development and progression of tumors, the study of this process is very important for the definition of new therapeutic strategies. In fact, the introduction of angiogenesis inhibitors has changed, in the last ten years, the natural history of locally advanced and metastatic renal cell carcinoma.23,24 However, clinical trials with these molecules in MBC did not produce the same results.25,26 In particular, treatment with bevacizumab, an anti-VEGF monoclonal antibody, in association with paclitaxel, resulted in a statistically significant increase in PFS compared with chemotherapy alone,27-29 but no significant difference in OS.30 For this reason, the FDA did not approve bevacizumab plus paclitaxel for the treatment of MBC.31 Prolongation of PFS with bevacizumab treatment highlights the importance of angiogenesis in tumor progression. However, the lack of OS benefit implies that the optimal way to inhibit this process or the right population in whom this drug should be used remains to be discovered. Considering that the VEGF and FGF pathways interact with each other in hyperneovascularization and disorganization of primitive tumor vasculature, it is thought that targeting both pathways may be more efficient than targeting one pathway alone at controlling cell proliferation and metastatic diffusion.

The FGF pathway as an emergent treatment target
As the FGFR signaling pathway may be a cause of breast cancer and related to poor prognosis,16 it represents an important development target for new treatments. Different agents— tyrosine kinase inhibitors (TKIs), monoclonal antibodies (mAb), and ligand traps—are currently being studied to evaluate their efficacy as inhibitors of the FGFR pathway.32 TKIs are small molecules targeting the ATP-binding site of the FGFR’s cytoplasmatic domain. They can be classified into two different families: nonselective or selective multikinase inhibitors.

The first family principally acts on other kinases, such as VEGFR, PDGFR (platelet-derived growth factor receptor), FLT3, RET (proto-oncogene encoding a receptor tyrosine kinase for members of the glial cell line-derived neurotrophic factor [GDNF] family), KIT (tyrosine-protein kinase Kit), and BCR-ABL (breakpoint cluster region–Abelson murine leukemia viral oncogene homolog), and induce a modest-but-significant inhibition of FGFR; the second family, selective inhibitors, specifically target FGFRs and are consequently more potent.

The first reported trial with a specific FGFR inhibitor evaluated dovitinib in metastatic breast cancer. André and colleagues suggested that dovitinib could have antitumor activity in FGFamplified tumors, but not in FGF-nonamplified tumors. In FGFR1-amplified tumors, with amplification detected not only by silver-enhanced in situ hybridization (SISH), but also with quantitative (real time) polymerase chain reaction (qPCR), a reduction in tumor size of up to 20% was observed. Furthermore, preclinical trials suggest that dovitinib is able to reverse endocrine resistance.17 These data indicate that dovitinib could be an important treatment in breast cancer, not only as a single agent, but also in combination with other therapies, such as endocrine therapy.33


Table I
Table I. Half maximal inhibitory concentration (IC50) of different nonselective multikinase
inhibitors.

Multikinase inhibitors currently being evaluated (yellow squares) are compared with molecules that have
already been approved in cancer treatment (red squares).
Abbreviations: FGFR, fibroblast growth factor receptor; PDGFR, platelet-derived growth factor receptor;
unk, unknown; VEGFR, vascular endothelial growth factor receptor.



Another important, recently published phase 1/2 trial evaluated the safety and efficacy of lucitanib in solid tumors. Lucitanib is a potent, highly selective inhibitor of the tyrosine kinase activity of FGFR types 1 and 2, VEGFR types 1 to 3, and PDGFR types α and β, which are essential kinases for tumor growth, survival, migration, and angiogenesis. This study started with a dose-escalation phase, in which the maximum tolerated dose (MTD) and the recommended dose (RD) were identified. MTD was 30 mg lucitanib once daily and RD for the next phase was 20 mg once daily, although this was subsequently adjusted to 15 mg because more than half of patients required dose reductions. The efficacy of lucitanib was in particular observed in FGF-aberrant breast cancers, for which the disease control rate was 100% (six patients with partial response and six with stable disease). In angiogenesis- sensitive patients, lucitanib is a potent inhibitor of FGFR and VEGFR, and it is probable that the double blockage of these pathways could explain the notable efficacy of lucitanib in FGFR1-amplified breast cancers.34 This contrasts with the limited activity of single inhibitors of VEGF or FGFR in breast cancers.35,36 Looking at the comparison between these nonselective multikinase inhibitors and those studied to date (for example, sunitinib, pazopanib, and axitinib, etc), the former have an IC50 (half maximal inhibitory concentration) inferior to the latter, suggesting that the former are more effective at inhibiting kinases (Table I). The spectrum of activity of lucitanib appears consistent, with clinical benefit in both FGFaberrant and angiogenesis-sensitive populations.

There are several ongoing phase 1 trials with selective TKIs, such as BGJ398, AZD4547, LY2874455, and JNJ-42756493. These TKIs in vitro are very potent inhibitors of FGFR1, FGFR2, and FGFR3. Preliminary results of phase 1 trials with AZD4547 and BGJ39836,37 don’t show a significant activity in breast cancers, but final data are still expected as well as the evaluation of these TKIs in association with hormone therapy (NCT01202591). Several mAbs against FGFR are in preclinical development: they can be highly specific for FGF ligand or FGFR isoform, and they are able to recruit the immune system via antibody-dependent cellular cytotoxicity or complement- dependent cytotoxicity, increasing antitumor activity. Until now no mAbs against FGFR have demonstrated activity in breast cancer and toxicity represents an important limit in evaluating this class of therapies.38 Future trials are needed to evaluate efficacy of this treatment strategy.

Another approach is the use of ligand traps, such as FP-1039, which consist of a modified extracellular domain of FGFR1 fused to the crystallizable fragment region of human immunoglobulin. It is thought that FP-1039 is able to sequester multiple FGF ligands, causing antiangiogenic and antitumor effects, as shown in preclinical in vivo studies.39 Unfortunately, a phase 2 study testing FP-1039 in endometrial cancer was deemed unfeasible because none of the 70 patients screened qualified, and nodata are available for breast cancers.

The development of these treatment strategies for the FGF pathway (Table II)32 demonstrates its importance in the cancer process. In breast cancer, FGFR1 amplification is thought to be the most important FGFR aberration responsible for tumor growth and progression. Therefore, the selection of patients with FGFR1 amplification probably represents the first step in identifying breast cancers that can benefit from target treatments of the FGFR pathway. FGFR1-amplified breast cancers are associated with poor prognosis, so developing a treatment that is able to improve prognosis of patients with these kind of tumors would be valuable. The first step is to find the most appropriate patients for this type of therapy by identifying, using SISH or qPCR, patients with FGFR1-amplified breast cancer.33 The second step is to identify the most effective types of treatment among those that are in development. Currently, nonselective multikinase inhibitors appear to be the most active treatment against FGFR1-amplified breast cancers, but these data need to be confirmed.

Furthermore, it is important to identify if TKIs should be used in monotherapy or in association with hormone therapy or chemotherapy. Data mentioned above demonstrate that FGFR1 amplification is related to endocrine resistance, so inhibition of this pathway could improve sensitivity to endocrine treatment. For this reason, the association of hormone therapy with FGFR inhibitors could be an efficient strategy. Positive responses at all these steps could lead to the definition of a new biological marker in breast cancer, as is the case with HER2, which would change the prognosis in a subgroup of our patients.


Table II
Table II. Fibroblast growth factor targeting therapies in clinical development.

Abbreviations: ABL, Abelson murine leukemia viral oncogene homolog; FGFR, fibroblast growth factor receptor; FLT, Fms-like tyrosine kinase; KIT, tyrosine-protein kinase Kit; PDGFR, platelet-derived growth factor receptor; RET, proto-oncogene encoding a receptor tyrosine kinase for members of the glial cell line-derived neurotrophic factor (GDNF) family; TKI, tyrosine-kinase inhibitor; VEGFR, vascular endothelial growth factor receptor.
From reference 32: Jain and Turner. Breast Cancer Res. 2012;14(3):208. © 2012, BioMed Central Ltd



Conclusion

At present, the identification of a target responsible for tumor cell proliferation, survival, and migration is a crucial goal in the development of a new treatment strategy. Hormone receptors in breast cancer were the first target in cancer treatment, until the delineation of the role of HER2 protein, which dramatically changed the treatment and prognosis in the 25% of breast cancers that are HER2 positive. In fact, the use of mAbs and TKIs that target the HER2 pathway have been shown to be effective in the neoadjuvant, adjuvant, and metastatic settings, which has led to significant increases in pathological complete response, PFS, and OS.40-42 Recent evidence has shown that agents targeting HER2 when used in association are more effective than monotherapy.4,43,44 Many other intracellular mechanisms of tumor growth are well known, and targeted therapies against different steps of cell proliferation are emerging. The PI3K/Akt/mTOR pathways and CDK pathways are crucial in tumor progression, and their blockade has promising efficacy in MBC. The FGF pathway, and in particular FGFR1 amplification, could also represent a new fundamental target in breast cancer management. FGFR1 seems to be responsible for endocrine resistance, so the association of hormone therapy with FGF-targeted therapy could be an effective strategy to overcome endocrine resistance and define a specific role for these agents. Also, the crucial role that FGF pathway plays in the angiogenic process is indicative of the importance that this new therapeutic target could represent. Several studies are warranted to define the most effective agents in FGFR-aberrant breast cancer and the best combinations to enhance antitumor activity. Moreover, predictive biomarkers are needed to facilitate the selection of the right population for these treatments, thus maximizing patient benefit. A further approach will be to act at different stages of these pathways to completely block mechanisms of tumor progression and avoid alternative ways for tumor cells to proliferate.

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Keywords: breast cancer; emergent; FGF pathway; FGFR; MBC; targeted therapy